Formulation containing essential oil of species of the genus Croton to inhibit oviposition of the Aedes aegypti mosquito.

Description

1 / 8 Formulation containing essential oil of species of the genus Croton to inhibit oviposition of the Aedes aegypti mosquito.

[001] The field of the invention relates to mosquito repellent. More specifically, to a natural repellent based on essential oil for protection against Aedes aegypti by means of oviposition deterrent action.

[002] In Brazil, more than a thousand cases of arbovirus infections are reported annually, with the highest number of infections and deaths related to dengue fever (Ministry of Health, 2023). In urban areas, the main form of transmission occurs through the bite of infected female mosquitoes of the species Aedes aegypti (Linnaeus, 1762), especially when they need blood for egg maturation. The mosquito is distributed throughout all federative units of the country (Foster; Walker, 2019), which shows the importance of methods of protection against vector bites.

[003] One of the methods of protection against mosquitoes is the use of repellents with spatial and topical action with synthetic active ingredients based on pyrethroids and N,N-diethyl-3-methylbenzamide. However, the scenario of high transmission rates involving dengue fever in urban areas in Brazil, for example, shows that the use of repellents alone has not been sufficient for protection against Aedes aegypti. Furthermore, studies show that the mosquito has been developing resistance to the aforementioned synthetic repellents (Yang et al., 2020), which demonstrates the importance of searching for complementary methods.

[004] In this context, the present patent application proposes complementary protection to the use of spatial and topical repellents against Aedes aegypti, based on an essential oil-based formulation to inhibit mosquito oviposition. This method is important not only for preventing or reducing oviposition by Aedes aegypti females in potential breeding sites, but also for leading to population reduction and consequently a decrease in the circulation of arboviruses transmitted by the mosquito. Petition 870250014934, dated 24 / 02 / 2025, page 7 / 20 2 / 8

[005] Studies show that DEET and transfluthrin can also act as oviposition deterrents against Ae. aegypti (Tikar et al., 2014; Bibbs et al., 2018), but as the mosquito has been showing resistance to their repellent actions, it is important to search for alternative active ingredients for oviposition deterrence. In this sense, essential oils have advantages, because in addition to already possessing different bioactive properties against mosquitoes, they are composed of numerous chemical components (Sousa et al., 2023), which can make it difficult for the vector to develop resistance.

[006] Among the plants that can synthesize essential oils are those of the genus Croton, including Croton sertanejus (Sodré & Da Silva, 2022) and Croton tetradenius (Baillon, 1864). The species C. sertanejus has a shrubby or arboreal physiognomy. It is endemic to the Caatinga biome and is widely dispersed throughout the Northeast, where it is popularly known as caatinga-branca, cassatinga, cassutinga, marmeleiro, marmeleiro-de-folha-miúda, marmeleiropreto and quebra-faca or quebra-facão (Sodré; Da Silva, 2022). C. tetradenius is a shrub endemic to Brazil with wide distribution also throughout the Northeast where it is known by the popular name caatinga de bode and velaminho da serra (Caruzo et al., 2020).

[007] After a review of scientific articles and patent databases, some articles and patents were found that used EOs in natural repellent preparations intended for vector mosquitoes. However, none described the use of C. sertanejus or C. tetradenius EO for the production of an oviposition deterrent repellent against mosquitoes, including Ae. aegypti, which shows the absolute novelty of the invention.

[008] Among the examples of repellents as oviposition deterrents is patent BR 102020015624-1, which presents the essential oil of Laurencia dendroidea containing acetone and water. However, caution should be exercised regarding the formulation, since acetone is a very volatile liquid (Lab synth, 2016), which may be more thermodynamically unstable, returning to its original state. Petition 870250014934, dated 24 / 02 / 2025, p. 8 / 20 3 / 8 oil / water and therefore result in the loss of solubility of the essential oil in water. The present patent application has the advantage of better stability and, consequently, improved solubility of the essential oil of C. sertanejus or C. tetradenius in aqueous medium, due to the surfactant Tween®80 being less volatile (Chemical Product Safety Data Sheet, 2007).

[009] Another patent, BR 102019010554-2, deals with a formulation based on the compound germacrene-D-4-ol as an oviposition deterrent aimed at inhibiting oviposition by female Ae. aegypti mosquitoes. The study that reveals that the formulation based on the essential oil of C. sertanejus or C. tetradenius proves to be more efficient because they have more than one chemical compound in their composition, which can delay the emergence of Ae. aegypti populations resistant to their deterrent action.

[010] Obtaining and preparing the oviposition deterrent repellent from the essential oils of C. sertanejus or C. tetradenius begins with collecting the leaves of these plants, which are then dried for subsequent extraction of the essential oil. Subsequently, different formulations based on these essential oils can be made, preferably, but not limited to, a solution containing a specific essential oil, the surfactant Tween®80, and distilled water. The formulation is important because it will ensure better solubility, dispersion, and effectiveness of its constituents, since the direct application of one of the essential oils as an oviposition deterrent becomes unfeasible due to the low solubility in water, rapid volatilization, and oxidation of the bioactive substances.

[011] To obtain the repellent as an oviposition deterrent, the process followed consists of the following steps: Step 1: Extraction of essential oils

[012] For the extraction of essential oils from the leaves of C. sertanejus or C. tetradenius immediately after collection in the morning, they were separately subjected to drying for 12 hours in a forced-air oven regulated at 45 °C. Each essential oil was obtained by hydrodistillation, from 100g of leaves, in 1.5 L of deionized water using a modified Clevenger apparatus, for 2 hours at 100 °C. The essential oils Petition 870250014934, dated 24 / 02 / 2025, page 9 / 20 4 / 8 of the obtained samples were dehydrated by adding anhydrous sodium sulfate and subsequently stored at a temperature of -4 ±1 °C in amber glass vials for later biological assay and chemical analysis. The collection and use of the botanical specimens in the research were in accordance with international and national guidelines on the use of biodiversity, followed by approval from the Brazilian Genetic Heritage, under number AF28031. Step 2: Biological assay: formulation preparation

[013] For the oviposition deterrence of the essential oil of each specimen, two concentrations were prepared, 5 and 2.5 mg / mL (0.5 and 0.25%, respectively) obtained from an adequate amount of EO dissolved in Tween®80, in a 1:1 ratio, and completed with 20mL of deionized water. Step 3: Preparing the assay for formulation evaluation.

[014] To evaluate oviposition deterrence, mated and nulliparous female mosquitoes ten days after emergence were used, originating from a stock population in which both sexes were maintained to allow mating. For the assay, goat blood (Capra aegagrus hircus L.) (Linnaeus, 1758) was used. The blood came from two male C. a. hircus from the Zootechnics sector of UESB - Itapetinga Campus, BA. The research was approved by the Animal Use Ethics Committee (CEUA-UFPE) and its complementary committees under registration number 23076.058811 / 2020.

[015] Until the experiments were set up, the mosquitoes were housed in polypropylene cages (16.5 x 25 cm) where a ten percent sugar solution was provided. 24 hours before the biological assay, the sugar solutions were removed and the females were transferred to test containers using an adapted suction device. The evaluations were carried out in a climate-controlled room at 27°C and 70% relative humidity.

[016] For evaluation, the methodology proposed by Santos et al. (2014) and Soonwera; Phasomkusolsil (2017) was used, with some modifications. As Petition 870250014934, dated 24 / 02 / 2025, page 10 / 20 5 / 8 concentrations evaluated were 5 and 2.5 mg / mL or 0.5 and 0.25%, with five replicates of 20 mosquitoes per cage, totaling 100 mosquitoes per treatment. Each concentration was evaluated as follows: with the 20 female mosquitoes in the polypropylene cage (16.5 cm x 25 cm), the artificial feeder with blood pre-warmed to 37 °C was offered for one hour. After this period, a 10% sugar solution was placed inside the cage. After three days, two 50 mL polystyrene containers, one with 20 mL of the solution containing the determined concentration of the essential oil and the other with 20 mL comprising the negative control, deionized water and Tween®80 equivalent to the higher concentration of the essential oils, were placed in opposite corners of the cage containing cone-shaped filter paper as a substrate for oviposition. After 96 hours, the papers were removed and the eggs were counted using a stereomicroscope.

[017] The average number of eggs deposited in the test and negative control cups was analyzed using a t-test (P < 0.05). The standard error for each mean was considered. The oviposition activity index (OAI) was also expressed, which was calculated using the formula established by Kramer; Mulla (1979). IAO = NT-Nc NT + Nc

[018] NT is the total number of eggs in the test solution and NC is the total number of eggs in the control solution. The oviposition activity index ranges from -1 to +1, with 0 indicating no response. A positive index value indicates that more eggs were laid and that there is no deterrence. Conversely, a negative value indicates that more eggs were laid and the test solution is deterrent.

[019] The percentage of effective repellency (RE) for each EO was also calculated using the following formula (Soonwera; Phasomkusolsil, 2017): Petition 870250014934, dated 24 / 02 / 2025, page 11 / 20 6 / 8 RE = Nc-NT Nc X100 where NC is the total number of eggs in the control solution and NT is the total number of eggs in the test solution. Step 5. Evaluation of the results of oviposition deterrence against Aedes aegypti

[020] Different concentrations of C. sertanejus and C. tetradenius EOs showed oviposition deterrent activity against Ae. aegypti, among which, containers containing only C. sertanejus EOs had a lower number of eggs. However, on average, the number of eggs from both EOs was significantly lower compared to the control group. The results can be seen in Table 1. Table 1. Oviposition deterrent of essential oils of Croton sertanejus and Croton tetradenius against Aedes aegypti after 96 hours. OEs Con ceo traction (Ή) Average of oves ± TP IÁO RE {%) P value Treatment Control C. íerianejuj 0.5 0.4'= 0.2 1551 = j22 -0.99 99.5 0.009 0.23 4.6· = 2.9 W =49.7 -0.95 97.4 0.004 C. isiradeníuz 0.5 42· ±2.5 184''=42 -0.95 97 0.013 0.25 13·= 8.8 2591 = 15.8 -0.90 74 0.000 =JEP = standard error. IAO = activity index of Dvtppsição. RE = effective repellency. Means followed by different letters in the rows differ significantly by the t-test, 7--0.05.

[021] Because the EO of C. sertanejus showed better egg inhibition at the highest concentration, the potential of the concentration of the specimen only when applied to the filter paper used as an oviposition substrate, and not to the water, was also investigated. It was observed that the concentration still inhibited oviposition, as described in Table 2. However, the average number of eggs deposited was lower when compared to Table 1, indicating that the product's effectiveness is best when applied to the water and not just to the sides of breeding tanks. Petition 870250014934, dated 24 / 02 / 2025, page 12 / 20 7 / 8 Tib ela. 2. Detection of oviposition of the best concentration of Croton sertanejas essential oil against Aedes aegypti applied only to the oviposition substrate, after 96 hours. OE Cod ceo traction Average of eggs ±EP IAO RE(%) P value Treatment Control C. zertanejur 0.5 45.8 = = 312 159^ = 22.9 -0.67 73.6 0.021 =EP = standard error. ΪAO = oviposition activity model. RE = repetition. Means followed by different letters in the rows differ significantly by the i-test P<0.05. Step 6. Chemical constituents of the essential oils of Croton sertanejus and Croton tetradenius

[022] The chemical composition analysis of the essential oils of C. sertanejus and C. tetradenius was performed by Gas Chromatography coupled to a Mass Spectrometer. The identification of the components was based on their retention indices (RI), calculated for each component by injecting a series of linear hydrocarbon standards (C8-C26) under the same conditions as the sample and compared with the values ​​tabulated in the literature (Adams, 2007), as well as through the library database (Nistl 1). Step 6.1: Results of the main constituents found

[023] Nineteen constituents were identified in the essential oil of C. sertanejus and 23 compounds in the essential oil of C. tetradenius, classified as monoterpenes and sesquiterpenes. However, the major compounds of the essential oils are shown in Table 3. Petition 870250014934, dated 24 / 02 / 2025, page 13 / 20 8 / 8 Tib ela. 3. Main chemical constituents of the essential oils of Croton sertanejas and Croton tetradenias Constituents Eucalyptus! C. sertanejas (j / df 13.8 C. tetradenias Ç:' ó)a IRb 1030 IKC 1031 Bi ci cio gemí acr eno 8.95 1510 1500 y-Cadinene 6.22 1493 1513 Spatulenol 6.16 1595 1578 Camphor 33.39 1150 1146 p-Cymene 19.29 1025 1024 Myrcene 5.05 991 990 a-Terpinene 4.81 1016 1017 'Constituents listed in order of elution in FourVF-5ms capillary column; retention balances with FourAT-jms capillary column; Kovats indices in DB-5 capillary column (Adans. 2007).

[024] This invention is not limited to the representations discussed or illustrated herein and should be understood in its broad scope. Many modifications and other representations of the invention will come to mind for those skilled in the art to which this invention pertains, having the benefit of the teaching set forth in the preceding descriptions and accompanying drawings. Furthermore, it is to be understood that the invention is not limited to the specific form disclosed, and that modifications and other forms are understood to be included within the scope of the appended claims. Although specific terms are employed herein, they are used only in a generic and descriptive sense and not for the purpose of limitation. Petition 870250014934, dated 24 / 02 / 2025, page 14 / 20

Claims

1 / 1 CLAIMS 1. Formulation characterized by containing essential oil of the species Croton sertanejus or Croton tetradenius.

2. Formulation according to claim 1, characterized by preferably using a solution of Tween®80 and deionized water as a vehicle.

3. Formulation according to claim 2, characterized in that the essential oils of Croton sertanejus or Croton tetradenius are formulated in any concentrations, preferably from 2.5 mg / mL.

4. Formulation, according to claim 3, characterized in that the formulation is applied to mosquito breeding sites, on the sides near the water level or directly into the water, preferably into the water. Petition 870250014934, dated 24 / 02 / 2025, page 15 / 20