Use of Croton sertanejus essential oil in formulations for repellent purposes against Aedes aegypti.

Description

1 / 13 Use of Croton sertanejus essential oil in formulations for repellent purposes against Aedes aegypti.

[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 through spatial and topical action.

[002] Every year in the Americas, high numbers of arboviral infections are recorded, such as dengue, chikungunya, and Zika. Among these arboviruses, the highest numbers are for dengue, as seen mainly in 2019 and 2023. In 2024, the incidence of dengue continues to increase on the continent, where Brazil is one of the epicenters of transmission, with the Aedes aegypti mosquito (Linnaeus, 1762) as the main vector (Pan American Health Organization, 2024).

[003] Among the types of mosquito repellents commonly used are those for spatial and topical use, based on synthetic active ingredients. Spatial repellent is dispersed in the air, in residences, and aims to prevent mosquitoes from entering the treated rooms. Topical repellent is applied mainly to the skin and aims at individual protection by contact. The active ingredients commonly used in such repellents are based on pyrethroids and benzamides, such as 2,3,5,6-tetrafluorobenzyl (lR,3S)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (transfluthrin) and N,N-diethyl-3-methylbenzamide (DEET), respectively (Norris; Coats, 2017). However, the indication of resistance of Ae. aegypti to these substances warns that their protections are compromised (Yang et al., 2020).

[004] In addition to the above, transfluthrin and DEET can cause harm to human health. Regarding transfluthrin, studies in rats via inhalation have shown that the compound has toxicological potential even at commercial concentrations, including toxicity to the trachea, lung, liver, kidney, dysregulation of the estrogen alpha receptor and alterations in the reproductive functions of male rats (Mahato et al., 2015; Kartikasari et al., 2020, Oyeniran et al., 2021). Petition 870250014937, dated 24 / 02 / 2025, page 6 / 24 2 / 13

[005] Regarding DEET, effects include encephalopathy in children, severe toxicity after inhalation exposure, anaphylaxis, hypotension, urticaria syndrome, and decreased heart rate (Tavares et al., 2019, Vama-Etienne et al., 2020). Also regarding the repellent, in-vitro assays indicate that DEET induces cytotoxicity in the nasosinusal epithelium (LEE; BASAK, 2021).

[006] Therefore, the search for new repellent alternatives against Ae. aegypti is important, such as repellents based on essential oils (EOs). EOs are volatile substances, essentially consisting of mixtures of terpenoids and related aromatic compounds originating from the secondary metabolism of plants (Dhifi et al., 2016), which makes them viable for the development of new repellents. Compared to the synthetic repellents mentioned, EOs are advantageous because they contain biocompatible properties and numerous chemical compounds (Unalan; Boccaccini, 2021; Sousa et al., 2023), which can provide greater health safety and make it more difficult for the vector to develop resistance.

[007] Some species of the genus Croton, considered the second largest in the Euphorbiaceae family, can synthesize essential oils, among them, Croton sertanejus (Sodré & Da Silva, 2022) (Sodré; Da Silva, 2022). The species has a shrubby or arboreal physiognomy. It is endemic to the Caatinga biome and has a wide distribution throughout the Northeast, where it is popularly known as caatinga-branca, cassatinga, cassutinga, marmeleiro, marmeleiro-de-folha-miúda, marmeleiro-preto and quebrafaca or quebra-facão (Sodré; Silva, 2022).

[008] The choice of a species from the genus Croton was based on the promising repellent results that some of its species have shown against Ae. aegypti (Sousa et al., 2023), which despite the potential, repellent studies against the vector involving the genus are still incipient, which demonstrates the importance of investigating new species aiming at more potent repellents, such as the EO of C. sertanejus.

[009] The use of essential oil from a plant native to the Caatinga by this patent application, in addition to valuing the national flora, also contributes to the Petition 870250014937, dated 24 / 02 / 2025, page 7 / 24 / 13 conservation of the biome and the source of income for residents of the region, by encouraging the cultivation of C. sertanejus specimens.

[010] 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 EO for the production of a repellent with spatial or topical activity against mosquitoes, including Ae. aegypti, demonstrating the absolute novelty of the invention.

[011] Among the examples of space repellents, there is patent BR1020120104520, which deals with a repellent candle containing as active ingredients EOs of four plant species of the genus Croton: Croton zenhtneri, Croton sonderianus, Croton nepetaefolius and Croton argyrophylloides, and patent PI0103766-8, which contains a cellulose pellet and EOs of Garapa guianensis and Eucalyptus citriodora as active ingredients. However, attention should be paid to some aspects of the inventions cited, because in addition to mixtures of EOs aiming at synergistic activities increasing the cost of repellent formulations, candles are dangerous due to the high risk of fire. The present patent application proves to be more efficient, as it presents a single essential oil as the active ingredient in the formulation, which may be more economically viable in obtaining a space repellent, in addition to being able to be incorporated into any type of repellent vehicle, without necessarily prioritizing the use of a candle.

[012] Regarding topical repellents, patent BR1020130337641 describes the use of Siparuma guianensis essential oil in different formulations, such as ointments and creams. The S. guianensis essential oil showed approximately 98% repellency against Ae. aegypti in a single evaluation after 15 minutes at the highest concentration of 20%. However, the present patent application proves to be more efficient, since the C. sertanejus essential oil at a lower concentration (10.9%) showed better protection with 99.4% repellency in 30 minutes of evaluation, that is, twice the time of the cited patent BR1020130337641. Petition 870250014937, dated 24 / 02 / 2025, p. 8 / 24 / 13

[013] In contrast to patent BR1020130337641, the present patent application reveals further protection at this concentration (10.9%), as the EO of C. sertanejus, in addition to protecting against landing, prevents the Ae. aegypti mosquito from feeding on host blood by 100% for 150 minutes, equivalent to 2 hours and 30 minutes. Considering that the Ae. aegypti mosquito transmits arboviruses through blood feeding, the protection offered by the EO of C. sertanejus against this activity demonstrates its potential as a natural alternative repellent to control the transmission of dengue, zika and chikungunya transmitted by Ae. aegypti in Brazil and worldwide.

[014] The obtaining and preparation process of the space and topical repellent from the essential oil of C. sertanejus begins with the collection of the leaves of this plant, which are then dried for subsequent extraction of the essential oil. Subsequently, formulations based on this essential oil can be made. For the space repellent, a solution containing the essential oil, surfactant Tween®80, and distilled water was prepared, preferably but not limited to. As for the topical repellent, the incorporation of the essential oil into a non-ionic emulsion was preferred but not limited to. Direct application of the essential oil to the air and skin becomes unfeasible due to its low solubility, rapid volatilization, and oxidation of bioactive substances. Also regarding the topical repellent, the essential oil formulated in an emulsion helps to spread better on the skin. Thus, the application of the essential oil in different formulations ensures solubility, better dispersion, and effectiveness of its constituents.

[015] To obtain the space and topical repellent, the process followed consists of the following steps: Step 1: Extraction of the essential oil

[016] To extract the essential oil from C. sertanejus leaves immediately after collection in the morning, they were subjected to drying for 12 hours in a forced-air oven regulated at 45 °C. The essential oil was obtained by hydrodistillation from 100g of leaves in 1.5 L of deionized water, using a Clevenger apparatus. Petition 870250014937, dated 02 / 24 / 2025, page 9 / 24 / 13 modified for 2 hours at 100 °C. The obtained essential oil was dehydrated by adding anhydrous sodium sulfate and subsequently stored at a temperature of -4 ± 1 °C in amber glass bottles for later biological assay and chemical analysis. The collection and use of the botanical specimen in the research was 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: preparation of repellent formulations

[017] For the space repellent, five concentrations were prepared: 5; 2.5; 1; 0.5 and 0.2 mg / mL, which were obtained from an appropriate amount of EO dissolved in Tween®80, in a 1:1 ratio, and completed with 10 mL of deionized water.

[018] Regarding the topical repellent, five concentrations were first prepared to estimate an effective repellent concentration of 50 and 99% repellency. The concentrations were: 125; 100; 75; 50 and 25 mg / cm2. Each concentration was diluted in 1g of a non-ionic emulsion. The effective concentration was also diluted in 1g of a non-ionic emulsion. Step 3: Preparation of biological assays for evaluating repellent formulations.

[019] To evaluate spatial repellency, seven-day-old non-mated female Ae. aegypti mosquitoes were used. Regarding topical repellency, mated and nulliparous female mosquitoes ten days after emergence were used, originating from a stock population where both sexes were maintained for mating. To estimate the effective repellent concentration and the protection time of the effective repellent concentration, which comprise the topical repellent, 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 Committee of Petition 870250014937, dated 24 / 02 / 2025, page 10 / 24 / 13 Ethics Committee on Animal Use (CEUA-UFPE) and its complementary guidelines under registration number 23076.058811 / 2020.

[020] 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 assays, the sugar solutions were removed and the females were transferred to test containers with the aid of an adapted suction device. The evaluations were carried out in a climate-controlled room, at 27°C and 70% relative humidity. Step 4: Spatial Repell

[021] The biological assay was performed according to the Guidelines for Efficacy Testing of Space Repellents (World Health Organization, 2013), using a horizontal double-choice olfactometer adapted by Sousa et al. (2023), which consists of a central chamber and two side chambers lined with black oxford fabric. To evaluate the concentrations (5; 2.5; 1; 0.5 and 0.2 mg / mL), ten replicates containing 20 mosquitoes were used, totaling 200 mosquitoes per treatment. Each concentration was evaluated as follows: with 20 mosquitoes in the central chamber, one side chamber was treated with 1 mL of the EO concentration on a strip of filter paper on a glass slide (26x76 mm) of the same dimensions. The other chamber was not treated. Subsequently, the side chambers were lined with black oxford fabric. After one minute, the lids of the central chamber were opened simultaneously for ten minutes.At the end of the time period, the lids were closed and the mosquitoes in the treated and untreated chambers were counted. These procedures were applied to the negative control, with deionized water containing 5 mg / mL of Tween®80, which is equivalent to the highest concentration of the essential oil.

[022] Spatial repellency was assessed using the spatial activity index (SAI) and the equation established by the (World Health Organization, 2013): Petition 870250014937, of 02 / 24 / 2025, p. 11 / 24 / 13IAEÁNc + NtJ / Nm\

[023] Nc means the number of mosquitoes present in the tube without the formulation; Nt is the number of mosquitoes in the tube with the formulation; Nm is the total number of mosquitoes at the ends of the two tubes; N is the total number of mosquitoes used in the assay. The spatial activity index ranges from -1 to 1, where zero indicates no response, -1 indicates no repellency, and 1 indicates a repellent spatial response. The results of the spatial activity index were subjected to analysis of variance (ANOVA), and the differences between the means were compared using Tukey's post-hoc test at a significance level of 5% (P < 0.05). The standard error of each index was considered. Step 4.1. Evaluation of the spatial repellency result

[024] Regarding the results, the EO of C. sertanejus showed spatial repellent activity against Ae. aegypti, indicating a significant difference between the average spatial activity indices (F = 3.08, GL = 5, P = 0.016). The EO indices that showed values ​​above zero were from the concentration of 0.5 mg / cm2, which represents a spatial repellent response. This differs from the negative control, which exhibited a negative index, corresponding to no repellency. However, the significantly higher EO index was observed for the highest concentration when compared to its lowest concentration and the negative control, which was not observed for the other concentrations, as shown in Table 1. Petition 870250014937, dated 24 / 02 / 2025, page 12 / 24 8 / 13 Tib ela 1. Spatial activity index of the essential oil from Croton sertanejus leaves against Aedes aegypti females in 10 minutes Treatment Concentrations mg-cnv IAE = EP C. serrafiejirs 5 0.47°±0.08 2.5 0.29iD±0.13 1 0.2lic ±0.11 0.5 0.20a1 ±0.12 -O,O2h±O,13 TweeiASO 5 -0.071 ±0.08 Means followed by different letters differ significantly by the T-key post-test. P < 0.05. IAE, Spatial Activity Index. SE. Standard Error. Step 5: Topical repellent: estimation of effective concentration.

[025] The biological assay aimed to estimate two effective concentrations to repel 50 and 99% of mosquitoes. To this end, it followed the Guidelines for Efficacy Testing of Mosquito Repellents for Human Skin (World Health Organization, 2009) and the methodology adapted by Sousa et al. (2023), which consists of using C. aegagrus hircus blood at 37 °C as a chemical stimulus in a polypropylene cage. The blood was supplied in the Glytube artificial feeder recommended by Silva et al. (2013).

[026] The experiment was conducted in polypropylene cages (16.5 cm x 25 cm) with an opening at the top of 59.4 cm2 covered with tulle, through which the five concentrations, 125; 100; 75; 50 and 25 mg / cm2, were applied successively. Each concentration was evaluated as follows: with 50 mosquitoes in the cage, five areas of 4.9 cm2 were marked on the top. A quantity of 0.1 g of the formulation containing the lowest concentration was applied over one of the markings.

[027] After one minute, the artificial feeder containing 1 mL of blood pre-warmed to 37 °C was placed over the concentration for three minutes. The evaluation criterion was the number of landings. At the end of the time, the feeder was Petition 870250014937, dated 24 / 02 / 2025, page 13 / 24 9 / 13 removed. These procedures were repeated for other concentrations. However, before evaluating the concentrations, the landing behavior of the mosquitoes with the artificial feeder on the screen was observed, both with and without the non-ionic emulsion; these were designated as the negative control. The biological assay contained seven replicates with 50 mosquitoes, totaling 350 mosquitoes per treatment.

[028] To calculate the 50% and 99% concentrations corresponding to the estimated effective repellent concentration, PROBIT regression analysis was used with a 95% confidence level (P < 0.05) (ROBERTSON; RUSSELL; SAVIN, 1980). Step 5.1. Evaluation of the result of the estimate of the effective repellent concentration

[029] Regarding the results, the EO of C. sertanejus showed contact repellent activity against female Ae. aegypti and that to repel 50 and 99% of mosquitoes, the estimate was 3 and 109 mg / cm2, respectively, as shown in table 2. Table 2. Effective concentration of 50% and 99% of the essential oil from Croton sertanejas leaves against female Aedes aegypti mosquitoes in up to three minutes of exposure. They treat affective concentration mg cm CE 56% Confidence Interval CE 99% Confidence Interval {DF} {1,7} 109 fBO, 195} 86.921(33} CE. Effective concentration, χ1, qt-squared. GL. Errors of freedom. Step 6: Protection time of the effective repellent concentration

[030] According to the “Guidelines for Efficacy Testing of Mosquito Repellents for Human Skin”, the protection time of the effective repellent concentration was evaluated at a concentration level of 99%. For this purpose, the methodology mentioned in the previous topic was also used.

[031] The biological assay consisted of seven replicates, with 50 mosquitoes per cage, totaling 350 mosquitoes. In each replicate, an area of ​​4.9 cm2 was made on the upper part of the tulle-screened cage, where 0.1g of the emulsion containing the effective repellent concentration of 109 mg / cm2 was applied. After 30 minutes of application, the artificial feeder was placed over the concentration for three minutes. Petition 870250014937, dated 24 / 02 / 2025, page 14 / 24 / 13 Subsequently, the feeder was removed and placed in a water bath until the next evaluation, which occurred at 30-minute intervals until the experiment totaled 360 minutes.

[032] The evaluation criterion was to quantify the percentage of repellency and blood meal. In this sense, the protection time was calculated as the number of minutes elapsed between the moment of repellent application and the first landing and blood meal. To confirm the total number of landings and blood meals at each evaluation time, previous records were considered. The same procedures were used for the evaluation of the control groups, positive and negative. DEET was used as the positive control. The negative control was the cage with and without non-ionic emulsion.

[033] The means for repellency and blood meal performance were also subjected to analysis of variance (ANOVA). The difference between the means in relation to repellency against landings was compared using Tukey's post-test at a significance level of 5% (P < 0.05). For the difference between the means of blood meal performance, Dunnett's post-test was used (P < 0.05). The standard error of each mean was considered. Step 6.1. Evaluation of the protection time result of the effective repellent concentration.

[034] The results showed that the effective concentration (109 mg / cm2) provided protection against landing and blood feeding against Ae. aegypti. Regarding repellency against landings, the concentration offered 99.4% protection in 30 minutes. Despite a decrease in protection over time, reaching 64.8% in 360 minutes, there was a significant difference with the positive control, the synthetic repellent DEET, only at the end of the experiment, which showed 99.1% protection throughout the period. Both treatments showed a significant difference with the negative controls in all periods evaluated. In the negative controls, screen with non-ionic emulsion and screen without emulsion, there was Petition 870250014937, dated 24 / 02 / 2025, page 15 / 24 / 13 increase in landings over time, with no significant difference between them in any of the periods, as described in table 3. Table 3. Cumulative percentage of repellent protection time against landings by female Aedes aegypti mosquitoes, as a function of the effective repellent concentration of the essential oil from Croton sertanejus leaves. Tra I a tnetilo: e EonEEntração. Rapalância a nunnla lira = EP Temper C. sertanejus 100 mg1 em* DEET 109 mgi'cnf TCENI TSENI ANDVA Fralar(GL)P valor 00:30 99.4==0.3 99.1= = 0..8 85.4*= 3.7 86.8*= 3.7 7.9(3)0,001 60 OS.Sa = 0.5 99.1= = 0.8 70.86* = 4.6 70.8* =6.2 16.9(3)0.000 90 97.4' = 0.8 99.1' = 0.8 58.5*= 7T7 55.8*= 8.5 16.8(3)0.000 120 94.8==1.6 99.1'= 0.8 48.8* = 10.4 44.5* =10.6 14.9(3)0,000 ISO 93.4'= 1.5 99. Ia =0.8 40.0^- = 11 32.0* = 12.5 173(3)0,000 180 91.4'= 1.8 99. P= 0.8 35,4*= 10.. 7 28.8*= 123 19.8(3)0,000 210 88.5'= 2.1 99.1'= 0.8 28.8*= 9.9 263*= 11.8 243(3)0,000 240 88.0= = 23 99.1 = 0.8 23.1* = 11.4 23.7* = 11.4 28 (3) 0.000 270 84.8a = 3.4 99.1 = 0.8 17.7b = 9.4 23 0*±U 31.7(3) 0.000 300 79.4 = 4.4 99.1 = 0.8 16.2* = 93 19.1* = 10 33.9(3) 0.000 330 71.4 = 6.5 99.1 = 0.8 13.4M.6 1?.1* = 9.8 32.5(3) 0.000 360 64.8 = 7.9 99. Pi 0.8 11 7C= 7.5 14,0= = 91 35(3)0.000 Means of different letters in the lines differ significantly from Tukey's post-test, P 0.05. TCENI. tulle with non-tonic emulsion. TSENL tulle without non-tonic emulsion. ANO VA. variable analysis. SE, standard error. GL, degrees of freedom.

[035] Although landings occurred in all evaluated periods, 100% protection against blood feeding was observed up to 150 minutes, which was similar to the DEET positive control. However, although blood feeding occurred at later times, there were few records, with only 2.8% until the end of the experiment, not differing significantly from the positive control, which showed 100% protection in all periods. Both treatments showed no significant difference from the negative controls only in the first minute of evaluation, which did not show protection against blood feeding in any period. In the negative controls, screen with non-ionic emulsion and screen without emulsion, there was an increase in blood feeding in all periods, with no significant difference between them, as shown in Table 4. Petition 870250014937, dated 24 / 02 / 2025, page 16 / 24 12 / 13 Table 4. Percentage of blood feeding by female Aedes aegypti™ mosquitoes as a function of protection time and effective repellent concentration of Croton sertanejas essential oil. Treatments and effective concentration. Effective repellency ±EP Teiupu C. seiianjus 109 mg / em.2 DEET 109 mg / cni2 *TCEM TSENI ANOVA F valor (GL)P vaiar 0030 0,0=00 0,0 = 0,0 0.20 = 028 1,4a = 22 23 (3) 0,098 60 0.0* = 0.0 0,0 = 0,0 1 l*=0.5 20=1.2 3.4(3)0.032 50 0,0 = 0,0 0.0 = 0,0 3.1* = 2.7 5.4*= 16 7.5(3)0.001 120 0.0=0,0 0.0· = 0,0 42* = 1.5 6.7* = 23 53(3)0.006 150 0.0=00 0,0 = 0,0 5,1* = 1,6 80 = 3> 52(3)0,006 ISO 0.2a = 02 0,0a =0.0 60 = 1.7 8.7*= 3.5 4.9 (3) 0.008 210 02a = 0,2. 0,0a = 0,0 7.l*=2.O 9,4* =3,8 4,7 (3) 0.009 240 0.2’= 0.2 0,0 = 0,0 8,0*= 2,0 11*= 4.8 4,7 (3) 0,010 270 0,2' =052 0.0 = 0,0 8.5*= 1,7 12.5*= 5,3 4.8 (3) 0.009 300 0.5a = 03 0.0 = 0,0 9.4*= 4.7 13.4*= 5.5 5.2(3)0.006 330 1,4a = 0,5 0.0 = 0,0 110=2.5 162*= 5,7 62(3)0.003 360 2.8a = 1.0 0.0 = 0,0 13,7*= 32 19.4*= 7.4 4.9 (3) 0,008 Means followed by different letters in the rows differ significantly by Dunnett's post-test. P < 0.05. TCENI. tulle with non-ionic emulsion. TSENI. tulle without non-ionic emulsion. ANO VA. experience analysis. SE, standard error. GL, degrees of freedom. Step 7. Chemical constituents of Croton sertanejus essential oil

[036] The chemical composition analysis of C. sertanejus essential oils 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 7.1: Results of the main constituents found

[037] Nineteen constituents were identified in the essential oil of C. sertanejus, classified as monoterpenes and sesquiterpenes. However, the major compounds of the essential oil are found in Table 3. Petition 870250014937, dated 24 / 02 / 2025, p. 17 / 24 / 13 Table 5. Main chemical constituents of Croton sertanejus essential oil Constituents C. sertanejus (%)1 IRC IKF Eucalyptol 13.5 1030 1031 Biciclogenna crene S.95 1510 1500 y.-Cadinene 6.22 1493 1513 Spatulenol 6.16 1595 157S 'C nnstttumtes listed by order of ílutção in capillary columnFpuiAT-5ms;bmdtces of retention tom capillary column Four VT-5ms; ^Kovats indices in capillary column DB-5 (Adams, 5 online ed. Gruver, TXUSArTexaists Publishing, 2017).

[038] 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 870250014937, dated 24 / 02 / 2025, page 18 / 24

Claims

1 / 1 CLAIMS 1. FORMULATIONS characterized by containing essential oil of Croton sertanejus, Tween®80, deionized water and emulsion.

2. Formulations according to claim 1, characterized by their use as a space repellent, at any concentration from 0.5 mg / cm2.

3. Formulations according to claim 2, characterized in that their preferred vehicle is a solution containing Tween®80 and deionized water.

4. Formulations according to claim 1, characterized by their use as a topical repellent against landings and blood meals at any concentration from 3 to 109 mg / cm2.

5. Formulations according to claim 4, characterized in that their preferred vehicle is a non-ionic emulsion.

6. Formulations, according to claim 5, characterized by being a product for topical use. Petition 870250014937, dated 24 / 02 / 2025, pp. 19 / 24