Combination of thymol and eugenol for the control of Rhipicephalus sanguineus sensu lato: Evaluation of synergism on immature stages and formulation development

Journal Pre-proof Combination of thymol and eugenol for the control of Rhipicephalus sanguineus sensu lato: Evaluation of synergism on immature stages and formulation development Let´ıcia Coelho, Luiza Gabriella Ferreira de Paula, Susy das Grac¸as Alecrim Alves, Aparecida Lorrany Nunes Sampaio, Giovana Pereira Bezerra, Fernanda Maria Pinto Vilela, Renata da Silva Matos, ´ Viviane Zeringota, L´ıgia Miranda Ferreira Borges, Caio Monteiro

PII:

S0304-4017(19)30270-5

DOI:

https://doi.org/10.1016/j.vetpar.2019.108989

Reference:

VETPAR 108989

To appear in:

Veterinary Parasitology

Received Date:

6 August 2019

Revised Date:

13 November 2019

Accepted Date:

14 November 2019

Please cite this article as: Coelho L, de Paula LGF, das Grac¸as Alecrim Alves S, Sampaio ´ V, Borges LMF, Monteiro C, ALN, Bezerra GP, Vilela FMP, da Silva Matos R, Zeringota Combination of thymol and eugenol for the control of Rhipicephalus sanguineus sensu lato: Evaluation of synergism on immature stages and formulation development, Veterinary Parasitology (2019), doi: https://doi.org/10.1016/j.vetpar.2019.108989

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1 Combination of thymol and eugenol for the control of Rhipicephalus sanguineus sensu lato: Evaluation of synergism on immature stages and formulation development

Letícia Coelhoa, Luiza Gabriella Ferreira de Paulaa, Susy das Graças Alecrim Alvesc, Aparecida Lorrany Nunes Sampaioc, Giovana Pereira Bezerrad, Fernanda Maria Pinto Vilelae, Renata da Silva Matosf, Viviane Zeringótaa, Lígia Miranda Ferreira Borgesa,b,

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Caio Monteiroa,b

a - Post-graduate program in Animal Science, Goiás Federal University. Avenida

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Esperança, s/n, Campus Samambaia, Goiânia, Goiás, Brazil, 74.690-900.

b - Institute of Tropical Pathology and Public Health, Goiás Federal University (UFG).

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Rua 235, s/n, Setor Universitário, Goiânia, Goiás, Brazil, 74605-050. Autor

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correspondente: Tel.: +55 32 8876 1228. E-mail: [email protected] c - Graduation in veterinary medicine in Goiás Federal University. Avenida Esperança, s/n, Campus Samambaia, Goiânia, Goiás, Brazil, 74.690-900.

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d - Graduation in Biological Science in Goiás Federal University. Avenida Esperança, s/n, Campus Samambaia, Goiânia, Goiás, Brazil, 74.690-900.

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e - Post-graduate program in Biological Science of Juiz de Fora Federal University. Rua

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José Lourenço Kelmer, s/n, Bairro Martelos, Juiz de Fora, MG, Brazil, 36036-330. f - Department of Pharmaceutical Science, Juiz de Fora Federal University. Rua José Lourenço Kelmer, s/n, Juiz de Fora, MG, Brazil, 36036-900.

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Graphical abstract

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Thymol+eugenol had a synergistic effect on unfed and engorged R. sanguineus

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larvae 

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Highlights

Thymol+eugenol had a synergistic effect on unfed and engorged R. sanguineus

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nymphs 

Synergistic effect increases activity and reduces cost to formulation production

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The formulation developed showed activity on larvae and nymphs, unfed and engorged

3 Abstract The aim of this study was to investigate whether the combination of thymol with eugenol has a synergistic effect on the immature life stages of Rhipicephalus sanguineus sensu lato (s.l.), to evaluate the cost-benefit ratio of using these compounds in combination, and to develop a formulation combining thymol with eugenol with activity on immature stages of R. sanguineus s.l. To evaluate synergism, thymol and eugenol, combined (ratio 1:1) or not, were tested at concentrations of 2.5, 5.0, 10.0, 15.0

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and 20.0 mg/mL on unfed larvae and nymphs using a larval packet test, and 0.625, 1.25, 2.5, 5.0 and 10 mg/mL on engorged larvae and nymphs using an immersion test. A cost estimate was calculated to produce 1 L of a solution containing a concentration of

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thymol and eugenol, combined or not, that could cause a tick mortality rate greater than

95%. Finally, a formulation was developed, consisting of a micellar dispersion

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containing polymers (MDP), with thymol + eugenol (1:1), at concentrations of 2.5, 5.0,

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10.0, 15.0 and 20.0 mg/mL, and the activity was evaluated on unfed and engorged larvae and nymphs. For unfed larvae and nymphs, concentrations of 2.5 and 5.0 mg/mL and 2.5, 5.0 and 10.0 mg/mL, respectively, presented synergistic effects. In tests with

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engorged larvae and nymphs, respective concentrations of 0.625, 1.25 and 2.5 mg/mL and 2.5 and 5.0 mg/mL had synergistic effects. The estimated costs for producing a

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solution of 1 L with efficacy greater than 95% was $5.97 using only thymol (15

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mg/mL), $ 5.93 using only eugenol (15 mg/mL), and $ 3.97 using thymol + eugenol (1:1 - 5,0 mg/mL). In tests with MDP, the combination of thymol + eugenol resulted in a mortality rate higher than 95% at concentration of 10 mg/mL for unfed and engorged larvae and nymphs. Thus, the combination of thymol + eugenol, depending on the concentration, has synergistic effects and this combination lowers the cost for the active

4 ingredients thymol and eugenol. The combination of thymol + eugenol in MDP had acaricidal activity against immature life stages of R. sanguineus s.l.

Keywords: Brown dog tick; essential oil, monoterpene, phenylpropanoid, botanical pesticide, botanical acaricidal.

1. Introduction

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Rhipicephalus sanguineus sensu lato (s.l.), popularly known as the brown dog tick, is an ixodid with a wide geographic distribution, and as the name suggests, dogs are its preferred host (Dantas-Torres, 2010; Gray et al., 2013). In addition to direct

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damage caused by parasitism, such as blood loss, skin lesions, irritation, discomfort, and

inflammatory and allergic reactions, R. sanguineus s.l. transmits several pathogens to

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dogs, such as Babesia vogeli, Ehrlichia canis, Anaplasma platys and Hepatozoon canis

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(Dantas-Torres, 2008; Dantas-Torres and Otranto, 2015). This tick can also be found parasitizing humans, causing public health concerns (Dantas-Torres, 2008; Reck et al, 2018).

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Considering its medical-veterinary importance, it is necessary to control this ixodid, and the use of acaricides is the most commonly used method. Several product

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are available containing different chemical groups for the control of R. sanguineus s.l.,

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but dog owners and veterinarians have reported ineffectiveness of some tick control agents, leading to difficulties in controlling this arthropod, possibly due to resistance (Borges et al., 2007; Rodriguez-Vivas et al., 2017a; Rodriguez-Vivas et al., 2017b; Miller et al., 2001; Eiden et al., 2015). There are reports of populations of R. sanguineuss.l. resistant to pyrethroids, amidines, phenylpyrazoles and macrocyclic lactones (Borges et al., 2007; Eiden et al., 2015; Rodrigues-Vivaz et al., 2017a;

5 Rodriguez-Vivas et al., 2017b; Becker et al., 2019). Given this scenario, new strategies for the control of ticks have been investigated seeking to develop products with low toxicity to vertebrates. The use of compounds derived from the secondary metabolism of plants has shown potential for the development of new acaricides (Else and Wall, 2014; Rosado-Aguilar et al., 2017; Pavela et al, 2016). Thymol (5-methyl-2-isopropyl-1-phenol) is a volatile monoterpene with a characteristic odor found in the essential oil of some plants of the families Lamiaceae

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(e.g., Thymus vulgaris) and Verbenaceae (e.g., - Lippia spp.). Eugenol (4-allyl-2methoxyphenol) is a phenylpropanoid, a yellowish oily liquid, found mainly in plants of

the families Myrtaceae, Lauraceae, and Lamiaceae (Neves, 2009, Senra et al., 2013a).

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These compounds are currently synthesized and marketed by different companies, and

their activity has been demonstrated against different development stages of

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Amblyomma sculptum (Berlese, 1888), Rhipicephalus microplus (Canestrini, 1888), R.

Novato et al., 2015).

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sanguineus s.l. and Dermacentor nitens (Neumann, 1897) (Senra et al., 2013, a, b;

Combined use of different terpenes and phenylpropanoids, such as thymol

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and eugenol, is an alternative that has been investigated in an attempt to develop new botanical pesticides using compounds with synergistic effects. Synergistic interactions

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can result in higher mortality rates, besides providing environmental, toxicological and

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production benefits by requiring lower concentrations of each compound (Gallardo et al., 2012; Berdejo et al., 2018). Using mixtures of two or more compounds with different modes of action can also delay selection for resistance (Tripathi et al., 2009). The synergistic effect of thymol and eugenol in combination has been documented against unfed larvae of R. microplus and R. sanguineus s.l. (Araújo et al., 2016). Further studies are needed about the effects of binary combinations of these compounds against

6 other stages of R. sanguineus s.l., since the response may vary according to development stage. The development of formulations also is an important aspect that needs to be investigated for the production of botanical acaricides (Delmonte et al., 2017). The formulation needs to satisfy several criteria, such as increased acaricidal activity, stability, reduction of degradation, ease of application, and increased duration of activity (Ferreira et al., 2017).

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The aim of this study was to investigate the synergistic action of thymol and eugenol on the immature life stages of R sanguineus s.l., to evaluate the cost-benefit ration using these compounds in combination, and to develop a suitable formulation of

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thymol and eugenol

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2. Materials and methods

2.1 Study site

The study was performed at the Laboratory of Biology, Ecology and Tick

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Goiás (UFG).

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Control (LABEC) of the Veterinary Parasitology Center (VPC) of Federal University of

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2.2 Ticks

Ticks were obtained from a colony of R. sanguineus s.l. kept by artificial

infestation on rabbits. The UFG Research Ethics Committee on Use of Animals (CEUA 033/17) approved this trial. The unfed larvae and nymphs were used 15 to 21 days after hatching or molting, respectively. For engorged ticks, larvae and nymphs recently detached from rabbits were used.

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2.3 Thymol and eugenol Thymol and eugenol were purchased from Sigma-Aldrich, with a certificate of 99% purity, and in all tests were used singly and in combination (ratio 1:1). For unfed larvae and nymphs, thymol and eugenol were tested at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL, using 50% ethanol (ethanol + distilled water v/v) as solvent. This dilution was called ethanolic solution (ES). For tests with engorged larvae

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and nymphs, the concentrations tested were of 0.625, 1.25, 2.5, 5.0 and 10.0 mg/mL, dissolved in 3% dimethyl sulfoxide (DMSO) (DMSO + distilled water v/v). This

formulation was called micellar dispersion in dimethylsulfoxide (MDD). Ethanol was

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previously described as the most suitable solvent for the larval packet test with unfed

ticks when using thymol and eugenol, while DMSO is the most suitable solvent for

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immersion tests with engorged ticks (Daemon et al, 2012b; Delmonte et al, 2017).

2.4 Interaction between thymol and eugenol against unfed larvae and nymphs The larval packet test (LPT) proposed by Stone and Haydock (1962) and

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adapted by Monteiro et al. (2012) was used. Approximately 100 larvae were placed at the center of a filter paper (6 x 6 cm). The paper was folded in half and its ends were

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sealed with binder clips. Subsequently, 90 μL of the sample formulation was pipetted on

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each side of the packet. Two control groups, one containing distilled water and the other containing the solvent (50% ethanol), were also formed, totaling 17 groups, 15 treatments and two controls. For each group, 10 replications were performed. After applying the solutions, the packets were kept under ambient conditions for 30 minutes for the solvent to evaporate, followed by placement in a climate-controlled incubator at 27±1 ºC and relative humidity (RH) of 85±5% for 24

8 hours. After this period, the number of dead and living ticks was counted. The mortality percentage was obtained according to the formula: (number of dead larvae/total number of larvae) x 100. In the test with unfed nymphs, the same method and the same concentrations were used, but only 10 unfed nymphs were used in each packet.

2.5 Interaction between thymol and eugenol against engorged larvae and nymphs

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The immersion test described by Drummond et al. (1973) and adapted by Daemon et al. (2009) was performed. Ten engorged larvae were immersed for five minutes in the test solutions. Afterward, the ticks were placed on a paper towel to

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absorb the excess solution and were transferred to a test tube sealed with hydrophilic cotton.

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Two control groups, one containing distilled water and other containing the

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solvent (DMSO 3%), were also formed, totaling 17 groups (15 treatments and two controls). For each group, 10 replications were performed. The experimental groups were kept in the incubator at 27±1 °C and RH >

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85±5%. After 20 days, the living and dead individuals were counted, and the percentage of mortality was obtained according to the formula: (number of dead ticks/total number

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of ticks) x 100.

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The same method and the same concentrations were used in the test with

engorged nymphs.

2.6 Cost evaluation For cost evaluation, the average price of 1,000 mg (1 g) of thymol and eugenol from five suppliers was calculated. Using these values, the cost was calculated

9 of producing 1 L of a solution of these compounds, alone or in combination (1:1), at a concentration capable of causing mortality higher than 95%, based on the results of the experiments described in 2.4 and 2.5. The cost of thymol and eugenol was also calculated, considering only the costs charged by Sigma Aldrich (origin of the compounds used in the present study).

2.7 - Formulation development and evaluation of efficacy the

development

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formulations,

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carboxymethylcellulose (CMC) were purchased from Sigma-Aldrich with 99% purity, and glycerin was purchased from Neon with 99.9% purity.

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After tests to determine interactions between thymol and eugenol, a formulation was developed using DMSO (3%), CMC (0.1%), glycerin (1%) and

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distilled water. This formulation was called micellar dispersion containing polymers

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(MDP). After the development of the base formula, thymol + eugenol were incorporated (1:1) at the concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL. In this formulation, DMSO acted as a penetration agent, CMC as an adhesion agent of the formulation

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under the cuticle of the arthropod, and glycerin acted as moisturizing agent. To evaluate the efficacy of the MDP against immature life stages of R.

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sanguineus s.l., the same methods described in sections 2.4 and 2.5 were used.

2.8 Data analysis For the statistical analysis of all experiments, the software Biostat version

5.0 was used. Because the data presented non-normal distribution, the means of treatments were compared using the Kruskal-Wallis test, followed by the StudentNewman-Keuls (SNK) test.

10 For the analysis of interactions between compounds, the software CompuSyn®, version 1.0, was used (Chou and Martin, 2005). The combination effects were classified according to the combination index (CI), following the classification proposed by Novato et al. (2015): synergism (<0.70), moderate synergism (0.70<0.90), additive effect (0.90- <1.10), moderate antagonism (1.10 - <1.45), and antagonism (≥1.45). For a better evaluation of the interactions between thymol and eugenol, the CI was calculated up to the first concentration where one of the isolated

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substances caused a mortality rate greater than 90%.

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3. Results

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3.1 Interaction between thymol and eugenol against unfed larvae and nymphs In tests with unfed larvae the, mortality percentage in the thymol treatment

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group at the lowest concentration (2.5 mg/mL) presented differences (p<0.05) in relation to the solvent control group, with mortality of 74.31%, reaching 100% at the

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concentration of 20.0 mg/mL, whereas in the solvent control group there was no mortality. For eugenol, differences (p <0.05), in relation to the solvent control group

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(mortality of 0.00%) were observed at the concentration of 5.0 mg/mL, with a mortality rate of 64.74%, and the mortality percentage was 99.96% at the highest concentration

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(20.0 mg/mL). In the combined thymol + eugenol group, the mortality was 99.89% at the lowest concentration (2.5 mg/mL) (p <0.05), reaching 100% at the concentration of 5.0 mg/mL. The CIs of thymol + eugenol at the concentrations of 2.5 and 5.0 mg/mL were 0.39 and 0.41, respectively, indicating synergistic action. At the concentration of 10 mg/mL, a moderate synergistic effect also was observed, with a CI of 0.82 (Table 1).

11 The mortality caused by thymol treatment for unfed nymphs presented differences (p <0.05) in relation to the solvent control group (mortality - 0.91%) from the concentration of 5.0 mg/mL, with mortality of 44.08%, reaching 100% at the concentration of 20.0 mg/mL. In groups treated with eugenol, differences (p <0.05) were also observed in relation to the solvent control group (mortality: 0.91%) from the concentration of 5.0 mg/mL, which resulted in mortality of 46.66%, reaching 100% at the concentration of 15.0 mg/mL. In treatments containing the combination (thymol +

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eugenol), mortality rates of 86.26% and 100% at concentrations of 2.5 and 5.0 mg/mL (p<0.05) were observed. The combination of thymol + eugenol resulted in a synergistic effect on unfed nymphs at the concentrations of 2.5, 5.0 and 10.0 mg/mL, with CIs of

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0.53, 0.25 and 0.50, respectively (Table 1).

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3.3 Interaction between thymol and eugenol against engorged larvae and nymphs

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In thymol tests with engorged larvae, significant differences (p <0.05) in relation to the solvent control group (mortality: 8.02%) were observed from the concentration of 1.25 mg/mL, with a mortality rate of 33.83%, reaching 100% at the

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concentration of 5.0 mg/mL. In eugenol treatments, a significant difference was observed in relation to the solvent control group (mortality 8.02%) from the

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concentration of 2.5 mg/mL, with a 92,00% mortality rate (p <0.05), reaching 100% in

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groups treated at the concentration of 5.0 mg/mL. In treatments with thymol + eugenol (1:1), the mortality percentage differed from the solvent control group (p <0.05) starting in the lowest concentration (0.625 mg/mL), with mortality of 90.67%, reaching at 100% at concentration of 2.5 mg/mL. In this development phase, the combination of thymol + eugenol presented synergic effects at the concentrations of 0.625, 1.25 and 2.5 mg/mL, with CI values of 0.41, 0.56 and 0.39, respectively (Table 2).

12 The mortality of engorged nymphs in the solvent control groups (mortality 1.01% for both) significantly differed (p < 0.05) from treatments with thymol and eugenol, from the concentration of 2.5 mg/mL, with mortality rates of 26.93% and 10.83%, respectively, reaching 100% for both compounds at concentration of 10.0 mg/mL. For treatments with thymol + eugenol, significant differences (p< 0.05) to the solvent control group were observed from 2.5 mg/mL (75.33%), reaching 100% at concentration of 5.0 mg/mL. For the combination of thymol and eugenol, there were

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moderate antagonistic and antagonistic effects at the concentrations of 0.625 mg/mL (CI = 1.42) and 1.25 mg/mL (CI = 2.84), respectively. An additive effect (CI = 0.91) was

observed at the concentration of 2.5 mg/mL and a synergistic effect (CI = 0.25) at the

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concentration of 5.0 mg/mL (Table 2).

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3.3 Cost evaluation

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In the simulation of the cost to produce a solution of 1 L considering the amount of thymol and eugenol, combined or not, necessary to cause a mortality rate higher than 95%, considering the average value of each compound, costs of $5.97 for

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thymol, $ 5.93 for eugenol and $ 3.97 for the combination (thymol + eugenol) were assumed (Table 3). Using these compounds with analytical reagent quality from Sigma

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Aldrich, the origin of thymol and eugenol used in this study, the values for thymol,

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eugenol and thymol + eugenol combination would be $11.67, $ 8.70 and $ 6.00 (Table 3).

3.4 Formulation development and evaluation of efficacy In tests with MDP containing different concentrations of thymol + eugenol (combined), for unfed larvae the mortality rate was greater than 95% from the

13 concentration of 10 mg/mL, while for engorged larvae, all concentrations resulted in 100% mortality. The combination of thymol and eugenol at the concentration of 5.0 mg/mL resulted in mortality higher than 95% (unfed nymphs) and 100% (engorged nymphs), as shown in Table 4. In the comparison of efficacy of the combined compounds (thymol + eugenol) at the concentrations of 2.5 and 5.0 mg/mL between formulations ES with MDP (unfed larvae and nymphs) and MDD with MDP (engorged larvae and nymphs),

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no differences (p> 0.05) were observed in the mortality percentages for engorged larvae and nymphs and unfed nymphs. However, for unfed larvae, the combination of thymol + eugenol in MDP resulted in mortality percentages of 18 and 73%, while in the

concentrations of 2,5 and 5,0 mg/mL (Figure 1).

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treatment with ES, the mortality rates were 99 and 100% (p <0.05), for the

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During the mortality assessment of engorged larvae and nymphs at different

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concentrations of thymol and eugenol in MDP, it was possible to observe specimens that died before the molting process, presenting dehydration and blackened or reddish appearance (Figure 2A and E). There were also specimens that died during (Figure 2B

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and F) or at the end of the molting process (Figure 2C and G). Finally, there were

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individuals that died shortly after molting (Figure 2D and H).

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4. Discussion

Although the acaricidal activity of the monoterpene thymol and the

phenylpropanoid eugenol have already been demonstrated on larvae and nymphs of R. sanguineus s.l. (Araújo et al., 2016; Senra et al., 2013b; Daemon et al., 2009; Delmonte et al., 2017; Monteiro et al., 2009), the synergistic effect of thymol + eugenol has only been demonstrated for unfed larvae of this species (Araújo et al., 2016). The present

14 study presents the first evidence of the synergism of thymol and eugenol on all immature life stages of R. sanguineus s.l., as well as data on a cost/benefit estimate using these compounds alone and in combination, and the development of a formulation containing this combination. Thymol and eugenol are compounds from essential oils of plants that have been studied for the development of new acaricides, due to their high activity, with efficacy near 100% at low concentrations against various tick species (ranging from 2.5

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to 15.0 mg/mL according to the species and/or development stage) (Monteiro et al., 2012; Daemon et al., 2012a; Senra et al., 2013a, b). The results found in this study

reinforce the potential of these compounds, where a mortality rate higher than 95% was

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observed at the concentration of 15.0 mg/mL (1.5%).

For unfed larvae, thymol + eugenol at concentrations of 2.5 mg/mL and 5.0

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mg/mL presented a synergistic effect, and the concentration of 10.0 mg/mL had a

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moderate synergistic effect. For unfed nymphs, the same treatment using those concentrations acted synergistically throughout. For engorged larvae, the synergistic effect was observed at concentrations of 0.625 mg/mL, 1.25 mg/mL and 2.5 mg/mL.

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Interestingly, for engorged nymphs, synergism was observed at the concentration of 5.0 mg/mL, while the concentration of 2.5 mg/mL presented an additive effect. The

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combination of phenolic monoterpenes, such as thymol, with a phenylpropanoid, such

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as eugenol, tends to increase the activity of these mixtures (Bassolé and Juliani, 2012; Delaquis et al., 2002). In addition, synergistic interactions with the combination of these compounds were also observed in studies of different organisms, such as bacteria (Didry et al., 1994; Pei et al., 2009), nematodes (Ntalli et al., 2011) and insects (Pavela, 2010; Lima et al., 2011).

15 Thymol is a hydrophobic compound able to enter the cell membrane, reducing its impermeability, both in prokaryotic and eukaryotic cells, facilitating the entry of other compounds into the cytoplasm (Delgado et al., 2004; Bakkali et al., 2008; Pei et al., 2009; Ahmad et al., 2011). This monoterpene has been found to have a cytotoxic effect on ovaries, synganglia, salivary glands and Gene's organ of R. sanguineus s.l. (Matos et al., 2014; Matos et al., 2019a, b). Eugenol has the ability to cause rupture of the cytoplasm membrane of bacteria cells, causing escape of ions and

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other cellular components (Devi et al., 2010). Thus, the action on the cell membrane may result in a synergistic effect when combining thymol with eugenol. This is

supported by the results reported by Yuan et al. (2019), who investigated the effects of

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the combination of thymol and eugenol on Escherichia coli. The authors assessed membrane integrity, membrane potential and glucose absorption capacity, and found

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that thymol + eugenol resulted in increased membrane damage compared to each

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compound on its own. Most treated cells showed signs of injury or death, in addition to increased depolarization and loss of glucose uptake activity. It is relevant to mention that eugenol tested alone did not result in antimicrobial activity, but in combination, it

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was able to increase thymol activity (Yuan et al., 2019). There is also evidence in the literature that thymol and eugenol has a

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neurotoxic effect on arthropods, with different mechanisms of action. Thymol has the

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ability to bind to GABA receptors located on the membrane of post-synaptic neurons and disrupt the functioning of synapses (Priestley et al., 2003), while eugenol binds to octopamine receptors (Enan, 2001). These differences in the mechanism of action may be responsible for synergistic effects. For engorged nymphs, the lowest concentrations presented a moderate antagonistic (0.625 mg/ml) and antagonistic effect (1.25 mg/ml), while the same

16 concentrations presented a synergistic effect on engorged larvae. The antagonistic effect in engorged nymphs is possibly enhanced by the higher resistance in this development phase. It became obvious that the combination of thymol and eugenol at low concentrations was not able to amplify the activity of these compounds. In in vitro studies on A. cajennense (= sculptum), nymphs are more resistant than larvae when exposed to different concentrations of synthetic pyrethroids (Bittencourt et al., 1989), plant extracts (of Acmella oleracea, Jambu) (Marchesini et al., 2018) and terpenes from

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the essential oils (thymol) (Mendes et al., 2011). Those authors suggested that this difference might be related to the composition and thickness of the cuticle at these stages.

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The search for synergistic mixtures aims to reduce the concentrations

needed by increasing the biological activity against the target organism. This reduction

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of concentration may also result in decreased toxicological and environmental risks, and

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lower production cost (Gallardo et al., 2012; Berdejo et al., 2018). The estimated average cost to produce a solution of 1 L containing only thymol or eugenol, considering only the average of price of the compounds, would be $ 5.97 or $ 5.93,

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while, considering the value of Sigma-Aldrich compounds, the price would be $ 11.76 and $ 8.70, respectively. When combining thymol and eugenol, the concentration of 5.0

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mg/mL (1:1) was able to cause mortality rates above 95%, resulting in costs of $ 3.97

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(average) and $ 6.00 (Sigma-Aldrich). For industrial scale production, the compounds would have even lower prices because they would be obtained from other suppliers and in much larger quantities. However, we performed this analysis just to have a quantitative parameter indicating the cost reduction from the combined use of thymol and eugenol.

17 To produce tick control agents using compounds of essential oils, the development of formulations is one of the most important steps, to confer stability, reduce degradation and contamination by microorganisms, increase persistence of efficacy, improve adhesion to the integument and penetration within the target organism, and enhance efficiency (Ferreira et al., 2017). In the present study, the combined formulation of thymol and eugenol (1:1) incorporated in MDP showed good activity, resulting in a mortality rate higher than

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95% at the concentration of 5.0 mg/mL for all development phases, except for unfed larvae. In that case, the combination of thymol and eugenol in ethanolic solution (ES) presented better activity than MDP. These results agree with studies that have shown

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that ethanolic solutions enhance thymol activity against R. sanguineus s.l. and R. microplus unfed larvae (Scoralik et al., 2012; Daemon et al., 2012b). However, ES

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reduces activity on engorged larvae and nymphs (Delmonte et al., 2017). Thus, it is

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necessary to develop formulations with high activity against larvae and nymphs, unfed and engorged. In the present study, for MDP this fact was observed for the concentration of 10 mg/mL (mortality > 95%). Even at the concentration of 5.0 mg/mL,

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the results for MDP can be also considered good, since the mortality rate was 73% for unfed larvae and over 95% for the other development stages.

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Thymol and eugenol are recognized as safe compounds (Generally

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Recognized as Safe- GRAS) by the Food and Drug Administration (FDA). Thymol was considered practically non-toxic in acute dermal application, with LD50 of 980 mg/kg for rats. For topical use, Tisserand and Young (2014) recommended the use of thymol up to the concentration of 1.0% (HSDB Thymol, 2019). For eugenol, skin toxicity was not detected (HSDB Eugenol, 2019). In this study, the combination of thymol and eugenol (1:1) resulted in a mortality rate higher than 95% for all immature life stages at

18 the concentration of 5 mg/mL (0.5%) in ES (unfed ticks) and in MDD (engorged ticks), while in MDP (engorged and unfed ticks), the concentration required to achieve the same effect was 10 mg/mL (1.0%). Thus, the combination thymol + eugenol allowed using compounds at concentrations considered safe for formulations intended for topical application. It is possible to conclude that the combination of thymol + eugenol, depending on the concentration, exerts a synergistic effect, resulting in an increase in

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activity at low concentrations. This low concentration with higher activity also causes a decrease in the cost of producing formulations. The formulation developed in the

present study was effective and showed satisfactory activity against immature life stages

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of R. sanguineus s.l.

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Conflict of interest

Acknowledgements

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The authors declared no conflict of interest.

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We thank the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the financial support (project financing and grants) and for the grants

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PQ to CMO. Monteiro (311889/2017-4). This study also was financed (scholarships) in

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part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001.

19 References

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Figure 1 - Comparison between the mortality percentage of different formulations containing thymol + eugenol (1:1), at concentrations of 2.5 and 5.0 mg/mL against immature life stages of Rhipicephalus sanguineus sensu lato. ES - Ethanolic solution; MDP - micellar dispersion containing polymers; MDD - micellar dispersion in dimethylsulfoxide. *Averages followed by different letters, for the same developmental stage, and concentration, differ significantly at the 5% level.

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30

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Figure 2 - Rhipicephalus sanguineus sensu lato, in different developmental stages, treated with thymol + eugenol (1: 1) formulation, in micellar dispersion containing polymers. A engorged larva dead before the molting process (larvae/nymph); B - engorged larvae dead during the molting process (larvae/nymph); C - nymph dead during final phase of the molting process (larvae/nymph); D - nymph dead after the molting process (larvae/nymph); E - engorged nymph dead before the molting process (nymph/adult); F - engorged nymph dead during the molting process (nymph/adult); G - adult dead during final phase of the molting process (nymph/adult); H adult dead after the molting process (nymph/adult).

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.

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Table 1 - Mortality percentage (mean ± standard deviation) of unfed larvae and nymphs, treated with different concentrations of thymol and eugenol, in combination (1:1) or not, and effect of these compounds on unfed immature stages of Rhipicephalus sanguineus sensu lato, in under

Tick

Concentrations

Thymol

(mg/mL)

0.48±0.00a

0.48±3.25a

CI

Effect

Solvent control*

0.00±0.00a

0.00±0.00a

0.00±0.00a

2.5

74.31±10.21b

1.30±1.21a

99.89±0.36b

0.39

Synergism

76.50±21.37b

64.74±27.82b

100±0.00b

0.41

Synergism

99.67±1.05c

95.12±8.60c

100±0.00b

0.82

Moderate synergism

99.59±0.98c

99.93±0.22d

100±0.00b

10.0

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15.0

e-

0.48±1.50a

5.0

20.0

100 ± 0.00c

99.96±0.14d

100±0.00b

Water H2O

0.00±0.00a

0.00±0.00a

0.00±0.00a

Solvent control*

0.91±0.00a

0.91±0.00a

0.91±2.87a

2.5

8.01±13.09a

4.73±8.09a

86.26±18.45b

0.53

Synergism

5.0

44.08±16.87b

46.66±35.05b

100±0.00c

0.25

Synergism

10.0

85±26.48c

89.29±17.29c

99.23±2.43c

0.50

Synergism

15.0

98.18±3.83d

100±0.00c

100±0.00c

20.0

100±0.00d

98.18±5.75c

100±0.00c

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Unfed nymphs

Thymol + Eugenol

Water H2O

Pr

Unfed larvae

Eugenol

pr

laboratory conditions (27 ± 1°C and RH 85 ± 5%).

* Solvent control - Ethanol 50%.

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Averages followed by different letters in the same column, for the same development stage, differ significantly at the 5% level. Combination index (CI): synergism (<0.70), moderate synergism (0.70 - <0.90), additive effect (0.90 - <1.10), moderate antagonism (1.10 - <1.45),

and antagonism (≥1.45).

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Table 2 - Mortality percentage (mean ± standard deviation) of engorged larvae and nymphs, treated with different concentrations of thymol and eugenol, in combination (1:1) or not, and effect of these compounds on engorged immature stages of Rhipicephalus sanguineus sensu lato, in

Tick

Concentrations

e-

under laboratory conditions (27 ± 1°C and RH 85 ± 5%). Thymol

Eugenol

Thymol + Eugenol

CI

Effect

8.23±12.08a

8.23±12.08a

8.23±12.08a

8.02±12.32a

8.02±12.32a

8.02±12.32a

9.45±16.60a

7.73±12.68a

90.67±13.29b

0.41

Synergism

1.25

33.83±12.54b

7.84±8.52a

97.09±4.69c

0.56

Synergism

2.5

85.00±20.43c

92.00±17.51b

100±0.00c

0.39

Synergism

5.0

100±0.00d

100±0.00b

100±0.00c

10.0

100±0.00d

100±0.00b

1000±0.00c

Water H2O

3.00±4.83a

3.00±4.83a

3.00±4.83a

Solvent control*

1.01±3.03a

1.01±3.03a

1.01±3.03a

0.625

2.00±4.22a

1.00±3.16a

2.00±6.32a

1.42

Moderate antagonism

1.25

6.21±14.75a

1.85±3.67a

2.04±4.06a

2.84

Antagonism

2.5

26.93±9.49b

10.83±13.99b

75.33±12.68b

0.91

Additive

Engorged Larvae

Water H2O Solvent control*

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0.625

Pr

(mg/mL)

Engorged nymphs

5.0

95.78±8.92c

41.76±24.44c

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10.0

100±0.00c

100±0.00c

100±0c

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100±0c

0.25

Synergism

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* Solvent control - DMSO 3%. Averages followed by different letters in the same column, for the same development stage, differ significantly at the 5% level. Combination index (CI): synergism (<0.70), moderate synergism (0.70 - <0.90), additive effect (0.90 - <1.10), moderate antagonism (1.10 - <1.45),

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Pr

e-

and antagonism (≥1.45)

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Value query

Company

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Table 3 - Average cost of thymol and eugenol for solution production resulting in greater than 95% efficacy for immature stages of Rhipicephalus sanguineus sensu lato. Purity

Thymol

14/11/2018

Sigma Aldrich

≥98%

14/11/2018

Alfa Aesar

≥98%

14/11/2018

Frontier Scientific. Inc.

14/11/2018

JK Scientific

14/11/2018

Fisher Scientific

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date

Eugenol

Thymol + Eugenol (1:1)

$ 0.58

$ 0.37

$ 0.42

≥98%

$ 0.26

$ 0.36

≥98%

$ 0.24

$ 0.32

$ 0.34

$ 0.28

$ 0.40

$ 0.39

15.0 mg/mL

15.0 mg/mL

5.0 + 5.0 mg/mL

15.0 g

15.0 g

5.0 + 5.0 g

Thymol and eugenol cost average for producing a 1 L of the solution

$ 5.97

$ 5.93

$ 3.97 - (1.99+1.97)

Thymol and eugenol cost for producing a 1 L of the solution, considering

$ 11.67

$ 8.70

$ 6.00 (3.89+2.90)

Pr

e-

$ 0.79

≥98%

na l

Average value - 1000 mg (= 1g) Concentration that resulted in mortality > 95% for all immature life stages Amount (g) of the thymol and eugenol required to produce 1 L of solution

Jo ur

with efficacy > 95%

only the Sigma Aldrich values

f

35

oo

Table 4 - Mortality percentage (mean ± standard deviation) of immature stages of Rhipicephalus sanguineus sensu lato, treated with different concentrations of thymol and eugenol (1:1) incorporated in formulation (micellar dispersion containing polymers - MDP), in laboratory (27 ±

Concentra

Unfed

Engorged

Unfed

Engorged

tions

larvae

larvae

nymphs

nymphs

0.00±0.00a

6.23±12.08a

3.65±4.86a

3.00±4.83a

0.08±0.26a

7.54±10.57a

0.74±2.25a

7.17±7.17a

18.18±10.56

100±0.00b

69.51±21.59

63.89±31.37

b

b

100±0.00b

98.75±3.54c

100±0.00c

100±0.00b

95.76±8.15c

100±0.00c

Water

e-

mg/mL

2.5

b 5.0

73.32±31.25 c

15.0 20.0

98.40±1.95d

Jo ur

10.0

na l

control*

Pr

H2O Solvent

pr

1°C and UR> 85 ± 5%).

100±0.00d

100±0.00b

98.28±3.43c

100±0.00c

100±0.00d

100±0.00b

95.38±10.38

100±0.00c

c

Averages followed by different letters in the same column, for the same development stage, differ significantly at the 5% level. * Solvent control – (Base formula) - glycerin 1%. carboxymethylcellulose (CMC) 0.1% e dimethylsulfoxide (DMSO) 3%.