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In vitro effects of Eucalyptus staigeriana nanoemulsion on Haemonchus contortus and toxicity in rodents
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Short communication
In vitro effects of Eucalyptus staigeriana nanoemulsion on Haemonchus contortus and toxicity in rodents Wesley Lyeverton Correia Ribeiro a , Ana Lourdes Fernandes Camurc¸a-Vasconcelos a , Iara Tersia Freitas Macedo a , Jessica Maria Leite dos Santos a , José Vilemar de Araújo-Filho a , Juliana de Carvalho Ribeiro a , Vanessa de Abreu Pereira b , Daniel de Araújo Viana c , Haroldo Cesar Beserra de Paula b , Claudia Maria Leal Bevilaqua a,∗ a b c
Programa de Pós-Graduac¸ão em Ciências Veterinárias, Universidade Estadual do Ceará, Brazil Departamento de Química Analítica e Físico-Química, Universidade Federal do Ceará, Brazil Faculdade de Veterinária, Universidade Estadual do Ceará, Brazil
a r t i c l e
i n f o
Article history: Received 13 January 2015 Received in revised form 14 July 2015 Accepted 18 July 2015 Keywords: Haemonchus contortus Anthelmintic Nanoemulsion Chitosan
a b s t r a c t Strategies for controlling gastrointestinal nematodes have been developed based on the use of numerous alternative methods, including the use of phytotherapy. New formulations of essential oils with anthelmintic activity have been proposed as a means to optimize their biological effects. Thus, the objective of this study was to formulate a nanoemulsion to optimize the nematicide effect of Eucalyptus staigeriana essential oil (EsEO). Initially, physico-chemical analyses were performed to verify the stability of the E. staigeriana nanoemulsion (EsNano). In vitro tests were conducted to evaluate the ovicidal and larvicidal activities of both EsNano and EsEO against Haemonchus contortus, and toxicology tests were then performed on rodents. The EsEO content in the nanoemulsion was 36.4% (v/v), and the mean particle size was 274.3 nm. EsNano and EsEO inhibited larval hatching by 99% and 96.3% at 1 and 2 mg/ml concentrations, respectively, and inhibited larval development by 96.3% and 97.3% at 8 mg/ml concentrations. The acute toxicity test revealed that the EsNano and EsEO doses required to kill 50% of the mice (LD50) were 1,603.9 and 3,495.9 mg/ml, respectively. EsNano did not alter the hematological parameters in the rats after treatment. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Inappropriate application of anthelmintics to control gastrointestinal nematodes (GINs) has promoted the selection of a Haemonchus contortus resistant population (Santos et al., 2014). Therefore, the development of effective and environmentally acceptable methods of nematode control has become a necessity. The nematicide action of Eucalyptus staigeriana essential oil (EsEO) has been described previously (Macedo et al., 2010). To protect and maximize the nematicidal effect of EsEO, encapsulation techniques employing chitosan have been investigated (Ribeiro et al., 2014). However, the hydrogel obtained in previous studies has not shown anthelmintic activity, and a suitable formulation
is still needed for administration under field conditions (Ribeiro et al., 2013). Thus, the use of a nanoemulsion formulation is recommended because of its greater capacity for entrapping the oil and maintaining a fluid consistency, which can facilitate administration of the drugs to target species (Chime et al., 2014). The objective of this study was to assess the anthelmintic activity of an E. staigeriana nanoemulsion (EsNano) against H. contortus, in addition to its toxicity in rodents and physico-chemical characteristics.
2. Materials and methods 2.1. Preparation and physico-chemical analysis of E. staigeriana nanoemulsion
∗ Corresponding author at: Programa de Pós-graduac¸ão em Ciências Veterinárias/FAVET/UECE, Av. Dr. Silas Munguba, 1700, Campus do Itaperi CEP 60714-903, Brazil. Fax: +55 85 31019840. E-mail address: [email protected] (C.M.L. Bevilaqua).
The chemical composition of the EsEO (Avondale Essências, Paraná, Brazil) was determined by gas chromatography and mass spectrometry (Adams, 2001).
http://dx.doi.org/10.1016/j.vetpar.2015.07.019 0304-4017/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Ribeiro, W.L.C., et al., In vitro effects of Eucalyptus staigeriana nanoemulsion on Haemonchus contortus and toxicity in rodents. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.07.019
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A mixture of EsEO and Tween 80 at a 4:1 ratio was added to a 1% chitosan (w/w) solution, under stirring at 188.5 rad/s for 5 min using a mechanical stirrer. The macroscopic characteristics of EsNano stability were observed over 72 h at 27 ◦ C. The size and distribution of nanoparticles in solution were determined using a beam of red light with a wavelength of 633 nm (ZetaSizer 3600, Malvern, United Kingdom). 2.2. Animal welfare The protocol was approved by the ethics committee for animal use of Universidade Estadual do Ceará (10461354-8/65). 2.3. Egg hatch test The egg hatch test (EHT) was performed based on the methodology described by Coles et al. (1992). Sheep experimentally infected with a H. contortus population resistant to benzimidazole was used as sources of eggs, which were recovered according to Hubert and Kerboeuf (1992). Aliquots of a suspension containing approximately 100 fresh H. contortus eggs were incubated with EsNano at concentrations of 0.06 to 2 mg/ml or with EsEO at concentrations of 0.125 to 2 mg/ml for 48 h at 27 ◦ C. Next, a drop of Lugol’s iodine was added to stop larvae hatching. The eggs and first-stage larvae (L1) were counted under a light microscope. The negative control for EsNano was a 1% chitosan solution and that for EsEO was 3% Tween 80. The positive control was 0.025 mg/ml thiabendazole. Three repetitions with five replicates for each treatment and for each control were performed. The hatching percentage was determined according to the following equation: number of hatched larvae/(number of hatched larvae + number of eggs) × 100.
dead animals was verified, and the lethal doses required to kill 50% (LD50) and 10% (LD10) were calculated. 2.6. Subchronic toxicity in rats Female Wistar albino rats (n = 24) weighing 150–185 g were allowed to acclimatize to the laboratory conditions for seven days and were maintained on standard animal feed for rodents (Labina® , Purina, São Paulo, Brazil) and provided water ad libitum. The animals were randomly divided into the following 3 groups: G1, in which they received a 1% chitosan solution (negative control), G2, in which they received the corresponding LD10 dose determined in the acute toxicity test; and G3, in which they received half of the dose of LD10. The treatments were administered daily by gavage for 30 days. The animals were weighed on days 0, 15 and 30 to measure weight gain. Blood samples were collected from all animals before and after the treatments. The hematological parameters were analyzed using species-specific smart cards for rats with a Vet ABCTM Hematology Analyzer (Scil Animal Care Company, Illinois, USA). After 30 days, the animals were euthanized. The liver, stomach, heart, lungs and kidneys were weighed, and samples were collected for histopathological analysis. Statistical analysis The results of the egg hatch, larval development and toxicity tests were analyzed by ANOVA and compared by Tukey’s test (P < 0.05) using GraphPad Prism® (GraphPad Software, Inc., California, USA) program. The effective concentrations for inhibiting 50% (EC50) of egg hatching and the larval development and the LD50 and LD10 for the mice were determined by probit analysis using SPSS 17.0 for Windows (IBM, New York, USA).
2.4. Larval development test An aliquot of egg suspension was incubated for 24 h at 27 ◦ C to obtain L1 (Hubert and Kerboeuf, 1992). Next, 500 l of a suspension containing approximately 250 L1 was incubated for 6 days at room temperature with the same volume of EsNano or EsEO at a concentration of 1 to 8 mg/ml in 1 g of feces collected from a gastrointestinal nematode-free sheep. Third-stage larvae (L3) were recovered according to Roberts and O’sullivan (1950) and counted under a light microscope. The negative control for EsNano was a 1% chitosan solution and that for EsEO was 3% Tween 80. The positive control was 0.008 mg/ml ivermectin. Three repetitions with five replicates for each treatment and for each control were performed. The inhibition of larval development was calculated based on the following equation: (number of L3 in the control—number of L3 in the treated group)/number of L3 in the control group × 100. 2.5. Acute toxicity in mice Female Swiss albino mice (n = 96) with an average weight of 25 g were allowed to acclimatize to the laboratory conditions for seven days, where they were kept in polypropylene boxes and provided commercial feed (Labina® , Purina, São Paulo, Brazil) and water ad libitum. The mice were randomly divided into the following 12 groups: G1 to G5, in which they received 2000, 3000, 4000, 5000 and 6000 mg/kg EsEO, respectively; G6 to G10, in which they received 1000, 1500, 2000, 2500 and 3000 mg/kg EsNano, respectively; G11, in which they received 3% Tween 80; and G12, in which they received a 1% chitosan solution. The treatments were administered in a single oral dose. The animals were observed carefully for any signs of toxicity during the first 24 h after the treatments and daily thereafter for a period of 14 days. The total number of
3.1. Results The major constituents of the EsEO were geranial (16%) and geraniol (14.8%) (Table 1), and its concentration in the nanoemulsion was 36.4% (v/v). Analysis of the resulting nanoemulsion particles demonstrated a mean size of 274.3 nm, with bimodal distribution and polydispersity. The nanoemulsion had a white color
Table 1 Composition of Eucalyptus staigeriana essential oil (%). KI
Compound
RT
Percentage
939 979 1026 1029 1031 1031 1097 1150 1153 1177 1180 1189 1226 1238 1253 1267 1325 1353 1362 1381 Total identified
˛-pinene ˇ-pinene o-cymene Limonene Eucalyptol ı-3-Carene Linalool Menth-3-en-8-ol Citronellal Terpinen-4-ol Cymen-8-ol (meta) ˛-terpineol Citronellol Z-citral Geraniol Geranial Methyl geranate Citronellol acetate Neryl acetate Geranyl acetate
5.85 7.04 8.59 8.69 8.86 9.97 11.32 13.45 13.58 14.66 14.78 15.21 16.80 17.32 17.95 18.62 21.19 22.41 22.81 23.70
1.8 1.3 0.7 7 5 2 1.1 0.6 5.5 1.7 0.8 2.2 6.3 9.7 14.8 16 11 0.8 2.5 9.2 99.9
KI – Kovats index. RT – Retention time (min).
Please cite this article in press as: Ribeiro, W.L.C., et al., In vitro effects of Eucalyptus staigeriana nanoemulsion on Haemonchus contortus and toxicity in rodents. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.07.019
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W.L.C. Ribeiro et al. / Veterinary Parasitology xxx (2015) xxx–xxx Table 2 Mean effects (±standard error) of Eucalyptus staigeriana nanoemulsion and Eucalyptus staigeriana essential oil on Haemonchus contortus egg hatching. E. staigeriana essential oil nanoemulsion
E. staigeriana essential oil
Concentration (mg/ml)
Efficacy (%)
Concentration (mg/ml)
Efficacy (%)
1 0.5 0.25 0.125 0.06 Negative control Positive control
99.0 ± 0.5Aa 87.9 ± 2.7Ba 59.1 ± 2.6Ca 16.2 ± 1.2Da 10.7 ± 1.2Da 10.4 ± 1.1Da
2 1 0.5 0.25 0.125 Negative control Positive control
96.3 ± 0.6Aa 66.2 ± 2.3Ba 18.5 ± 1.1Cb 7.8 ± 0.8Db 4.9 ± 0.6Da 4.2 ± 0.9Da
98.3 ± 0.3Aa
98.3 ± 0.3Aa
The capital letters indicate comparisons of the means in the columns, and the lowercase letters denote comparisons of the means in the rows. The different letters indicate significant differences (P < 0.05). A chitosan solution (1%) was used as a negative control for E. staigeriana nanoemulsion, and 3% Tween 80 was the negative control for E. staigeriana essential oil. Thiabendazole (0.025 mg/ml) used as a positive control.
Table 3 Mean effects (± standard error) of Eucalyptus staigeriana nanoemulsion (EsNano) and Eucalyptus staigeriana (EsEO) essential oil on Haemonchus contortus larval development. Concentration (mg/ml)
Effect (%) EsNano
EsEO
96.3 ± 1.3 75.5 ± 4.5Ba 31.9 ± 2.9Ca 17.3 ± 2.2Da 9.1 ± 1.6Da 1.2 ± 0.6Da 99.9 ± 0.1Aa Aa
8 4 2 1 0.5 Negative control Positive control
97.4 ± 0.3Aa 85.2 ± 1.1Bb 40.4 ± 1.8Ca 25.9 ± 1.5Da 11.3 ± 2.0Ea 2.1 ± 0.9Ea 99.9 ± 0.1Aa
The capital letters indicate comparisons of the means in the columns, and the lowercase letters denote comparisons of the means in the rows. The different letters indicate significant differences (P < 0.05). A chitosan solution (1%) was used as a negative control for E. staigeriana nanoemulsion, and 3% Tween 80 was used as a negative control for E. staigeriana essential oil. Ivermectin (0.008 mg/ml) was used as a positive control.
and milky consistency. No phase separation was visually observed after 72 h. The results of the EHT and LDT for EsNano and EsEO are presented in Tables 2 and Table 3, respectively. The EHT revealed EC50 values (95% confidence intervals) of 0.2 (0.16–0.24) and 0.7 mg/ml (0.4–1.3) for EsNano and EsEO, respectively, and the LDT showed values of 2.3 (1.4–3.8) and 1.8 mg/ml
3
(1.2–2.8), respectively. The inhibition of egg hatching and the larval development was dose-dependent. In the acute toxicity test, EsNano and EsEO presented LD50 values of 1,603.9 (1,222.2–1,991.1) mg/ml and 3,495.9 (2,598–4,507.7) mg/ml, respectively. The hematological parameters of the rats are presented in Table 4. No significant differences were found in the body weights or the histological morphologies of organs between the treatment and control groups.
4. Discussion The composition of EsEO may vary substantially. That used by Ribeiro et al. (2013) contained (+)-limonene (73%) and cineole (9.5%), whereas that used in current study contained geranial (16%) and geraniol (14.8%). These chemical variations may result in a modification of this essential oil’s efficacy against GINs. The preparation of a chitosan-based matrix for encapsulation has been proposed to promote the active protection of volatile compounds and to maximize the biological effects of EsEO. Chitosan is a biopolymer suitable for biomedical applications such as encapsulation matrices due to its low toxicity and biodegradability (Balan and Verestiuc, 2014). Therefore, it was selected as the encapsulating matrix for EsEO for evaluation of the activity of essential oil H. contortus using in vitro tests. The nanoemulsion obtained had similar physico-chemical characteristics as those of an E. citriodora nanoemulsion evaluated by Ribeiro et al. (2014). The EHT results indicated that the EC50 of EsNano was approximately one-third of that of EsEO, and the LDT results revealed that it was greater than that of EsEO. This discrepancy in results may have been due to the presence of feces in the medium used to promote larval development. An aqueous system is necessary for the proper release of oil; the diffusion of the drugs retained in nanoemulsions occurs when water enters the polymer system, resulting in its swelling and the subsequent release of the encapsulated drugs (Dash et al., 2011). The acute toxicity test results indicated that EsNano was more toxic than EsEO. These results differ from those described by Ribeiro et al. (2014) for an E. citriodora nanoemulsion, in which the encapsulation process caused a rise in the LD50 for mice. The relative surface area and size of a nanosystem is important for promoting the intermolecular interactions of a the nanometric substance with gastric mucosa. These properties can result in the generation of adhesive interactions (Sosnik et al., 2014), which can cause retention of the nanoemulsion in the gastrointestinal tract, with increased intestinal absorption and consequently, increased toxicity.
Table 4 Hematological parameters (± standard error) of albino Wistar rats (n=24) before and after treatments with 866.5 mg/kg (LD10) and 433.3 mg/kg (Half LD10) Eucalyptus staigeriana nanoemulsion. Parameter (Unit)
WBC (103 /mm3 ) RBC (106 /mm3 ) Hb (g/dl) Ht (%) Plt (103 /mm3 ) MCV (m3 ) MCH (g3 ) MCHC (%)
Negative Control
LD10 (866.5 mg/kg)
Half LD10 (433.3 mg/kg)
Day 0
Day 30
Day 0
Day 30
Day 0
Day 30
1.6 ± 0.2Aa 7.4 ± 0.1Aa 14.4 ± 0.1Aa 39.1 ± 0.6Aa 765.4 ± 11.8Aa 52.7 ± 0.4Aa 18.7 ± 0.8Aa 36.8 ± 0.4Aa
1.8 ± 0.1Aa 7.3 ± 0.1Aa 14.3 ± 0.2Aa 39.2 ± 0.5Aa 767.5 ± 21.1Aa 53. 5 ± 0.5Aa 19.6 ± 0.2Aa 36.6 ± 0.3Aa
1.3 ± 0.1Aa 7.8 ± 0.1Aa 14.9 ± 0.1Aa 40.6 ± 0.6Aa 809.2 ± 12.1Aa 52.1 ± 0.37Aa 19.0 ± 0.2Aa 36. 8 ± 0.2Aa
1.6 ± 0.4Aa 7.5 ± 0.2Aa 14.6 ± 0.3Aa 38.8 ± 1.0Aa 732.8 ± 37.3Aa 45.7 ± 6.6Ab 19.5 ± 0.4Aa 37.2 ± 0.6Aa
1.8 ± 0.3Aa 7.7 ± 0.2Aa 15.1 ± 0.15Aa 39.96 ± 0.4Aa 775.3 ± 16.5Aa 51.50 ± 0.4Aa 19.48 ± 0.2Aa 37.81 ± 0.2Aa
3.3 ± 0.7Ab 7.6 ± 0.1Aa 14.7 ± 0.2Aa 39.0 ± 0.6Aa 757.5 ± 24.1Aa 51.45 ± 0.4Aa 19.5 ± 0.2Aa 37.9 ± 0.1Aa
Reference1
8 ± 4.1 7.4 ± 0.3 13.6 ± 0.4 38.0 ± 1.31 638.5 ± 42.4 51.0 ± 0.3 18.4 ± 0.2 36.1 ± 0.2
The capital letters indicate comparisons of the means of the same parameter on day 0 and 30 for the same group. The lowercase letters denote comparisons of the means between different groups at the same sampling time. The different letters indicate significant differences (P < 0.05). A 1% chitosan solution was used as a negative control. The hematological parameters analyzed included the white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), platelets count (Plt), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC); 866.5 mg/kg = LD10; and 433.3 mg/kg = half LD10. 1 The reference values established by Diniz et al. (2006).
Please cite this article in press as: Ribeiro, W.L.C., et al., In vitro effects of Eucalyptus staigeriana nanoemulsion on Haemonchus contortus and toxicity in rodents. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.07.019
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The subchronic toxicity test revealed that the hematological parameters were not altered, except for the white blood cell count (WBC), after treatment of the mice (P > 0.05). The highest dose of EsNano caused a significant increase in the WBC (P < 0.05); however, WBC reference values for rats in different animal facilities have varied from 1.9 to 17 × 103 /mm3 . This discrepancy may be due to the use of different methodologies for hematological analysis, as well as different equipment and reagents for preparing doses (Melo et al., 2012). As reported by Macedo et al. (2010) for EsEO, no histopathological tissue changes and body weight gain of the animals were observed. EsNano has ovicidal and larvicidal effects on H. contortus,and it did not alter the hematological parameters in the rats after treatment with the product. In vivo studies are needed to validate its anthelmintic activity Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgements The authors would like to thank FUNCAP for their financial support (CI3-0093-01020100/14) and CAPES for a the scholarship. Dr. Bevilaqua was supported by a grant from CNPq (303018/2013-5). References Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectrometry. Allured Publishing Corp.: Carol Stream, Illinois, pp. 456. Balan, V., Verestiuc, L., 2014. Strategies to improve chitosan hemocompatibility: a review. Eur. Polym. J. 53, 171–188. Chime, S.A., Kenechukwu, F.C., Attama, A.A., 2014. Nanoemulsions–advances in formulation, characterization and applications in drug delivery. In: Sezer, D.
(Ed.), Application of Nanotechnology in Drug Delivery. InTech, Rijeka, pp. 76–126. Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World Association for the advancement of veterinary parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44. Dash, M., Chiellini, F., Ottenbrite, R.M., Chiellini, E., 2011. Chitosan—a versatile semi-synthetic polymer in biomedical applications. Prog. Polym. Sci. 36, 981–1014. Diniz, M.F.F., Medeiros, I.A., Santos, H.B., de Oliveira, K.M., Vasconcelos, T.H.C., Aguiar, F.B., Toscano, M.G., Ribeiro, E.N., 2006. Haematological and biochemical parameter standardization of swiss mice and Wistar rats. Rev. Bras. de Cienc. Saúde 10, 171–176. Hubert, J., Kerboeuf, D., 1992. A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes. Vet. Rec. 130, 442–446. Macedo, I.T.F., Bevilaqua, C.M.L., Oliveira, L.M.B., Camurc¸a-Vasconcelos, A.L.F., Vieira, L.S., Oliveira, F.R., Queiroz-Júnior, E.M., Tome, A.R., Nascimento, N.R.F., 2010. Anthelmintic effect of Eucalyptus staigeriana essential oil against goat gastrointestinal nematodes. Vet. Parasitol. 173, 93–98. Melo, M.G.D., Dória, G.A.A., Serafini, M.R., Araújo, A.A.S., 2012. Valores de referência hematológicos e bioquímicos de ratos (Rattus novergicus linhagem Wistar) provenientes do biotério central da Universidade Federal de Sergipe. Sci. Plena. 8, 1–6. Ribeiro, J.C., Ribeiro, W.L.C., Camurc¸a-Vasconcelos, A.L.F., Macedo, I.T.F., Santos, J.M.L., Paula, H.C.B., Araújo-Filho, J.V., Magalhães, R.D., Bevilaqua, C.M.L., 2014. Efficacy of free and nanoencapsulated Eucalyptus citriodora essential oils on sheep gastrointestinal nematodes and toxicity for mice. Vet. Parasitol. 204, 243–248. Ribeiro, W.L.C., Macedo, I.T.F., Dos Santos, J.M.L., de Oliveira, E.F., Camurc¸a-Vasconcelos, A.L.C., de Paula, H.C.B., Bevilaqua, C.M.L., 2013. Activity of chitosan-encapsulated Eucalyptus staigeriana essential oil on Haemonchus contortus. Exp. Parasitol. 135, 24–29. Roberts, F.H.S., O’sullivan, P.J., 1950. Methods for egg counts and larval cultures for strongyles infecting the gastrointestinal tract of cattle. Aust. J. Agric. Res. 1, 99–102. Santos, J.M.L., Monteiro, J.M.L., Ribeiro, W.L.C., Macedo, I.T.F., Camurc¸a-Vasconcelos, A.L.C., Vieira, L.S., Bevilaqua, C.M.L., 2014. Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil. Vet. Parasitol. 199, 160–164. Sosnik, A., Neves, J., Sarmento, B., 2014. Mucoadhesive polymers in the design of nano-drug delivery systems for administration by non-parenteral routes: a review. Prog. Polym. Sci. 39, 2030–2075.
Please cite this article in press as: Ribeiro, W.L.C., et al., In vitro effects of Eucalyptus staigeriana nanoemulsion on Haemonchus contortus and toxicity in rodents. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.07.019
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Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
In vitro effects of Eucalyptus staigeriana nanoemulsion on Haemonchus contortus and toxicity in rodents Wesley Lyeverton Correia Ribeiro a , Ana Lourdes Fernandes Camurc¸a-Vasconcelos a , Iara Tersia Freitas Macedo a , Jessica Maria Leite dos Santos a , José Vilemar de Araújo-Filho a , Juliana de Carvalho Ribeiro a , Vanessa de Abreu Pereira b , Daniel de Araújo Viana c , Haroldo Cesar Beserra de Paula b , Claudia Maria Leal Bevilaqua a,∗ a b c
Programa de Pós-Graduac¸ão em Ciências Veterinárias, Universidade Estadual do Ceará, Brazil Departamento de Química Analítica e Físico-Química, Universidade Federal do Ceará, Brazil Faculdade de Veterinária, Universidade Estadual do Ceará, Brazil
a r t i c l e
i n f o
Article history: Received 13 January 2015 Received in revised form 14 July 2015 Accepted 18 July 2015 Keywords: Haemonchus contortus Anthelmintic Nanoemulsion Chitosan
a b s t r a c t Strategies for controlling gastrointestinal nematodes have been developed based on the use of numerous alternative methods, including the use of phytotherapy. New formulations of essential oils with anthelmintic activity have been proposed as a means to optimize their biological effects. Thus, the objective of this study was to formulate a nanoemulsion to optimize the nematicide effect of Eucalyptus staigeriana essential oil (EsEO). Initially, physico-chemical analyses were performed to verify the stability of the E. staigeriana nanoemulsion (EsNano). In vitro tests were conducted to evaluate the ovicidal and larvicidal activities of both EsNano and EsEO against Haemonchus contortus, and toxicology tests were then performed on rodents. The EsEO content in the nanoemulsion was 36.4% (v/v), and the mean particle size was 274.3 nm. EsNano and EsEO inhibited larval hatching by 99% and 96.3% at 1 and 2 mg/ml concentrations, respectively, and inhibited larval development by 96.3% and 97.3% at 8 mg/ml concentrations. The acute toxicity test revealed that the EsNano and EsEO doses required to kill 50% of the mice (LD50) were 1,603.9 and 3,495.9 mg/ml, respectively. EsNano did not alter the hematological parameters in the rats after treatment. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Inappropriate application of anthelmintics to control gastrointestinal nematodes (GINs) has promoted the selection of a Haemonchus contortus resistant population (Santos et al., 2014). Therefore, the development of effective and environmentally acceptable methods of nematode control has become a necessity. The nematicide action of Eucalyptus staigeriana essential oil (EsEO) has been described previously (Macedo et al., 2010). To protect and maximize the nematicidal effect of EsEO, encapsulation techniques employing chitosan have been investigated (Ribeiro et al., 2014). However, the hydrogel obtained in previous studies has not shown anthelmintic activity, and a suitable formulation
is still needed for administration under field conditions (Ribeiro et al., 2013). Thus, the use of a nanoemulsion formulation is recommended because of its greater capacity for entrapping the oil and maintaining a fluid consistency, which can facilitate administration of the drugs to target species (Chime et al., 2014). The objective of this study was to assess the anthelmintic activity of an E. staigeriana nanoemulsion (EsNano) against H. contortus, in addition to its toxicity in rodents and physico-chemical characteristics.
2. Materials and methods 2.1. Preparation and physico-chemical analysis of E. staigeriana nanoemulsion
∗ Corresponding author at: Programa de Pós-graduac¸ão em Ciências Veterinárias/FAVET/UECE, Av. Dr. Silas Munguba, 1700, Campus do Itaperi CEP 60714-903, Brazil. Fax: +55 85 31019840. E-mail address: [email protected] (C.M.L. Bevilaqua).
The chemical composition of the EsEO (Avondale Essências, Paraná, Brazil) was determined by gas chromatography and mass spectrometry (Adams, 2001).
http://dx.doi.org/10.1016/j.vetpar.2015.07.019 0304-4017/© 2015 Elsevier B.V. All rights reserved.
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A mixture of EsEO and Tween 80 at a 4:1 ratio was added to a 1% chitosan (w/w) solution, under stirring at 188.5 rad/s for 5 min using a mechanical stirrer. The macroscopic characteristics of EsNano stability were observed over 72 h at 27 ◦ C. The size and distribution of nanoparticles in solution were determined using a beam of red light with a wavelength of 633 nm (ZetaSizer 3600, Malvern, United Kingdom). 2.2. Animal welfare The protocol was approved by the ethics committee for animal use of Universidade Estadual do Ceará (10461354-8/65). 2.3. Egg hatch test The egg hatch test (EHT) was performed based on the methodology described by Coles et al. (1992). Sheep experimentally infected with a H. contortus population resistant to benzimidazole was used as sources of eggs, which were recovered according to Hubert and Kerboeuf (1992). Aliquots of a suspension containing approximately 100 fresh H. contortus eggs were incubated with EsNano at concentrations of 0.06 to 2 mg/ml or with EsEO at concentrations of 0.125 to 2 mg/ml for 48 h at 27 ◦ C. Next, a drop of Lugol’s iodine was added to stop larvae hatching. The eggs and first-stage larvae (L1) were counted under a light microscope. The negative control for EsNano was a 1% chitosan solution and that for EsEO was 3% Tween 80. The positive control was 0.025 mg/ml thiabendazole. Three repetitions with five replicates for each treatment and for each control were performed. The hatching percentage was determined according to the following equation: number of hatched larvae/(number of hatched larvae + number of eggs) × 100.
dead animals was verified, and the lethal doses required to kill 50% (LD50) and 10% (LD10) were calculated. 2.6. Subchronic toxicity in rats Female Wistar albino rats (n = 24) weighing 150–185 g were allowed to acclimatize to the laboratory conditions for seven days and were maintained on standard animal feed for rodents (Labina® , Purina, São Paulo, Brazil) and provided water ad libitum. The animals were randomly divided into the following 3 groups: G1, in which they received a 1% chitosan solution (negative control), G2, in which they received the corresponding LD10 dose determined in the acute toxicity test; and G3, in which they received half of the dose of LD10. The treatments were administered daily by gavage for 30 days. The animals were weighed on days 0, 15 and 30 to measure weight gain. Blood samples were collected from all animals before and after the treatments. The hematological parameters were analyzed using species-specific smart cards for rats with a Vet ABCTM Hematology Analyzer (Scil Animal Care Company, Illinois, USA). After 30 days, the animals were euthanized. The liver, stomach, heart, lungs and kidneys were weighed, and samples were collected for histopathological analysis. Statistical analysis The results of the egg hatch, larval development and toxicity tests were analyzed by ANOVA and compared by Tukey’s test (P < 0.05) using GraphPad Prism® (GraphPad Software, Inc., California, USA) program. The effective concentrations for inhibiting 50% (EC50) of egg hatching and the larval development and the LD50 and LD10 for the mice were determined by probit analysis using SPSS 17.0 for Windows (IBM, New York, USA).
2.4. Larval development test An aliquot of egg suspension was incubated for 24 h at 27 ◦ C to obtain L1 (Hubert and Kerboeuf, 1992). Next, 500 l of a suspension containing approximately 250 L1 was incubated for 6 days at room temperature with the same volume of EsNano or EsEO at a concentration of 1 to 8 mg/ml in 1 g of feces collected from a gastrointestinal nematode-free sheep. Third-stage larvae (L3) were recovered according to Roberts and O’sullivan (1950) and counted under a light microscope. The negative control for EsNano was a 1% chitosan solution and that for EsEO was 3% Tween 80. The positive control was 0.008 mg/ml ivermectin. Three repetitions with five replicates for each treatment and for each control were performed. The inhibition of larval development was calculated based on the following equation: (number of L3 in the control—number of L3 in the treated group)/number of L3 in the control group × 100. 2.5. Acute toxicity in mice Female Swiss albino mice (n = 96) with an average weight of 25 g were allowed to acclimatize to the laboratory conditions for seven days, where they were kept in polypropylene boxes and provided commercial feed (Labina® , Purina, São Paulo, Brazil) and water ad libitum. The mice were randomly divided into the following 12 groups: G1 to G5, in which they received 2000, 3000, 4000, 5000 and 6000 mg/kg EsEO, respectively; G6 to G10, in which they received 1000, 1500, 2000, 2500 and 3000 mg/kg EsNano, respectively; G11, in which they received 3% Tween 80; and G12, in which they received a 1% chitosan solution. The treatments were administered in a single oral dose. The animals were observed carefully for any signs of toxicity during the first 24 h after the treatments and daily thereafter for a period of 14 days. The total number of
3.1. Results The major constituents of the EsEO were geranial (16%) and geraniol (14.8%) (Table 1), and its concentration in the nanoemulsion was 36.4% (v/v). Analysis of the resulting nanoemulsion particles demonstrated a mean size of 274.3 nm, with bimodal distribution and polydispersity. The nanoemulsion had a white color
Table 1 Composition of Eucalyptus staigeriana essential oil (%). KI
Compound
RT
Percentage
939 979 1026 1029 1031 1031 1097 1150 1153 1177 1180 1189 1226 1238 1253 1267 1325 1353 1362 1381 Total identified
˛-pinene ˇ-pinene o-cymene Limonene Eucalyptol ı-3-Carene Linalool Menth-3-en-8-ol Citronellal Terpinen-4-ol Cymen-8-ol (meta) ˛-terpineol Citronellol Z-citral Geraniol Geranial Methyl geranate Citronellol acetate Neryl acetate Geranyl acetate
5.85 7.04 8.59 8.69 8.86 9.97 11.32 13.45 13.58 14.66 14.78 15.21 16.80 17.32 17.95 18.62 21.19 22.41 22.81 23.70
1.8 1.3 0.7 7 5 2 1.1 0.6 5.5 1.7 0.8 2.2 6.3 9.7 14.8 16 11 0.8 2.5 9.2 99.9
KI – Kovats index. RT – Retention time (min).
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W.L.C. Ribeiro et al. / Veterinary Parasitology xxx (2015) xxx–xxx Table 2 Mean effects (±standard error) of Eucalyptus staigeriana nanoemulsion and Eucalyptus staigeriana essential oil on Haemonchus contortus egg hatching. E. staigeriana essential oil nanoemulsion
E. staigeriana essential oil
Concentration (mg/ml)
Efficacy (%)
Concentration (mg/ml)
Efficacy (%)
1 0.5 0.25 0.125 0.06 Negative control Positive control
99.0 ± 0.5Aa 87.9 ± 2.7Ba 59.1 ± 2.6Ca 16.2 ± 1.2Da 10.7 ± 1.2Da 10.4 ± 1.1Da
2 1 0.5 0.25 0.125 Negative control Positive control
96.3 ± 0.6Aa 66.2 ± 2.3Ba 18.5 ± 1.1Cb 7.8 ± 0.8Db 4.9 ± 0.6Da 4.2 ± 0.9Da
98.3 ± 0.3Aa
98.3 ± 0.3Aa
The capital letters indicate comparisons of the means in the columns, and the lowercase letters denote comparisons of the means in the rows. The different letters indicate significant differences (P < 0.05). A chitosan solution (1%) was used as a negative control for E. staigeriana nanoemulsion, and 3% Tween 80 was the negative control for E. staigeriana essential oil. Thiabendazole (0.025 mg/ml) used as a positive control.
Table 3 Mean effects (± standard error) of Eucalyptus staigeriana nanoemulsion (EsNano) and Eucalyptus staigeriana (EsEO) essential oil on Haemonchus contortus larval development. Concentration (mg/ml)
Effect (%) EsNano
EsEO
96.3 ± 1.3 75.5 ± 4.5Ba 31.9 ± 2.9Ca 17.3 ± 2.2Da 9.1 ± 1.6Da 1.2 ± 0.6Da 99.9 ± 0.1Aa Aa
8 4 2 1 0.5 Negative control Positive control
97.4 ± 0.3Aa 85.2 ± 1.1Bb 40.4 ± 1.8Ca 25.9 ± 1.5Da 11.3 ± 2.0Ea 2.1 ± 0.9Ea 99.9 ± 0.1Aa
The capital letters indicate comparisons of the means in the columns, and the lowercase letters denote comparisons of the means in the rows. The different letters indicate significant differences (P < 0.05). A chitosan solution (1%) was used as a negative control for E. staigeriana nanoemulsion, and 3% Tween 80 was used as a negative control for E. staigeriana essential oil. Ivermectin (0.008 mg/ml) was used as a positive control.
and milky consistency. No phase separation was visually observed after 72 h. The results of the EHT and LDT for EsNano and EsEO are presented in Tables 2 and Table 3, respectively. The EHT revealed EC50 values (95% confidence intervals) of 0.2 (0.16–0.24) and 0.7 mg/ml (0.4–1.3) for EsNano and EsEO, respectively, and the LDT showed values of 2.3 (1.4–3.8) and 1.8 mg/ml
3
(1.2–2.8), respectively. The inhibition of egg hatching and the larval development was dose-dependent. In the acute toxicity test, EsNano and EsEO presented LD50 values of 1,603.9 (1,222.2–1,991.1) mg/ml and 3,495.9 (2,598–4,507.7) mg/ml, respectively. The hematological parameters of the rats are presented in Table 4. No significant differences were found in the body weights or the histological morphologies of organs between the treatment and control groups.
4. Discussion The composition of EsEO may vary substantially. That used by Ribeiro et al. (2013) contained (+)-limonene (73%) and cineole (9.5%), whereas that used in current study contained geranial (16%) and geraniol (14.8%). These chemical variations may result in a modification of this essential oil’s efficacy against GINs. The preparation of a chitosan-based matrix for encapsulation has been proposed to promote the active protection of volatile compounds and to maximize the biological effects of EsEO. Chitosan is a biopolymer suitable for biomedical applications such as encapsulation matrices due to its low toxicity and biodegradability (Balan and Verestiuc, 2014). Therefore, it was selected as the encapsulating matrix for EsEO for evaluation of the activity of essential oil H. contortus using in vitro tests. The nanoemulsion obtained had similar physico-chemical characteristics as those of an E. citriodora nanoemulsion evaluated by Ribeiro et al. (2014). The EHT results indicated that the EC50 of EsNano was approximately one-third of that of EsEO, and the LDT results revealed that it was greater than that of EsEO. This discrepancy in results may have been due to the presence of feces in the medium used to promote larval development. An aqueous system is necessary for the proper release of oil; the diffusion of the drugs retained in nanoemulsions occurs when water enters the polymer system, resulting in its swelling and the subsequent release of the encapsulated drugs (Dash et al., 2011). The acute toxicity test results indicated that EsNano was more toxic than EsEO. These results differ from those described by Ribeiro et al. (2014) for an E. citriodora nanoemulsion, in which the encapsulation process caused a rise in the LD50 for mice. The relative surface area and size of a nanosystem is important for promoting the intermolecular interactions of a the nanometric substance with gastric mucosa. These properties can result in the generation of adhesive interactions (Sosnik et al., 2014), which can cause retention of the nanoemulsion in the gastrointestinal tract, with increased intestinal absorption and consequently, increased toxicity.
Table 4 Hematological parameters (± standard error) of albino Wistar rats (n=24) before and after treatments with 866.5 mg/kg (LD10) and 433.3 mg/kg (Half LD10) Eucalyptus staigeriana nanoemulsion. Parameter (Unit)
WBC (103 /mm3 ) RBC (106 /mm3 ) Hb (g/dl) Ht (%) Plt (103 /mm3 ) MCV (m3 ) MCH (g3 ) MCHC (%)
Negative Control
LD10 (866.5 mg/kg)
Half LD10 (433.3 mg/kg)
Day 0
Day 30
Day 0
Day 30
Day 0
Day 30
1.6 ± 0.2Aa 7.4 ± 0.1Aa 14.4 ± 0.1Aa 39.1 ± 0.6Aa 765.4 ± 11.8Aa 52.7 ± 0.4Aa 18.7 ± 0.8Aa 36.8 ± 0.4Aa
1.8 ± 0.1Aa 7.3 ± 0.1Aa 14.3 ± 0.2Aa 39.2 ± 0.5Aa 767.5 ± 21.1Aa 53. 5 ± 0.5Aa 19.6 ± 0.2Aa 36.6 ± 0.3Aa
1.3 ± 0.1Aa 7.8 ± 0.1Aa 14.9 ± 0.1Aa 40.6 ± 0.6Aa 809.2 ± 12.1Aa 52.1 ± 0.37Aa 19.0 ± 0.2Aa 36. 8 ± 0.2Aa
1.6 ± 0.4Aa 7.5 ± 0.2Aa 14.6 ± 0.3Aa 38.8 ± 1.0Aa 732.8 ± 37.3Aa 45.7 ± 6.6Ab 19.5 ± 0.4Aa 37.2 ± 0.6Aa
1.8 ± 0.3Aa 7.7 ± 0.2Aa 15.1 ± 0.15Aa 39.96 ± 0.4Aa 775.3 ± 16.5Aa 51.50 ± 0.4Aa 19.48 ± 0.2Aa 37.81 ± 0.2Aa
3.3 ± 0.7Ab 7.6 ± 0.1Aa 14.7 ± 0.2Aa 39.0 ± 0.6Aa 757.5 ± 24.1Aa 51.45 ± 0.4Aa 19.5 ± 0.2Aa 37.9 ± 0.1Aa
Reference1
8 ± 4.1 7.4 ± 0.3 13.6 ± 0.4 38.0 ± 1.31 638.5 ± 42.4 51.0 ± 0.3 18.4 ± 0.2 36.1 ± 0.2
The capital letters indicate comparisons of the means of the same parameter on day 0 and 30 for the same group. The lowercase letters denote comparisons of the means between different groups at the same sampling time. The different letters indicate significant differences (P < 0.05). A 1% chitosan solution was used as a negative control. The hematological parameters analyzed included the white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), platelets count (Plt), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC); 866.5 mg/kg = LD10; and 433.3 mg/kg = half LD10. 1 The reference values established by Diniz et al. (2006).
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The subchronic toxicity test revealed that the hematological parameters were not altered, except for the white blood cell count (WBC), after treatment of the mice (P > 0.05). The highest dose of EsNano caused a significant increase in the WBC (P < 0.05); however, WBC reference values for rats in different animal facilities have varied from 1.9 to 17 × 103 /mm3 . This discrepancy may be due to the use of different methodologies for hematological analysis, as well as different equipment and reagents for preparing doses (Melo et al., 2012). As reported by Macedo et al. (2010) for EsEO, no histopathological tissue changes and body weight gain of the animals were observed. EsNano has ovicidal and larvicidal effects on H. contortus,and it did not alter the hematological parameters in the rats after treatment with the product. In vivo studies are needed to validate its anthelmintic activity Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgements The authors would like to thank FUNCAP for their financial support (CI3-0093-01020100/14) and CAPES for a the scholarship. Dr. Bevilaqua was supported by a grant from CNPq (303018/2013-5). References Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectrometry. Allured Publishing Corp.: Carol Stream, Illinois, pp. 456. Balan, V., Verestiuc, L., 2014. Strategies to improve chitosan hemocompatibility: a review. Eur. Polym. J. 53, 171–188. Chime, S.A., Kenechukwu, F.C., Attama, A.A., 2014. Nanoemulsions–advances in formulation, characterization and applications in drug delivery. In: Sezer, D.
(Ed.), Application of Nanotechnology in Drug Delivery. InTech, Rijeka, pp. 76–126. Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World Association for the advancement of veterinary parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44. Dash, M., Chiellini, F., Ottenbrite, R.M., Chiellini, E., 2011. Chitosan—a versatile semi-synthetic polymer in biomedical applications. Prog. Polym. Sci. 36, 981–1014. Diniz, M.F.F., Medeiros, I.A., Santos, H.B., de Oliveira, K.M., Vasconcelos, T.H.C., Aguiar, F.B., Toscano, M.G., Ribeiro, E.N., 2006. Haematological and biochemical parameter standardization of swiss mice and Wistar rats. Rev. Bras. de Cienc. Saúde 10, 171–176. Hubert, J., Kerboeuf, D., 1992. A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes. Vet. Rec. 130, 442–446. Macedo, I.T.F., Bevilaqua, C.M.L., Oliveira, L.M.B., Camurc¸a-Vasconcelos, A.L.F., Vieira, L.S., Oliveira, F.R., Queiroz-Júnior, E.M., Tome, A.R., Nascimento, N.R.F., 2010. Anthelmintic effect of Eucalyptus staigeriana essential oil against goat gastrointestinal nematodes. Vet. Parasitol. 173, 93–98. Melo, M.G.D., Dória, G.A.A., Serafini, M.R., Araújo, A.A.S., 2012. Valores de referência hematológicos e bioquímicos de ratos (Rattus novergicus linhagem Wistar) provenientes do biotério central da Universidade Federal de Sergipe. Sci. Plena. 8, 1–6. Ribeiro, J.C., Ribeiro, W.L.C., Camurc¸a-Vasconcelos, A.L.F., Macedo, I.T.F., Santos, J.M.L., Paula, H.C.B., Araújo-Filho, J.V., Magalhães, R.D., Bevilaqua, C.M.L., 2014. Efficacy of free and nanoencapsulated Eucalyptus citriodora essential oils on sheep gastrointestinal nematodes and toxicity for mice. Vet. Parasitol. 204, 243–248. Ribeiro, W.L.C., Macedo, I.T.F., Dos Santos, J.M.L., de Oliveira, E.F., Camurc¸a-Vasconcelos, A.L.C., de Paula, H.C.B., Bevilaqua, C.M.L., 2013. Activity of chitosan-encapsulated Eucalyptus staigeriana essential oil on Haemonchus contortus. Exp. Parasitol. 135, 24–29. Roberts, F.H.S., O’sullivan, P.J., 1950. Methods for egg counts and larval cultures for strongyles infecting the gastrointestinal tract of cattle. Aust. J. Agric. Res. 1, 99–102. Santos, J.M.L., Monteiro, J.M.L., Ribeiro, W.L.C., Macedo, I.T.F., Camurc¸a-Vasconcelos, A.L.C., Vieira, L.S., Bevilaqua, C.M.L., 2014. Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil. Vet. Parasitol. 199, 160–164. Sosnik, A., Neves, J., Sarmento, B., 2014. Mucoadhesive polymers in the design of nano-drug delivery systems for administration by non-parenteral routes: a review. Prog. Polym. Sci. 39, 2030–2075.
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