Acaricide activity of leaves extracts of Sambucus australis Schltdl. (Caprifoliaceae) at 2% on engorged females of Rhipicephalus (Boophilus) microplus/ Atividade acaricida de extratos de folhas de Sambucus australis Schltdl (Caprifoliaceae) a 2% sobre teleoginas de Rhipicephalus (Boophilus) microplus.
Rhipicephalus (Boophilus) microplus is the most important haematophagous ectoparasite of cattle (FORTES, 1993); it has larger geographic distribution and economic importance for producing- cattle countries in tropical and subtropical areas in the world. The economic losses caused by tick cattle infestation in Brazil overcome 2 billion dollars a year (GRISI et al., 2002). In addition, there are direct damages as well as decreases in the production of milk and meat (SUTHERST et al., 1983), a lot of injuries on the leather due to inflammatory reactions in the spots where the ticks were fixed on the animal skin (SEIFERT et al., 1968); besides this mite is an important carrier of infectious agents to farm animals such as Anaplasma spp. and Babesia spp., responsible for Tick Fever (GRISI et al., 2002). R (B.) microplus is found in all Brazilian territory, being the south region the most affected by the occurrence of ticks on cattle mainly during the warmer seasons. Two factors seem to contribute significantly for this: the predominance of animals of European origin and the large population density of cattle (HORN, 1983).
In Brazil, regular acaricide usage constitutes the main control method for this tick (FARIAS, 1999; VARGAS et al., 2003). However, historically, after some time of use of the majority of the pesticides available in the marketplace, the development of a population of resistant ticks was detected (FURLONG et al., 2002; GRAF et al., 2004). The foremost issues in the selection of resistant individuals involve defects in conservation, dilution and application of the pesticide, as well as mistaken interval and application methods that lead to the use of the products in non-lethal concentration for the ticks. All of these aspects contribute directly to the emergence of R. (B) microplus resistant strains (VARGAS et al., 2003). Due to those problems, the alternative use of plants with medical potential emerges as an important choice in the parasite control. Advantages for using this control include lower prices than industrialized chemical products, it does not leaving toxic residues in the animal-food products (AVANCINI, 1994; HEIMERDINGER et al., 2006) and it is biodegradable, avoiding severe damages to the environment (ROEL, 2001). Unfortunately, researches aiming the use of plant extracts in the control of parasites are still rare. Additionally, there is a lack of further information concerning to various aspects as well as variety of plant will be usefulness, the adequate conditions for plants cultivate, the correct period of harvest, the quantity needed to produce extracts with potential therapeutic use, and the specific parts of the plants will be employed to elaborate the plant extracts for medicinal purposes (HEIMERDINGER et al., 2006).
Sambucus australis Schltdl. (Caprifoliaceae) is a native plant in Brazil and its occurrence is reported in the Northeast, Southeast and South Regions (TORRES et al., 2005). S. australis is a many-branched shrub or a small tree, up to 4.0m high, rarely more, irregular crown leaves are petiolate, opposite, composed of seven (rarely five) to 13 leaflets, oval- lanceolate form, asymmetric, membranous, 4.0 to 7.5cm long, serrated margin, acuminate at the apex, and glabrous. The flowers present white to yellowish-white color, they are small, arranged in large terminal corymbs summits morphologically monoclinic or tetramer pentamerous, and actinomorphic (NUNES et al., 2007). The flowers have been employed in popular medicine, in the form of infusion or decoction, as a diuretic, antipyretic, anti-inflammatory, laxative, diaphoretic, demulcent; as well as in the treatment of respiratory diseases, analgesic and anti-rheumatic agent (SIMOES et al., 1988; JORGE et al., 1999; SCOPEL, 2005; NUNES et al., 2007).
The central goal of this study was to evaluate the potential acaricidal activity in vitro of extracts of S. australis against R. (B). microplus.
MATERIAL AND METHODS
Leaves of S. australis Schltdl. were collected in Santa Maria city, Rio Grande do Sul state in Brazil. The exsiccates were deposited in the Herbarium of the Botanic Department at Federal University of Santa Maria (UFSM) as HDFI 136 code (NUNES et al., 2007). The leaves were submitted to dehydration and stabilization in an incubator at 40[degrees]C, grounded in a knife mill and stocked in an amber bottle. This plant material was macerated in ethanol at 70% to total depletion and filtered to obtain the crude extract. The leaves extract was lead to dry crude extract ([DCE.sub.L]) by evaporation of the solvent into a rotary evaporator with temperature around 30[degrees]C overnight. Later on, in order to carry the in vitro tests out, the [DCE.sub.L] was suspended in two fractions, one in ethanol at 70% and another one in distilled water. All tests were performed with both solutions adjusted to the final concentration of 2% (0.2 [mg.sup.-1]).
Engorged females ticks of R (B). microplus measuring 4.5 to 7.5mm in length were manually collected of crossbred European cattle naturally infested from a rural property situated in Cacequi town, in Rio Grande do Sul state, in Brazil, during December of 2009. The cattle received acaricidal treatment with commercial products during the summer season. The engorged females were placed in a strip cup, washed in distilled water, dried on absorbent wipes and separated in ten groups containing ten engorged females in each group. All of the ticks were selected based on the mobility and their noticeable features such as intact body and maximum of engorgement (LEITE et al., 1995). All of the engorged females were weighed in order to maintain the uniformity along with each tested group.
The groups individually were submitted to the immersion test and maintaining the liquid (20ml of solution test) in constant agitation for five minutes. The excess of solution was removed from the engorged females with absorbent wipes, and the ticks from each group were fixed on a Petri dish on a double-sided adhesive tape and kept in the incubator at 27[degrees]C and humidity of 80% per 40 days (DRUMMOND et al., 1973).
Fourteen days later the weighing of the egg mass from each group was proceeded and 0.3g was transferred from each group into test tubes. These tubes were closed with cotton and incubated in the same conditions previously described. After 26 days the percentage of hatchability was evaluated by using a stereoscope and the visual verification was applied as parameter for this test (DRUMMOND et al., 1973). Aqueous and ethanol at 70% solutions were used in the tests, using the [DCE.sub.L] of the leaves of S. australis adjusted to a final concentration of 2%. As positive control (acaricide solution), aqueous solution containing Cypermethrin at 15% (150[mg.sup.-1]), Chlorpyrifos at 25% (250[mg.sup.-1]) and Citronella at 1% (10[mg.sup.-1]) diluted at 1:1000 was employed. As control groups, distilled water and 70% ethanol solution were used. The method developed to analyze the effectiveness of the solutions used the respective control for every tested diluent.
The effectiveness tests of the plant were performed in triplicate, calculating the average of the results. The calculi of the effectiveness of the products (EP) were executed applying the formula described by DRUMMOND et al. (1973).
The statistical analysis of the observed results was carried out by the Chi-square test (SAS, 1997), in 5% level of probability (P<0.05).
RESULTS AND DISCUSSION
The results obtained in vitro, referring to the efficacy of the treatments are represented in table 1. The effectiveness of S. australis [DCE.sub.L] in aqueous solution demonstrated results ranging from 22 to 50%, with an average rate of 34% and the standard error (SE) for its value [+ or -] 8.3%. The percentage of effectiveness of S. australis [DCE.sub.L] at 70% ethanol solution ranged 50 to 83%, with the average of 66%, where the SE established was of [+ or -] 9.5% (Table 1).The positive control used in this research presented 100% of efficacy (Table 1), indicating that this pesticide association is efficient facing the ticks of the rural property where they were collected, according to BRASIL (1990).
CLEMENTE et al. (2007) tested, in similar conditions, the in vitro effectiveness of the crude extract of Sapindus saponaria diluted in distilled water, in four different concentrations, and no significant values for any acaricide action on engorged females or on larvae were observed. On the other hand, in the present study it was possible to verify any acaricidal effect of the S. australis leaves extract on the ticks, although it displayed low rates of effectiveness. HEIMERDINGER et al. (2006) obtained an effectiveness of46.56% on the control of R. (B.) microplus in Holstein dairy cattle, through aspersion, using alcoholic extracts of Cymbopogon citratus (lemongrass). However, in that study, the concentration used it was superior (2.72%) than employed in our study (2%). In addition, both results do not demonstrate high effectiveness on R (B.) microplus control. Conversely, MANSINGH & WILLIAMS (1998), applying in vitro ethanol extract of leaves of Simarouba glauca on engorged females of R. (B.) microplus, achieved 100% of effectiveness. Differences in efficacy in the results could be justified by varying the concentration of active substances in the different plants that hold distinct origin and diverse extraction methods used in the extract preparation. In relation to the Chi-square test, there was a significant difference (P<0.0163) among the treatments employed in this study. It was possible to observe that the plant extract of S. australis diluted in ethanol at 70% was higher (66%) than the extract diluted in distilled water (34%) if they are compared to the control groups (distillated water and 70% ethanol). Probably the S. australis [DCE.sub.L] effect was potentialized by the ethanol used during the extraction procedures that might enhance the concentration of active acaricidal ingredients in the plant extract.
In this study, an average hatchability obtained with S. australis [DCE.sub.L] recovered in distilled water and 70% ethanol was 84% and 28% respectively (Table 2). It was possible that the effectiveness produced by the S. australis [DCE.sub.L] at 70% ethanol does not apply only to the action of the plant, but also the ethanol action. SILVA et al. (2008) observed similar results studying Cymbopogon citratus, indicating that the ethanol could potentiate the effect by presenting a degree of acaricidal efficacy and/or facilitating the release of the active substances of the plant extract. CHAGAS et al. (2003) evaluated the susceptibility of larvae and engorged females of R. (B.) microplus to several solvents and observed that only the ethanol at 100% led to boost of mortality rates. According to those authors, the ethanol demonstrated low toxicity; in contrast, in the present study, it was found that ethanol interfered in the hatchability of the eggs (Control 2) (Table 2). Thus, the results suggested that ethanol could potentiate the effectiveness of the treatments which it was used as solvent.
The results obtained in this study demonstrated that S. australis [DCE.sub.L] presented any acaricidal effect in vitro. However, other studies testing different solvents and extraction procedures, several concentrations, other parts of the plant, as well as further in vivo experiments are required. Additionally, phytochemical studies are needed for evaluating the chemical composition effective this plant on the tick.
The obtained results suggest that plant extracts of S. australis presented acaricide potential, although it displayed low rates of effectiveness. This study was the first report that demonstrated any acaricidal activity of this plant against R. (B.) microplus. To incorporate high effectiveness on acaricidal effect of S. australis, there is the need to perform additional studies. Analysis to identify the active metabolites in different physiological stages of S. australis will provide important data in order to optimize and apply the extracts of this plant in the control of R. (B.) microplus.
AVANCINI, C.A.M. Sanidade animal na agroecologia-- atitudes ecologicas de sanidade animal e plantas medicinais em medicina veterinaria. Porto Alegre: Fundacao Gaia, 1994. p.33, 46p.
BRASIL. Ministerio da Agricultura. Portaria n. 90, de 04 dez. 1989. Normas para producao, controle e utilizacao de produtos antiparasitarios. Diario Oficial, 22 jan. 1990, secao 1, coluna. 2.
CHAGAS, A.C.S. et al. Sensibility of Boophilus microplus tick to solvents. Ciencia Rural, v.33, n.1, p.109-114, 2003. Available from: <http://www.scielo.br/pdf/cr/v33n1/14151.pdf>. Accessed: sept. 26, 2011. doi: 10.1590/S0103- 84782003000100017.
CLEMENTE, et al. Avaliacao do potencial de plantas medicinais no controle de Boophilus microplus (Acari: Ixodidae). Revista Brasileira de Biociencias, v.5, n.2, p.516-518, 2007.
DRUMMOND, R.O. et al. Boophilus annulatus and Boophilus microplus: laboratory tests for insecticides. Journal of Economic Entomology, v.66, p.130-133, 1973. Available from: <http://www6.ufrgs.br/seerbio/ojs/index.php/rbb/article/ viewFile/460/404>. Accessed: sept. 26, 2011.
FARIAS, N.A.R. Situacion de la resistencia de la garrapata Boophilus microplus em la region sur de Rio Grande Del Sur, Brazil. In: SEMINARIO INTERNACIONAL DE PARASITOLOGIA ANIMAL, 4., 1999, Puerto Vallarta, Mexico. Proceedings... Puerto Vallarta: CONASAG, 1999. p.25-30.
FORTES, E. Parasitologia veterinaria. 2 ed. Porto Alegre: Sulina.1993. 606p.
FURLONG, J. et al. CL50 e CL90 dos extratos alcoolico e aquoso de nim indiano (Azadirachta indica) em larvas de Boophilus microplus. In: CONGRESSO BRASILEIRO DE PARASITOLOGIA VETERINARIA, 12., 2002, Rio de Janeiro. Proceedings ... Rio de Janeiro: Universidade Federal Rural do Rio de Janeiro, 2002. CD-Rom.
GRAF et al. Tick control: an industry point of view. Parasitology, v.129, p.427-442, 2004. Available from: <http:/ /www.cbpv.com.br/artigos/CBPV_artigo_020.pdf>. Accessed: sept. 25, 2011. doi: 10.1017/S0031182004006079.
GRISI, L. et al. Impacto economico das principais ectoparasitoses em bovinos no Brasil. A Hora Veterinaria, v.21, n.115, p.8-10, 2002.
HORN, S.C. Provaveis prejuizos causados pelos carrapatos. 2.ed. Brasilia: Ministerio da Agricultura, 1983. 79p. (. Boletim de Defesa Sanitaria Animal).
HEIMERDINGER, A. et al. Extrato alcoolico de capim-cidreira no controle do Boophilus microplus em bovinos. Revista Brasileira de Parasitologia Veterinaria, v.15, n.1, p.37- 39, 2006. Available from: <http://www.cbpv.com.br/rbpv/ documentos/1512006/c15137_39.pdf>. Accessed: sept. 25, 2011.
JORGE, L.F. et al. Identificacao histologica de Sambucus australis Cham. & Schlecht. (Sabugueiro). Revista de Ciencias Farmacologicas, v.20, p.117-123, 1999.
LEITE, R.C. et al. Efficacy of doramectin against natural infestations of Boophilus microplus (Canestrini, 1887) (Acari: Ixodidade) in cattle. Revista Brasileira de Parasitologia Veterinaria, v.4, n.1, p.53-56, 1995.
MANSINGH, A.; WILLIAMS, L.A.D. Pesticidal potential of tropical plants - II. Acaricidal activity of crude extracts of several Jamaican plants. Insect Science and its Application, v.18, n.3, p.658-664, 1998.
NUNES et al. Caracterizacao farmacobotanica das especies de Sambucus (Caprifoliaceae) utilizadas como medicinais no Brasil. Parte II. Sambucus australis Cham. & Schltdl. Revista Brasileira de Farmacognosia, v.17, n.3, p.414-425, 2007. Available from: <http://www.scielo.br/pdf/rbfar/v17n3/16.pdf>. Accessed: sept. 28, 2011. doi: 10.1590/S0102- 695X2007000300017.
ROEL, A.R. Utilizacao de plantas com propriedades inseticidas: uma contribuicao para o desenvolvimento rural sustentavel. Revista Internacional de Desenvolvimento Local, v.1, n.2, p.43-50, 2001. Available from: <http://www3.ucdb.br/mestrados/ RevistaInteracoes/n2_railda_2001a.pdf>. Accessed: sept. 28, 2011.
SAS, Statistical Analisys System. User's guide Stat. 2.ed. Cary, 1997. 456p.
SCOPEL. M. Analise botanica, quimica e biologica comparativa entre flores das especies Sambucus nigra L. e Sambucus australis cham. & Schltdt. e avaliacao preliminar da sua estabilidade. 2005. 260f. Dissertacao (Mestrado em Ciencias Farmaceuticas) - Universidade Federal do Rio Grande do Sul, RS. Available from: <http:// www.lume.ufrgs.br/bitstream/handle/10183/6559/ 000531771.pdf?sequence=1>. Accessed: sept. 28, 2011.
SEIFERT, GW. et al. Radioactive studies on the feeding of larvae, nymphs and adults of the cattle tick Boophilus microplus (Canestrini). Parasitology, v.58, p.415-430, 1968. Available from: <http:// journals.cambridge.org/ download.php?file = %2FPAR%2FPAR58_02% 2FS003 1 182000069444a. pdf&code =ad185763010b225d559d97c60a9e146d>. Accessed: sept. 28, 2011.
SILVA, F. F. et al. Avaliacao comparativa da eficacia de fitoterapicos e produtos quimicos carrapaticidas no controle do Boophilus microplus (Canestrini, 1887) por meio do biocarrapaticidograma. Revista Medicina Veterinaria do Departamento de Medicina Veterinaria da Universidade Federal Rural de Pernambuco, v.2, n.3, p.1-8, 2008.
SIMOES, C.M.O. et al. Plantas da Medicina Popular no Rio Grande do Sul. Porto Alegre: Universidade Federal do Rio Grande do Sul, 1988. 173p.
SUTHERST, R.W. et al. The effect of the cattle tick (Boophilus microplus) on the growth of Bos indicus x Bos taurus steers. Australian Journal Agricultural Research, v.34, p.317- 327, 1983.
TORRES, A.R. et al. Estudo sobre o uso de plantas medicinais em criancas hospitalizadas da cidade de Joao Pessoa: riscos e beneficios. Revista Brasileira de Farmacognosia, v.15, n.4, p.373-380, 2005.
VARGAS, M.S. et al. Avaliacao in vitro de uma cepa de campo de Boophilus microplus (Acari: Ixodidae) resistente a Amitraz. Ciencia Rural, v.33, n.4, p.737-742, 2003. Available from: <http://www.scielo.br/pdf/%0D/cr/v33n4/16698.pdf>. Accessed: sept. 26, 2011. doi: 10.1590/S0103-84782003000400024.
Felipe da Silva Krawczak (I) * Andreia Buzatti (I) Felipe Lamberti Pivoto (I) Luis Antonio Sangioni (I) Fernanda Silveira Flores Vogel (I) Sonia de Avila Botton (I) Gilberto Dolejal Zanetti (II) Melania Palermo Manfron (II)
(I) Departamento de Medicina Veterinaria Preventiva (DMVP), Centro de Ciencias Rurais (CCR), Universidade Federal de Santa Maria (UFSM), Av. Roraima, 1000, Predio 44, Sala 5149, Bairro Camobi, 97105-900, Santa Maria, RS, Brasil. E-mail: firstname.lastname@example.org. *Autor para correspondencia.
(II) Departamento Farmacognosia (DF), Centro de Ciencias da Saude (CCS), UFSM, Santa Maria, RS, Brasil.
Received 04.16.11 Approved 09.26.11 Returned by the author 10.06.11 CR-5086
Table 1--In vitro results of the average effectiveness [+ or -] Standard Error (SE) of the dry crude extract ([DCE.sub.L]) of Sambucus australis at 2% in distilled water or ethanol at 70%, on engorged females of Rhipicephalus (B) microplus and laying average of the engorged female facing the treatments. Treatment Laying Average effectiveness average (g) [+ or -] SE (%) Aqueous solution of the 0.66 34 (b) [+ or -] 8.3 [DCE.sub.L] of S. australis at 2% (0.2 [mg.sup.-1]) Ethanol solution at 70% of 0.50 66 (a) [+ or -] 9.5 DCEl of S. australis at 2% (0.2 [mg.sup.-1]) Control 1: distilled water 0.85 -- Control 2: ethanol at 70% 0.87 -- Positive control: solution of 0.11 100 [+ or -] 0 Cypermethrin at 15%, Clorpyrifos at 25% and Citronella at 1% in the dilution of 1:1000. (a, b): Average followed by different letters in the column, differ by the Chi-square test (P<0.0163). Table 2--Average hatchability [+ or -] Standard Error (SE) and reproductive effectiveness obtained in vitro facing the exposure of the Rhipicephalus (B) microplus to the dry crude extract ([DCE.sub.L]) of Sambucus australis at 2% recovered in distilled water or ethanol at 70% compared to the control groups. Treatment Average hatchability Reproductive [+ or -] SE(%) average effectiveness Aqueous solution of 84 (a) [+ or -] 4.7 613883.40 [DCE.sub.L] of S. australis at 2% (0.2 [mg.sup.-1]) Ethanol solution at 70% of 28 (b) [+ or -] 7.3 144864.51 [DCE.sub.L] of S. australis at 2% (0.2 [mg.sup.-1]) Control 1 : distilled water 98 [+ or -] 0 941242.94 Control 2: ethanol at 70% 50 [+ or -] 0 430693.07 Positive control: solution of 0 [+ or -] 0 0 Cypermethrin at 15%, Chlorpyrifos at 25% and Citronella at 1% in the dilution 1:1000 (a, b): Average followed by different letters in the column, differ by the Chi-square test (P < 0.0001).