Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.17 no.4 João Pessoa Oct./Dec. 2007
Efeitos do óleo essencial de Pilocarpus spicatus Saint-Hilaire (Rutaceae) no desenvolvimento de ninfas de Rhodnius prolixus
Cícero B. MelloI; Cléber D. UzedaI; Marana V. BernardinoI; Duclécio Mendonça-LopesI; Alphonse KelecomI; Paulo C. A. FevereiroI; Marcelo S. GuerraII; Adriana P. OliveiraIII; Leandro M. RochaIV; Marcelo S. GonzalezI, *
IDepartamento de Biologia Geral, instituto de Biologia, Universidade Federal Fluminense, Morro do Valonguinho s/n, 24001-970, Niterói, RJ, Brazil
IIDepartamento de Ciências, Faculdade de Formação de Professores, Universidade do Estado do Rio de Janeiro, Rua Dr. Francisco Portela, 24435-000, São Gonçalo, RJ, Brazil
IIINúcleo de Pesquisa de Produtos Naturais, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21941-590, Rio de janeiro, RJ, Brazil
IVLaboratório de Tecnologia de Produtos Naturais, Departamento de Tecnologia Farmacêutica, Universidade Federal Fluminense, Rua Mário Viana 523, Santa Rosa, Niterói, RJ, Brazil
Bioassays against fifth-instar nymphae of Rhodnius prolixus were conducted with essential oil of Pilocarpus spicatus extracted by hydrodistillation. The main results may be summarized as follows: (i) high levels of toxicity and paralysis together with discrete moulting inhibition were caused by topical application of either 0.5 µL or 1.0 µL per insect of the crude essential oil; (ii) partial fagoinhibition, high moulting inhibition, prolonged intermoulting period and high number of paralyzed insects, but no toxicity were observed after oral treatment using either 5 µL or 10 µL of Pilocarpus spilcatus essential oil per mL of ingested blood meal. The importance of these results in relation to the relevant biological events in R. prolixus is herein discussed.
Keywords: Rhodnius prolixus, Pilocarpus spicatus, essential oil.
Bioensaios contra ninfas de 5º estádio de Rhodnius prolixus foram conduzidos utilizando-se óleo essencial de Pilocarpus spicatus extraído por hidrodestilação. Os principais resultados podem ser resumidos como se segue: (i) altos níveis de toxicidade e paralisia associados à discreta inibição da muda foram induzidos pela aplicação tópica de 0,5 µL ou 1,0 µL do óleo essencial por inseto; (ii) fagoinibição parcial, altos níveis de inibição da muda, período intermuda prolongado e alto número de insetos paralisados mas ausência de toxicidade foram observados após tratamento oral com 5,0 µL ou 10 µL de óleo essencial de P. spicatus por mL de sangue ingerido. A importância destes resultados em relação a eventos biológicos relevantes em R. prolixus é aqui discutida.
Unitermos: Rhodnius prolixus, Pilocarpus spicatus, óleo essencial.
it is long known that secondary plant metabolites are important molecules involved in the coevolutionary mechanisms of interaction with other organisms, mainly insects (Fraenkel, 1959; Ehrlich; Raven, 1964; Jermy, 1966; Bowers, 1984; Edwards; Wratten, 1981). Some of these secondary metabolites are able to directly disrupt specific physiological routes related with neuroendocrine and feeding systems, metamorphosis, reproduction, diapausis and behavior of arthropods, constituting vulnerable points for the population control based on the life cycle of insects vectors (Stoka, 1987, kelecom et al., 2002a,b, Garcia; Azambuja, 2004). Undoubtedly, the defense mechanisms of plants includes antifeedant and repellent as well as hormones and antihormones principles acting as insect growth regulators that could be useful for both the protection of agricultural plants and the control of insect vectors with medical and veterinary importance, such as mosquitoes, flies, triatomines and fleas (Mulla; Su, 1999; Garcia; Azambuja, 2004).
Although many plant families have been screened, little is known about the effects of compounds derived from the family Rutaceae on the development of arthropods (Craveiro et al., 1981; Guenter, 1972; Lahiou, 2004). Pilocarpus is a neotropical genus of arboreous plants, comprising 16 species, ranging from southern Mexico and Central America to the south of south America. Thirteen species occur in Brazil, 11 of them endemic. Most species are found in the North, Northeast and East of Brazil (Skopura, 1996). Species of Pilocarpus represents in Brazil one of the most important sources of pharmacologically active compounds (see discussion) and also accumulate other groups of secondary metabolites such as essential oils (Skopura et al., 1998). Thus, species of the genus Pilocarpus are interesting candidates to utilization in control programs against insect vector populations.
Rhodnius prolixus, an important Chagas disease vector in America, is traditionally used as a model for studies of insect physiology (Wigglesworth, 1934a,b, 1943; 1972; Garcia et al., 1990), control vector strategies (Garcia; Azambuja, 2004) and vector parasite interactions (Chagas, 1909; Dias, 1943; Garcia; Azambuja, 1991; Garcia et al., 1999; Gonzalez et al., 1999, 2000; Kollien; Schaub, 2000; Cortez et al., 2002; kelecom et al., 2002a). In this work, studies were carried out with the essential oil obtained from Pilocarpus spicatus plants (popularly know as " Jaborandi da restinga" ) - collected in natura in the state of Rio de Janeiro (Brazil) - looking for effects produced on blood ingestion, mortality, molt and movement of fifth-instar nynphae of R. prolixus.
MATERIAL AND METHODS
Insect rearing and feeding procedures
Fifth-instar nymphae of male Rhodnius prolixus (Hemiptera: Reduviidae) were reared and maintained in the laboratory at 28 ºC and relative humidity of 60-70%, as described by Azambuja and Garcia (1997). Randomly chosen insects from different experimental groups were then allowed to feed upon a membrane apparatus (Garcia et al., 1984) and submitted to the biological assays (see below).
Collection of Pilocarpus spicatus
The specimens of P. spicatus Saint-Hilaire (Rutaceae) were harvested during springtime (October) in the afternoon (5 p.m.) from the border of the periodically flooded forest formation of the sandbank coastal plain of Restinga de Jurubatiba National Park (county of Carapebus), located on the Atlantic coast of Rio de Janeiro State-Brazil (22º30-33S, 42º15-19W). The dried herbarium specimens were both identified and deposited concomitantly in the Herbarium of the Faculdade de Formação de Professores (Universidade do Estado do Rio de Janeiro-UERJ) and in the Herbarium of the Departamento de Biologia Geral (Universidade Federal Fluminense-UFF) under the signature of M. Guerra Santos and P. Ayres Fevereiro (nº 1824). The specimens of Pilocarpus spicatus were identified according kaastra (1982) by comparison with others specimens in the Herbarium of the Museu Nacional (Universidade Federal do Rio de Janeiro).
Extraction of essential oil from Pilocarpus spicatus
The essential oil extract (5.5 mL) was obtained from whole P. spicatus fresh leaves (741 g) by hydrodistillation during 6 hours at room temperature using a modified Clevenger s equipment (Gotlieb; Magalhães, 1960).
Following ecdysis, fifth-instar nymphae of male R. prolixus were starved for 25 days. Insects were then submitted to three different approaches. In the control group I insects were only fully engorged on citrated whole human blood. Doses of 0.5 µL and 1.0 µL per insect of the crude P. spicatus essential oil were used for the topical treatment and applied directly to the ventral surface of the abdomen immediately after feeding. Feeding treatment with P. spicatus oil was performed by adding different amounts of the crude essential oil to reach final concentrations of 5 µL and 10 µL /mL to the blood meal. To compare the effect of blood meal alone on the moult process during feeding treatment assays, partially-fed insects (control II) had a deliberated interrupted feeding in such a way to ingest the same amount of human blood than P. spicatus-fed insects. Except for these groups, only fully engorged insects were used throughout the experiments. The biological evaluation of the different treatments were recorded by weight of ingested blood, toxicity (i.e., 24 h mortality), intermoulting period (range), ecdysis and paralysis, which was determined by the absence of response to checking withdrawal reflex after application of a delicate pressure to the legs with forceps and watching the capacity of the antennae to be extended as well as by the absence of nymph movement (Garcia et al., 1990). All experiments were repeated at least in triplicate with batches of 50 insects.
Significance of the results was analyzed using ANOVA and Turkey's test (Armitage et al., 2002) according to Stats Direct Statistical Software, version 2.2.7 for Windows 98. Differences between treated and control insects were considered not statistically significant when p > 0.05. Probability levels are specified in the text.
Table 1 shows that topical treatment with P. spicatus essential oil caused high mortality of 90.5 and 91.1% in 24 hours at doses of 0.5 µL and 1.0 µL/insect, respectively, whereas only 5.5% of control insects died at the end of the experiments (p < 0.0001). For both doses, the intermoulting cycle (4 days) was not altered and 81% of surviving insects moult to adult stage while control group presented 100% of ecdysis (p < 0.01). No paralysis was observed in the control group. On the contrary, doses of 0.5 µL and 1.0 µL/insect induced paralysis in 89% and 92% of surviving treated insects, respectively, lasting 15 days after starting the experiments (p < 0.0001). After that period, all treated-nymphae recovered normal movement.
As seen in Table 2, orally given P. spicatus essential oil had significant feeding inhibition effect at concentrations of 5.0 µL and 10.0 µL/mL of blood meal (168.3 and 171.7 mg of ingested blood, respectively) when comparing with fully engorged control group insects (239,7 mg) (p < 0.01). No significant difference was determined between partially-fed control insects (170,9 mg) and Pilocarpus-fed insects (p > 0.05). Also, no significant mortality effects were detected by any applied treatment used (p > 0.05). Complete ecdysis in fully engorged control group occurred between 12-16 days after feeding whereas the intermoulting period had been prolonged to 6 days in partially-fed control insects and in all Pilocarpus-fed experimental groups (p < 0.01). While only 57.4% of survived partially-fed insects reached the adult stage if compared with 100%, in surviving fully engorged nymphs (p < 0.001), the higher inhibition of ecdysis (5.3 and 3.2% of molt in surviving nymphs) were detected for both concentrations of P. spicatus essential oil tested, respectively (p < 0.0001). Despite they had ingested approximately the same amount of blood than partially-fed insects (170.9 mg), Pilocarpus-fed insects displayed significant less ecdysis (p < 0.001). in the same way, high level of lethargic Pilocarpus-fed insects (91-93%) were observed along 15 days after starting the experiments when compared with control groups where no paralysis was detected (p < 0.0001).
The most pronounced biological effects obtained after topical or feeding treatment with P. spicatus essential oil on R. prolixus nymphs are summarized in Table 3.
DISCUSSION AND CONCLUSION
Whole plants or different parts as well as isolated compounds obtained from the genus Pilocarpus have been traditionally used in medical therapy for a variety of ophthalmic and geriatric human diseases (Craveiro et al., 1979, 1981; Link et al., 1974; Avancini et al., 2003; Taveira et al., 2003; Andrade-Neto et al., 2002; Bertrand et al., 2001; Krause et al., 1986; Bohm; Fabel, 1987; Wamil, et al. 1989, Leto, 1991, Yermakova et al., 1991; Riedler, 1992; Bodner; Gorsky, 1996; Rosche et al., 1997; Hoffman; Zang, 1999; Auffarth et al., 2002; Agra et al., 2007). In the same way, some Pilocarpus-obtained compounds also act as antidotes on intoxication by Datura stramonium (Salyi; Abonyi, 1994), induce spittling (Kempf, 1999) and have in vitro action against Pseudomonas aeruginosa, Staphylococcus aereus and Trypanosoma cruzi (Santos et al., 1997; Vieira et al., 2001; Mafezoli et al., 2000; Pavão et al., 2002). Essential oil extracted from a variety of host plants (Guenter, 1972) have been recently reported on account of their effects on the development and behavior of insects as Trialeurodes vaporariorum (Choi et al., 2003), Tribolium confusum (Tunc; Erler, 2003), mosquito species (Tuetun et al., 2004; Costa et al., 2005; Pimenta et al., 2006), Triatoma infestans and Rhodnius neglectus (Fournet et al., 1996; Laurent et al., 1997; Vilaseca et al., 2004), Pediculus humanus (Cestari et al., 2004), Oxyops vitiosa (Wheeler, 2005), Meligethes aeneus (Mauchline et al., 2005) and Lobesia botrana (katerinopoulos et al., 2005). Despite the well-known use of Rutaceae plants for a diversity of diseases, only the feeding deterrence induced in Spodoptera litoralis by some furanocoumarins obtained from Pilocarpus goudotianus (Calcagno et al., 2002) were studied in insects with economic importance. From our results, only a single topical application of 0.5 or 1.0 µL of the crude essential oil of P. spicatus per nymph induced high mortality (90.5 and 91.1% respectively) in R. prolixus. On the other hand, an oral concentration of up to 10 µL of the essential oil per ml of blood meal (dose nearby 1.72 µL per nymph) had no significant effect on insects mortality. Moreover, considering the survived treated-nymph, a high number of insects (around 90%) were induced to temporary paralysis by both topical and oral assays at all concentrations tested. As neither mortality nor paralysis were observed after topical application of non-toxic vegetable oil on R. prolixus nymph (not shown), it is reasonable to suppose that the P. spicatus topical effects are not due to physical properties of the oil (i.e., inducing of impervious cuticle which blocks insect respiration and/or movement, etc) but, perhaps the substances in the essential oil ingested during feeding treatment may be better metabolized by the triatomine digestive system than by the cutaneous route before reaching the haemolymph and/or other target-organs. However, nothing is known about cuticle or gut absorption of any Pilocarpus-derived compound in insects. in the feeding treatment, both doses of P. spicatus essential oil had a feeding deterrent effect. In the same way, only 57.4% of the on purpose partially-fed insects had molt when compared with 100% of ecdysis observed in the control group. However, despite feeding is considered a fundamental pre-requisite for ecdysis (Wigglesworth, 1934a,b), the antifeedant effect of oral administered P. spicatus essential oil does not explain alone the high moult inhibition observed since partially-fed insects, which ingested the same amount of blood, molted nearly 10 times more than Pilocarpus-fed insects. On the contrary, the intermoulting period was extended in both Pilocarpus-fed and partially-fed insects pointing out the importance of the amount of ingested blood for the molting range as described by Wigglesworth (1943) and Garcia (1987). On the other side, only a discrete molt inhibition without alteration of intermoulting period was induced by the topical treatment. Therefore, both molt inhibition and transitory paralysis triggered by topical and feeding treatments are related not only to toxicity but, probably also due interference of Pilocarpus compounds on the triatomine neuroendocrine system, once ecdysis and insect movement are under neurological control (Wigglesworth, 1934a,b, 1972; Lazzari, 1992; Takano-Lee; Edman, 2001). Unfortunately, despite many essential oils have been extracted and analyzed, and a number of components identified, little is know about their useful biological activities (Lahiou, 2004). Thus, chemical substances such as episesamin, hesperidin, pilocarpin, imidazole, 2-tridecadone, alpha-cadinol, spathulenol, eugenol, and classes of metabolites such as flavonoids, terpenoids, coumarins, alkaloids and others have been described for different species of the genus Pilocarpus (Craveiro et al., 1979; Amaro Luis et al., 1990; Andrade-Neto et al., 1996, 2000; Bertrand et al., 2001, Lúcio et al., 2002; Santos et al., 2004) and deserve biological screening. Specially, volatile constituents of P. spicatus essential oil, such as aliphatic ketones have been recently described but, without data about their biological effects (Andrade-Neto et al., 2002). However, it has well been established that P. spicatus essential oil components display in vitro antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aereus (Santos et al., 1997). Moreover, chalepin - purified from P. spicatus essential oil - complexes in vitro with glycosomal glyceraldehyde-3-phosphate dehydrogenase of T. cruzi - a protozoan exclusively transmitted by haematophagous triatomines like R. prolixus (Chagas, 1909) - disrupting the flagellate development (Mafezoli et al., 2000, Pavão et al., 2002). These observations also indicate P. spicatus essential oil to studies concerning the disruption of parasite development even into insect vector. Thus, it may be concluded from this preliminary study that the variety of effects of Pilocarpus spicatus essential oil on R. prolixus development herein described indicates its secondary metabolites, nowadays under investigation in our laboratory, as good candidates for the study of insect physiology, vector control population and perhaps, blockage of protozoan development in triatomine hosts.
We thank Universidade Federal Fluminense for the grants to the scientific initiation students as well as Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) and Fundação de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for the financial support.
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