Print version ISSN 0103-5053
J. Braz. Chem. Soc. vol.20 no.2 São Paulo 2009
Edinilza M. A. FeitosaI; Ângela M. C. ArriagaI, *; Gilvandete M. P. SantiagoI, II; Telma L. G. de LemosI; M. Conceição F. de OliveiraI; Jackson Nunes e VasconcelosI; Jefferson Q. LimaI; Grazielle T. MalcherI; Ronaldo F. do NascimentoIII; Raimundo Braz-FilhoIV
IDepartamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CP 6044, 60455-970 Fortaleza-CE, Brazil
IIDepartamento de Farmácia, Universidade Federal do Ceará, Rua Capitão Francisco Pedro 1210, 60430-370 Fortaleza-CE, Brazil
IIIDepartamento de Química Analítica e Físico-Química, Universidade Federal do Ceará, CP 12100, 60451-670 Fortaleza-CE, Brazil
IVSetor de Química de Produtos Naturais, LCQUI-CCT, Universidade Estadual do Norte Fluminense, 28013-603 Campus-RJ, Brazil
The aim of present study was to describe the chemical composition of the essential oils from the leaf and stem of Rollinia leptopetala R. E. Fries (Annonaceae) and to evaluate the larvicidal activities of these essential oils, of the methanol extract from roots of this plant and of the oxoaporphine alkaloid, liriodenine (1) against the third-instar of Aedes aegypti larvae. The methanol extract from the roots showed larvicidal activity with LC50 64.6 ± 1.5 ppm. Higher activity was observed for the isolated alkaloid liriodenine (1), LC50 3.6 ± 0.4 ppm. The essential oils from the leaves and stems, also exhibited larvicidal activity with LC50 104.7 ± 0.2 and 34.7 ± 0.3 ppm, respectively. These results suggest R. leptopetala as a source of natural larvicidal compounds. This is the first report about the chemical composition and larvicidal activity of the leaf and stem essential oils of R. leptopetala.
Keywords: Rollinia leptopetala, Annonaceae, essential oils, liriodenine, Aedes aegypti
No presente trabalho descreve-se a composição química dos óleos essenciais de Rollinia leptopetala R.E. Fries (Annonaceae) e as atividades larvicidas dos óleos essenciais, do extrato metanólico das raízes desta espécie e do alcalóide oxoaporfinico, liriodenina (1), frente às larvas no terceiro estágio do mosquito Aedes aegypti. O extrato metanólico mostrou-se ativo com CL50 64,6 ± 1,5 ppm e uma forte atividade foi exibida para o composto (1), CL50 3,6 ± 0,4 ppm. Os óleos essenciais das folhas e galhos também mostraram atividade com CL50 104,7 ± 0,2 and 34,7 ± 0,3 ppm, respectivamente. Estes dados sugerem que R. leptopetala é fonte potencial de larvicidas naturais. A composição química do óleo essencial e as atividades descritas são comunicadas pela primeira vez.
Aedes aegypti is the principal mosquito vector of dengue fever, including the hemorrhagic form, which is endemic to South East Asia, Central and South America, and West Africa.1 Between 50 and 100 million of cases are registered each year, causing thousands of deaths.2 The disease has high levels of mortality, and also inflicts great economic losses and social disruption in Brazil.3 Although some viral diseases, such as yellow fever, have been brought reasonably under control with a vaccine, no vaccine is so far available for dengue. Today, the only way of decreasing the incidence of this disease is by controlling the Aedes aegypti proliferation. This is not an easy task because the mosquitoes have developed resistance to the current synthetic insecticides that, in addition, are toxic to humans and the environment. The mosquito population control in the larval stage is much easier than in the adult stage, and then new strategies are needed for controlling the proliferation of the larvae of A. aegypti. Several studies have focused on natural products as insecticides for controlling A. aegypti larvae. Alkaloids and essential oils from herbal plants have demonstrated larvicidal activity,4-8 which motivated our group to search for new insecticides from Brazilian plants.
Rollinia leptopetala R. E. Fries, popularly known as pinha brava, is an endemic shrub in Brazil and shows strong toxicity when eaten by animals.9 The phytochemical investigation of Rollinia species has been reported in the literature revealing acetogenins, steroids, lignans and alkaloids, as secondary metabolites.10-13 Previous studies on liriodenine showed prominent antibacterial and cytotoxic biological activities.14,15 In this paper, the larvicidal activities of R. leptopetala and of the alkaloid liriodenine (1) against the larvae of Aedes aegypti are reported.
Results and Discussion
The methanol extract from the roots of R. leptopetala showed activity against A.aegypti larvae with LC50 64.6 ± 1.5 ppm. Fractionation of this extract by column chromatography gave the alkaloid liriodenine (1), which was identified by comparing its NMR and MS data with those previously reported in the literature.13,16-18
The alkaloid was evaluated and showed strong larvicidal activity against A. aegypti, with LC50 3.6 ± 0.4 ppm. Although no similar investigation was found in the literature for oxoaporphine alkaloids, some nitrogenated compounds have been presented as promising larvicides.19-22
The chemical composition of the essential oils from the leaves and stems of R. leptopetala showed quantitative and qualitative variation. The leaf oil consisted mainly of oxygenated monoterpenes (47.7%), with linalool and 1,8-cineole as the major compounds. The stem oil was devoid of monoterpenes, whereas sesquiterpenes, mainly oxygenated, were abundant (76.2%). Spathulenol (63.9%) was found in high concentration. The retention indices and relative percentages of the constituents of the oils are shown in Table 1.
The essential oil from the leaves showed moderate activity against the third-instar of Aedes aegypti larvae (LC50 104.7 ± 0.2 ppm) but higher larvicidal activity (LC50 34.7 ± 0.3 ppm) was found for essential oil from the stems, which confirmed a higher concentration of oxygenated sesquiterpenes. As no reports on the larvicidal activity were found for spathulenol, and this sesquiterpene was not isolated from the oil to be assayed, we can not suggest the higher activity found in the essential oil from stems is associated to spathulenol.
The quite high larvicidal activity of liriodenine (1) suggests this alkaloid as a model for development of new insecticides for A. aegypti control. The biological activity found for the essential oils, particularly for the essential oil from the stems, corroborates the importance of the investigation of essential oils from plants as potential insecticides and larvicides for controlling Aedes mosquitoes.5,8,23
R. leptopetala was collected, at the flowering stage, in Guaraciaba do Norte, in the Northeast of Brazil, in February 2004. The plant was identified by Dr. F. S. Cavalcanti and Prof. E. P. Nunes from the Herbário Prisco Bezerra (EAC), Universidade Federal do Ceará, Brazil where a voucher specimen, # 33496, is deposited.
Extraction and isolation of liriodenine (1)
The air-dried and pulverized roots (500 g) of R. leptopetala were washed with 20% (v/v) aqueous NH4OH, and extracted with CH2Cl2 in a Soxhlet apparatus. The organic solution was distilled under reduced pressure giving a brown viscous syrup (50 g). This material was chromatographed on a silica gel column (63-200 µm, Vetec), previously washed with NaHCO3, and eluted with a gradient system (0→30%) of CH2Cl2/MeOH. Fractions were pooled according to TLC analysis. From the combined fractions eluted with an 80:20 mixture of CH2Cl2:MeOH, a residue was obtained which was submitted to exclusion chromatography on SephadexTM LH-20 (Amersham Biosciences, Sweden) by elution with a mixture of MeOH/CH2Cl2 to yield liriodenine (1, 78.0 mg, 0.16% yield), 278.0-280.4 °C. Liriodenine (1) was also isolated from the methanol extract (18.5 g) of the roots (500 g), which was obtained by extraction of roots with cold MeOH for 72 h at room temperature. This material was chromatographed on a silica gel column (63-200 µm, Vetec) using hexane, CHCl3, EtOAc and MeOH as eluents. The combined fractions eluted with CHCl3 (2.23 g) were chromatographed using a gradient system (0→30%) of CH2Cl2/MeOH. The fractions pooled with CH2Cl2/MeOH (90:10 v/v), provided 1 (12.7 mg, 0.07% yield). Recrystallization of 1 was from MeOH and structural determination was performed by spectral analysis, including comparison with literature data.16 1H and 13C NMR spectra were recorded on a Bruker Advance DRX 500 spectrometer and the mass spectrum was obtained from a VG Auto M Spec Fisions Instruments spectrometer.
Essential oil isolation and chemical analysis
The oils from the fresh leaves (500 g) and stems (500 g) of R. leptopetala were extracted by hydrodistillation for 4 h, using a modified Clevenger-type apparatus with a water-cooled oil receiver to reduce distillation overheating artifacts. Pale yellowish oils were obtained in 0.03 and 0.05% yield from the leaves and stems, respectively, which were dried over anhydrous sodium sulfate and kept at 4 °C until GC-MS and GC analysis.
The essential oil constituents were quantified by Analytical GC-FID, that was carried out on a Shimadzu GC-17A gas chromatograph using dimethylpolysiloxane DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 µm film thickness). Hydrogen was used as carrier gas at a flow rate of 1 mL min-1 and 30 psi inlet pressure; split, 1:30; temperature programmed, 35-180 °C at 4 °C min-1, then heated at a rate of 17 °C min-1 to 280 °C and held isothermal for 10 min; injector temperature, 250 °C; detector used FID, detector temperature, 250 °C. The analysis of the oils was performed by GC-MS analysis on a Hewlett-Packard 5971 GC-MS instrument employing the following conditions: column: dimethylpolysiloxane DB-5 fused silica capillary column (30 m × 0.25 mm × 0.1 µm film thickness); carrier gas: helium (1mL min-1); injector temperature: 250 °C; detector temperature: 200 °C; column temperature: 35°-180 °C at 4 °C min-1 then 180-250 °C at 10 °C min-1; mass spectra: electron impact 70 eV. Individual components were identified using the Wiley L-built library and two other computer libraries24,25 MS searches using retention indices as a pre-selection routine, as well as by visual comparison of the fragmentation patterns with those reported in the literature.26,27 No linear retention index was calculated and no standard was used. The chemical components identified in the essential oils are presented in Table 1.
Screening for larvicidal activity
Liriodenine (1), essential oils, and the methanol extract were placed in beakers (50 mL) and dissolved in H2O/DMSO 1.5% (v/v) at concentrations of 1-500 ppm, followed by the addition of 50 larvae at the third-instar. For each experiment, both positive (Temephos® at 3.22 ppm) and negative (distilled water containing 1.5% DMSO) control assays were carried out. Mortality was recorded after 24 h of exposure, during which no nutritional supplement was added. The experiments were carried out at 28 ± 2 °C. Each test was performed in triplicate. Data were evaluated through regression analysis. From regression line, the LC50 values were read representing the lethal concentration for 50% larval mortality of A. aegypti. The bioassays were performed at the Laboratório de Entomologia, Núcleo de Endemias, Secretaria de Saúde do Estado do Ceará, Brazil.
Authors are indebted to Olga Ramos (Parque de Desenvolvimento Tecnológico do Ceará) for recording the GC-MS data, to Laboratório de Entomologia - Núcleo de Endemias da Secretaria de Saúde do Estado do Ceará for providing larvae, and to FUNCAP, FAPERJ, CAPES and CNPq for financial support and fellowship research (CNPq).
Supplementary information are available free of charge at http://jbcs.sbq.org.br, as PDF file.
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Received: June 21, 2006
Web Release Date: December 4, 2008