Camara et al.
Composition and Insecticidal Activity of the Essential Oil of Croton grewioides Baill. against Mexican Bean Weevil (Zabrotes subfasciatus Boheman). Cléia G. V. Silva, Hugo B. Zago, Hugo J. G. S. Júnior and Claudio A. G. da Camara*, Laboratory for Natural Bioactive Products, Department of Chemistry – Federal Rural University of Pernambuco, Av. Dom Manoel de Medeiros, s/n, 52171-900 - Recife-PE, Brasil,
[email protected].
José Vargas de Oliveira and Reginaldo Barros, Department of Agronomy, Federal Rural University of Pernambuco, Av. Dom Manoel de Medeiros, s/n, 52171-900 Recife-PE, Brazil.
Manfred O. E. Schwartz, Department of Fundamental Chemistry, Federal University of Pernambuco, Av. Prof. Nelson Chaves, s/n, 50670-420-Recife –PE, Brazil.
Maria F. A. Lucena, Post-Graduation Program in Vegetal Biology of the Department of Botanics, Federal University of Pernambuco. Av. Prof. Nelson Chaves, s/n, 50670-420-Recife –PE, Brasil. Abstract The essential oils of leaves and stems of Croton grewioides Baill. of the “agreste” region of Pernambuco, Brazil, were obtained by hydrodistillation and analyzed by GC and GC/MS. Twenty-two components were identified, which represented 99.8% and 98.6% of the total constituents in the leaf and stem oil, respectively. Croton grewioides produced an oil with a predominance of phenylpropanoid compounds, whose principal component was (E)-anethole (65.5% in the leaf and 47.8% in the stem). Other major components found in the leaf oil were methyl eugenol (10.6%) and (E)-methyl isoeugenol (4.7%). Other components identified in the stem oil were (E)-methyl isoeugenol (30.0%), cadalene (8.4%) and methyl eugenol (4.6%). The insecticidal activity of C. grewioides was evaluated against Mexican bean weevils, Zabrotes subfaciatus (Boheman) resulting in a LC50 for the leaf oil, which was 3.4 times less than that obtained from the stem oil. Key Word Index Croton grewioides, Euphorbiaceae, essential oil composition, (E)-anethole, (E)-methyl isoeugenol, methyl eugenol, insecticidal activity, Zabrotes subfasciatus.
Introduction The Bioma caatinga is a characteristic flora of the Northeast of Brazil. The principal vegetation type is composed basically by tree and herb species that are well adapted to the drastic climatic conditions (1). These forests are rich in aromatic bushes, vines and trees from many plant families, like: Eugenia (2); Piper (3) and Cordia (4), which were previously investigated by our research group. Other species widely distributed in the restinga and the semiarid are the ones belonging to the Croton family (Euphorbiaceae), which is one of the major plant families with about 1,300 species of which 300 can be found in Brazil (5). Species of Croton are reported to be rich in terpenoids (essential oils and diterpenes) and alkaloids (6). Due to the great diversity of biological properties shown by these chemical constituents, the Croton family is one of the most riches
source for bioactive compounds of the family Euphorbiaceae (7). In Brazil, specifically in the caatinga region in the “agreste” of Pernambuco, species of the Croton family are popularly known as “velame” or “marmeleiro” and are used for the most diverse purposes (8). The search for insecticidal properties in medicinal plants has grown steadily in the last few years, which could result in the discovery of new plant species with fumigant activity against infestation in stored grains (9). Species which have shown to be an excellent source of insecticidal compounds, are the ones of the Croton family (10–12). In general, Croton species are aromatic and are characterized by their essential oil production, which due to their volatility and possible fumigant action, could be used in the control of pests on stored products as an alternative for commonly used fumigant agents, such as phosphine Received: November 2006
*Address for correspondence
Revised: February 2007 1041-2905/08/0002-0179$14.00/0—© 2008 Allured Publishing Corp. Vol. 20, March/April 2008
Accepted: February 2007 Journal of Essential Oil Research/179
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Table I. Relative percentages of chemical constituents in leaf and stem oils of Croton grewioides. Compound
RIa
Leaf
Stem
Compound
RIa
Leaf
Stem
1,8-cineole linalool camphor α-terpineol methyl chavicol (Z)-anethole p-anisaldehyde (E)-anethole α-copaene β-elemene methyl eugenol
1031 1103 1145 1192 1201 1254 1256 1287 1379 1396 1405 Total
0.1 - - - 1.9 4.6 - 65.5 0.1 - 10.6 98.8
- 0.2 1.5 0.9 - - 1.4 47.8 - 0.3 4.6 98.6
β-caryophyllene trans-α-bergamotene (Z)-methyl isoeugenol germacrene D (E)-methyl isoeugenol cubebol δ-cadinene spathulenol caryophyllene oxide β-acorenol cadalene
1423 1439 1458 1484 1497 1518 1528 1579 1585 1636 1679
4.5 0.3 2.9 0.4 4.7 0.5 1.3 1.6 0.8 - -
0.2 30.0 0.2 0.5 2.6 8.4
Retention indices. Values are calculated from retention times in relation the ones of n-alkanes on the non-polar DB-5, capillary column, 30 m.
a
Table II. Mortality (mean / replicate)1 of Zabrotes subfaciatus exposed for 24h to leaf and stem oils of Croton grewioides and Lethal concentration values2. Dosage (μL) Control
Mortality Leaf
Efficiency (%)
Stem
Efficiency (%)
0.33 + 0.4 cA
-
1.00 + 0.8 dA
-
14.00 + 0.8 aA 6.67 + 0.9 b B 4.33 + 0.9 bcB 2.33 + 0.4 cdB 1.53+ 0.4 dB
70.0 33.3 21.7 11.7 7.7
13.7 µL/L air (10.43-58.75)
-
6 19.00 + 1.4 aA 95.0 5 16.00 + 2.1 abA 80.0 4 15.67 + 1.2 abA 78.4 3 14.00 + 1.6 bA 70.0 2 12.33 + 1.2 bA 61.5 4.0 µL/L air LC50 (1.98-5.26) -
Mean values (± SE) followed by the same minor letter in the column and major letter in the line, for each oil, do not differ significantly between themselves based on Tukey test (P = 0,05); 2Numbers in parenthesis are 95% fiducially limit values.
1
and methyl bromide, which have undesired effects on natural enemies on the environment and on human health. Insect infestation are the major problems in the worldwide bean production (13). The increase of bean plantations gave an increase for the incidence of infestation. These include the bruchids, which attack various seeds causing economic losses. Within the bruchids, Zabrotes subfasciatus (Boheman), commonly known as Mexican bean weevil, is a major infestor of stored grains, responsible for considerable losses of stored bean seeds in the Northeast of Brazil. It is estimated that in Northeast Brazil the losses are higher than 35% while across Brazil the losses are between 7 and 17% (13). Despite the fact that the Mexican been weevil is well distributed worldwide it is responsible for causing large losses in bean seeds, only a few studies on the use of essential oils for its control can be found (14,15). Only a limited number of plants of different families have been studied with respect to their insecticidal potential against Z. subfasciatus. Various plant derived materials have been used to constrain the loss caused by this infestation in stored grains, such as essential oils (14,15), seed oils (16,17) and powders and extracts of different plant parts (18–20). Between the aromatic plants which are abundant in the caatinga vegetation in Pernambuco is the species Croton grewioides Baill., which is a bush growing to a height of 1.5 m naturally in areas of grass between jungles and rocky vegetation in the “agreste” of Pernambuco at a height of 740 m. In 180/Journal of Essential Oil Research
the caatinga, it is characterized by the pleasant smell emitted principally from the leaves. To the best of our knowledge, C. grewioides has not previously been investigated for identification of its chemical components and insecticidal action. Therefore, as a part of a systematic study of the chemical composition and the insecticidal potential of aromatic plants in the bioma caatinga in Pernambuco, the present work describes the chemical composition of the oils of the leaves and stems of C. grewioides. Furthermore, the fumigant potential against Z. subfasciatus (Boheman) was evaluated.
Experimental Plant material: Fresh leaves and stems of Croton grewioides Baill. were collected in the caatinga region in the municipality of Bezerro in Pernambuco, Brazil at an altitude of 740 m. The plant was identified by the specialist (Maria de Fátima de Araújo Lucena) and a voucher specimen was deposited with the number 42193 in the Herbarium Professor Geraldo Mariz of the Federal University of Pernambuco. Biological material: Zabrotes subfasciatus (Boheman) (Mexican bean weevils) used for the bioassay were bred in glass containers (0.3 L) at a temperature of 27º ± 0.5ºC, relative humidity of 75 ± 5% and a light phase of 12 h. The glass containers were topped with 40 mesh copper wire net and as alimentary substrate bean seeds Phaseolus vulgaris L. cv. carioca were used. Vol. 20, March/April 2008
Camara et al.
Oil Isolation: The oils from the fresh leaves and stems (ca. 100 g for each part) were obtained by separate hydrodistillation using a modified Clevenger-type apparatus for 2 h. The oils layers were separated and dried with anhydrous sodium sulphate, stored in glass recipients hermitically sealed under cooling at +8ºC before analysis. The yield calculations were realized by the relation of oil volume isolated in the modified Clevenger-type apparatus and the mass of plant material used for extraction. GC and GC/MS: The oils obtained from leaves and stems of Croton grewioides Baill. were analyzed by GC and GC/MS. Identification was initially made using mass spectra library search with comparison of retention indices obtained by co-injection of the oils and a linear hydrocarbons C11-C24 calculated according of the Van den Dool and Kratz equation (21) followed by computerized matching of the acquired mass spectra with those stored in the Wiley/NBS mass spectral library of the GC/MS data system and other published mass spectra (22). GC: The oils were analyzed using a Hewlett-Packard 5890 SERIES II GC apparatus equipped with a flame ionisation detector (FID) and a J & W Scientific DB-5 fused silica capillary column (30m x 0.25mm x 0.25mm); column temperatures were programmed from 35°C for 2 min, raised to 180°C at 4°C/min, then increased to 280°C at 20 °C/min for integrating purposes. Injector and detector temperatures were 250°C and 280°C, respectively. Hydrogen was used as carrier gas, flow rate 1.5 mL/min, split mode (1:100). Injection volume, 1.5 mL solution of about 10 mg of oil in ethyl acetate. GC/MS: The oil analyses were carried out using a HewlettPackard GC/MS (CG: 5890 SERIES II/ CG-MS: MSD 5971) fitted with the same column and temperature programme as that for the GC experiments. The carrier gas was He, flow rate 1 mL/min, split mode (1:100). Injected volume: 1 mL of 1/100 diluted solution in ethyl acetate. Mass spectra were taken at 70 eV. Scanning speed was 0.5 scan/sec from m/z of 40 to 550. Fumigant assay: The method for evaluating the vapor toxicity of oils of the leaves and stems was modified based on Aslan et al. (23). The tests were made at a temperature of 25 ± 2ºC, relative humidity of 70 ± 8 % and light phase of 12 h. Glass vessels of 2.5 L were used as the fumigation chambers. Each replica consisted of 10 couples of Zabrotes subfasciatus (Boheman) (0 and 24h of age). For each dose, three replicas were used. The oil quantities, applied by an automatic pipette on a paper of 5 x 2 cm2, which was fixed in the middle of the internal surface of the tap of the fumigation chamber, were: 5, 7.5, 10, 12.5 and 15µL, corresponding to 2, 3, 4, 5 and 6 μL/L of air, respectively. To obtain a direct contact of the insect with the paper used for diffusion of the oils a piece of 40 mesh copper wire net was put on the fumigation chamber before closing. A similar procedure was used for the control, where nothing was applied on the paper of glass recipient used in the fumigation chamber control. After 24 h, the mortalities of the insects were reported. An insect was considered dead when its extremities did not move after a contact with a fine brush. The obtained mortality data obtained in the fumigation experiments were submitted to a variance analysis comparing the mean values with the Tukey test (P = 0.05) calculated by Software SANEST 3.0. The data obtained from the bio tests of dose-response were submitted to the probit analysis (24) for estimation the mean lethal dose for the population of the Mexican bean weevils (LC50) with confidence limit of 95% for each oil. Vol. 20, March/April 2008
Results and Discussion The yield of the oils from leaves and stems of C. grewioides was 2.4% and 0.5% (v/w), respectively. The retention index and the percentage of identified compounds of each oil are shown in Table I. In total, 22 compounds were identified making 99.9% and 98.6% of the leaf and stem oils respectively. From the 22 compounds, only (E)-anethole, methyl eugenol, (E)-methyl isoeugenol, β-caryophyllene, δ-cadinene and caryophyllene oxide can be found simultaneously in the leaf and stem oils. Both oils showed a predominance of benzenoid compounds (92.2 % for the stems and 90.2% in the leaves), while terpenoid compounds represented only 9.6% in the leaf oil and 6.4% in the stem oil. The study of the oils of C. grewioides showed phenyl propanoids as the principal compound class for the benzenoids found in the leaves and stems. In the leaf oil all the identified benzenoid compounds were phenyl propanoids, while in the stem oil, besides the phenyl propanoids, other benzenoid compounds could be found like p-anisaldehyde (1.4%) and cadalene (8.4%) (Table I). (E)-anethole was the principal identified constituent in the leaf oil (65.5%) and the stem oil (47.8%). Others principal components found in the stem oil were: (E)-methyl isoeugenol (30.0%) and cadalene (8.44%). Smaller amounts of others compounds present in the leaf oil were: methyl eugenol (10.6%), β-caryophyllene (4.5%), (Z)-anethole (4.7%) and (E)-methyl isoeugenol (4.7%). The oils of various species of Croton are characterized by the predominance of chemical constituents of the class of monoterpenes and sesquiterpenes as principal components (25). Phenyl propanoids, like anethole and eugenol-derivatives, which are common in oils of fennel, basil and clove have been reported as principal components of the oils of others Croton species found in different parts of the world like for example: C. zehntneri and C. nepetaefolius, in Brazil (26); C. malambo and Croton cuneatus in Venezuela (27); Croton pseudoniveus and C suberosus in Mexico (28). Table II shows the mortality of Z. subfaciatus applying different concentrations of leaf and stem oils of C. grewioides. Both oils were found to be toxic for the Mexican bean weevil. Toxicity was determined to vary with the applied concentration. Zabrotes subfaciatus is more susceptible to the leaf oil. After 24 h exposure to the major dose of the leaf oil (15µL (6µL/L of air) insects had of 95% mortality, while at the same dose and exposure time the stem oil caused 70% mortality (Table II). As mortality of the control was less than 10%, the other concentrations did not need a correction before they could be submitted to the probit analysis for the estimation of the LC50 values. From the tested oils, the leaf oil revealed a major fumigant activity against of Z. subfasciatus. The LC50 value for the leaf oil (4.0 µL/L of air) was 3.4 times lower than the one of the calculated value for the stem oil (LC50 = 13.7 µL/L of air). In general, former studies showed that the toxicity of an oil isolated from different plant parts against stored grain infestations was a direct relations to its major components (23). As a result the observed toxicity in this work of the oils of C. grewioides could be related to the phenyl propanoids, which were the principal components identified in the leaf oil: (E)anethole (65.5% in the leaves and 47.8% in the stems), methyl eugenol (10.6% in the leaves), and (E)-methyl isoeugenol (30.0%) in the stems. Using the same line of argument we can suggest that the major toxicity found for the leaf oil could be Journal of Essential Oil Research/181
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attributed to its principal component, which was present in major quantity (ca. 1.4 times more in the leaf oil than in the stems) and its shown insecticidal activity (26, 29–31) without taking into account a probable synergistic and antagonist effects by other components present in the oil in smaller quantities. Phenyl propanoids identified in the oil of C. grewioides as principal components like (E)-anethole, methyl eugenol and their derivatives are known to possess insecticidal properties. There are some studies showing that the insecticidal properties of essential oils from some plants, including Croton, were related to the presence of phenyl propanoids in the oils, like: the oil of C. zenhtneri showed high larvae activity against Aedes aegypti and the bioactive constituent was anethole (26); Chang and Ahn (29) also showed that (E)-anethole possessed fumigant activity against Blattella germânica, and Huang and Ho (30, 31) showed that phenyl propanoids: (E)-anethole, eugenol, isoeugenol and methyl eugenol, which were the main components identified in the oil Illicium verum exhibited insecticidal properties against two infestation of stored grains, Tribolium castaneum and Sitophilus zeamais. These results and ours obtained in the fumigation tests of oils from C. grewioides against Mexican bean weevils clearly revealed that their essential oils were potential fumigant agents, which could be used in a systematic way to limit caused losses by various infestations in stored grains including the control of Zabrotes subfasciatus. Consequently, the possibility to use these natural fumigants for infestation control makes major investigations and cost/benefit studies necessary. Acknowledgments
The authors would like to thank Jorge Braz Torre of the Departamento de Agronomia da Universidade Federal Rural de Pernambuco for the statistical analysis. We also thanks the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) giving scholarships and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support. References 1. E.V.S.B. Sampaio, Overview of the Brazilian Caatinga. In: Seasonally dry tropical forests. Edits., S. H. Bullock, H. Mooney and E. Medina, pp 35–63, Cambridge University Press, Cambridge, UK (1995). 2. R.N. de Oliveira, I.J.M. Dias and C.A.G. da Camara, Estudo comparativo do óleo essencial de Eugenia punicifolia (HBK) DC. de diferentes localidades de Pernambuco. Rev. Bras. Farmacogn., 15, 45–46 (2005). 3. J.C.S. de Oliveira, I.J.M. Dias, M.O.E. Schwartz and C.A.G. da Camara, Volatile constituents of the leaf oils of Piper aduncum L. from different region of Pernambuco (Northeast of Brazil). J. Essent. Oil Res., 18, 557–559 (2006). 4. J.C.S. de Oliveira, C.A.G. da Camara and M.O.E. Schwartz, Volatile constituents of the stem and leaves of Cordia species from mountain forests of Pernambuco (North-eastern Brazil). J. Essent. Oil Res., in the press (2006). 5. V.H. Heywood, Flowering Plants of the World. Oxford University Press, New York, NY (1993). 6. D.J. Milanowski, R.E.K. Winter, M.P.F. Elvin-Lewis and W.H. Lewis, Geographic Distribution of Three Alkaloid Chemotypes of Croton lechleri. J. Nat. Prod., 65, 814–819 (2002). 7. M.F. Guerrero, P. Puebla, R. Carrón, M.L. Martín and L. San Román, Vasorelaxant effect of new neo-clerodane diterpenoids isolated from Croton schiedeanus. J. Ethnpharmacol., 94, 185–189 (2004). 8. N.R. Farnsworth, R.N. Blomster, W.M. Messmer, J.C. King, G.J. Persinos and J.D. Wilkes, A phytochemical and biological review of the genus Croton. Lloydia 32, 1–28 (1969). 9. D.K. Tewary, A. Bhardwaj and A. Shanker, Pesticidal activities in five medicinal plants collected from mid hills of western Himalayas. Ind. Crops Prod., 22, 241–247 (2005). 182/Journal of Essential Oil Research
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