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Structure–activity relationships of larvicidal monoterpenes and derivatives against Aedes aegypti Linn
Chemosphere 84 (2011) 150–153
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Structure–activity relationships of larvicidal monoterpenes and derivatives against Aedes aegypti Linn Sandra R.L. Santos a, Manuela A. Melo a, Andrea Valença Cardoso b, Roseli L.C. Santos b, Damião P. de Sousa c, Sócrates C.H. Cavalcanti a,⇑ a b c
Medicinal Chemistry Laboratory, Pharmacy Department, Federal University of Sergipe, Av. Marechal Rondon S/N, Rosa Elze, São Cristóvão, Sergipe, Brazil ParasitologyLaboratory, Morphology Department, Federal University of Sergipe, Av. Marechal Rondon S/N, Rosa Elze, São Cristóvão, Sergipe, Brazil Natural Products and Synthetic Chemistry Laboratory, Pharmacy Department, Federal University of Sergipe, Av. Marechal Rondon S/N, Rosa Elze, São Cristóvão, Sergipe, Brazil
a r t i c l e
i n f o
Article history: Received 4 November 2010 Received in revised form 2 February 2011 Accepted 5 February 2011 Available online 3 March 2011 Keywords: Structure–activity relationships Monoterpene Larvicidal activity Aedesaegypti Essential oil Limonene
a b s t r a c t In the search for larvicidal compounds against Aedes aegypti L. (Diptera: Culicidae), a collection of monoterpenes were selected and evaluated. R- and S-limonene exhibited the highest larvicidal potency (LC50 = 27 and 30 ppm, respectively), followed by c-terpinene (LC50 = 56 ppm) and RS-carvone (LC50 = 118 ppm). Structural characteristics which may contribute to the understanding of the larvicidal activity of monoterpenes were empirically identified. The presence of heteroatoms in the basic hydrocarbon structure decreases larvicidal potency. Conjugated and exo double bonds appear to increase larvicidal potency. Replacement of double bonds by more reactive epoxides decreases the larvicidal potency. The presence of hydroxyls in the cyclic structure resulted in decreased potency, probably due to increased polarity indicanting that lipophilicity seems to play an important role in increasing the larvicidal potency in this set of compounds. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The incidence of dengue has increased around the world in the last years. According to the World Health Organization (WHO) two fifths of the world’s population is now at risk of dengue with an estimated 50 million dengue infection worldwide every year (WHO, 2009). Dengue is transmitted to humans through the bite of the mosquito Aedes aegypti (Diptera: Culicidae). After virus infection, it causes a severe, flu-like illness. Since there is no specific treatment or vaccine for dengue, the only method of controlling or preventing dengue virus transmission is to combat the vector mosquito by using environmental management and chemical methods. The application of larvicides, such as temephos, or broad application of insecticides as space sprays, such as malathion, resulted in resistance to larvicides and insecticides in subtropical and tropical regions of the world (Braga et al., 2004; Polson et al., 2011). Additionally, non-selective chemical usage has caused damage to non-target organisms other than the dengue transmitting vector, as well as, environmental damage (Bhanti and Taneia, 2007). Therefore, resistance to pesticides has persuaded researchers to find new methods intended to control Ae. aegypti proliferation.
⇑ Corresponding author. Tel./fax: +55 79 2105 6641. E-mail address: [email protected] (S.C.H. Cavalcanti). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.02.018
In the past years there have been an increased number of studies aiming to find novel larvicidal agents in natural products, particularly in essential oil of plants (Silva et al., 2008; Doria et al., 2010; Santos et al., 2010). Naturally occurring monoterpenes, constituents of plant essential oils, are widely described and presents significant biological activities (Erasto and Viljoen, 2008; Lim et al., 2009; Nerio et al., 2010). For example, limonene is a monoterpene derived from the peels of citrus fruits. Its fragrance has long been known to be a strong natural insect repellent and insecticide. Various terpenes, including R-limonene, are found in bark, which repel and kill insects which attack living trees (Rod, 1997). Such terpenes are further used in insecticidal formulations (Tejima et al., 1995). Menthol and its derivatives have been evaluated as insecticides and larvicide against Culex quinquefasciatus Say, Ae. aegypti, Anopheles maculates Theobald, and Anopheles tessellatus Theobald. The evaluated compounds exhibited better knockdown effect and mortality rate against adult female mosquitoes than larvicidal activity (Samarasekera et al., 2008; Yang, 2008). A set of compounds representing many classes of natural products including polyacetylenes, phytosterols, flavonoids, sesquiterpenoids, and triterpenoids have been evaluated against Ae. aegypti larvae (Cantrell et al., 2010). Among these compounds, two eudesmanolides, alantolactone, and isoalantolactone showed larvicidal activities. Further structural modification followed by structure–activity relationships showed a relationship between the Ae. aegypti larvicidal activity and the number of C-atoms in
S.R.L. Santos et al. / Chemosphere 84 (2011) 150–153
the linear amine of alantolactone and isoalantolactone form by a Michael addition. We have recently surveyed 21 compounds, including monoterpenes, for larvicidal activity against third-instar larvae of Ae. aegytpi and have observed the structural characteristics of such compounds in the modulation of larvicidal activity (Santos et al., 2010). The presence of lipophilic groups resulted in increased potency. The presence of hydroxyls resulted in decreased potency. The stereochemistry of selected compounds plays an important role on modulating the potency. Additionally, lipophilicity seems to play a significant role on larvicidal activity. As part of our continuing effort to understand the structural characteristics which modulate the larvicidal activity of monoterpenes and derivatives, we now carried out present the structure–activity relationships studies involving a set of fourteen monoterpenes.
2. Materials and methods 2.1. Chemicals The following compounds were used: R-carvone (98.0%), S-carvone (96.0%), RS-carvone (97.0%), R-limonene (99.2%), S-limonene (99.0%), RS-menthol (99.0%), c-terpinene (98.8%), 3-carene (96.1%), and temephos (97.5%) purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Isopulegol and neoisopulegol were separated and obtained by column chromatography from technical grade isopulegol/Dierberger – Brazil. 1,2-carvone oxide (Klein and Ohloff,
151
1963), (1R,2R,5R)-2-Methyl-5-(1-methylethenyl)-3-oxo-cyclohexanecarbonitrile(Cocker et al., 1995), RS-menthone(Corey and Suggs, 1975), and limonene oxide, mixture of cis and trans (Thomas and Bessiere, 1989) were synthesized according to the literature. The structures of the evaluated compounds are shown in Fig. 1. 2.2. Rearing of Ae. aegypti Eggs of Ae. aegypti were field collected in Aracaju city, Sergipe State, Brazil and laboratory-reared at the Federal University of Sergipe insectary, at 27 °C and 80–85% relative humidity under a 12:12 h light: dark cycle. Ae. aegypti collected in this neighborhood are known to be resistant to temephos. Adults were provided with a 10% sucrose solution ad libitum. Assays eggs were obtained attached to paper strips. The paper strips (1000 eggs L 1) were placed in a rectangular polyethylene container with natural mineral water. The container was kept in the insectary for hatching and monitoring of larvae development for 3 to 4 d. Larvae were fed with cat food (Purina™) to allow regular development. All bioassays were conducted in a walk-in environmental chamber with the above environmental conditions. 2.3. Larvicidal assay Third-instar larvae were used in the experiment (Santos et al., 2010). The concentration ranges were determined by a previous curve concentration–response with 20 larvae. A 20 000 ppm stock
Fig. 1. Structures of evaluated compounds.
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S.R.L. Santos et al. / Chemosphere 84 (2011) 150–153 Table 1 Larvicidal activities (LC50) and 95% confidence intervals (CI) of evaluated compounds on third-instar larvae of Ae.aegypti. Compound
LC50 ppm (CI)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Temephos
152 (136 to 171) 124 (117 to 130) 118 (113 to 124) 412 (306 to 451) 219 (212 to 233) 517 (463 to 570) 27 (23 to 31) 30 (26 to 33) 56 (43 to 70) 150 (112 to 189) 297 (273 to 321) 554 (476 to 633) 404 (371 to 433) 508 (465 to 557) 0.042 (0.035 to 0.050)
Probit analysis was conducted on mortality data collected after 24 h exposure to different concentration of testing solutions to establish the lethal concentration for 50% mortality (LC50) and 95% confidence interval values (CI) for the respective compounds and temephos. Compounds activity is considered significantly different when the 95% CI fail to overlap.
solution was prepared using each compound (20 mg mL 1), Tween-80 (10% vv 1), DMSO (30% vv 1), and natural mineral water (60% vv 1). The stock solution was used to make 20 mL water solutions ranging from 10 to 1000 ppm. Twenty larvae were collected with a Pasteur pipette, placed on a 25 mL graduated cylinder. The volume was completed to 20 mL with natural mineral water and transferred to disposable cups containing variable volumes of the stock solution. A mortality count was conducted 24 h after treatment. Controls were prepared with Tween-80 (0.1 ml), DMSO (0.3 ml), and water (19.6 ml). Three replicates were used for each concentration and the control. Positive control with the organophosphatetemephos, a commonly used insecticide for larvae control, was used on final concentrations ranging from 0.015 to 0.135 ppm. 2.4. Statistics Probit analysis (Finney, 1971) was conducted on mortality data collected after 24 h exposure to different concentration of testing solutions to establish the lethal concentration for 50% mortality (LC50) and 95% confidence intervals (CI) values for the respective compounds and temephos (Table 1). In all cases where deaths had occurred in the control experiment, the data was corrected using Abbott’s formula (%Deaths = [1-(test/control)] 100). Compounds activity is considered significantly different when the 95% CI fail to overlap. 3. Results and discussion 3.1. Structures Structures of the investigated compounds are given in Fig. 1. A diverse set of cyclic aliphatic monoterpenes bearing hydroxyls, ketones, epoxides, and double bonds were selected. (1R,2R,5R)-2Methyl-5-(1-methylethenyl)-3-oxo-cyclohexanecarbonitrile was additionally synthesized and evaluated with the goal to examine the effects of an electron with drawing group in the aliphatic ring. 3.2. Larvicidal effects Compounds listed in Fig. 1 were tested for their in vivo larvicidal activities against Ae. aegypti and were able to induce larvae
mortality to a higher or lower degree. The set of 14 compounds and their evaluated lethal concentration for 50% mortality (LC50), along with 95% confidence intervals (CI) expressed as ppm, are presented in Table 1. At higher concentrations, the larvae showed restless movement for some time and then settled at the bottom of the cups with abnormal wagging and died. The rates of mortality were directly proportional to concentration. According to the data shown in Table 1, the LC50 value of 1,2-epoxycarvone (5) was significantly different from all the other evaluated compounds, LC50 = 219 ppm (212 to 233), since the CI value failed to overlap with the CI of the remaining compounds in Table 1. Interestingly, the larvicidal activity values of the enantiomers R-carvone (1) and S-carvone (2) were significantly different, LC50 = 152 ppm (136 to 171) and 124 ppm (117–130). In contrast, the larvicidal activities of R-limonene (7) and S-limonene (8) were similar LC50 = 27 ppm (23–31) and 30 ppm (26–33), respectively. Additionally, R- and S-limonene were the most potent compounds in this series, inducing 100% mortality of Ae. aegypti larvae after 24 h at 100 ppm, followed by c-terpinene (9), LC50 = 56 ppm (43– 70). The racemic mixture of carvone (3) exhibited higher potency than the individual enantiomers LC50 = 118 ppm (113–124). Neoisopulegol (12) exhibited the lowest larvicidal potency inducing 100% mortality only at 1000 ppm. Other compounds exhibited intermediate potencies.
3.3. Structure–activity The structurally related compounds exhibited different potency profiles, varying from 27 to 554 ppm. The most potent compounds, R- and S-limonene (LC50 = 27 and 30 ppm, respectively), are unsaturated cyclic hydrocarbons possessing endo and exo double bonds. Shifting the exo double bond in limonene results in nearly two fold less potent c-terpinene (9) (LC50 = 56 ppm). Similarly, lack of the exo double bond in limonene along with the presence of a three membered ring, resulting in the bicyclic compound 3-carene (10) (LC50 = 150 ppm) led to five times decrease in potency. Similar results were found in previous work, when the potency of bicyclic norbornenes were lower than aromatic compounds (Santos et al., 2010). The importance of double bonds in the larvicidal activity of natural products has been reported. The reduction of double bonds of sodium salts of cashew nut shell extracts, cardanol, cardol, and anacardic acids isolated from Anacardium occidentale resulted in decrease in larvicidal potency against Ae. aegypti (Laurens et al., 1997; Lomonaco et al., 2009). Belzile et al. (2000) reported the synergistic activity of dillapiol in combination with the phototoxin alpha-terthienyl against Aedesatropalpus larvae. Modification of the allyl side chain resulted in either reduction or elimination of the synergistic activity. The addition of heteroatoms to the cyclic hydrocarbon structure of limonene results in an overall decrease in potency, which may be related to a decrease in lipophilicity. Within the heteroatombearing compounds, the racemic mixture of carvone exhibited the highest potency (3), (LC50 = 118 ppm), followed by S-carvone (2) (LC50 = 124 ppm) and R-carvone (1) (LC50 = 152 ppm). Lack of the a,b-unsaturated ketone of carvone with the addition of groups containing heteroatoms, resulting in (1R,2R,5R)-2-methyl-5-(1methylethenyl)-3-oxo-cyclohexanecarbonitrile (4) and 1,2-carvone oxide (5) results in two to three fold decrease in potency, LC50 = 412 and 219 ppm, respectively. The number of conjugated double bonds appear to contribute to an increase in potency, since aromatic compounds, such as, thymol, carvacrol, and eugenol exhibit higher larvicidal potencies than carvone (Santos et al., 2010). Additionally, fully conjugated cinnamaldehyde exhibited high potency compared to other monoterpenes evaluated against Culexpipiens (Diptera: Culicidae) larvae (Radwan et al., 2008). These data
S.R.L. Santos et al. / Chemosphere 84 (2011) 150–153
show that the influence of electronic density and aromatic ring planarity may also play an important role on potency. More reactive epoxides 5 and 6 did not contribute to an increase in potency, instead a decrease up to19 fold in potency was observed. In general, the presence of hydroxyl in the aliphatic ring resulted in less active compounds. Changing hydroxyl (13, LC50 = 404 ppm) to carbonyl (14, LC50 = 508) lead to a slight decrease in potency for menthol. A reasonable explanation for this result may be the presence of hydroxyl groups preventing the substance penetration in the larvae cuticle and reaching its targets (Lopez et al., 2005). The diastereoisomers isopulegol (11, LC50 = 297 ppm) and neoisopulegol (12, LC50 = 554 ppm) exhibited significantly different potency profiles. Neoisopulegol exhibited much weaker potency than its diastereoisomer. Some of our reported LC50 values differ from reported data (Perumalsamy et al., 2009) which may be the result of different methodologies and analysis. Additionally, different species, from different ecological niches, appear to be more susceptible or resistant to specific compounds (Waliwitiya et al., 2009). 4. Conclusions Limonene exhibited the highest larvicidal activity against Ae. aegypti, while neoisopulegol exhibited the lowest potency. Structural characteristics which may contribute to the understanding of the larvicidal activity of monoterpenes were identified. The presence of heteroatoms in the basic monoterpene structure decreases larvicidal potency. Conjugated and exo double bonds appear to increase larvicidal potency. Replacement of double bonds by epoxides decreases the larvicidal potency. The presence of hydroxyls in the cyclic structure resulted in decreased potency. One area of interest in the production of environmentally safe larvicides is the use of Generally Regarded As Safe (GRAS) food flavorings. Most compounds herein evaluated are approved for use as a GRAS food flavoring additive in foods eaten by humans (United States. Office of the Federal Register, 2009). Therefore, such class of compounds is probably safe for use as larvicides. However, additional studies are required and are being conducted by our research group. Acknowledgements The authors are deeply grateful to the technical assistance of Mr. Osvaldo Andrade Santos. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Grant nuber 550 116/2010-9) and Fundação de Apoio a Pesquisa e Inovação Tecnológica do Estado de Sergipe (FAPITEC/SE). References Belzile, A.S., Majerus, S.L., Podeszfinski, C., Guillet, G., Durst, T., Arnason, J.T., 2000. Dillapiol derivatives as synergists: structure-activity relationship analysis. Pest Biochem. Physiol. 66, 33–40. Bhanti, M., Taneia, A., 2007. Contamination of vegetables of different seasons with organophosphorous pesticides and related health risk assessment in northern India. Chemosphere 69, 63–68.
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Braga, I.A., Lima, J.B.P., Soares, S.D., Valle, D., 2004. Aedes aegypti resistance to temephos during 2001 in several municipalities in the states of Rio de Janeiro, Sergipe, and Alagoas, Brazil. Mem. Inst. Oswaldo Cruz. 99, 199–203. Cantrell, C.L., Pridgeon, J.W., Fronczek, F.R., Becnel, J.J., 2010. Structure-activity relationship studies on derivatives of eudesmanolides from Inula helenium as toxicants against Aedes aegypti larvae and adults. Chem. Biodivers. 7, 1681– 1697. Cocker, W., Grayson, D.H., Shannon, P.V.R., 1995. Hydrocyanation of Some a,bunsaturated ketones, and the synthesis of some unusual isoxazoles. J. Chem. Soc. Perk T 1, 1153–1162. Corey, E.J., Suggs, J.W., 1975. Pyridinium chlorochromate – efficient reagent for oxidation of primary and secondary alcohols to carbonyl-compounds. Tetrahedron Lett. 16, 2647–2650. Doria, G.A.A., Silva, W.J., Carvalho, G.A., Alves, P.B., Cavalcanti, S.C.H., 2010. A study of the larvicidal activity of two Croton species from northeastern Brazil against Aedes aegypti. Pharm. Biol. 48, 615–620. Erasto, P., Viljoen, A.M., 2008. Limonene – a review: biosynthetic, ecological and pharmacological relevance. Nat. Prod. Commun. 3, 1193–1202. Finney, D.J., 1971. Probit Analysis. 3rd ed. University Press, Cambridge. Klein, E., Ohloff, G., 1963. Der stereochemische verlauf der alkalischen epoxydation von alpha, beta-ungesattigten carbonylverbindungen der cyclischen monoterpenreihe. Tetrahedron 19, 1091–1099. Laurens, A., Fourneau, C., Hocquemiller, R., Cave, A., Bories, C., Loiseau, P.M., 1997. Antivectorial activities of cashew nut shell extracts from Anacardium occidentale L. Phytother. Res. 11, 145–146. Lim, P.F.C., Liu, X.Y., Chan, S.Y., 2009. A review on terpenes as skin penetration enhancers in transdermal drug delivery. J. Essent. Oil Res. 21, 423–428. Lomonaco, D., Santiago, G.M.P., Ferreira, Y.S., Arriaga, A.M.C., Mazzetto, S.E., Mele, G., Vasapollo, G., 2009. Study of technical CNSL and its main components as new green larvicides. Green Chem. 11, 31–33. Lopez, O., Fernandez-Bolanos, J.G., Gil, M.V., 2005. New trends in pest control: the search for greener insecticides. Green Chem. 7, 431–442. Nerio, L.S., Olivero-Verbel, J., Stashenko, E., 2010. Repellent activity of essential oils: a review. Bioresour. Technol. 101, 372–378. Perumalsamy, H., Kim, N.J., Ahn, Y.J., 2009. Larvicidal activity of compounds isolated from Asarum heterotropoides against Culex pipiens pallens, Aedes aegypti, and Ochlerotatus togoi (Diptera: Culicidae). J. Med. Entomol. 46, 1420–1423. Polson, K.A., Brogdon, W.G., Rawlins, S.C., Chadee, D.D., 2011. Characterization of insecticide resistance in Trinidadian strains of Aedes aegypti mosquitoes. Acta Trop. 117, 31–38. Radwan, M.A., El-Zemity, S.R., Mohamed, S.A., Sherby, S.M., 2008. Larvicidal activity of some essential oils, monoterpenoids and their corresponding N-methyl carbamate derivatives against Culex pipiens (Diptera: Culicidae). Int. J. Trop. Insect Sci. 28, 61–68. Rod, R.L., 1997. Process for repelling and killing insects and compositions to effect the same comprising a monoterpene. In: Patent, US (Ed.). USPTO Patent full-text and image database. The Rod Family Trust United States, p. 11. Samarasekera, R., Weerasinghe, I.S., Hemalal, K.D.P., 2008. Insecticidal activity of menthol derivatives against mosquitoes. Pest Manage. Sci. 64, 290–295. Santos, S.R.L., Silva, V.B., Melo, M.A., Barbosa, J.D.F., Santos, R.L.C., Sousa, D.P., Cavalcanti, S.C.H., 2010. Toxic effects on and structure–toxicity relationships of phenylpropanoids, terpenes, and related compounds in Aedes aegypti larvae. Vector Borne Zoonotic Dis. 10, 1049–1054. Silva, W.J., Doria, G.A.A., Maia, R.T., Nunes, R.S., Carvalho, G.A., Blank, A.F., Alves, P.B., Marcal, R.M., Cavalcanti, S.C.H., 2008. Effects of essential oils on Aedes aegypti larvae: alternatives to environmentally safe insecticides. Bioresour. Technol. 99, 3251–3255. Tejima, I., Kusumi, Y., Matsunaga, T., 1995. Control of insects by volatilization of insecticide solutions containing terpenoids by heat and the insecticide solutions. In: Koho, K.T., (Ed.). Japan, p. 5. Thomas, A.F., Bessiere, Y., 1989. Limonene. Nat. Prod. Rep. 6, 291–309. United States, 2009. Office of the Federal Register. Code of federal regulations. 21, Food and drugs, Part 170-199, Washington. Waliwitiya, R., Kennedy, C.J., Lowenberger, C.A., 2009. Larvicidal and ovipositionaltering activity of monoterpenoids, trans-anethole and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae). Pest Manag. Sci. 65, 241–248. WHO, 2009. Dengue and dengue haemorrhagic fever. Geneva, p. Fact sheet 117. Yang, W., 2008. Larvicidal activity of menthol derivatives. Shijie Nongyao (World pesticides) 30, 33–36.
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Structure–activity relationships of larvicidal monoterpenes and derivatives against Aedes aegypti Linn Sandra R.L. Santos a, Manuela A. Melo a, Andrea Valença Cardoso b, Roseli L.C. Santos b, Damião P. de Sousa c, Sócrates C.H. Cavalcanti a,⇑ a b c
Medicinal Chemistry Laboratory, Pharmacy Department, Federal University of Sergipe, Av. Marechal Rondon S/N, Rosa Elze, São Cristóvão, Sergipe, Brazil ParasitologyLaboratory, Morphology Department, Federal University of Sergipe, Av. Marechal Rondon S/N, Rosa Elze, São Cristóvão, Sergipe, Brazil Natural Products and Synthetic Chemistry Laboratory, Pharmacy Department, Federal University of Sergipe, Av. Marechal Rondon S/N, Rosa Elze, São Cristóvão, Sergipe, Brazil
a r t i c l e
i n f o
Article history: Received 4 November 2010 Received in revised form 2 February 2011 Accepted 5 February 2011 Available online 3 March 2011 Keywords: Structure–activity relationships Monoterpene Larvicidal activity Aedesaegypti Essential oil Limonene
a b s t r a c t In the search for larvicidal compounds against Aedes aegypti L. (Diptera: Culicidae), a collection of monoterpenes were selected and evaluated. R- and S-limonene exhibited the highest larvicidal potency (LC50 = 27 and 30 ppm, respectively), followed by c-terpinene (LC50 = 56 ppm) and RS-carvone (LC50 = 118 ppm). Structural characteristics which may contribute to the understanding of the larvicidal activity of monoterpenes were empirically identified. The presence of heteroatoms in the basic hydrocarbon structure decreases larvicidal potency. Conjugated and exo double bonds appear to increase larvicidal potency. Replacement of double bonds by more reactive epoxides decreases the larvicidal potency. The presence of hydroxyls in the cyclic structure resulted in decreased potency, probably due to increased polarity indicanting that lipophilicity seems to play an important role in increasing the larvicidal potency in this set of compounds. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The incidence of dengue has increased around the world in the last years. According to the World Health Organization (WHO) two fifths of the world’s population is now at risk of dengue with an estimated 50 million dengue infection worldwide every year (WHO, 2009). Dengue is transmitted to humans through the bite of the mosquito Aedes aegypti (Diptera: Culicidae). After virus infection, it causes a severe, flu-like illness. Since there is no specific treatment or vaccine for dengue, the only method of controlling or preventing dengue virus transmission is to combat the vector mosquito by using environmental management and chemical methods. The application of larvicides, such as temephos, or broad application of insecticides as space sprays, such as malathion, resulted in resistance to larvicides and insecticides in subtropical and tropical regions of the world (Braga et al., 2004; Polson et al., 2011). Additionally, non-selective chemical usage has caused damage to non-target organisms other than the dengue transmitting vector, as well as, environmental damage (Bhanti and Taneia, 2007). Therefore, resistance to pesticides has persuaded researchers to find new methods intended to control Ae. aegypti proliferation.
⇑ Corresponding author. Tel./fax: +55 79 2105 6641. E-mail address: [email protected] (S.C.H. Cavalcanti). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.02.018
In the past years there have been an increased number of studies aiming to find novel larvicidal agents in natural products, particularly in essential oil of plants (Silva et al., 2008; Doria et al., 2010; Santos et al., 2010). Naturally occurring monoterpenes, constituents of plant essential oils, are widely described and presents significant biological activities (Erasto and Viljoen, 2008; Lim et al., 2009; Nerio et al., 2010). For example, limonene is a monoterpene derived from the peels of citrus fruits. Its fragrance has long been known to be a strong natural insect repellent and insecticide. Various terpenes, including R-limonene, are found in bark, which repel and kill insects which attack living trees (Rod, 1997). Such terpenes are further used in insecticidal formulations (Tejima et al., 1995). Menthol and its derivatives have been evaluated as insecticides and larvicide against Culex quinquefasciatus Say, Ae. aegypti, Anopheles maculates Theobald, and Anopheles tessellatus Theobald. The evaluated compounds exhibited better knockdown effect and mortality rate against adult female mosquitoes than larvicidal activity (Samarasekera et al., 2008; Yang, 2008). A set of compounds representing many classes of natural products including polyacetylenes, phytosterols, flavonoids, sesquiterpenoids, and triterpenoids have been evaluated against Ae. aegypti larvae (Cantrell et al., 2010). Among these compounds, two eudesmanolides, alantolactone, and isoalantolactone showed larvicidal activities. Further structural modification followed by structure–activity relationships showed a relationship between the Ae. aegypti larvicidal activity and the number of C-atoms in
S.R.L. Santos et al. / Chemosphere 84 (2011) 150–153
the linear amine of alantolactone and isoalantolactone form by a Michael addition. We have recently surveyed 21 compounds, including monoterpenes, for larvicidal activity against third-instar larvae of Ae. aegytpi and have observed the structural characteristics of such compounds in the modulation of larvicidal activity (Santos et al., 2010). The presence of lipophilic groups resulted in increased potency. The presence of hydroxyls resulted in decreased potency. The stereochemistry of selected compounds plays an important role on modulating the potency. Additionally, lipophilicity seems to play a significant role on larvicidal activity. As part of our continuing effort to understand the structural characteristics which modulate the larvicidal activity of monoterpenes and derivatives, we now carried out present the structure–activity relationships studies involving a set of fourteen monoterpenes.
2. Materials and methods 2.1. Chemicals The following compounds were used: R-carvone (98.0%), S-carvone (96.0%), RS-carvone (97.0%), R-limonene (99.2%), S-limonene (99.0%), RS-menthol (99.0%), c-terpinene (98.8%), 3-carene (96.1%), and temephos (97.5%) purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Isopulegol and neoisopulegol were separated and obtained by column chromatography from technical grade isopulegol/Dierberger – Brazil. 1,2-carvone oxide (Klein and Ohloff,
151
1963), (1R,2R,5R)-2-Methyl-5-(1-methylethenyl)-3-oxo-cyclohexanecarbonitrile(Cocker et al., 1995), RS-menthone(Corey and Suggs, 1975), and limonene oxide, mixture of cis and trans (Thomas and Bessiere, 1989) were synthesized according to the literature. The structures of the evaluated compounds are shown in Fig. 1. 2.2. Rearing of Ae. aegypti Eggs of Ae. aegypti were field collected in Aracaju city, Sergipe State, Brazil and laboratory-reared at the Federal University of Sergipe insectary, at 27 °C and 80–85% relative humidity under a 12:12 h light: dark cycle. Ae. aegypti collected in this neighborhood are known to be resistant to temephos. Adults were provided with a 10% sucrose solution ad libitum. Assays eggs were obtained attached to paper strips. The paper strips (1000 eggs L 1) were placed in a rectangular polyethylene container with natural mineral water. The container was kept in the insectary for hatching and monitoring of larvae development for 3 to 4 d. Larvae were fed with cat food (Purina™) to allow regular development. All bioassays were conducted in a walk-in environmental chamber with the above environmental conditions. 2.3. Larvicidal assay Third-instar larvae were used in the experiment (Santos et al., 2010). The concentration ranges were determined by a previous curve concentration–response with 20 larvae. A 20 000 ppm stock
Fig. 1. Structures of evaluated compounds.
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S.R.L. Santos et al. / Chemosphere 84 (2011) 150–153 Table 1 Larvicidal activities (LC50) and 95% confidence intervals (CI) of evaluated compounds on third-instar larvae of Ae.aegypti. Compound
LC50 ppm (CI)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Temephos
152 (136 to 171) 124 (117 to 130) 118 (113 to 124) 412 (306 to 451) 219 (212 to 233) 517 (463 to 570) 27 (23 to 31) 30 (26 to 33) 56 (43 to 70) 150 (112 to 189) 297 (273 to 321) 554 (476 to 633) 404 (371 to 433) 508 (465 to 557) 0.042 (0.035 to 0.050)
Probit analysis was conducted on mortality data collected after 24 h exposure to different concentration of testing solutions to establish the lethal concentration for 50% mortality (LC50) and 95% confidence interval values (CI) for the respective compounds and temephos. Compounds activity is considered significantly different when the 95% CI fail to overlap.
solution was prepared using each compound (20 mg mL 1), Tween-80 (10% vv 1), DMSO (30% vv 1), and natural mineral water (60% vv 1). The stock solution was used to make 20 mL water solutions ranging from 10 to 1000 ppm. Twenty larvae were collected with a Pasteur pipette, placed on a 25 mL graduated cylinder. The volume was completed to 20 mL with natural mineral water and transferred to disposable cups containing variable volumes of the stock solution. A mortality count was conducted 24 h after treatment. Controls were prepared with Tween-80 (0.1 ml), DMSO (0.3 ml), and water (19.6 ml). Three replicates were used for each concentration and the control. Positive control with the organophosphatetemephos, a commonly used insecticide for larvae control, was used on final concentrations ranging from 0.015 to 0.135 ppm. 2.4. Statistics Probit analysis (Finney, 1971) was conducted on mortality data collected after 24 h exposure to different concentration of testing solutions to establish the lethal concentration for 50% mortality (LC50) and 95% confidence intervals (CI) values for the respective compounds and temephos (Table 1). In all cases where deaths had occurred in the control experiment, the data was corrected using Abbott’s formula (%Deaths = [1-(test/control)] 100). Compounds activity is considered significantly different when the 95% CI fail to overlap. 3. Results and discussion 3.1. Structures Structures of the investigated compounds are given in Fig. 1. A diverse set of cyclic aliphatic monoterpenes bearing hydroxyls, ketones, epoxides, and double bonds were selected. (1R,2R,5R)-2Methyl-5-(1-methylethenyl)-3-oxo-cyclohexanecarbonitrile was additionally synthesized and evaluated with the goal to examine the effects of an electron with drawing group in the aliphatic ring. 3.2. Larvicidal effects Compounds listed in Fig. 1 were tested for their in vivo larvicidal activities against Ae. aegypti and were able to induce larvae
mortality to a higher or lower degree. The set of 14 compounds and their evaluated lethal concentration for 50% mortality (LC50), along with 95% confidence intervals (CI) expressed as ppm, are presented in Table 1. At higher concentrations, the larvae showed restless movement for some time and then settled at the bottom of the cups with abnormal wagging and died. The rates of mortality were directly proportional to concentration. According to the data shown in Table 1, the LC50 value of 1,2-epoxycarvone (5) was significantly different from all the other evaluated compounds, LC50 = 219 ppm (212 to 233), since the CI value failed to overlap with the CI of the remaining compounds in Table 1. Interestingly, the larvicidal activity values of the enantiomers R-carvone (1) and S-carvone (2) were significantly different, LC50 = 152 ppm (136 to 171) and 124 ppm (117–130). In contrast, the larvicidal activities of R-limonene (7) and S-limonene (8) were similar LC50 = 27 ppm (23–31) and 30 ppm (26–33), respectively. Additionally, R- and S-limonene were the most potent compounds in this series, inducing 100% mortality of Ae. aegypti larvae after 24 h at 100 ppm, followed by c-terpinene (9), LC50 = 56 ppm (43– 70). The racemic mixture of carvone (3) exhibited higher potency than the individual enantiomers LC50 = 118 ppm (113–124). Neoisopulegol (12) exhibited the lowest larvicidal potency inducing 100% mortality only at 1000 ppm. Other compounds exhibited intermediate potencies.
3.3. Structure–activity The structurally related compounds exhibited different potency profiles, varying from 27 to 554 ppm. The most potent compounds, R- and S-limonene (LC50 = 27 and 30 ppm, respectively), are unsaturated cyclic hydrocarbons possessing endo and exo double bonds. Shifting the exo double bond in limonene results in nearly two fold less potent c-terpinene (9) (LC50 = 56 ppm). Similarly, lack of the exo double bond in limonene along with the presence of a three membered ring, resulting in the bicyclic compound 3-carene (10) (LC50 = 150 ppm) led to five times decrease in potency. Similar results were found in previous work, when the potency of bicyclic norbornenes were lower than aromatic compounds (Santos et al., 2010). The importance of double bonds in the larvicidal activity of natural products has been reported. The reduction of double bonds of sodium salts of cashew nut shell extracts, cardanol, cardol, and anacardic acids isolated from Anacardium occidentale resulted in decrease in larvicidal potency against Ae. aegypti (Laurens et al., 1997; Lomonaco et al., 2009). Belzile et al. (2000) reported the synergistic activity of dillapiol in combination with the phototoxin alpha-terthienyl against Aedesatropalpus larvae. Modification of the allyl side chain resulted in either reduction or elimination of the synergistic activity. The addition of heteroatoms to the cyclic hydrocarbon structure of limonene results in an overall decrease in potency, which may be related to a decrease in lipophilicity. Within the heteroatombearing compounds, the racemic mixture of carvone exhibited the highest potency (3), (LC50 = 118 ppm), followed by S-carvone (2) (LC50 = 124 ppm) and R-carvone (1) (LC50 = 152 ppm). Lack of the a,b-unsaturated ketone of carvone with the addition of groups containing heteroatoms, resulting in (1R,2R,5R)-2-methyl-5-(1methylethenyl)-3-oxo-cyclohexanecarbonitrile (4) and 1,2-carvone oxide (5) results in two to three fold decrease in potency, LC50 = 412 and 219 ppm, respectively. The number of conjugated double bonds appear to contribute to an increase in potency, since aromatic compounds, such as, thymol, carvacrol, and eugenol exhibit higher larvicidal potencies than carvone (Santos et al., 2010). Additionally, fully conjugated cinnamaldehyde exhibited high potency compared to other monoterpenes evaluated against Culexpipiens (Diptera: Culicidae) larvae (Radwan et al., 2008). These data
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show that the influence of electronic density and aromatic ring planarity may also play an important role on potency. More reactive epoxides 5 and 6 did not contribute to an increase in potency, instead a decrease up to19 fold in potency was observed. In general, the presence of hydroxyl in the aliphatic ring resulted in less active compounds. Changing hydroxyl (13, LC50 = 404 ppm) to carbonyl (14, LC50 = 508) lead to a slight decrease in potency for menthol. A reasonable explanation for this result may be the presence of hydroxyl groups preventing the substance penetration in the larvae cuticle and reaching its targets (Lopez et al., 2005). The diastereoisomers isopulegol (11, LC50 = 297 ppm) and neoisopulegol (12, LC50 = 554 ppm) exhibited significantly different potency profiles. Neoisopulegol exhibited much weaker potency than its diastereoisomer. Some of our reported LC50 values differ from reported data (Perumalsamy et al., 2009) which may be the result of different methodologies and analysis. Additionally, different species, from different ecological niches, appear to be more susceptible or resistant to specific compounds (Waliwitiya et al., 2009). 4. Conclusions Limonene exhibited the highest larvicidal activity against Ae. aegypti, while neoisopulegol exhibited the lowest potency. Structural characteristics which may contribute to the understanding of the larvicidal activity of monoterpenes were identified. The presence of heteroatoms in the basic monoterpene structure decreases larvicidal potency. Conjugated and exo double bonds appear to increase larvicidal potency. Replacement of double bonds by epoxides decreases the larvicidal potency. The presence of hydroxyls in the cyclic structure resulted in decreased potency. One area of interest in the production of environmentally safe larvicides is the use of Generally Regarded As Safe (GRAS) food flavorings. Most compounds herein evaluated are approved for use as a GRAS food flavoring additive in foods eaten by humans (United States. Office of the Federal Register, 2009). Therefore, such class of compounds is probably safe for use as larvicides. However, additional studies are required and are being conducted by our research group. Acknowledgements The authors are deeply grateful to the technical assistance of Mr. Osvaldo Andrade Santos. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Grant nuber 550 116/2010-9) and Fundação de Apoio a Pesquisa e Inovação Tecnológica do Estado de Sergipe (FAPITEC/SE). References Belzile, A.S., Majerus, S.L., Podeszfinski, C., Guillet, G., Durst, T., Arnason, J.T., 2000. Dillapiol derivatives as synergists: structure-activity relationship analysis. Pest Biochem. Physiol. 66, 33–40. Bhanti, M., Taneia, A., 2007. Contamination of vegetables of different seasons with organophosphorous pesticides and related health risk assessment in northern India. Chemosphere 69, 63–68.
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