Parasitol Res (2014) 113:2647–2654 DOI 10.1007/s00436-014-3917-6
ORIGINAL PAPER
Essential oils with insecticidal activity against larvae of Aedes aegypti (Diptera: Culicidae) Sharon Smith Vera & Diego Fernando Zambrano & Stelia Carolina Méndez-Sanchez Fernando Rodríguez-Sanabria & Elena E. Stashenko & Jonny E. Duque Luna
Received: 18 March 2014 / Accepted: 11 April 2014 / Published online: 30 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Insecticidal activity of the essential oils (EOs) isolated from Tagetes lucida, Lippia alba, Lippia origanoides, Eucalyptus citriodora, Cymbopogon citratus, Cymbopogon flexuosus, Citrus sinensis, Swinglea glutinosa, and Cananga odorata aromatic plants, grown in Colombia (Bucaramanga, Santander), and of a mixture of L. alba and L. origanoides EOs were evaluated on Aedes (Stegomyia) aegypti Rockefeller larvae. The EOs were extracted by microwave-assisted hydrodistillation and characterized by gas chromatography–mass spectrometry (GC-MS). The main components of the EOs were identified using their linear retention indices and mass spectra. The lethal concentrations (LCs) of the EOs were determined between the
third and fourth instar of A. aegypti. LC50 was determined by probit analysis using mortality rates of bioassays. All essential oils tested showed insecticidal activity. The following values were obtained for C. flexuosus (LC50 =17.1 ppm); C. sinensis (LC50 =20.6 ppm); the mixture of L. alba and L. origanoides (LC50 =40.1 ppm); L. alba (LC50 =42.2 ppm); C. odorata (LC 50 = 52.9 ppm); L. origanoides (LC 50 = 53.3 ppm); S. glutinosa (LC50 =65.7 ppm); T. lucida (LC50 =66.2 ppm); E. citriodora (LC50 =71.2 ppm); and C. citratus (LC50 = 123.3 ppm). The EO from C. flexuosus, with citral (geranial+neral) as main component, showed the highest larvicidal activity.
S. S. Vera : D. F. Zambrano : F. Rodríguez-Sanabria : J. E. Duque Luna Center for Research on Tropical Diseases (Centro de Investigación en Enfermedades Tropicales-CINTROP), Faculty of Health, School of Medicine, Department of Basic Sciences, Universidad Industrial de Santander, Bucaramanga, Colombia
Keywords Essential oil . Larvicidal activity . Mosquito control . Geranial
F. Rodríguez-Sanabria : J. E. Duque Luna (*) Group for Research on Infectious and Metabolic Diseases (Grupo de Investigación en Enfermedades Infecciosas y Metabólicas-GINEM), Faculty of Health, School of Medicine, Department of Basic Sciences, Universidad Industrial de Santander, Bucaramanga, Colombia e-mail: [email protected] S. C. Méndez-Sanchez Group for Research in Biochemistry and Microbiology (Grupo de Investigación en Bioquímica Y Microbiología-GIBIM), School of Chemistry, Universidad Industrial de Santander, Bucaramanga, Colombia E. E. Stashenko Center for Research in Biomolecules (Centro de Investigación en Biomoléculas-CIBIMOL) and the National Research Center for the Agro-industrialization of Tropical Medicinal and Aromatic Plants (Centro Nacional de Investigación para la Agroindustrialización de Plantas Aromáticas y Medicinales Tropicales-CENIVAM), Universidad Industrial de Santander, Bucaramanga, Colombia
Introduction Dengue is the most important disease transmitted by arthropods in the world; more than 2.5 billion people are at risk of contracting the virus by sharing an area of spatial distribution with the vector Aedes (Stegomyia) aegypti (L., 1762) (Guzman et al. 2010). Although this mosquito can also transmit other arboviral infections such as encephalitis, yellow fever, and hemorrhagic fevers, among others, none of these ailments compares to dengue when it comes to morbidity and mortality (Bhatt et al. 2013). A phenomenon of the problem faced with this vector is that the number of cases increases every year as a result of the simultaneous circulation of the four serotypes DENV-1, DENV-2, DENV-3, and DENV-4 (Laughlin et al. 2012; Simmons et al. 2012). Since more than 50 million people are infected with dengue each year, and this leads to the death of a significant percentage of the people infected due to dengue complications, there are periodic health campaigns to find solutions for the
2648
problem. Nevertheless, these activities are carried out in accordance with the policies of the countries affected by the problem. However, in one way or another, they are all focused on the population decline of the vector. Actions involve environmental sanitation, removal of breeding areas, elimination of potential containers, and the application of chemical and biological insecticides (Duque et al. 2009; Guzman et al. 2010). Despite the efforts in environmental sanitation and vector control, it has been observed that what is being done does not have a significant impact on case reduction, which is why it is said that the problem remains unresolved (Guzman et al. 2010; Simmons et al. 2012). A consensus among experts on the topic is that dengue will not be eliminated until the vaccine is created to act against the four serotypes of the dengue virus (Simmons et al. 2012). To this effect, the discovery of other alternatives for protecting from the mosquito is crucial, as is the discovery of new insecticides to replace the traditional organophosphates (or) and pyrethroids (Pr) that cause adverse effects on man and the environment. Particularly, since there are countless records that these insecticides have generated populations of A. aegypti resistant to these commercial products (Grisales et al. 2013; Harris et al. 2010; Lima et al. 2011). Under this perspective, new molecules must be found to control insect pests that will replace synthetic insecticides. You could say that there is now a good source of information on products that have an effect on A. aegypti. Thanks to the studies of many researchers on the semiochemical manifestations of different plants in terms of their extracts and essential oils (EOs), we now have knowledge of insecticidal activity, growth inhibition, and repellent, antifeedant, and deterrent actions, among others (Bakkali et al. 2008; Isman 2006; Shaalan et al. 2005). In addition, there are a number of benefits that make the exploration of botanical insecticides for pest control attractive, such as low environmental persistence, little or no toxicity to mammals, and the different forms of action on the insect (Kishore et al. 2011; Mullai et al. 2008; Silva et al. 2008). Although the use of plant-based products has been known for centuries, many of the studies that evaluate plants and their by-products applied on mosquitoes are no more than one hundred years old. That is because most of this research has been conducted on agricultural pests. However, current literature shows an increasing interest in discovering new plants with insecticidal action, which is reflected in at least 44 plant families with confirmed insecticidal activity, including an effect on the reduction of the dengue virus (Abd et al. 2013; Kishore et al. 2011; Neiro et al. 2010). That is why we intend to contribute information about plants and majority compounds that have biocidal action on mosquitoes by evaluating the insecticidal effect of the EO of Tagetes lucida Cav (Asteraceae), Lippia alba (Mill.) N.E.Br.
Parasitol Res (2014) 113:2647–2654
ex Britton & P. Wilson, Lippia origanoides Kunth (Verbenaceae), Eucalyptus citriodora (Hook.) K.D.Hill & L.A.S. Johnson (Myrtaceae), Cymbopogon citratus (DC.) Stapf, Cymbopogon flexuosus (Nees ex Steud) Wats (Poaceae), Citrus sinensis Osbeck, Swinglea glutinosa (Blanco) Merr. (Rutaceae), Cananga odorata (Lam.) Hook. F & Thomson (Annonaceae), and a combination of L. alba and L. origanoides on larvae of A. aegypti.
Materials and methods Colony of mosquitoes To conduct the experiments on insecticidal action, A. aegypti mosquitoes Rockefeller colony were kept in breeding cages (40×40×40 cm) in an insectary at 25±5 °C, humidity of 70± 5 %, and photoperiod (12:12). Female A. aegypti were permanently offered a sugary solution of honey at 10 %, and when larvae were needed for the experiments, they were fed by inserting the forearm of one of the researchers for 15 minu with prior knowledge of the ethics committee as per CEINCIUIS, Minutes No. 3 2013. Once the females were fed, they rested in the same cage allowing them to carry out oviposition in a container kept inside the cage lined internally with filter paper, which served as an oviposition substrate. Obtaining the essential oils All the plants used in the study were collected in the Department of Santander, Colombia. The material was identified up to the species level, and the exsiccatae were deposited in the National Herbarium (Table 1). The extraction of the essential oils, as well as the chemical composition thereof, was carried out based on the protocol reported by Stashenko et al. (2004). The essential oils were obtained by simple hydrodistillation (HD) and microwave-assisted hydrodistillation (MWHD). HD was conducted in a round-bottomed flask (5 L) with 500 g of plant material and 4 L of water using an electric heater (boiling water) for 2 h. The oil was decanted from the condensate, previously saturated with NaCl, and dried with anhydrous sodium sulfate. For MWHD, the hydrodistillation unit was placed in a domestic microwave oven (2.45 GHz, 800 W) with a hole on the side through which an external glass condenser was connected to the round-bottomed flask containing the plant material (500 g) and water (0.2 L) inside the oven. The unit was operated for 30 min at maximum power, which caused the water to boil vigorously, maintaining the reflux thereof. The EO was accumulated in a Dean-Stark trap and was later decanted from the condensate and dried with anhydrous sodium sulfate.
Parasitol Res (2014) 113:2647–2654 Table 1 Yield of essential oil, place of collection of the plant, and registration number (voucher)
2649
Scientific name
Common name
Voucher no.
Collected in
Yield % (P/P)
Cymbopogon flexuosus Eucalyptus citriodora Cananga odorata Swinglea glutinosa Citrus sinensis
Lemon grass Eucalyptus Ylang-Ylang African lemon Orange
531013 C-446 531012 521530 C-455
Bucaramanga Bucaramanga Bucaramanga Bucaramanga Bucaramanga
0.4 0.7 0.4 0.2 0.2
Tagetes lucida Lippia origanoides Lippia alba Cymbopogon citratus
Winter tarragon Wild oregano Quick relief Lemon grass
512074 517741 512078 519986
Bucaramanga Bucaramanga Bucaramanga Bucaramanga
0.4 1.4 0.6 0.5
Determination of major components To establish the chemical composition of the essential oils, an Agilent Technologies 6890 Plus gas chromatograph equipped with an HP-5 MS capillary column (30 m×0.25 mm ID× 0.25 m, df) was used, along with a mass selective detector Agilent Technologies MSD 5973. An electron impact ionization system at 70 eV was used for detection by gas chromatography–mass spectrometry (GC-MS). Helium was the carrier gas at a flow rate of 1 mL/min. The temperatures of the injector and the transfer line were set at 250 and 285 °C, respectively. Column temperature was initially set to 50 °C and then gradually increased up to 150 °C, at a speed of 3 °C per minute; it was maintained for 10 min and finally increased to 250 °C at 10 °C per minute. The diluted samples (1:20v/v in CH2Cl2) were injected at 1 μL in splitless mode. The components were identified by comparing their relative retention times and mass spectra with those of standard compounds; data from NIST, Wiley, and ADAMS of the GC-MS system were used along with data from literature. Determination of insecticidal activity After feeding the female mosquitoes with blood for 3 days, the oviposition containers that were in the breeding cages were removed. To synchronize hatching, the filter paper was removed and left to dry for 3 days at room temperature for the embryos to mature; following this period, hatching was stimulated to obtain the larvae for the experiments. When they reached the larval stage, between the third and fourth instar, they were counted, separated, and transferred with Pasteur pipettes to plastic cups with 100 mL of the same water without chlorine, where a diagnostic assay (DA) was conducted, which determined the concentrations that cause mortality between 2 and 98 % in the larvae of A. aegypti. For the experiment DA, a total of 120 larvae distributed in three concentrations (1,000, 300, and 30 ppm) were used with four replicas each, along with control treatment without the evaluated oil and with DMSO at 0.5 %. Mortality was assessed by recording the larvae that were unable to reach the surface of
the water when the beaker of the experiment was tapped; they were considered dead (WHO 1981, 1992). Subsequently, five concentrations were established for each oil. In each, four replicas were conducted per concentration in addition to the control treatment, which was the same as that employed to determine those of the DA. Each completed experiment was repeated three times on different days. Larval mortality readings were taken at two times, one at 24 h and the other at 48 h. The results of mortality and survival of the bioassay were subjected to probit analysis (Finney 1971).
Results The EO obtained by simple hydrodistillation of T. lucida, L. alba, L. origanoides, E. citriodora, C. citratus, C. flexuosus, C. sinensis, S. glutinosa, and C. odorata leaves had different yields of extraction. Among them, L. origanoides had the highest performance with 1.4 % of the EO, and the ones with the lowest yield were S. glutinosa and C. sinensis, with 0.2 % each. Of all the compounds identified, methyl chavicol was obtained with the highest percentage (92.1) while linalol was the least abundant (0.3 %); they are both components of EO T. lucida (Table 2). The lowest concentration established in the experiments of mortality in larvae of A. aegypti was 5 ppm at 4.7 and 7.5 %, at 24 and 48 h, respectively, for C. flexuosus. The maximum concentration established was 132 ppm, at 24 and 48 h, at 100 % for C. citratus. The percentage of mortality of all EO evaluated showed insecticidal activity when compared with each of the control treatments (Table 3). Lethal concentrations are LC50 17.1 and LC95 49.9 and LC50 14.6 and LC95 55.5 at 24 and 48, respectively, indicating that the EO of C. flexuosus showed the most insecticidal activity, when compared to other oils. The mixture of L. alba and L. origanoides showed lower LC50 than when the EO of L. alba and L. origanoides were evaluated separately (Table 4). The major compounds identified from the AE of C. flexuosus were geranial, neral, geranyl acetate, geraniol, and trans-β-caryophyllene. On the other hand, the EO of C. citratus was less effective as it presented the highest LC
2650
Parasitol Res (2014) 113:2647–2654
Table 2 Chemical composition of the essential oils Metabolite
% of oil C. odorata C. flexuosus C. citratus C. sinensis E. citriodora L. alba L. origanoides
Benzyl acetate Geranyl acetate Benzyl benzoate Methyl benzoate Bicyclosesquiphellandrene β-Bourbonene transβ-caryophyllene Carvacrol Carvone P-cymene 1,8-Cineol Citronellal Citronellol P-cresol methyl ether Geranial Geraniol Germacrene D α-Humulene isopulegol Limonene Linalol Methyl chavicol β-Myrcene Neral Nerol Trans-β-ocimene n-octanol α-Pinene
S. glutinosa T. lucida
18.2 10.0 12.3 10.0 3.8
9.0 1.3
2.0
2.5
1.5
32.3 38.3 12.0 3.4 49.3 13.0
3.0
8.4 37.5 9.0
26.0 2.9
7.8
8.1 12.9
14.1
1.8
28.2
71.3 5.4
20.2 29.0 3.0
1.4 31.8
4.4
2.9
5.0
0.3 92.1 5.9
2.8
2.0
1.3
1.5
β-Pinene piperitenone Piperitone Sabinene
of the entire study LC50 123.3 and 94.3 and LC95 242.6 and 163 ppm at 24 and 48 h, respectively: the compounds identified in this oil were limonene, β-myrcene, linalol, sabinene, γterpinene, and n-octanol.
Discussion According to the results of this study, it can be concluded that all the plants collected in the city of Bucaramanga enabled us to obtain EO that showed high insecticidal activity against the A. aegypti mosquito. One explanation for this effect is the major compounds found in each of the oils that were extracted. To this effect, a contribution has been made to the knowledge of new molecules with insecticidal action, which can serve as a replacement for the traditional synthetic insecticides (Kishore et al. 2011).
12.0 4.8 1.6
4.4 2.6
49.6 11.0
In a strict order of effectiveness, C. flexuosus had the best larvicidal activity against A. aegypti, when compared with the other EOs evaluated. This confirmed that the plant provides insecticidal action against mosquitoes as was originally reported by Kumar and Dutta (1987) and Osmani and Sighamony (1980); even the estimated LC50 are lower than those of Kumar and Dutta (1987) with LC50 91.4 ppm in Anopheles stephensi Liston, 1901 (Diptera: Culicidae). In more recent publications, it was also noted that the plant has promising insecticidal activity, compared to the results of Manimaran et al. (2012) with an 84 % mortality at 1,000 ppm in Culex quinquefasciatus Say, 1823 (Diptera: Culicidae). Tennyson et al. (2013) observed that LC50 was eight times higher (138.36 ppm) for A. aegypti than that presented herein. The high level of insecticidal activity of the EO of C. flexuosus collected in Bucaramanga is possibly due to the higher concentration of the major compounds
Parasitol Res (2014) 113:2647–2654
2651
Table 3 Mortality rate of each concentration of each EO evaluated in larvae of A. aegypti at 24 and 48 h
Table 3 (continued) Essential oil
Essential oil
a
Concentration (ppm)
b
Mortality rate (% ±DS) 24 h
Lippia alba
Lippia origanoides
Eucalyptus citriodora
Cymbopogon citratus
Cymbopogon flexuosus
Citrus sinensis
Cananga odorata
Swinglea glutinosa
23
Concentrationa Mortality rateb (% ±DS) (ppm) 24 h 48 h
48 h
3±1.1
6±2.9
30 48 65 90 Control 23 32 43 58 70 Control 9 23 45 70 80 97 Control
30±12 60±16 79±7.6 91±4.0 0 2.50±0.6 9±3.1 21±7.5 78±3.5 80±13 0 5±2.3 11±4.2 26±3.2 50±7.6 53±12 72±17 0
23±14 60±17 85±6.2 95±2.1 0 5±1.5 14±3.5 30±8.0 78±5.0 82±13 0 13±5.8 30±14 50±16 50±19 60±13 80±17 0
60 95 120 128 132 Control 5 11 16 25 39 Control 12 18 23 45 53 Control
5±1.5 29±4.7 49±8.6 81±9.3 100±23 0 4±1.2 16±1.2 51±4.5 77±4.9 88±1.0 0 10±4.6 40±14 60±10 79±8.0 100±23 0
32 40 65 79 90 Control 23 34 45
2.5±0.58 28±4.7 70±16 90±10 91±6.4 0 0.8±0.58 9±2.5 27±2.9
11±3.6 45±7.0 80±7.8 89±4.5 100±23 0 8±2.0 17.500±0.001 67±2.1 84±3.5 90±1.7 0 14±6.4 50±16 58±9.5 80±18 100±23 0 2.5±0.58 39±9.1 86±9.1 98±1.7 99.2±0.58 0 6±2.1 10±3.1 46±7.8
Tagetes lucida
Lippia alba-L. origanoides mixture
a
70 82 90 Control 32 54 65 82 95 Control 18 23 29 37 45 Control
49±6.0 60±18 70±20 0 0.0±0.0 21±5.1 41±9.5 80±19 100±23 0 1.3±0.58 13±6.3 21±9.8 30±14 70±12 0
72±6.5 80±18 80±20 0 3±1.2 23±5.6 50±12 80±19 100±23 0 1.3±0.58 20±3.6 20±10 40±14 70±11 0
Each of the dilutions made with the solvent DMSO
b
Mortality rate and standard deviation of the replicas of each concentration evaluated
geranial, neral, and geranyl acetate found in the analysis by GC-MS. The difference between the LC50 of the EOs can be attributed to the fact that the C. flexuosus Bucaramanga has different major compounds than the C. flexuosus of India (Citral a and Citral b), according to Makhaik et al. (2005). C. sinensis was the second EO in terms of larvicidal activity, as was C. flexuosus. When compared to similar studies, it is also noted that lower LCs were found. This was observed in a study in India conducted by Ramar et al. (2013) with a LC100 of 500 ppm. Tennyson et al. (2013) reported with the LC50 of 85.93 ppm, Warikoo et al. (2012), a LC50 of 446.84 ppm, and Murugan et al. (2012) with a LC50 of 891.63 ppm in larvae of A. aegypti. The same effect was observed in reports of the same continent such as the case of C. sinensis of Brazil with a LC 50 of 538 ppm, at least 26 times higher than ours (Cavalcanti et al. 2004). In this case, the difference in action is not explained by place of origin, as both the Brazilian plant and the Colombian have the same limonene component (Brazil=98 %, Colombia=71.3 %). There may be a synergist effect in the other components of the EO of Bucaramanga that boosts the insecticide action. However, Amusan et al. (2005) showed that the C. sinensis of Nigeria was more effective than that of Colombia. This result can be attributed to the fact that in Nigeria, the extract from the peel of the fruit was evaluated, rather than the EO from the leaves as was the case in this study. Another explanation of this effect according to Amusan
2652
Parasitol Res (2014) 113:2647–2654
Table 4 Larvicidal activity (in ppm) of the different EOs against larvae of A. aegypti Essential oil
24 h LC50
48 h LC95
Cymbopogon flexuosus 17.16 (13.78–21.37) 49.9 (30.92–80.66) Citrus sinensis 20.61 (16.49–23.82) 99.07 (91.30–402.53) L. alba and L. origanoides mixture 40.13 (37.76–43.26) 79.77 (68.63–99.23) Lippia alba 44.26 (41.58–47.01) 99.61 (89.24–114.70) Cananga odorata 52.96 (49.91–55.79) 101.52 (93.29–113.47) Lippia origanoides 53.37 (50.60–56.60) 93.05 (83.57–107.86) Swinglea glutinosa 65.71 (61.64–70.46) 151.01 (131.01–182.72) Tagetes lucida 66.27 (63.7–68.7) 95.13 (89.52–103.49) Eucalyptus citriodora 71.22 (63.91–81.62) 288.0 (208.53–479.27) Cymbopogon citratus 123.30 (114.17–138.60) 242.69 (195.97–363.81)
χ2
LC50
3.08 2.22 6.63 7.40 5.8 3.41 2.3 2.98 2.66 0.96
14.67 (10.13–21.23) 55.59 (22.75–183.45) 18.84 (14.03–22.22) 102.12 (68.19–254.49) 37.55 (30.90–45.74) 62.48 (38.29–110.55) 42.79 (40.35–45.27) 89.05 (106.05–142.32) 45.92 (43.55–48.13) 74.71 (70.11–80.94) 38.06 (33.41–41.98) 112.1 (94.81–196.05) 59.23 (55.20–63.76) 148.73 (128.16–181.36) 64.86 (62.24–67.32) 93.92 (100.72–123.75) 52.51 (44.49–62.50) 312.34 (225.40–660.13) 94.31 (89.33–99.30) 163.0 (147.22–192.23)
LC95
χ2 0.99 3.87 6.51 1.65 0.91 0.93 3.47 1.4 2.86 3.08
LC50 is the lethal concentration that causes mortality to the 50 % exposed to treatment. LC95 is the lethal concentration that causes mortality to the 50 % exposed to treatment 95 % exposed to treatment. The confidence interval is given in parentheses. The statistical analysis was well adjusted to the probit model (Finney 1971) χ2 chi-square
et al. (2005) is that the major components of the African species were limonene and linalol. Another aspect that highlights the importance of this plant as a possible insecticide is the fact that in Asian countries, it has been registered as an adulticide in Aedes albopictus Skuse, 1894 (Diptera: Culicidae). In Pakistan, a LC50 of 22.3 % (223,000 ppm) was r ep o r t e d , i n t h e H i n ck l ey va r i e t y a nd 21 . 1 4 % (211,400 ppm) in the Cassa grandis variety. This confirms the potential that can be offered by this plant in mosquito control (Hafeez et al. 2010). Unlike the EO of C. flexuosus and C. sinensis, the EO of L. alba has not been evaluated with the same intensity as an insecticide for mosquitoes. However, the LC obtained in this study indicate that the plant has strong biocidal action when compared to other EO and extracts from other medicinal and aromatic plants in several countries (Kishore et al. 2011; Gleiser et al. 2011). On the other hand, the EO of L. alba, C. sinensis, C. odorata, L. origanoides, and E. citriodora can provide protection against other mosquitoes of medical interest, other than A. aegypti, such as C. quinquefasciatus and Anopheles dirus Peyton & Harrison, 1978 (Diptera: Culicidae). This effect is significant, because it helps in the fight against the vector (Amer and Mehlhorn 2006b; Jaramillo et al. 2012; Phasomkusolsil and Soonwera 2011). To this effect, products that offer protection against mosquito bite should also be studied because up to now, there is no product that commercially equalizes N,N-diethyl-m-toluamide (DEET) as an effective mosquito repellent (Isman 2006). As for the EO of T. lucida, it may be the first record published on its action as a larvicide against A. aegypti. Therefore, information of a new plant has been contributed, whose EO serves as a model of study and can be used in the fight against the vector. Also, there is the hypothesis that its
effect may be attributed to its major compound, methyl chavicol, which was present in the sample at 92.1 %. It is no surprise that the EO had an insecticidal effect, as there are records of other species of the Tagetes genus with biocidal potential in insects such as Tagetes minuta L. and Tagetes erectes L. (Asteraceae) in A. aegypti and A. stephensi as shown by Shaalan et al. (2005). E. citriodora has not been considered promising from the insecticidal standpoint in many studies, probably because it never showed low LC50; however, in this study, the LC50 of the EO were less than 100 ppm, which indicates that it is promising. This is clear in several studies such as Amer and Mehlhorn (2006a), where the larvae of A. aegypti had a 76.7 % mortality at 50 ppm. Basically, the study of Amer and Mehlhorn had LC similar to those presented herein, although the E. citriodora used by them was collected in Germany. On the other hand, E. citriodora from India resulted in a LC50 of 1.5 % (15,000 ppm) at 60 min. For this specific case, the difference is not explained by the major compound, because the species in India and in Colombia have citroneral (Makhaik et al. 2005). Therefore, there should not be such large differences between the LCs. In this paper, the insecticidal activity of the compounds was not analyzed separately from the EO. Unfortunately, many papers do not include a chemical analysis of the components of the plants evaluated as insecticides, and this makes it more difficult to understand the differences shown between studies. C. citratus was the least effective EO according to the data of the study, since its LC50 were the highest when compared with the other EO analyzed. However, compared to a chemotype from the same country, C. citratus collected in the city of Bogotá by Cárdenas et al. (2013) shows that the LC in this study are significantly lower, as Cárdenas et al. (2013)
Parasitol Res (2014) 113:2647–2654
had a LC50 of 1.07 % (10,700 ppm). In addition, we do not know the chemical composition of the EO from Bogotá, because the major compounds presented are from another paper (Bassolé et al. 2011). In the case of C. citratus, it has been extensively researched in various arthropods, and based on its activity, it is considered an effective insecticide against other mosquitoes such as Anopheles arabiensis Patton, 1905 (Diptera: Culicidae) in extract LC50 74.02 ppm. Moreover, it is a repellent (Karunamoorthi and Ilango 2010; Phasomkusolsil and Soonwera 2011). In the results shown in this research, the insecticidal activity of C. citratus was contrary to C. flexuosus, although they are from the same genus. This difference is normally between EOs of plants from the same genus, family, or species, and it is due to the fact that they contain diverse compounds that can act on the insect in a different way. Similarly, the compounds can be found in different proportions, as a result of the conditions of production such as harvest date, storage time, climate, and soil factors (Bakkali et al. 2008) When the EOs of L. alba + L. origanoides were mixed, the larvicidal effect was boosted. This effect was due to the different forms of action of the components of these plants that act on the mosquito. So far, the synergistic roles of the various EOs, in comparison with the action of one or two main components of the oil, are not understandable, as research is required in order to evaluate the action of the mixture, with only components and with variations in accordance with the concentrations of each compound. This is also the case because the insecticide effect can be modulated by the other minor components that may be in the EO. Finally, and as a recommendation of study, few studies have analyzed EOs in relation to the mechanism of action. They have been shown to have a cytotoxic, mutagenic effect, they are generators of apoptosis, and they inhibit cellular respiration and other forms of action at the cellular level (Bakkali et al. 2008; Rattan 2010). This action should take the lead in creating new molecules to replace traditional insecticides.
Conclusions The larvae of A. aegypti were susceptible to all the EOs evaluated in this study, in particular the essential oil of C. flexuosus. To this effect, they can be considered insecticides. The LCs of this study were low in most cases when compared with other studies that evaluate EOs against A. aegypti. Acknowledgments This study was conducted thanks to the research support program of the “Vicerrectoría de Investigación of the Universidad Industrial de Santander for Project 5680” and the contributions of the “Patrimonio Autónomo, Fondo Nacional de Financiamiento para la
2653 Ciencia, Francisco Jose de Caldas,” contract RC-0572-2012- Bio-Red CENIVAM. We would also like to thank Dr. German Eduardo Matiz of the University of Cartagena Colombia, for providing the Rockefeller strain.
References Abd SL, Yaakob H, Mohamed RZ (2013) Potential anti-dengue medicinal plants: a review. J Nat Med 67:677–689 Amer A, Mehlhorn H (2006a) Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae). Parasitol Res 99:466–472 Amer A, Mehlhorn H (2006b) Repellency effects of forty-one essential oils against Aedes, Anopheles, and Culex mosquitoes. Parasitol Res 99:478–490 Amusan AA, Idowu AB, Arowolo FS (2005) Comparative toxicity effect of bush tea leaves (Hyptis suaveolens) and orange peel (Citrus sinensis) oil extract on larvae of the yellow fever mosquito Aedes aegypti. Tanzan Health Res Bull 7:174–178 Bakkali F, Averbeck S, Averbeck D, Idaomar M (2008) Biological effects of essential oils—a review. Food Chem Toxicol 46:446–475 Bassolé IH, Lamien-Meda A, Bayala B, Obame LC, Ilboudo AJ, Fransz C, Novak J, Nebié RC, Dicko MH (2011) Chemical composition and antimicrobial activity of Cymbopogon citratus and Cymbopogon giganteus essential oils alone and in combination. Phytomedicine 18:107–1074 Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, Geroge DB, Jaenisch TJ, Wint GR, Simmons CP, Scott TW, Farrar JJ, Hay SI (2013) The global distribution and burden of dengue. Nature 496: 504–507 Cárdenas EC, Riveros IT, Lugo LV (2013) Efecto insecticida de cuatro aceites esenciales sobre adultos de Aedes aegypti y Anopheles albimanus en condiciones experimentales. Entomotropica 28:1–10 Cavalcanti ESB, Morais SM, Lima MAA, Santana EWP (2004) Larvicidal Activity of essential oils from Brazilian plants against Aedes aegypti L. Mem Inst Oswaldo Cruz 99:541–544 Duque JEL, Navarro-Silva MA, Trejos ADY (2009) Simulando manejo de Aedes aegypti (Diptera: Culicidae) y sus efectos en una epidemia de dengue. Rev Colomb Entomol 35:66–72 Finney DJ (1971) Probit analysis. Cambridge University Press, 3rd edn. 174 p Gleiser RM, Bonino MA, Zygadlo JA (2011) Repellence of essential oils of aromatic plants growing in Argentina against Aedes aegypti (Diptera: Culicidae). Parasitol Res 108:69–78 Grisales N, Poupardin R, Gomez S, Fonseca-Gonzalez I, Rason H, Lenhart A (2013) Temephos resistance in Aedes aegypti in Colombia compromises dengue vector control. PLoS Negl Trop Dis 7:1–10 Guzman MG, Hal-Stead SB, Artsob H, Buchy P, Farrar J, Gubler DJ, Hunsperger E, Kroeger A, Margolis HS, Martínez E, Nathan MB, Pelegrino JL, Simmons C, Yoksan S, Peeling RW. (2010). Dengue: a continuing global threat. Nat Rev Micro S7-S16, doi:10.1038/ nrmicro2460 Hafeez F, Akram W, Suhail A, Knan MA (2010) Adulticidal action of ten citrus oils against Aedes albopictus (Diptera: Culicidae). Pak J Agric Sci 47:241–244 Harris AF, Rajatileka S, Rason H (2010) Pyrethroid resistance in Aedes aegypti from Grand Cayman. Am J Trop Med Hyg 83:277–284 Isman MB (2006) Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu Rev Entomol 51:45–66
2654 Jaramillo GIR, Logan JG, Loza-Reyes E, Stashenko E, Moores GD (2012) Repellents inhibit P450 enzymes in Stegomyia (Aedes) aegypti. PLoS ONE 7:e48698. doi:10.1371/journal.pone.0048698 Karunamoorthi K, Ilango K (2010) Larvicidal activity of Cymbopogon citratus (DC) Stapf. and Croton macrostachyus Del. against Anopheles arabiensis Patton, a potent malaria vector. Eur Rev Med Pharmacol Sci 14:57–62 Kishore N, Mishra BB, Tiwari VK, Tripathi V (2011) A review on natural products with mosquitosidal potentials. In: Tiwari VK (ed) Opportunity, challenge and scope of natural products in medicinal chemistry, Kerala Research Signpost 335–365 Kumar A, Dutta GP (1987) Indigenous plant oils as larvicidal agent against Anopheles stephensi mosquitoes. Curr Sci 56:959–960 Laughlin CA, Morens DM, Cassetti MC, Denis AC, Martin JL, Whitehead SS, Fauci AS (2012) Dengue research opportunities in the Americas. J Infect Dis 206:1121–1127 Lima EP, Paiva ME, Araújo AP, Silva EVG, Silva UM, Oliveira LN, Santana AE, Barbosa CN, Neto CP, Goulart MO, Wilding CS, Ayres CFJ, Santos MAVM (2011) Insecticide resistance in Aedes aegypti populations from Ceará, Brazil. Parasit Vectors 4:1–12 Makhaik M, Narayan SN, Tewary DK (2005) Evaluation of antimosquito properties of essential oils. J Sci Ind Res 64:129–133 Manimaran A, Cruz MJJ, Muthu C, Vicent S, Ignacimuthu S (2012) Larvicidal and knockdown effects of some essential oils against Culex quinquefasciatus Say, Aedes aegypti (L.) and Anopheles stephensi (Listos). Adv Biosci Biotechnol 3:855–862 Mullai K, Jebanesan A, Pushpanathan T (2008) Effect of bioactive fractions of Citrullus vulgaris Schrad. leaf extract against Anopheles stephensi and Aedes aegypti. Parasitol Res 102: 951–955 Murugan K, Kumar PM, Kovedan K, Amerasan D, Subrmaniam J, Hwang JS (2012) Larvicidal, pupicidal, repellent and adulticidal activity of Citrus sinensis orange peel extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae). Parasitol Res. doi:10.1007/s00436-012-3021-8 Neiro LS, Olivero-Verbel J, Stashenko E (2010) Repellent activity of essential oils: a review. Bioresour Technol 101:372–378
Parasitol Res (2014) 113:2647–2654 Osmani Z, Sighamony S (1980) Effects of certain essential oils on mortality and metamorphosis of Aedes aegypti. Pesticides 14:15–16 Phasomkusolsil S, Soonwera M (2011) Comparative mosquito repellency of essential oils against Aedes aegypti (Linn.) Anopheles dirus (Peyton and Harrison) and Culex quinquefasciatus (Say). Asian Pac J Trop Biomed S113-S118 Ramar M, Paulraj MG, Ignacimutgu S (2013) Preliminary screening of plant essential oils against larvae of Culex quinquefasciatus Say (Diptera: Culicidae). Afr J Biotechnol 12:6480–6483 Rattan RS (2010) Mechanism of action of insecticidal secondary metabolites of plant origin. Crop Prot 29:913–920 Shaalan EAS, Canyon D, Faried MWY, Abdel-Wahab H, Mansour AH (2005) A review of botanical phytochemicals with mosquitocidal potential. Environ Int 31:149–1166 Silva WJ, Dória GA, Maia RT, Nunes RS, Carvalho GA, Blank AF, Alves PB, Marçal RM, Cavalcanti SC (2008) Effects of essential oils on Aedes aegypti larvae: alternatives to environmentally safe insecticides. Bioresour Technol 99:3251–3255 Simmons CP, Farrar JJ, Vinh Chau V, Wills B (2012) Current concepts Dengue. N Engl J Med 366:1423–1432 Stashenko EE, Jaramillo BE, Martínez JR (2004) Comparison of different extraction methods for the analysis of volatile secondary metabolites of Lippia alba (Mill.) N.E. Brown, grown in Colombia and evaluation of its in vitro antioxidant activity. J Chromatogr A 1025:93–103 Tennyson S, Samraj DA, Jeyasundar D, Chalieu K (2013) Larvicidal efficacy of plant oils against the dengue vector Aedes aegypti (L.) (Diptera: Culicidae). Middle East J Sci Res 13:64–68 Warikoo R, Ray A, Sandhu JK, Samal R, Wahab N, Kumar S (2012) Larvicidal and irritant activities of hexane leaf extracts of Citrus sinensis against dengue vector Aedes aegypti L. Asian Pac J Trop Biomed 2:152–155 (WHO)-World Health Organization (1981) Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides. Geneva. 6 p (WHO)- World Health Organization (1992) Vector resistance to pesticides. Fifteenth report of the WHO Expert Committee on Vector Biology and Control. WHO Tech Rep Ser 818:1–62
ORIGINAL PAPER
Essential oils with insecticidal activity against larvae of Aedes aegypti (Diptera: Culicidae) Sharon Smith Vera & Diego Fernando Zambrano & Stelia Carolina Méndez-Sanchez Fernando Rodríguez-Sanabria & Elena E. Stashenko & Jonny E. Duque Luna
Received: 18 March 2014 / Accepted: 11 April 2014 / Published online: 30 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Insecticidal activity of the essential oils (EOs) isolated from Tagetes lucida, Lippia alba, Lippia origanoides, Eucalyptus citriodora, Cymbopogon citratus, Cymbopogon flexuosus, Citrus sinensis, Swinglea glutinosa, and Cananga odorata aromatic plants, grown in Colombia (Bucaramanga, Santander), and of a mixture of L. alba and L. origanoides EOs were evaluated on Aedes (Stegomyia) aegypti Rockefeller larvae. The EOs were extracted by microwave-assisted hydrodistillation and characterized by gas chromatography–mass spectrometry (GC-MS). The main components of the EOs were identified using their linear retention indices and mass spectra. The lethal concentrations (LCs) of the EOs were determined between the
third and fourth instar of A. aegypti. LC50 was determined by probit analysis using mortality rates of bioassays. All essential oils tested showed insecticidal activity. The following values were obtained for C. flexuosus (LC50 =17.1 ppm); C. sinensis (LC50 =20.6 ppm); the mixture of L. alba and L. origanoides (LC50 =40.1 ppm); L. alba (LC50 =42.2 ppm); C. odorata (LC 50 = 52.9 ppm); L. origanoides (LC 50 = 53.3 ppm); S. glutinosa (LC50 =65.7 ppm); T. lucida (LC50 =66.2 ppm); E. citriodora (LC50 =71.2 ppm); and C. citratus (LC50 = 123.3 ppm). The EO from C. flexuosus, with citral (geranial+neral) as main component, showed the highest larvicidal activity.
S. S. Vera : D. F. Zambrano : F. Rodríguez-Sanabria : J. E. Duque Luna Center for Research on Tropical Diseases (Centro de Investigación en Enfermedades Tropicales-CINTROP), Faculty of Health, School of Medicine, Department of Basic Sciences, Universidad Industrial de Santander, Bucaramanga, Colombia
Keywords Essential oil . Larvicidal activity . Mosquito control . Geranial
F. Rodríguez-Sanabria : J. E. Duque Luna (*) Group for Research on Infectious and Metabolic Diseases (Grupo de Investigación en Enfermedades Infecciosas y Metabólicas-GINEM), Faculty of Health, School of Medicine, Department of Basic Sciences, Universidad Industrial de Santander, Bucaramanga, Colombia e-mail: [email protected] S. C. Méndez-Sanchez Group for Research in Biochemistry and Microbiology (Grupo de Investigación en Bioquímica Y Microbiología-GIBIM), School of Chemistry, Universidad Industrial de Santander, Bucaramanga, Colombia E. E. Stashenko Center for Research in Biomolecules (Centro de Investigación en Biomoléculas-CIBIMOL) and the National Research Center for the Agro-industrialization of Tropical Medicinal and Aromatic Plants (Centro Nacional de Investigación para la Agroindustrialización de Plantas Aromáticas y Medicinales Tropicales-CENIVAM), Universidad Industrial de Santander, Bucaramanga, Colombia
Introduction Dengue is the most important disease transmitted by arthropods in the world; more than 2.5 billion people are at risk of contracting the virus by sharing an area of spatial distribution with the vector Aedes (Stegomyia) aegypti (L., 1762) (Guzman et al. 2010). Although this mosquito can also transmit other arboviral infections such as encephalitis, yellow fever, and hemorrhagic fevers, among others, none of these ailments compares to dengue when it comes to morbidity and mortality (Bhatt et al. 2013). A phenomenon of the problem faced with this vector is that the number of cases increases every year as a result of the simultaneous circulation of the four serotypes DENV-1, DENV-2, DENV-3, and DENV-4 (Laughlin et al. 2012; Simmons et al. 2012). Since more than 50 million people are infected with dengue each year, and this leads to the death of a significant percentage of the people infected due to dengue complications, there are periodic health campaigns to find solutions for the
2648
problem. Nevertheless, these activities are carried out in accordance with the policies of the countries affected by the problem. However, in one way or another, they are all focused on the population decline of the vector. Actions involve environmental sanitation, removal of breeding areas, elimination of potential containers, and the application of chemical and biological insecticides (Duque et al. 2009; Guzman et al. 2010). Despite the efforts in environmental sanitation and vector control, it has been observed that what is being done does not have a significant impact on case reduction, which is why it is said that the problem remains unresolved (Guzman et al. 2010; Simmons et al. 2012). A consensus among experts on the topic is that dengue will not be eliminated until the vaccine is created to act against the four serotypes of the dengue virus (Simmons et al. 2012). To this effect, the discovery of other alternatives for protecting from the mosquito is crucial, as is the discovery of new insecticides to replace the traditional organophosphates (or) and pyrethroids (Pr) that cause adverse effects on man and the environment. Particularly, since there are countless records that these insecticides have generated populations of A. aegypti resistant to these commercial products (Grisales et al. 2013; Harris et al. 2010; Lima et al. 2011). Under this perspective, new molecules must be found to control insect pests that will replace synthetic insecticides. You could say that there is now a good source of information on products that have an effect on A. aegypti. Thanks to the studies of many researchers on the semiochemical manifestations of different plants in terms of their extracts and essential oils (EOs), we now have knowledge of insecticidal activity, growth inhibition, and repellent, antifeedant, and deterrent actions, among others (Bakkali et al. 2008; Isman 2006; Shaalan et al. 2005). In addition, there are a number of benefits that make the exploration of botanical insecticides for pest control attractive, such as low environmental persistence, little or no toxicity to mammals, and the different forms of action on the insect (Kishore et al. 2011; Mullai et al. 2008; Silva et al. 2008). Although the use of plant-based products has been known for centuries, many of the studies that evaluate plants and their by-products applied on mosquitoes are no more than one hundred years old. That is because most of this research has been conducted on agricultural pests. However, current literature shows an increasing interest in discovering new plants with insecticidal action, which is reflected in at least 44 plant families with confirmed insecticidal activity, including an effect on the reduction of the dengue virus (Abd et al. 2013; Kishore et al. 2011; Neiro et al. 2010). That is why we intend to contribute information about plants and majority compounds that have biocidal action on mosquitoes by evaluating the insecticidal effect of the EO of Tagetes lucida Cav (Asteraceae), Lippia alba (Mill.) N.E.Br.
Parasitol Res (2014) 113:2647–2654
ex Britton & P. Wilson, Lippia origanoides Kunth (Verbenaceae), Eucalyptus citriodora (Hook.) K.D.Hill & L.A.S. Johnson (Myrtaceae), Cymbopogon citratus (DC.) Stapf, Cymbopogon flexuosus (Nees ex Steud) Wats (Poaceae), Citrus sinensis Osbeck, Swinglea glutinosa (Blanco) Merr. (Rutaceae), Cananga odorata (Lam.) Hook. F & Thomson (Annonaceae), and a combination of L. alba and L. origanoides on larvae of A. aegypti.
Materials and methods Colony of mosquitoes To conduct the experiments on insecticidal action, A. aegypti mosquitoes Rockefeller colony were kept in breeding cages (40×40×40 cm) in an insectary at 25±5 °C, humidity of 70± 5 %, and photoperiod (12:12). Female A. aegypti were permanently offered a sugary solution of honey at 10 %, and when larvae were needed for the experiments, they were fed by inserting the forearm of one of the researchers for 15 minu with prior knowledge of the ethics committee as per CEINCIUIS, Minutes No. 3 2013. Once the females were fed, they rested in the same cage allowing them to carry out oviposition in a container kept inside the cage lined internally with filter paper, which served as an oviposition substrate. Obtaining the essential oils All the plants used in the study were collected in the Department of Santander, Colombia. The material was identified up to the species level, and the exsiccatae were deposited in the National Herbarium (Table 1). The extraction of the essential oils, as well as the chemical composition thereof, was carried out based on the protocol reported by Stashenko et al. (2004). The essential oils were obtained by simple hydrodistillation (HD) and microwave-assisted hydrodistillation (MWHD). HD was conducted in a round-bottomed flask (5 L) with 500 g of plant material and 4 L of water using an electric heater (boiling water) for 2 h. The oil was decanted from the condensate, previously saturated with NaCl, and dried with anhydrous sodium sulfate. For MWHD, the hydrodistillation unit was placed in a domestic microwave oven (2.45 GHz, 800 W) with a hole on the side through which an external glass condenser was connected to the round-bottomed flask containing the plant material (500 g) and water (0.2 L) inside the oven. The unit was operated for 30 min at maximum power, which caused the water to boil vigorously, maintaining the reflux thereof. The EO was accumulated in a Dean-Stark trap and was later decanted from the condensate and dried with anhydrous sodium sulfate.
Parasitol Res (2014) 113:2647–2654 Table 1 Yield of essential oil, place of collection of the plant, and registration number (voucher)
2649
Scientific name
Common name
Voucher no.
Collected in
Yield % (P/P)
Cymbopogon flexuosus Eucalyptus citriodora Cananga odorata Swinglea glutinosa Citrus sinensis
Lemon grass Eucalyptus Ylang-Ylang African lemon Orange
531013 C-446 531012 521530 C-455
Bucaramanga Bucaramanga Bucaramanga Bucaramanga Bucaramanga
0.4 0.7 0.4 0.2 0.2
Tagetes lucida Lippia origanoides Lippia alba Cymbopogon citratus
Winter tarragon Wild oregano Quick relief Lemon grass
512074 517741 512078 519986
Bucaramanga Bucaramanga Bucaramanga Bucaramanga
0.4 1.4 0.6 0.5
Determination of major components To establish the chemical composition of the essential oils, an Agilent Technologies 6890 Plus gas chromatograph equipped with an HP-5 MS capillary column (30 m×0.25 mm ID× 0.25 m, df) was used, along with a mass selective detector Agilent Technologies MSD 5973. An electron impact ionization system at 70 eV was used for detection by gas chromatography–mass spectrometry (GC-MS). Helium was the carrier gas at a flow rate of 1 mL/min. The temperatures of the injector and the transfer line were set at 250 and 285 °C, respectively. Column temperature was initially set to 50 °C and then gradually increased up to 150 °C, at a speed of 3 °C per minute; it was maintained for 10 min and finally increased to 250 °C at 10 °C per minute. The diluted samples (1:20v/v in CH2Cl2) were injected at 1 μL in splitless mode. The components were identified by comparing their relative retention times and mass spectra with those of standard compounds; data from NIST, Wiley, and ADAMS of the GC-MS system were used along with data from literature. Determination of insecticidal activity After feeding the female mosquitoes with blood for 3 days, the oviposition containers that were in the breeding cages were removed. To synchronize hatching, the filter paper was removed and left to dry for 3 days at room temperature for the embryos to mature; following this period, hatching was stimulated to obtain the larvae for the experiments. When they reached the larval stage, between the third and fourth instar, they were counted, separated, and transferred with Pasteur pipettes to plastic cups with 100 mL of the same water without chlorine, where a diagnostic assay (DA) was conducted, which determined the concentrations that cause mortality between 2 and 98 % in the larvae of A. aegypti. For the experiment DA, a total of 120 larvae distributed in three concentrations (1,000, 300, and 30 ppm) were used with four replicas each, along with control treatment without the evaluated oil and with DMSO at 0.5 %. Mortality was assessed by recording the larvae that were unable to reach the surface of
the water when the beaker of the experiment was tapped; they were considered dead (WHO 1981, 1992). Subsequently, five concentrations were established for each oil. In each, four replicas were conducted per concentration in addition to the control treatment, which was the same as that employed to determine those of the DA. Each completed experiment was repeated three times on different days. Larval mortality readings were taken at two times, one at 24 h and the other at 48 h. The results of mortality and survival of the bioassay were subjected to probit analysis (Finney 1971).
Results The EO obtained by simple hydrodistillation of T. lucida, L. alba, L. origanoides, E. citriodora, C. citratus, C. flexuosus, C. sinensis, S. glutinosa, and C. odorata leaves had different yields of extraction. Among them, L. origanoides had the highest performance with 1.4 % of the EO, and the ones with the lowest yield were S. glutinosa and C. sinensis, with 0.2 % each. Of all the compounds identified, methyl chavicol was obtained with the highest percentage (92.1) while linalol was the least abundant (0.3 %); they are both components of EO T. lucida (Table 2). The lowest concentration established in the experiments of mortality in larvae of A. aegypti was 5 ppm at 4.7 and 7.5 %, at 24 and 48 h, respectively, for C. flexuosus. The maximum concentration established was 132 ppm, at 24 and 48 h, at 100 % for C. citratus. The percentage of mortality of all EO evaluated showed insecticidal activity when compared with each of the control treatments (Table 3). Lethal concentrations are LC50 17.1 and LC95 49.9 and LC50 14.6 and LC95 55.5 at 24 and 48, respectively, indicating that the EO of C. flexuosus showed the most insecticidal activity, when compared to other oils. The mixture of L. alba and L. origanoides showed lower LC50 than when the EO of L. alba and L. origanoides were evaluated separately (Table 4). The major compounds identified from the AE of C. flexuosus were geranial, neral, geranyl acetate, geraniol, and trans-β-caryophyllene. On the other hand, the EO of C. citratus was less effective as it presented the highest LC
2650
Parasitol Res (2014) 113:2647–2654
Table 2 Chemical composition of the essential oils Metabolite
% of oil C. odorata C. flexuosus C. citratus C. sinensis E. citriodora L. alba L. origanoides
Benzyl acetate Geranyl acetate Benzyl benzoate Methyl benzoate Bicyclosesquiphellandrene β-Bourbonene transβ-caryophyllene Carvacrol Carvone P-cymene 1,8-Cineol Citronellal Citronellol P-cresol methyl ether Geranial Geraniol Germacrene D α-Humulene isopulegol Limonene Linalol Methyl chavicol β-Myrcene Neral Nerol Trans-β-ocimene n-octanol α-Pinene
S. glutinosa T. lucida
18.2 10.0 12.3 10.0 3.8
9.0 1.3
2.0
2.5
1.5
32.3 38.3 12.0 3.4 49.3 13.0
3.0
8.4 37.5 9.0
26.0 2.9
7.8
8.1 12.9
14.1
1.8
28.2
71.3 5.4
20.2 29.0 3.0
1.4 31.8
4.4
2.9
5.0
0.3 92.1 5.9
2.8
2.0
1.3
1.5
β-Pinene piperitenone Piperitone Sabinene
of the entire study LC50 123.3 and 94.3 and LC95 242.6 and 163 ppm at 24 and 48 h, respectively: the compounds identified in this oil were limonene, β-myrcene, linalol, sabinene, γterpinene, and n-octanol.
Discussion According to the results of this study, it can be concluded that all the plants collected in the city of Bucaramanga enabled us to obtain EO that showed high insecticidal activity against the A. aegypti mosquito. One explanation for this effect is the major compounds found in each of the oils that were extracted. To this effect, a contribution has been made to the knowledge of new molecules with insecticidal action, which can serve as a replacement for the traditional synthetic insecticides (Kishore et al. 2011).
12.0 4.8 1.6
4.4 2.6
49.6 11.0
In a strict order of effectiveness, C. flexuosus had the best larvicidal activity against A. aegypti, when compared with the other EOs evaluated. This confirmed that the plant provides insecticidal action against mosquitoes as was originally reported by Kumar and Dutta (1987) and Osmani and Sighamony (1980); even the estimated LC50 are lower than those of Kumar and Dutta (1987) with LC50 91.4 ppm in Anopheles stephensi Liston, 1901 (Diptera: Culicidae). In more recent publications, it was also noted that the plant has promising insecticidal activity, compared to the results of Manimaran et al. (2012) with an 84 % mortality at 1,000 ppm in Culex quinquefasciatus Say, 1823 (Diptera: Culicidae). Tennyson et al. (2013) observed that LC50 was eight times higher (138.36 ppm) for A. aegypti than that presented herein. The high level of insecticidal activity of the EO of C. flexuosus collected in Bucaramanga is possibly due to the higher concentration of the major compounds
Parasitol Res (2014) 113:2647–2654
2651
Table 3 Mortality rate of each concentration of each EO evaluated in larvae of A. aegypti at 24 and 48 h
Table 3 (continued) Essential oil
Essential oil
a
Concentration (ppm)
b
Mortality rate (% ±DS) 24 h
Lippia alba
Lippia origanoides
Eucalyptus citriodora
Cymbopogon citratus
Cymbopogon flexuosus
Citrus sinensis
Cananga odorata
Swinglea glutinosa
23
Concentrationa Mortality rateb (% ±DS) (ppm) 24 h 48 h
48 h
3±1.1
6±2.9
30 48 65 90 Control 23 32 43 58 70 Control 9 23 45 70 80 97 Control
30±12 60±16 79±7.6 91±4.0 0 2.50±0.6 9±3.1 21±7.5 78±3.5 80±13 0 5±2.3 11±4.2 26±3.2 50±7.6 53±12 72±17 0
23±14 60±17 85±6.2 95±2.1 0 5±1.5 14±3.5 30±8.0 78±5.0 82±13 0 13±5.8 30±14 50±16 50±19 60±13 80±17 0
60 95 120 128 132 Control 5 11 16 25 39 Control 12 18 23 45 53 Control
5±1.5 29±4.7 49±8.6 81±9.3 100±23 0 4±1.2 16±1.2 51±4.5 77±4.9 88±1.0 0 10±4.6 40±14 60±10 79±8.0 100±23 0
32 40 65 79 90 Control 23 34 45
2.5±0.58 28±4.7 70±16 90±10 91±6.4 0 0.8±0.58 9±2.5 27±2.9
11±3.6 45±7.0 80±7.8 89±4.5 100±23 0 8±2.0 17.500±0.001 67±2.1 84±3.5 90±1.7 0 14±6.4 50±16 58±9.5 80±18 100±23 0 2.5±0.58 39±9.1 86±9.1 98±1.7 99.2±0.58 0 6±2.1 10±3.1 46±7.8
Tagetes lucida
Lippia alba-L. origanoides mixture
a
70 82 90 Control 32 54 65 82 95 Control 18 23 29 37 45 Control
49±6.0 60±18 70±20 0 0.0±0.0 21±5.1 41±9.5 80±19 100±23 0 1.3±0.58 13±6.3 21±9.8 30±14 70±12 0
72±6.5 80±18 80±20 0 3±1.2 23±5.6 50±12 80±19 100±23 0 1.3±0.58 20±3.6 20±10 40±14 70±11 0
Each of the dilutions made with the solvent DMSO
b
Mortality rate and standard deviation of the replicas of each concentration evaluated
geranial, neral, and geranyl acetate found in the analysis by GC-MS. The difference between the LC50 of the EOs can be attributed to the fact that the C. flexuosus Bucaramanga has different major compounds than the C. flexuosus of India (Citral a and Citral b), according to Makhaik et al. (2005). C. sinensis was the second EO in terms of larvicidal activity, as was C. flexuosus. When compared to similar studies, it is also noted that lower LCs were found. This was observed in a study in India conducted by Ramar et al. (2013) with a LC100 of 500 ppm. Tennyson et al. (2013) reported with the LC50 of 85.93 ppm, Warikoo et al. (2012), a LC50 of 446.84 ppm, and Murugan et al. (2012) with a LC50 of 891.63 ppm in larvae of A. aegypti. The same effect was observed in reports of the same continent such as the case of C. sinensis of Brazil with a LC 50 of 538 ppm, at least 26 times higher than ours (Cavalcanti et al. 2004). In this case, the difference in action is not explained by place of origin, as both the Brazilian plant and the Colombian have the same limonene component (Brazil=98 %, Colombia=71.3 %). There may be a synergist effect in the other components of the EO of Bucaramanga that boosts the insecticide action. However, Amusan et al. (2005) showed that the C. sinensis of Nigeria was more effective than that of Colombia. This result can be attributed to the fact that in Nigeria, the extract from the peel of the fruit was evaluated, rather than the EO from the leaves as was the case in this study. Another explanation of this effect according to Amusan
2652
Parasitol Res (2014) 113:2647–2654
Table 4 Larvicidal activity (in ppm) of the different EOs against larvae of A. aegypti Essential oil
24 h LC50
48 h LC95
Cymbopogon flexuosus 17.16 (13.78–21.37) 49.9 (30.92–80.66) Citrus sinensis 20.61 (16.49–23.82) 99.07 (91.30–402.53) L. alba and L. origanoides mixture 40.13 (37.76–43.26) 79.77 (68.63–99.23) Lippia alba 44.26 (41.58–47.01) 99.61 (89.24–114.70) Cananga odorata 52.96 (49.91–55.79) 101.52 (93.29–113.47) Lippia origanoides 53.37 (50.60–56.60) 93.05 (83.57–107.86) Swinglea glutinosa 65.71 (61.64–70.46) 151.01 (131.01–182.72) Tagetes lucida 66.27 (63.7–68.7) 95.13 (89.52–103.49) Eucalyptus citriodora 71.22 (63.91–81.62) 288.0 (208.53–479.27) Cymbopogon citratus 123.30 (114.17–138.60) 242.69 (195.97–363.81)
χ2
LC50
3.08 2.22 6.63 7.40 5.8 3.41 2.3 2.98 2.66 0.96
14.67 (10.13–21.23) 55.59 (22.75–183.45) 18.84 (14.03–22.22) 102.12 (68.19–254.49) 37.55 (30.90–45.74) 62.48 (38.29–110.55) 42.79 (40.35–45.27) 89.05 (106.05–142.32) 45.92 (43.55–48.13) 74.71 (70.11–80.94) 38.06 (33.41–41.98) 112.1 (94.81–196.05) 59.23 (55.20–63.76) 148.73 (128.16–181.36) 64.86 (62.24–67.32) 93.92 (100.72–123.75) 52.51 (44.49–62.50) 312.34 (225.40–660.13) 94.31 (89.33–99.30) 163.0 (147.22–192.23)
LC95
χ2 0.99 3.87 6.51 1.65 0.91 0.93 3.47 1.4 2.86 3.08
LC50 is the lethal concentration that causes mortality to the 50 % exposed to treatment. LC95 is the lethal concentration that causes mortality to the 50 % exposed to treatment 95 % exposed to treatment. The confidence interval is given in parentheses. The statistical analysis was well adjusted to the probit model (Finney 1971) χ2 chi-square
et al. (2005) is that the major components of the African species were limonene and linalol. Another aspect that highlights the importance of this plant as a possible insecticide is the fact that in Asian countries, it has been registered as an adulticide in Aedes albopictus Skuse, 1894 (Diptera: Culicidae). In Pakistan, a LC50 of 22.3 % (223,000 ppm) was r ep o r t e d , i n t h e H i n ck l ey va r i e t y a nd 21 . 1 4 % (211,400 ppm) in the Cassa grandis variety. This confirms the potential that can be offered by this plant in mosquito control (Hafeez et al. 2010). Unlike the EO of C. flexuosus and C. sinensis, the EO of L. alba has not been evaluated with the same intensity as an insecticide for mosquitoes. However, the LC obtained in this study indicate that the plant has strong biocidal action when compared to other EO and extracts from other medicinal and aromatic plants in several countries (Kishore et al. 2011; Gleiser et al. 2011). On the other hand, the EO of L. alba, C. sinensis, C. odorata, L. origanoides, and E. citriodora can provide protection against other mosquitoes of medical interest, other than A. aegypti, such as C. quinquefasciatus and Anopheles dirus Peyton & Harrison, 1978 (Diptera: Culicidae). This effect is significant, because it helps in the fight against the vector (Amer and Mehlhorn 2006b; Jaramillo et al. 2012; Phasomkusolsil and Soonwera 2011). To this effect, products that offer protection against mosquito bite should also be studied because up to now, there is no product that commercially equalizes N,N-diethyl-m-toluamide (DEET) as an effective mosquito repellent (Isman 2006). As for the EO of T. lucida, it may be the first record published on its action as a larvicide against A. aegypti. Therefore, information of a new plant has been contributed, whose EO serves as a model of study and can be used in the fight against the vector. Also, there is the hypothesis that its
effect may be attributed to its major compound, methyl chavicol, which was present in the sample at 92.1 %. It is no surprise that the EO had an insecticidal effect, as there are records of other species of the Tagetes genus with biocidal potential in insects such as Tagetes minuta L. and Tagetes erectes L. (Asteraceae) in A. aegypti and A. stephensi as shown by Shaalan et al. (2005). E. citriodora has not been considered promising from the insecticidal standpoint in many studies, probably because it never showed low LC50; however, in this study, the LC50 of the EO were less than 100 ppm, which indicates that it is promising. This is clear in several studies such as Amer and Mehlhorn (2006a), where the larvae of A. aegypti had a 76.7 % mortality at 50 ppm. Basically, the study of Amer and Mehlhorn had LC similar to those presented herein, although the E. citriodora used by them was collected in Germany. On the other hand, E. citriodora from India resulted in a LC50 of 1.5 % (15,000 ppm) at 60 min. For this specific case, the difference is not explained by the major compound, because the species in India and in Colombia have citroneral (Makhaik et al. 2005). Therefore, there should not be such large differences between the LCs. In this paper, the insecticidal activity of the compounds was not analyzed separately from the EO. Unfortunately, many papers do not include a chemical analysis of the components of the plants evaluated as insecticides, and this makes it more difficult to understand the differences shown between studies. C. citratus was the least effective EO according to the data of the study, since its LC50 were the highest when compared with the other EO analyzed. However, compared to a chemotype from the same country, C. citratus collected in the city of Bogotá by Cárdenas et al. (2013) shows that the LC in this study are significantly lower, as Cárdenas et al. (2013)
Parasitol Res (2014) 113:2647–2654
had a LC50 of 1.07 % (10,700 ppm). In addition, we do not know the chemical composition of the EO from Bogotá, because the major compounds presented are from another paper (Bassolé et al. 2011). In the case of C. citratus, it has been extensively researched in various arthropods, and based on its activity, it is considered an effective insecticide against other mosquitoes such as Anopheles arabiensis Patton, 1905 (Diptera: Culicidae) in extract LC50 74.02 ppm. Moreover, it is a repellent (Karunamoorthi and Ilango 2010; Phasomkusolsil and Soonwera 2011). In the results shown in this research, the insecticidal activity of C. citratus was contrary to C. flexuosus, although they are from the same genus. This difference is normally between EOs of plants from the same genus, family, or species, and it is due to the fact that they contain diverse compounds that can act on the insect in a different way. Similarly, the compounds can be found in different proportions, as a result of the conditions of production such as harvest date, storage time, climate, and soil factors (Bakkali et al. 2008) When the EOs of L. alba + L. origanoides were mixed, the larvicidal effect was boosted. This effect was due to the different forms of action of the components of these plants that act on the mosquito. So far, the synergistic roles of the various EOs, in comparison with the action of one or two main components of the oil, are not understandable, as research is required in order to evaluate the action of the mixture, with only components and with variations in accordance with the concentrations of each compound. This is also the case because the insecticide effect can be modulated by the other minor components that may be in the EO. Finally, and as a recommendation of study, few studies have analyzed EOs in relation to the mechanism of action. They have been shown to have a cytotoxic, mutagenic effect, they are generators of apoptosis, and they inhibit cellular respiration and other forms of action at the cellular level (Bakkali et al. 2008; Rattan 2010). This action should take the lead in creating new molecules to replace traditional insecticides.
Conclusions The larvae of A. aegypti were susceptible to all the EOs evaluated in this study, in particular the essential oil of C. flexuosus. To this effect, they can be considered insecticides. The LCs of this study were low in most cases when compared with other studies that evaluate EOs against A. aegypti. Acknowledgments This study was conducted thanks to the research support program of the “Vicerrectoría de Investigación of the Universidad Industrial de Santander for Project 5680” and the contributions of the “Patrimonio Autónomo, Fondo Nacional de Financiamiento para la
2653 Ciencia, Francisco Jose de Caldas,” contract RC-0572-2012- Bio-Red CENIVAM. We would also like to thank Dr. German Eduardo Matiz of the University of Cartagena Colombia, for providing the Rockefeller strain.
References Abd SL, Yaakob H, Mohamed RZ (2013) Potential anti-dengue medicinal plants: a review. J Nat Med 67:677–689 Amer A, Mehlhorn H (2006a) Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae). Parasitol Res 99:466–472 Amer A, Mehlhorn H (2006b) Repellency effects of forty-one essential oils against Aedes, Anopheles, and Culex mosquitoes. Parasitol Res 99:478–490 Amusan AA, Idowu AB, Arowolo FS (2005) Comparative toxicity effect of bush tea leaves (Hyptis suaveolens) and orange peel (Citrus sinensis) oil extract on larvae of the yellow fever mosquito Aedes aegypti. Tanzan Health Res Bull 7:174–178 Bakkali F, Averbeck S, Averbeck D, Idaomar M (2008) Biological effects of essential oils—a review. Food Chem Toxicol 46:446–475 Bassolé IH, Lamien-Meda A, Bayala B, Obame LC, Ilboudo AJ, Fransz C, Novak J, Nebié RC, Dicko MH (2011) Chemical composition and antimicrobial activity of Cymbopogon citratus and Cymbopogon giganteus essential oils alone and in combination. Phytomedicine 18:107–1074 Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, Geroge DB, Jaenisch TJ, Wint GR, Simmons CP, Scott TW, Farrar JJ, Hay SI (2013) The global distribution and burden of dengue. Nature 496: 504–507 Cárdenas EC, Riveros IT, Lugo LV (2013) Efecto insecticida de cuatro aceites esenciales sobre adultos de Aedes aegypti y Anopheles albimanus en condiciones experimentales. Entomotropica 28:1–10 Cavalcanti ESB, Morais SM, Lima MAA, Santana EWP (2004) Larvicidal Activity of essential oils from Brazilian plants against Aedes aegypti L. Mem Inst Oswaldo Cruz 99:541–544 Duque JEL, Navarro-Silva MA, Trejos ADY (2009) Simulando manejo de Aedes aegypti (Diptera: Culicidae) y sus efectos en una epidemia de dengue. Rev Colomb Entomol 35:66–72 Finney DJ (1971) Probit analysis. Cambridge University Press, 3rd edn. 174 p Gleiser RM, Bonino MA, Zygadlo JA (2011) Repellence of essential oils of aromatic plants growing in Argentina against Aedes aegypti (Diptera: Culicidae). Parasitol Res 108:69–78 Grisales N, Poupardin R, Gomez S, Fonseca-Gonzalez I, Rason H, Lenhart A (2013) Temephos resistance in Aedes aegypti in Colombia compromises dengue vector control. PLoS Negl Trop Dis 7:1–10 Guzman MG, Hal-Stead SB, Artsob H, Buchy P, Farrar J, Gubler DJ, Hunsperger E, Kroeger A, Margolis HS, Martínez E, Nathan MB, Pelegrino JL, Simmons C, Yoksan S, Peeling RW. (2010). Dengue: a continuing global threat. Nat Rev Micro S7-S16, doi:10.1038/ nrmicro2460 Hafeez F, Akram W, Suhail A, Knan MA (2010) Adulticidal action of ten citrus oils against Aedes albopictus (Diptera: Culicidae). Pak J Agric Sci 47:241–244 Harris AF, Rajatileka S, Rason H (2010) Pyrethroid resistance in Aedes aegypti from Grand Cayman. Am J Trop Med Hyg 83:277–284 Isman MB (2006) Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu Rev Entomol 51:45–66
2654 Jaramillo GIR, Logan JG, Loza-Reyes E, Stashenko E, Moores GD (2012) Repellents inhibit P450 enzymes in Stegomyia (Aedes) aegypti. PLoS ONE 7:e48698. doi:10.1371/journal.pone.0048698 Karunamoorthi K, Ilango K (2010) Larvicidal activity of Cymbopogon citratus (DC) Stapf. and Croton macrostachyus Del. against Anopheles arabiensis Patton, a potent malaria vector. Eur Rev Med Pharmacol Sci 14:57–62 Kishore N, Mishra BB, Tiwari VK, Tripathi V (2011) A review on natural products with mosquitosidal potentials. In: Tiwari VK (ed) Opportunity, challenge and scope of natural products in medicinal chemistry, Kerala Research Signpost 335–365 Kumar A, Dutta GP (1987) Indigenous plant oils as larvicidal agent against Anopheles stephensi mosquitoes. Curr Sci 56:959–960 Laughlin CA, Morens DM, Cassetti MC, Denis AC, Martin JL, Whitehead SS, Fauci AS (2012) Dengue research opportunities in the Americas. J Infect Dis 206:1121–1127 Lima EP, Paiva ME, Araújo AP, Silva EVG, Silva UM, Oliveira LN, Santana AE, Barbosa CN, Neto CP, Goulart MO, Wilding CS, Ayres CFJ, Santos MAVM (2011) Insecticide resistance in Aedes aegypti populations from Ceará, Brazil. Parasit Vectors 4:1–12 Makhaik M, Narayan SN, Tewary DK (2005) Evaluation of antimosquito properties of essential oils. J Sci Ind Res 64:129–133 Manimaran A, Cruz MJJ, Muthu C, Vicent S, Ignacimuthu S (2012) Larvicidal and knockdown effects of some essential oils against Culex quinquefasciatus Say, Aedes aegypti (L.) and Anopheles stephensi (Listos). Adv Biosci Biotechnol 3:855–862 Mullai K, Jebanesan A, Pushpanathan T (2008) Effect of bioactive fractions of Citrullus vulgaris Schrad. leaf extract against Anopheles stephensi and Aedes aegypti. Parasitol Res 102: 951–955 Murugan K, Kumar PM, Kovedan K, Amerasan D, Subrmaniam J, Hwang JS (2012) Larvicidal, pupicidal, repellent and adulticidal activity of Citrus sinensis orange peel extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae). Parasitol Res. doi:10.1007/s00436-012-3021-8 Neiro LS, Olivero-Verbel J, Stashenko E (2010) Repellent activity of essential oils: a review. Bioresour Technol 101:372–378
Parasitol Res (2014) 113:2647–2654 Osmani Z, Sighamony S (1980) Effects of certain essential oils on mortality and metamorphosis of Aedes aegypti. Pesticides 14:15–16 Phasomkusolsil S, Soonwera M (2011) Comparative mosquito repellency of essential oils against Aedes aegypti (Linn.) Anopheles dirus (Peyton and Harrison) and Culex quinquefasciatus (Say). Asian Pac J Trop Biomed S113-S118 Ramar M, Paulraj MG, Ignacimutgu S (2013) Preliminary screening of plant essential oils against larvae of Culex quinquefasciatus Say (Diptera: Culicidae). Afr J Biotechnol 12:6480–6483 Rattan RS (2010) Mechanism of action of insecticidal secondary metabolites of plant origin. Crop Prot 29:913–920 Shaalan EAS, Canyon D, Faried MWY, Abdel-Wahab H, Mansour AH (2005) A review of botanical phytochemicals with mosquitocidal potential. Environ Int 31:149–1166 Silva WJ, Dória GA, Maia RT, Nunes RS, Carvalho GA, Blank AF, Alves PB, Marçal RM, Cavalcanti SC (2008) Effects of essential oils on Aedes aegypti larvae: alternatives to environmentally safe insecticides. Bioresour Technol 99:3251–3255 Simmons CP, Farrar JJ, Vinh Chau V, Wills B (2012) Current concepts Dengue. N Engl J Med 366:1423–1432 Stashenko EE, Jaramillo BE, Martínez JR (2004) Comparison of different extraction methods for the analysis of volatile secondary metabolites of Lippia alba (Mill.) N.E. Brown, grown in Colombia and evaluation of its in vitro antioxidant activity. J Chromatogr A 1025:93–103 Tennyson S, Samraj DA, Jeyasundar D, Chalieu K (2013) Larvicidal efficacy of plant oils against the dengue vector Aedes aegypti (L.) (Diptera: Culicidae). Middle East J Sci Res 13:64–68 Warikoo R, Ray A, Sandhu JK, Samal R, Wahab N, Kumar S (2012) Larvicidal and irritant activities of hexane leaf extracts of Citrus sinensis against dengue vector Aedes aegypti L. Asian Pac J Trop Biomed 2:152–155 (WHO)-World Health Organization (1981) Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides. Geneva. 6 p (WHO)- World Health Organization (1992) Vector resistance to pesticides. Fifteenth report of the WHO Expert Committee on Vector Biology and Control. WHO Tech Rep Ser 818:1–62
