Chemical composition and larvicidal activity of edible plant-derived essential oils against the pyrethroid-susceptible and -resistant strains of Aedes aegypti (Diptera: Culicidae)

Abstract

The chemical compositions and larvicidal potential against mosquito vectors of selected essential oils obtained from five edible plants were investigated in this study. Using a GC/MS, 24, 17, 20, 21, and 12 compounds were determined from essential oils of Citrus hystrix, Citrus reticulata, Zingiber zerumbet, Kaempferia galanga, and Syzygium aromaticum, respectively. The principal constituents found in peel oil of C. hystrix were β-pinene (22.54%) and d-limonene (22.03%), followed by terpinene-4-ol (17.37%). Compounds in C. reticulata peel oil consisted mostly of d-limonene (62.39%) and γ-terpinene (14.06%). The oils obtained from Z. zerumbet rhizome had α-humulene (31.93%) and zerumbone (31.67%) as major components. The most abundant compounds in K. galanga rhizome oil were 2-propeonic acid (35.54%), pentadecane (26.08%), and ethyl-p-methoxycinnamate (25.96%). The main component of S. aromaticum bud oil was eugenol (77.37%), with minor amounts of trans-caryophyllene (13.66%). Assessment of larvicidal efficacy demonstrated that all essential oils were toxic against both pyrethroid-susceptible and resistant Ae. aegypti laboratory strains at LC50, LC95, and LC99 levels. In conclusion, we have documented the promising larvicidal potential of essential oils from edible herbs, which could be considered as a potentially alternative source for developing novel larvicides to be used in controlling vectors of mosquito-borne disease. Diseases transmitted by blood-feeding mosquitoes, such as dengue fever, dengue hemorrhagic fever, Japanese encephalitis, malaria, and filariasis, are increasing in prevalence, particularly in tropical and subtropical zones. To control mosquitoes and mosquito-borne diseases, which have a worldwide health and economic impacts, synthetic insecticide-based interventions are still necessary, particularly in situations of epidemic outbreak and sudden increases of adult mosquitoes (Yaicharoen et al. 2005, Nathan et al. 2006). However, the indiscriminate use of conventional insecticides is fostering multifarious problems like widespread development of insecticide resistance, toxic hazards to mammals, undesirable effects on non-target organisms, and environmental pollution (Yang et al. 2002, Kiran et al. 2006, Senthikumar et al. 2008). Each year, larger quantities of synthetic insecticides are applied, leading to increased dangers for humans and other organisms and progressively greater environmental damage. Even though plants and their preparations were the only pest management agents available before the advent of synthetic organic chemicals, only a few insecticides of plant origin are now commercially available. Furthermore, most of them have lower and shorter-lived efficacy than the synthetic substances. Due to the high degree of biodegradation, however, plant-derived bioproducts are currently attractive as replacements for synthetic insecticides or for use in integrated management programs to minimize human health hazards and reduce the accumulation of harmful residues in the environment. Furthermore, insect resistance to mosquitocidal botanical agents has not previously been documented (Shaalan et al. 2005). Numerous products of botanical origin, especially essential oils, have received considerable renewed attention as potent bioactive compounds against various species of mosquitoes. Due to the fact that application of adulticides may only temporarily diminish the adult population (El Hag et al. 1999, 2001), a more efficient and attractive approach in mosquito control programs is to target the larval stage in their breeding sites with larvicides (Amer and Mehlhorn 2006a, Knio et al. 2008). Essential oils are potentially suitable for application in larval control management because they constitute a rich source of bioactive compounds that are effective and naturally biodegradable into non-toxic products (Lucia et al. 2007, Cheng et al. 2008, 2009a). Considerable research on the larvicidal potential of volatile oils against Aedes aegypti mosquitoes has been conducted and the findings contribute an insight into the possibility of developing novel larvicides from essential oils for use in mosquito control programs. The promising essential oils, with larvicidal activity demonstrating LC50 ranging between 1–258.5 ppm, are derived from a large number of plants, including Cymbopogon proximus, Lippia multiflora, and Ocimum canum (Bassolé et al. 2003); Ipomoea cairica (Thomas et al. 2004); Juniperus macropoda and Pimpinella anisum (Prajapati et al. 2005); Citrus bergamia, Cuminum myrrham, and Pimenta racemosa (Lee 2006); Cinnamomum camphora, Boswellia carteri, Anethum graveolens, and Myrtus communis (Amer and Mehlhorn 2006a,b); Chloroxylon swietenia (Kiran et al. 2006); Carum carvi, Apium graveolens, Foeniculum vulgare, Zanthoxylum limonella, and Curcuma zedoaria (Pitasawat et al. 2007); Zanthoxylum armatum (Tiwary et al. 2007); Eucalyptus camaldulensis and E. urophylla (Cheng et al. 2009b). Thailand is a source of a large diversity of medicinal herbs. It is reassuring to ascertain plant-derived products that are fully potential, safe, and eco-friendly. The edible plants used as vegetables, spices, and traditional medicine are therefore encouraging targets. Citrus plants such as Citrus hystrix DC and C. reticulata Blanco are familiar through use of their fruit for culinary purposes and traditional medicine (Gimlette and Thomson 1983, Yeung 1985). Apart from use as food flavoring and appetizer, Zingiber zerumbet Smith is also commonly used in folkloric medicine (Burkill 1966, Habsah et al. 2000). In Thailand and China, the rhizome of Kaempferia galanga Linn is well-known for its popular use as a food spice and traditional medicine (Huang et al. 2008). The flower bud of Syzygium aromaticum Linn is a well-known food flavor in exotic food preparations and a popular remedy in the traditional medicines of Australia and Asian countries (Gurib-Fakim 2006). The present study attempted to investigate the chemical composition and larvicidal efficacy of essential oils derived from five edible plants against both the pyrethroid-susceptible and -resistant strains of the mosquito Ae. aegypti with the purpose of identifying effective indigenous bioproducts to control the vector of mosquito-borne diseases, particularly in cases where the vector's susceptibility to conventional synthetics is decreasing. Five plant species, including Citrus hystrix DC., Citrus reticulata Blanco., Zingiber zerumbet Smith., Kaempferia galanga Linn., and Syzygium aromaticum Linn. (Table 1) were commercially obtained from traditional herb suppliers in Chiang Mai province. Taxonomic identification of the plants was performed by botanists and taxonomists in the Department of Biology, Faculty of Science, Chiang Mai University, Thailand. A voucher specimen of each plant was deposited at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand. Each plant material was shade-dried at room temperature, mechanically ground by an electrical blender, and steam distilled at 100° C for at least three h to obtain essential oils. The oil layer was separated from the aqueous phase, dried over anhydrous sodium sulfate (Na2So4), and kept in an amber-colored bottle under refrigeration at 4° C until further chemical analysis and larvicidal bioassays. In each case, the yield of oil was averaged over three experiments and calculated according to the dry weight of the plant materials. Analysis of essential oils was performed by gas chromatography coupled with mass spectrometry (GC/MS) in a Hewlett-Packard 6850 gas chromatograph (Agilent Technologies) equipped with a split-splitless injector and HP-5MS (30 m × 0.25 mm ID and 0.25 μm film thickness) columns directly coupled to a quadrupole mass selective detector, MSD 5973 (Agilent Technologies). The injector temperature was set at 250° C and the oven temperature was initially at 50° C, and programmed to reach 230° C at the rate of 6° C/min and held at 230° C for 10 min, then increased from 230° C to 250° C at the rate of 10° C/min. Helium was used as the carrier gas with a flow rate of 1.0 ml/min. The sample (0.2 μl) was injected neat with a split ratio of 250:1. The mass spectrometer (MSD 5973) was operated in the electron impact (EI) mode at 70eV. The ion source and quadrupole temperatures were set at 230° C and 150° C, respectively. The oil components were identified by comparison with standards, by spiking and on the basis of their mass spectral fragmentation using the NIST 05, NIST 98, Wiley 7N, and Wiley 275 GC/MS libraries. Percentage of the identified compound was computed from a total ion chromatogram. Mosquito populations tested in the larvicidal bioassays comprised both pyrethroid-susceptible and -resistant Ae. aegypti laboratory strains. The pyrethroid susceptible Ae. aegypti colony was established from specimens collected in Muang district, Chiang Mai province, and maintained continuously since 1995. The pyrethroid-resistant Ae. aegypti colony was collected from various places in Mae Tang district, Chiang Mai province, and maintained under selective pressure (Chareonviriyaphap et al. 2002) to establish a pyrethroid-resistant laboratory colony. Both pyrethroid-susceptible and -resistant Ae. aegypti were maintained and reared separately without exposure to any insecticides or pathogens in an insectary (6.2 × 7.3 × 3 m) at 25±2° C and 80%±10% RH under a 14:10 light-and-dark cycle, following standard operating procedures for mosquito maintenance (Limsuwan et al. 1987). Tests of susceptibility of adult Ae. aegypti mosquitoes to the synthetic pyrethroids, permethrin and lambdacyhalothrin, were regularly conducted using WHO test kits with some modifications (WHO 1998). Newly molted 4th instar Ae. aegypti larvae were continuously available for the larvicidal bioassays. The mosquito larvicidal assays were carried out under laboratory conditions by a slight adaptation of the standard protocol recommended by the World Health Organization (WHO 1981). Each essential oil was dissolved in dimethylsulphoxide (DMSO) to prepare graded concentrations of tested material. In preparing a series of aqueous solutions with different concentrations of tested oil, 1 ml of DMSO solution containing the desired essential oil was completely mixed with 249 ml of distilled water in an enamel bowl of 10 cm in diameter and 8 cm in depth. Four batches of 25 early 4th instar larvae of Ae. aegypti were maintained in 250 ml of aqueous solutions with the final total number of 100 larvae for each concentration. A total of five essential oils were tested in this manner with a range of concentrations (four to six concentrations, ppm), yielding a range of 0–100% mortality. Control tests receiving DMSO-distilled water were performed in parallel for comparison. Mortality and survival of larvae were determined after 24 h of exposure and the larvae were starved within this period. Observations were also made on the behavior of larvae. Larvae were considered dead when they did not respond to stimuli such as probing with a needle in the siphon or cervical region. Moribund larvae were those incapable of rising to the surface of the water (within a reasonable period of time) or showing a characteristic diving reaction when the water was disturbed. They might also show discoloration, unnatural positions, tremors, incoordination, or rigor. The moribund and dead larvae in each concentration were combined in quadruplicate and expressed as percentage mortalities. Every bioassay was carried out in environmentally controlled conditions (temperature ∼ 25±2° C; humidity ∼ 80%±10% RH; 14-h light and 10-h dark cycle), and replicated four times with mosquitoes from different rearing batches. The percentage mortality was reported from the average of four replicates. It was important to obtain no less than three mortality counts of between 10% and 90%. In cases where the control mortality was between 5–20%, the observed percentage mortality (%M) was corrected by Abbott's formula (Abbott 1925): Data for larvicidal potential were analyzed by means of computerized probit analysis (Harvard Programming; Hg1, 2), yielding the lethal concentrations LC50, LC95, LC99, and 95% confidence intervals (CI) of upper and lower confidence levels. Significant differences were determined by comparing the CI of each plant oil. The yields of volatile oils ranged from a minimum of 0.30% to a maximum of 3.36% (v/w) according to dry weight (Table 1). The highest oil content was found in C. hystrix (3.36%), followed by S. aromaticum (1.50%), C. reticulata (1.40%), K. galanga (0.76%), and Z. zerumbet (0.30%). It is generally known that the yield of essential oil depends not only on the plant species and their climatic or geographical areas, but also other variables such as method of extraction and plant-related factors, including parts of plant, rearing condition, maturation of the harvested plant, and plant storage or preservation (Vieira and Simon 2000, Tawatsin et al. 2001). In order to achieve the best yield, it is therefore necessary to establish the most appropriate combination of these variable factors. However, in addition to the yield of essential oil, much consideration was given to the quality and quantity of chemical constituents, particularly the major active ingredients. In this study, GC/MS characterization was performed to show the profile of constituents in the selected essential oils, of which gas chromatograms and percentage compositions are presented in Figure 1 and Table 2, respectively. A total of 68 compounds were identified from five essential oils, representing 96.01- 100% of the oil obtained. The essential oil of C. hystrix peel contained 24 identified components, amounting to 99.52% of the whole oil with β-pinene (22.54%) and d-limonene (22.03%) as the principal constituents, followed by terpinene-4-ol (17.37%), together with trace amounts of α-terpineol (6.29%) and sabinene (5.49%). For C. reticulata peel oil, 17 compounds were identified, representing 100.00% of the whole oil with the rich constituents of d-limonene (62.39%), followed by γ-terpinene (14.06%), with minor contents of 1-methyl-2-(1-methylethyl) (6.46%), α-humulene (5.22%), and methyl-n-methyl anthranilate (3.25%). The rhizome oil of Z. zerumbet showed the presence of 20 compounds, accounting for 96.01% of the whole oil with α-humulene (31.93%) and zerumbone (31.67%) as the main constituents, followed by minor quantities of o-menth-8-ene (8.46%), santolina triene (5.38%), β-caryophyllene (3.36%), and camphor (3.05%). A total of 21 compounds were identified in K. galanga rhizome oil, corresponding to 98.89% of the total oil. The most abundant compounds were 2-propeonic acid (35.54%), pentadecane (26.08%), and ethyl-p-methoxycinnamate (25.96%), whereas 3-carene (2.47%) and eucalyptol (2.12%) were minor constituents. Twelve compounds constituting 100.00% of all the volatile compositions were characterized from S. aromaticum bud oil containing the chief constituent of eugenol (77.37%), followed by minor amounts of trans-caryophyllene (13.66%) and eugenol acetate (4.60%). GC-MS total ion chromatograms for essential oils of five plants. In the larvicidal assessment, all essential oils demonstrated efficacy in both the pyrethroid-susceptible and -resistant strains of Ae. aegypti with dose dependent and different degrees among plant species (Tables 3 and 4). When exposed to the higher oil concentrations, more larvae showed toxic symptoms that led to an increase in mortality values. Correspondingly, the treated larvae tended to show toxic symptoms and die earlier at increasing oil concentrations. These findings suggest that concentrations of test substance affected degree of toxicity, mortality speed, and mortality rates. Although toxic symptoms in larvae treated with each essential oil were observed during different periods of time, the symptoms in larvae treated by these oils seemed to be similar, depending on dosage and oil variety. The incapacitated larvae showed abnormal behaviors such as restlessness, sluggishness, and coiling movement, and subsequently settled at the bottom of the bowl with abnormal wagging, tremors, convulsions, and paralysis, and later died slowly. However, no mortality was observed in the control groups. Even though effects on both strains of Ae. aegypti were relatively similar, all the oils tested proved to be slightly more toxic against the pyrethroid-susceptible strain than the resistant one. The highest potential was established from C. reticulata, followed by C. hystrix, Z. zerumbet, K. galanga, and S. aromaticum, with an LC50 of 15.42, 30.07, 48.88, 53.64, and 124.69 ppm, respectively, in the pyrethroid susceptible strain, and 19.38, 34.78, 53.08, 59.03, and 143.89 ppm, respectively, in the pyrethroid-resistant strain. The susceptibility to essential oils between the two strains of Ae. aegypti was slightly different but statistically significant. This performance has been initially observed and documented in our study. However, the relevance of these findings, which are probably due to mosquito tolerance or resistance to botanical agents, cannot be explained at the moment and requires more extensive studies. In Thailand, due to the higher cost and lower efficacy compared with conventional synthetics, the plant-derived mosquitocides have been subjected to use in the vector control programs with lower frequency, for shorter periods, and on smaller scales. However, the increasing utilization of plants and plant products for pest management in agriculture possibly led to the cross-resistance effect. The similarity in chemical structure and/or mechanism of action between the pyrethroid insecticides and used plant products might be a key to the development of tolerance or resistance in natural populations of mosquitoes. In order to clarify this suspicion, isolation and identification of the active ingredients responsible for such larvicidal activity need to be performed. In recent years, the active insecticidal compounds isolated from plants have received much attention due to their pronounced larvicidal efficacy. The bioactive component, β-thujaplicin, derived from Chamaecyparis obtusa leaf extract demonstrated strong larvicidal potential against Ae. aegypti, Ae. togoi, and Culex pipiens pallens, with LC50 of 2.91, 2.60, and 1.33 ppm, respectively (Jang et al. 2005). Larvicidal investigation of Eucalyptus grandis essential oil and its major components on Ae. aegypti revealed that the most effective was β-pinene, followed by α-pinene, and 1,8-cineole with the LC50 of 12.1, 15.4 ppm, and 57.2 ppm, respectively (Lucia et al. 2007). The essential oils of Cryptomeria japonica leaf and their effective constituents, including α–terpinene, γ-terpinene, ρ-cymene, 3-carene, terpinolene, and β–myrcene, provided an excellent larvicidal effect against both Ae. aegypti and Ae. albopictus, with an LC50 below 40 μg/ml. Among the pure constituents, 3-carene and terpinolene exhibited the best inhibitory action against Ae. aegypti (LC50= 25.3 μg/ml) and Ae. albopictus (LC50= 22.0 μg/ml), respectively (Cheng et al. 2008, 2009a). The toxicities of ethyl cinnamate and ethyl p-methoxycinnamate identified in K. galanga rhizome and another 12 known compounds were evaluated against 3rd instar larvae of laboratory-reared Cx. pipiens pallens, Ae. aegypti, and Ae. togoi, and field-collected Cx. pipiens pallens. Results were compared with those for fenthion and temephos (Kim et al. 2008). Ethyl p-methoxycinnamate, the most effective of the plant-derived compounds (LC50=12.3–20.7 mg/l), was found to be less toxic than either fenthion (LC50=0.0096–0.021 mg/l) or temephos (LC50=0.0039–0.0079 mg/l). Ethyl cinnamate and 3-carene were highly active against Cx. pipiens pallens (LC50=24.1 and 21.6 mg/l, respectively) but less toxic to Ae. aegypti and Ae. togoi. Variations in toxicity of essential oils against different species of mosquitoes are common (Sukumar et al. 1991, Amer and Mehlhorn 2006a), due to qualitative and quantitative variations of chemical constituents. Interestingly, the active larvicidal compounds in these works, including α-pinene, β-pinene, 1,8-cineole, α–terpinene, γ-terpinene, terpinolene, 3-carene, β–myrcene, ethyl cinnamate, and ethyl p-methoxycinnamate, were also detected in the essential oils investigated in this study. Therefore, other compounds such as d-limonene, α-humulene, zerumbone, 2-propeonic acid, pentadecane, and eugenol identified as the major components in the effective essential oils derived from C. hystrix, C. reticulata, Z. zerumbet, K. galanga, or S. aromaticum should not be neglected. Isolation and purification of the active compounds that might be responsible for the larvicidal activity against Ae. aegypti could be an important next step in the development of novel mosquitocidal agents. Production of larvicides from the locally available edible plants, which could be a new acceptable alternative to employ in the water supplies for drinking and daily use, may lead to decreasing dependence on imported synthetic insecticides and be beneficial for developing countries such as Thailand. Despite essential oils likely having less potential than synthetic pyrethroids, their natural biodegradation and remarkable activity on pyrethroid-resistant mosquitoes make them promising candidates for further study in controlling dengue and other mosquito-borne diseases. This work is supported by the Faculty of Medicine Research Fund, Faculty of Medicine, Chiang Mai University, Thailand. The authors acknowledge Assoc. Prof. Dr. Chusie Trisonthi and Assist. Prof. Paritat Trisonthi, botanists and taxonomists at the Department of Biology, Faculty of Science, Chiang Mai University, Thailand, for their kindness in identification of the plant samples.