Chemical composition and larvicidal activity of essential oil of Cupressus arizonica E.L. Greene against malaria vector Anopheles stephensi Liston (Diptera: Culicidae).

BACKGROUND: Using botanical insecticides as an alternative biocontrol technique for vector control is considered by some scientists.
MATERIALS AND METHODS: Chemical composition of the essential oil was analyzed using gas chromatography-mass spectrometry (GC-MS). In addition, the mosquito larvicidal activity of leaf essential oil of Cupressus arizonica was investigated against fourth instar larvae of laboratory-reared An. stephensi according to the method of the World Health Organization.
RESULTS: Of 46 constituents in the oil, limonene (14.44%), umbellulone (13.25%) and α-pinene (11%) were determined as the main constituents. Cupressus arizonica volatile oil showed significant larvicidal activity against An. stephensi with LC(50) and LC(90) values 79.30 ppm and 238.89 ppm respectively. Clear dose-response relationships were established with the highest dose of 160 ppm essential oil with almost 100% mortality. DISCUSSION: The results from this study revealed that C. arizonica essential oil could be considered as a natural larvicide against An. stephensi. However, the field evaluation of the formulation is necessary.

INTRODUCTION

Anopheles mosquitoes are bloodsucking insects that are responsible for the transmission of malaria, filariasis and arboviruses; on the other hand they are important as nuisance mosquitoes in particular areas while they may not be vectors of any disease.[1] Human malaria is considered as the most important disease among the vector-borne diseases. Malaria still remains an endemic disease in the southeastern corner of the country.[23] There are more than 25 currently recognized Anopheles species out of which seven of them have important roles in malaria transmission. Among these species, Anopheles stephensi is considered as a primary vector of malaria in the southern parts of the country.[24-9]

There are several methods for malaria control, most of which have been applied in Iran. Synthetic insecticides which are generally used have side-effects on human and animal health and also the environment. The side-effects of synthetic organophosphorus compounds on fish and other organisms in the environment are being increasingly reported.[1011] A lot of attention is being paid to natural products in vector control as they are environmentally safe, degradable and target-specific. Recent studies have demonstrated the insecticidal properties of plant essential oils and their efficacies against larvae of different species of mosquito.[412-20] The repellent effect of the extract and essential oil of plants has been investigated on some mosquito species in Iran.[21-23]

The purpose of this paper is to investigate thecomposition and larvicidal activity of the leaf essential oilof Cupressus arizonica E.L. Greene. This species is a medium-sized evergreen tree with a conic to ovoid-conic crown known as a native plant in the southwestern United States.[2425] It was introduced to Iran in 1954 and has been commonly cultivated in many parts of the country.[26] The biological effect of C. arizonica have been investigated and its effects as a potential mosquito repellent have been reported earlier.[2728] Previous studies indicated that α-pinene, limonene and umbellulone were obtained as major components from the oil of C. arizonica leaves in Iran,[29] Italy,[30] USA,[31] Algeria,[32] France,[33] Tunisia[27] and Argentina.[34]

This study was conducted to find out the efficacy of the leaf essential oil of C. arizonica against fourth instar larvae of An. stephensi under laboratory conditions and determine the chemical composition of the essential oil.

MATERIALS AND METHODS

Plant materials

A fresh leaf from an eight-year-old C. arizonica tree was collected in May 2009 from Tehran, Iran (51° 23′E, 35° 43′N, elevation: 1329 Meter). The plant was identified and authenticated and the voucher specimen was deposited at the Herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

Essential oil isolation

Fresh leaves (200 g) of C. arizonica were subjected to hydrodistillation using a modified clevenger-type apparatus for 3 h, dried over anhydrous sodium sulphate and transferred into amber-colored vials at 5°C for further work. Essential oil was stored in an airtight container prior to analysis by gas chromatography-mass spectrometry (GC and GC–MS).

Gas chromatography-mass spectroscopy

Analytical gas chromatography was carried out using an HP gas chromatograph. The separation was achieved by use of a HP1 (Fused silica) capillary column (30 m × 0.25 mm; film thickness 0.25 μm); split ratio, 1:25, and using a flame ionization detector. The GC settings were programmed as follows: initial oven temperature was held at 40°C for 1 min, rising to 250°C at 5°C /min. The injector temperature was maintained at 250°C. The detector temperature was at 230°C. Helium was used as the carrier gas at a flow rate of 1 ml/min.

GC-MS was performed on Agilent Technology 5973 mass selective detector connected with a HP 6890 gas chromatograph. The separation was achieved by use of a HP1 MS (Fused silica) capillary column (30 m × 0.25 mm; film thickness 0.25 μm); split ratio, 1:25, and using a flame ionization detector. The MS operated at 70 eV ionization energy. Quantitative data were obtained from the electronic integration of the Flame Ionization Detector (FID) peak areas.

Determination of oil composition

Identification of the oil components was based on retention indices which were calculated by using retention times of n-alkanes that were injected after the oil at the same chromatographic conditions. The components of the oils were identified by comparison of their mass spectra and retention indices with Wiley library and those published in the literature.[2735] The percentage of each component is presented in Table 1.

Table 1

Chemical constituents of leaf essential oil from Cupressus arizonica

Mosquito rearing

The fourth-instar larvae of An. stephensi Bandar-Abass strain was obtained from the Department of Medical Entomology, Tehran University Medical Sciences. The mosquito colony was maintained continuously at 27°C with 12:12 light and dark photoperiod in 80 ± 10% relative humidity. Larvae of An. stephensi were continuously available for the mosquito larvicidal experiments.

Bioassays and larval mortality

Bioassays were performed according to the standard method recommended by the World Health Organization (WHO).[36] The fourth-instar larvae of An. stephensi, collected from the maintained laboratory stock served as the test organisms in this study. Methanol 99.0% was used as co-solvent and then different concentrations of the essential oil in distillated water were prepared. A 400-ml glass beaker was used for each experiment or control. Controls included batches of mosquitoes from the colony exposed to water and the solvent alone.

The larvae were exposed to the concentrations of 10, 20, 40, 80 and 160 ppm of essential oil extract in distilled water for 24 h. In the control beakers only 1 ml of solvent was dissolved into the water. Mortality was counted after 24 h recovery period. The percentage of mortality was reported from the average for the five replicates taken.

Statistical analysis

LC50 (lethal concentration to cause 50% mortality in the population) and LC90 (lethal concentration to cause 90% mortality in the population) were determined by the use of regression line employed by Finney.[37] In case of mortality in control beakers, it was corrected by Abbott's correction.[38] Differences between means were considered significant at P ≤ 0.05.[39]

RESULTS

Yields and chemical constituents of essential oil

The hydrodistillation of the C. arizonica leave gave oil in 0.8% (w/w) yield on fresh weight material. The essential oil was yellow with a distinct sharp odor.

Table 1 shows constituents of the oil. Forty-six constituents in the leaf essential oil of C. arizonica were identified corresponding to 97.33% of the total oil. The main constituents in the C. arizonica leaf essential oil were limonene (14.44%), umbellulone (13.25%) and α-pinene (11%) respectively. The results revealed that monoterpene hydrocarbons in the leaf oil were predominant (40.72%), whereas oxygenated sesquiterpenes were present in low amounts.

Mosquito larvicidal activity of essential oil

The larvicidal potency of different concentrations of leaf essential oil of C. arizonica against An. stephensi is given in Table 2. Among the five concentrations tested, the dosage of 160 ppm was found to be the most toxic with 100% larval mortality. Cupressus arizonica leaf essential oil showed toxicity against the larvae. There was no mortality in the control groups. The LC50 and LC90 values against An. stephensi larvae were 79.30 ppm and 238.89 ppm respectively.

Table 2

Probit regression line parameters of Anopheles stephensi to leaf essential oil extraction of Cupressus arizonica at different interval concentrations

DISCUSSION

The efficacy of several plant essential oils and extracts as natural larvicides has been reported.[131416-2040] In this study, major constituents and bioactivity of leaf essential oil of C. arizonica were considered. Limonene (14.44%), umbellulone (13.25%) and α-pinene (11%), cis-muurola-4(14),5-diene (7.36%) and terpinen-4-ol (7.29%) were known as the main compounds of the oil.

In previous studies, various constituents of the oil of C. arizonica were reported.[2729-34] Although umbellulone is the most abundant constituent in the oils obtained from all mentioned studies, the percentage of the compound was varied, based on the origins of the plant. While it has been reported as 45.1% from C. arizonica cultivated in Italy, 37.3% in Algeria, 18.4% in Tunisia, 16.5% in Argentine and 5.4% in Texas, it was recorded as 13.25% in this study which is close to the result obtained from Argentinean samples. Although the oil obtained in this study has a moderate amount of limonene (14.44%) and known as the most abundant constituent, it was recorded 5.8% in Tunisia, 8.5% in Argentina, 8.7% in France and 14% in Texas.

The amount of limonene in this study is similar to the US samples. In the previous researches, α-pinene has been reported from the Texas (7.6%), Algeria (10.5%), Tunisia (20.0%) and Argentina samples (22.9%). In a similar study, it was discovered as the main constituent of the leaf oil (19.2%) in Isfahan, Iran, which is similar to the results of our study.[2729-34]

According to the results of the larvicidal tests, the essential oil C. arizonica was effective against An. stephensi with LC50 and LC90 values of 79.30 and 238.89 ppm, respectively. The bioactivity of different extracts from the plants has been investigated on An. stephensi larvae. There is a report about the efficacy of the methanolic extract of Tagetes minuta L. on the An. stephensi in which, the LC50 and LC90 values were obtained as 2.5 mg/l and 11.0 mg/l, respectively.[17] The larvicidal activity of Tagetes patula oil has been reported on the larvae of An. stephensi with the LC50 and LC90 values of 12.08 mg/l and 57.62 mg/l, respectively.[41] Vatandoost and Moein-Vaziri reported LC50 and LC90 values of about 0.35 mg/l and 1.81 mg/l respectively of neem tree (Azadirachta indica) extract against An. stephensi larvae.[40] Our previous studies demonstrated the efficacy of Eucalyptus camaldulensis oil against An. stephensi larvae with the LC50 and LC90 values of 89.85 mg/l and 397.75 mg/l respectively.[20] The comparison of the efficacy of essential oils of C. arizonica and E. camaldulensis against An. stephensi larvae revealed that the oil of C. arizonica is more potent than E. camaldulensis. It seems that the presence of a high amount of limonene and α-pinene as larvicidal compounds in C. arizonica essential oil, could demonstrate its efficacy against An. stephensi larvae.[42]

The usage of plant essential oils as larvicides in vector control can be considered as a substitute method to reduce the side-effects of synthetic insecticides on the environment. Extensive efforts have been made to find new plants and their oils as natural and eco-friendly larvicides. In this way, our finding suggests that the leaf essential oil of C. arizonica has a potential larvicide effect. It is essential to conduct more research in this area in order to find good candidates and isolate the effective compounds.