Chemical composition and larvicidal activity of Zanthoxylum gilletii essential oil against Anopheles gambiae

Malaria is a serious health problem in many African countries. The Anopheles gambiae mosquito which is the major vector for this disease has developed resistance against synthetic pyrethroids which are the main stay of insecticide treated bed nets. The development of insecticide resistance and side effects associated with synthetic pesticides has triggered intense research efforts towards natural products (for vector control) such as essential oils because of their efficacy and safety. In this study, larvicidal potential of essential oil from Zanthoxylum gilletii was evaluated against malaria vector mosquito, A. gambiae. The essential oil was extracted by hydro-distillation, and its chemical compositions determined by gas chromatography mass spectrometry. The oil was dominated by sesquiterpenes and monoterpenes which accounted for 38.30 and 34.00%, respectively. The oil showed good activity against A. gambiae and recorded LC50 and LC90 values of 57.73 and 140.24 ×10 mg/ml, respectively. The results obtained show that the essential oil isolated from Z. gilletii is a promising mosquito larvicide.


INTRODUCTION
Mosquitoes are known vectors of various diseases which are life threatening.Anopheles gambiae mosquitoes are known to transmit malaria (Cheng et al., 2003;Das and Ansari, 2003;Magalhaes et al., 2010).According to the latest WHO estimates, there were approximately 219 million cases of malaria globally in 2010 and 660,000 fatal cases: approximately 90% of these fatal cases occurred in Africa (WHO, 2012).
Currently there is no effective available vaccine for malaria (Matasyoh et al., 2008).Among the efforts that have been made in recent decades in seeking to reduce mosquito bites and transmission of malaria include the use of insecticide treated nets (ITNs) and larviciding.The insecticide treated nets rely solely on pyrethroids to enhance their protective utility (Chavasse et al., 1999).Larviciding has the greatest control impact on mosquito populations because the larvae are concentrated, immobile and accessible, and it employs the use of synthetic insecticides (Tiwary et al., 2007).However, the overreliance on these synthetic chemicals for mosquito control has resulted in the development of insecticide resistance over time (Hemingway and Ranson, 2000).The *Corresponding author.E-mail: jeffombito@gmail.com.Tel: +254 729 020575.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License spread in resistance to majority of present synthetic insecticides by A. gambiae and the environmental pollution coupled with the safely risks for both human and domestic animals posed by these insecticides has highlighted the need for novel strategies for control of A. gambiae (Cheng et al., 2009b).Hence, there is a renewed interest in the exploration and use of plant products with insecticidal properties for mosquito control.
Aromatic plants and their essential oils are very important sources of many compounds that are used for different applications (Abduelrahman et al., 2009).Essential oils are simply volatile fractions obtained by either steam or water distillation of medicinal and aromatic plants (Rabha et al., 2012).Essential oils have received considerable renewed attention as potent bioactive compounds against various species of mosquitoes.They 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;Cheng et al., 2009a).
Zanthoxylum gilletii is a tropical rainforest species, distributed between altitudes ranging from 900 to 2400 m.It is a valued forest tree that grows naturally but planted in Western Kenya for timber and medicinal properties (Kokwaro et al., 1976).The Luhya community; that is, a major habitat of this region uses the bark of Z. gilletii in traditional anti-malaria preparations (Nyunja et al., 2009).
The present study attempted to investigate the larvicidal efficacy of essential oils derived from Z. gilletii leaves against the medically important malaria vector mosquito, A. gambiae with the purpose of identifying effective indigenous bio-products to control the vector of mosquitoborne diseases, particularly in cases where the vector's susceptibility to conventional synthetics is decreasing.

Sample collection
The leaves of Z. gilletii were collected from Kakamega forest, a tropical rain forest in Kenya which stretches from 0° 10 ʹ to 0° 21 ʹ N and longitude 34° 44 ʹ to 34° 58 ʹ E and an altitude of 1524 m above the sea level.The leaves were identified with the help of a taxonomist.Voucher specimens were deposited at the Department of Biological Sciences, Egerton University, Kenya.

Extraction of essential oil
Fresh leaves of Z. gilletii were cut into pieces less than 2 × 2 cm within 12 h after collection and 1000 g hydro-distilled in a modified type-Clevenger apparatus for 4 h.The essential oil obtained was dried over anhydrous Na 2 SO 4 and kept in glass vial under refrigeration at 4°C.

Essential oil analysis
Samples of essential oils were diluted in methyl-t-butyl ether (MTBE) (1:100) and analyzed on an Agilent GC-MSD apparatus equipped with an Rtx-5SIL MS ('Restek') (30 m × 0.25 mm, 0.25 µm film thickness) fused-silica capillary column.Helium (at 0.8 ml/min) was used as a carrier gas.Samples were injected in the split mode at a ratio of 1:10 to 1:100.The injector was kept at 250°C and the transfer line at 280°C.The column was maintained at 50°C for 2 min and then programmed to 260°C at 5°C/min and held for 10 min at 260°C.The MS was operated in the electron impact ionization (EI) mode at 70 eV, in m/z ranging from 42 to 350.The identification of the compounds was performed by comparing their retention indices and mass spectra with those found in literature (Adams, 2007) and supplemented by Wiley 7N.l, HPCH 1607.L and FLAVORS.L GC-MS libraries.The relative proportions of the essential oil constituents are expressed as percentages obtained by peak area normalization, all relative response factors being taken as one.

Mosquito larvicidal bioassays
The larval toxicity tests were carried out following the standard World Health Organization larval bioassay method (WHO, 2005), with slight modifications.Since oil does not dissolve in water, it was first solubilized in dimethyl-sulphoxide (DMSO, analytical reagent, Lobarchemi) and diluted with spring river water to make a stock solution of 1000 mg/L.Serial dilutions of the stock solution were done at different concentrations which included 500, 250, 200, 150, 125, 100, 62.5, 55, 45, 40, 31.25, 15.6 and 7.8 ×10 -3 mg/ml.The concentration of DMSO was kept below 1% since at this level it does not affect larval mortality.The bioassays were conducted at the Kenya Medical Research Institute (KEMRI), Centre for Global Health Research (CGHR), Kisumu, Kenya, where the insects were reared in plastic and enamel trays in spring river water.They were maintained and all experiments were carried out at 26 ± 3°C and the humidity ranged between 70 and 75%.The bioassays were performed with third instar larvae of A. gambiae and carried out in triplicate using 20 larvae for each replicate assay.The replicates were run simultaneously yielding a final total of 60 larvae for each concentration.The larvae were collected by direct pipetting from the enamel trays and transferred to 25 ml disposable plastic cups containing 10 ml of test solution and fed on tetramin fish feed during all testing.Mortality and survival was established after 24 h of exposure.Larvae were considered dead if they were unrousable within a period of time, even when gently prodded with a micropipette.The dead larvae in the three replicates were combined and expressed as the percentage mortality for each concentration.The negative control was 1% DMSO in spring river water while the positive control was the pyrethrum based larvicide, pylarvex.

Statistical analysis
The average larval mortality data were subjected to probit analysis for calculating LC 50 and LC 90 at 95% fiducial limits of upper confidence limit and lower confidence limit (Finney, 1971) using IBM SPSS software version 15.
The essential oil of Z. gilletii was active against third instar larvae of A. gambiae with LC 50 and LC 90 values of 57.73 and 140.24 ×10 -3 mg/ml respectively (Table 2).The negative control showed no activity against third instar larvae of A. gambiae.At a concentration of 100×10 -3 mg/ml, the positive control pyrethrin EC 0.5% w/v (Pylarvex ™ ) recorded 100 % larval mortality (Table 2).
Compared to essential oils from other plants, the essential oil of Z. gilletii exhibits significant activity against malaria vector A. gambiae.Reports from earlier studies indicate that larvicidal activity of essential oil from F. angolensis (Rutaceae) against third instar larvae of A. gambiae exhibited LC 50 and LC 90 values of 83.7 and 324.0 mg/L, respectively (Mudalungu et al., 2013).Kweka et al. (2012) reported larvicidal activity of P. amboinicus essential oil against late third instar larvae of A. gambiae and observed LC 50 and LC 90 values of 67.53 and 107.60 ppm, respectively.The essential oil of C. citratus was observed to have an LC 50 of 69 ppm against Aedes aegypti larvae (Cavalcanti et al., 2004).The same oil was reported to possess larvicidal activity against Culex quinquefasciatus larvae with LC 50 of 165.7 ppm (Pushpanathan et al., 2008) (Table 3).
γ-Terpinene, the major component of Z. gilletii essential oil has been found to possess larvicidal activity against three mosquito species (Cheng et al., 2009c;Zhu and Tian., 2011).This compound had LC 50 values of 26.8 µg/ml, 22.8 µg/ml and 29.21 mg/L against A. aegypti, Aedes albopictus and A. anthropophagus larvae,  1996).Sabinene which was also in appreciable amount in the oil has been reported to exhibit larvicidal activity against third instar larvae of C. quinquefasciatus, A. aegypti and Anopheles stephensi (Govindarajan, 2010).The LC 50 values recorded against the three mosquito species were 25.01, 21.20 and 19.67 ppm, respectively.The compound also recorded LC 90 values of 45.15 ppm against C. quinquefasciatus, 39.22 ppm against A. aegypti and 36.45 ppm against A. stephensi.Sabinene was also isolated from the essential oil of Clausena dentata and found to be active against Spodoptera litura with LC 50 and LC 90 values of 21.42 and 40.39 ppm respectively (Krishnappa et al., 2010).Another monoterpene also present in appreciable amount in the oil was β-myrcene.Previous studies have documented the activity of β-Myrcene against the larvae of A. aegypti and A. albopictus (Cheng et al., 2009c).The LC 50 values recorded were 27.9 and 23.5 µg/ml respectively.
Trans-Caryophyllene, which occurs in appreciable amounts in this oil, is also reported to show activity against A. aegypti larvae with LC 50 of 104 ppm (Morais et al., 2006).Its oxygenated form caryophyllene oxide is known to exhibit larvicidal activity against the fourth instar larvae of A. anthropophagus (Zhu and Tian, 2013) with LC 50 and LC 90 values of 49.46 and 115.38 mg/L, respectively.α-Cadinol although in small quantity in Z. gilletii oil, is known to possess larvicidal activity against A. aegypti with LC 50 value of 76.1 ppm (Chun et al., 2008).Germacrene D is known to be effective against larvae of A. aegypti and A. stephensi (Kiran et al., 2006).This sesquiterpene hydrocarbon, isolated from the essential oils of Chloroxylon swietenia, had LC 50 values of 63.6 and 59.5 µg/ml against A. aegypti and A. stephensi respectively.The LC 90 values recorded for this compound were 100.7 µg/ml against A. aegypti and 96.4 µg/ml against A. stephensi.
The high larvicidal activity of Z. gilletii can therefore be attributed to the presence of γ-terpinene, β-myrcene, sabinene, trans-caryophyllene, caryophyllene oxide, α-cadinol and germacrene D which have been documented to possess larvicidal activity against different species of mosquito.

Conclusion
Plants are rich source of bioactive organic chemicals and offer an advantage over synthetic pesticides as they are less toxic, less prone to development of resistance, and easily biodegradable.The findings of this study show that the essential oil isolated from Z. gilletii holds great promise as potential mosquito larvicides.Furthermore these outcomes could be useful in the search for newer more selective, biodegradable and natural larvicidal compounds.These findings also offer an opportunity for developing alternatives to inorganic insecticides.

Table 1 .
Identified compounds of the essential oil from Zanthoxylum gilletii.

Table 2 .
Larvicidal activity of essential oil against third instar larvae of An. gambiae after 24 h of exposure.
X Positive control, Y Negative control.

Table 3 .
Comparison of relative toxicity of essential oil from Zanthoxylum gilletii with three previously isolated essential oils tested against A. gambiae, A. aegypti and C. Quinquefasciatus.