Chemical Composition of Zanthoxylum avicennae Essential Oil and its Larvicidal Activity on Aedes albopictus Skuse

Purpose: To determine the larvicidal activity of the essential oil derived from Zanthoxylum avicennae (Lam.) DC. (Rutaceae) leaves and stems against the larvae of Aedes albopictus Skuse ． Methods: Essential oil of Z. avicennae leaves and stems were obtained by hydrodistillation and analyzed by gas chromatography (GC) and gas chromaotography-mas spectrometry (GC-MS). The activity of the essential oil was evaluated, using World Health Organization (WHO) procedures, against the fourth larvae of A. albopictus for 24 h and larval mortality recorded at various essential oil concentrations ranging from 12.5 - 200 μg/mL. Results: A total of 31 components of the essential oil of Z. avicennae were identified. The essential oil had higher content of monoterpenoids (65.70 %) than sesquiterpenoids (33.45 %). The principal compounds of the essential oil were 1,8-cineol (53.05 %), β-elemene (6.13 %), α-caryophyllene (5.96 %), β-caryophyllene (5.09 %) and caryophyllene oxide (4.59 %). The essential oil exhibited larvicidal activity against A. albopictus with a median lethal concentration (LC 50 ) value of 48.79 μg/mL. Conclusion: The findings obtained indicate that the essential oil of Z. avicennae has potentials for use in the control of A. albopictus larvae and could be useful in the search for newer, safer and more effective natural compounds as larvicides.


INTRODUCTION
Beyond their itchy, irritating bites, mosquitoes are vectors for some of humanity's most deadly illnesses, such as yellow fever, dengue fever, malaria, several forms of encephalitis and filariasis. The Asian tiger mosquito (Aedes albopictus Skuse) and the yellow fever mosquito (A. aegypti L.) are two main species of mosquito responsible for dengue fever and malaria in China.
All mosquitoes need water to breed. The control of mosquito larvae worldwide depends currently on continued application of synthetic insecticides and insect growth regulators [1]. However, repeated use of these synthetic insecticides has caused several environmental and health concerns, including disruption of natural biological control systems, outbreaks of other insect species, widespread development of resistance and undesirable effects on non-target organism [2]. Thus, there is urgent need to look for new strategies for mosquito control.
From this point of view, botanical pesticides, including essential oils, are promising since they are effective, environmentally friendly, easily biodegradable, and often inexpensive [2]. It is suggested that many essential oils and constituent compounds derived from various essential oils can exert toxic activity against mosquito species [3][4][5]. During our mass screening program for new agrochemicals from wild plants and Chinese medicinal herbs, the essential oil of Zanthoxylum avicennae (Lam.) DC. (Family: Rutaceae) leaves and stems, was found to possess larvicidal activity against the Asian tiger mosquito, A. albopictus.
Zanthoxylum avicennae is an evergreen shrub distributed in India, Indonesia, Malaysia, Philippines, Thailand, Vietnam, southern China (Fujian, Guangdong, Guangxi, Hainan, and Yunnan Province) and Taiwan [6]. It is used as a folk medicine for treatment of rheumatism, abdominal pain, jaundice, chronic hepatitis, and common cold in China [7]. The chemical constituents of this medicinal herb have been studied. Alkaloids, coumarins, terpenoids, flavonoids, and neolignans, and their derivatives were isolated from this plant in previous studies [8][9][10][11][12][13]. Chemical composition of the essential oil of Z. avicennae has also been determined [14][15][16]. However, a literature survey has shown that there is no report on larvicidal activity of Z. avicennae essential oil against mosquitoes, thus we decided to investigate the chemical constituents and larvicidal activity of the essential oil against the Asian tiger mosquito.

EXPERIMENTAL Plant collection and identification
Fresh aerial parts (leaves and stems) of Z. avicennae (5 kg) were harvested from Qujing City (25 The samples were air-dired for two weeks, ground to powder using a grinding mill (Retsch Muhle, Germany), subjected to hydrodistillation using a modified Clevenger-type apparatus for 6 h and then extracted with n-hexane. The oil was dried over anhydrous Na 2 SO 4 and kept in a refrigerator (4 C) pending subsequent experiments.

Analysis of the essential oil
Capillary gas chromatography was performed using Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector and fused silica capillary column HP-5 (5 % diphenyl and 95 % dimethylpolysyloxane, 30 m × 0.25 mm, 0.25 μm film thickness), at a flow rate of 1 mL min −1 . Temperature was programmed from 60 to 280 °C (at a rate of 2 °C min −1 ); injector and detector temperatures were 270 and 300 C, respectively. The components of the essential oil were separated and identified by gas chromatography-mass spectrometry (GC-MS) using Agilent 6890N gas chromatography coupled to Agilent 5973N mass selective detector. The system was equipped with a flame ionization detector and capillary column with HP-5MS (30 m × 0.25 mm × 0.25 μm). GC settings were as follows: the initial oven temperature was held at 60 C for 1 min and ramped at 10 C min −1 to 180 C where it was held for 1 min, and then ramped at 20 C min −1 to 280 C and held there for 15 min. The injector temperature was maintained at 270 C. The samples (1 μL, diluted to 100:1 with acetone) were injected, with a split ratio of 1:10. The carrier gas was helium at a flow rate of 1.0 ml min −1 . Spectra were obtained over the scan range 20 to 550 m/z at 2 scans s -1 . Most constituents were identified by gas chromatography by comparison of their retention indices with those published in the literature or with those of authentic compounds available in our laboratories. The retention indices were determined in relation to a homologous series of n-alkanes (C 8 -C 24 ) under the same operating conditions. Further identification was made by comparison of their mass spectra with those stored in National Institute of Standards and Technology 05 (NIST 05) and Wiley 275 libraries or with mass spectra from literature [17]. Relative contents of the oil components were calculated based on GC peak areas without applying correction factors.

Insect cultures and rearing conditions
The eggs of A. albopictus utilized in bioassays were obtained from a laboratory colony maintained in the Department of Vector Biology and Control, Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The dehydrated eggs were placed on a plastic tray containing tap water to hatch and yeast pellets served as food for the emerging larvae. The eggs batches, collected daily, were kept wet for 24 h and then placed in distilled water in the laboratory at 24-26 °C and natural summer photoperiod for hatching. The newly emerged larvae were then isolated in 150 groups of ten specimens in 100 ml tubes with 50 mL of mineral water and a small amount of cat food (3:1, dried bread and pounder of pork liver). Larvae were daily controlled until they reached the fourth instar stage, when they were utilized for bioassay (within 12 h).

Larvicidal bioassay
Range-finding studies were run to determine the appropriate testing concentrations. Concentrations of 200, 100, 50, 25, and 12.5 μg/mL of essential oil were tested. The larval mortality bioassay was carried out according to the test method for larval susceptibility proposed by the World Health Organization (WHO) [18]. Twenty larvae were placed in glass beaker with 250 ml of aqueous suspension of tested material at various concentrations and an emulsifier, dimethyl sulfoxide (DMSO) was added in the final test solution (<0.05%). Five replicates per concentration were run simultaneously and with each experiment, a set of controls using 0.05 % DMSO and untreated sets of larvae in tap water, were also run for comparison. For comparison, commercial chlorpyrifos (purchased from National Center of Pesticide Standards, Tiexi District, Shenyang 110021, China) was used as positive control. The toxicity of chlorpyrifos was determined at concentrations of 5, 2.5, 1.25, 0.6, and 0.3 μg/mL. The assay was carried out in a growth chamber (Ningbo Jiangnan Instrument Factory, Ningbo 315012, China. http://www.nbjn.com) with L16:D9, 26-27 o C, 78-80 % relative humidity. Mortality was recorded after 24 h of exposure to essential oil and the larvae were starved of food over this period.

Statistical analysis
Percent mortality was corrected for control mortality using Abbott's formula [19]. Results from all replicates for the pure compounds/oil were subjected to probit analysis using PriProbit Program V1.6.3 to determine LC 50 values and their 95 % confidence intervals [20]. Samples for which the 95 % fiducial limits did not overlap were considered to be significantly different.
The essential oil possessed strong larvicidal activity against the 4 th instar larvae of A. albopictus with a LC 50 value of 48.79 μg/mL ( Table 2).
In previous reports, one of the main constituent compounds of the essential oil, β-caryophyllene was demonstrated to possess larvicidal activity against A. aegypti larvae with a 48 h LC 50 value of 34 μg/mL [28]. Caryophyllene oxide exhibited strong larvicidal activity against A. albopictus larvae with a 24 h LC 50 value of 65.6 μg/mL while another main constituent, 1,8-cineol, exhibited weaker larvicidal activity (LC 50 > 100 μg/mL) [22]. Although 1,8-cineole did not exhibit any significant mosquito larvicidal activity, it was moderately effective as a feeding repellent and highly effective as an ovipositional repellent against adult yellow fever mosquito (A. aegypti) [29]. However, another two of main constituents, β-elemene and α-caryophyllene have not been evaluated for larvicidal activity against mosquitoes so far. The isolation and identification of the bioactive compounds in the essential oil of Z. avicennae are of utmost importance to determine if their potential application in controlling mosquito pests can be fully exploited. Considering that the currently used larvicides are synthetic insecticides, larvicidal activity of the crude essential oil is quite promising and it shows its potential for use in the control of A. albopictus larvae and could be useful in the search for newer, safer and more effective natural compounds as larvicides.
For the actual use of Z. avicennae essential oil and its constituents as novel larvicides or insecticides to be realized, further research is needed to establish their human and environmental safety. In traditional Chinese medicine, the plant is used to treat abdominal pain, chronic hepatitis, and common cold [7] and appears to be safe for human consumption. However, no experimental data on its toxicity to human is available. Additionally, their larvicide modes of action have to be established, and formulations for improving larvicidal potency and stability need to be developed. Furthermore, field evaluation and further investigation of the effects of the essential oil on non-target organisms are necessary.

CONCLUSION
The essential oil of Z. avicennae demonstrates some activity against Aedes albopictus mosquito larvae but needs to be further evaluated for safety in human and to enhance its activity.