Larvicidal Activity of Essential Oil Derived from Illicium henryi Diels (Illiciaceae) Leaf

Purpose: To determine larvicidal activity of the essential oil derived from Illicium henryi Diels (Illiciaceae) leaf and stem against the larvae of Aedes albopictus Skuse ． Methods: The essential oil of I. henryi leaves was obtained by hydrodistillation and analyzed by gas chromatography (GC) and gas chromaotography-mass spectrometry (GC-MS). The activity of the oil was evaluated, using World Health Organization (WHO) procedures, against the fourth instar larvae of A. albopictus for 24 h, and larval mortalities recorded at various essential oil concentrations ranging from 12.5 - 200 μg/mL. Results: A total of 25 components of the essential oil of I. henryi were identified and the principal compounds in the essential oil were safrole (54.09 %), myristicin (22.24 %), and 1, 8-cineole (5.43 %). The essential oil has higher content of (76.48 %) of phenylpropanoids than monoterpenoids (10.79 %) and sesquiternoids (11.72 %). The essential oil exhibited larvicidal activity against A. albopictus with a median lethal concentration (LC 50 ) of 35.43 μg/mL. Conclusion: The findings obtained indicate that the essential oil of I. henryi leaves has potentials for use in the control of A. albopictus larvae and may be useful in the search for newer, safer and more effective natural compounds as larvicides.


INTRODUCTION
The Asian tiger mosquito, Aedes albopictus Skuse (Stegomyia albopictus) (Diptera: Culicidae), is one of two main species of mosquito responsible for dengue fever in China [1]. Currently, light mineral oils and insect-growth regulators and some organophosphates such as temephos, fenthion, and Malathion are used as mosquito larvicides in many countries. However, these mineral oils and organophosphates can also kill other aquatic animals such as fish and crustaceans in the water body [2]. Thus, there is an urgent need to search new materials for controlling mosquitoes in an environmentally safe way, using biodegradable and target-specific insecticides against them. Fortunately, essential oils and their constituents have been suggested as alternative sources for insect control. Many studies have demonstrated that essential oils and their individual components possess promising larvicidal activities against mosquito vectors [3][4][5][6][7][8]. During our mass screening program for new agrochemicals from the wild plants, essential oil of Illicium henryi Diels leaves (Family: Illiciaceae) was found to possess larvicidal activity against the Asian tiger mosquito, A. albopictus Skuse.
Illicium henryi is an evergreen shrub or tree, 3 to 8 m (up to 12 m) tall and distributed in the southwestern part of China (Anhui, Fujian, Gansu, Guangdong, Guangxi, Guizhou, Henan, Hubei, Hunan, Jiangxi, Shaanxi, Sichuan, and Yunnan Province). Its root bark is locally used as a folk medicinal herb for repelling pathogenic wind and assuaging pain in China, and the fruits are poisonous [9]. In the previous phytochemical investigations, various sesquiterpene lactones, lignans, flavonoids, triterpenoids, and prenylated C6-C3 compounds have been isolated and identified from the plant [10][11][12]. Chemical composition of the essential oil of I. henryi fruits has been analyzed previously [13]. However, a literature survey has shown that there is no report on chemical composition and larvicidal activity of the essential oil of I. henryi leaves against mosquitoes, thus we decided to investigate chemical composition and larvicidal activity of the essential oil of I. henryi leaves against the Asian tiger mosquito.

Plant collection and identification
Fresh leaves of I. henryi (

Extraction of the essential oil
The leaves (10 kg) were air-dried, ground to powder using a grinding mill (Retsch Muhle, Haan, Germany). Each portion of the powder (600 g) was soaked in water at a ratio of 1:3 (w/v) for 3 h, prior to hydrodistillation using a round bottom flask (3000 mL) over a period of 6 h. The volatile essential oil was collected in a flask. Separation of the essential oil from the aqueous layer was done in a separating funnel using n-hexane and the n-hexane layer was then dried over anhydrous Na 2 SO 4 . The solvent was evaporated at 40 o C using a Buchi Rotavapor R-124 vacuum rotary evaporator. The oil was kept in a refrigerator at 4 o C pending subsequent experiments.

Analysis of the essential oils
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 o 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 o C for 1 min and ramped at 10 o C min −1 to 180 o C where it was held for 1 min, and then ramped at 20 o C min −1 to 280 o C and held there for 15 min. The injector temperature was maintained at 270 o 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 NIST 05 and Wiley 275 libraries or with mass spectra from literature [14]. Relative contents of the oil components were calculated based on GC peak areas without applying correction factors.

Insect cultures and rearing conditions
Mosquito 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 original eggs of A. albopictus were collected from Nanjing, Jiangsu province, China in 1997. Adults were maintained in a cage (60 × 30 × 30 cm) at 28 -30 °C and 75 -85 % RH. The females were fed with blood every alternate day whereas the males were fed with 10 % glucose solution soaked on cotton pad, which were hung in the middle of the cage. A beaker with strips of moistened filter paper was kept for oviposition. The eggs laid on paper strips were kept wet for 24 h and then dehydrated at room temperature. The dehydrated eggs were put into plastic tray containing tap water in our laboratory at 26 -28 °C and natural summer photoperiod for hatching and yeast pellets served as food for the emerging larvae. The newly emerged larvae were then isolated in groups of ten specimens in 100 ml tubes with tap water and a small amount of cat food. Larvae were daily controlled until they reached the fourth instar, when they were utilized for bioassays (within 12 h).

Larvicidal bioassay
Range-finding studies were run to determine the appropriate testing concentrations. The essential oil was first diluted to 10 % (v/v) using acetone as a solvent and final concentrations of 200, 100, 50, 25, and 12.5 μg/mL of the essential oil were tested. The larval mortality bioassay was carried out according to the test method for larval susceptibility recommended by the World Health Organization (WHO) [15]. 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 acetone 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 (L16:D9, 26 -27 o C, 78 -80 % relative humidity). Mortality was recorded after 24 h of exposure.

Statistical analysis
Percent mortality was corrected for control mortality using Abbott's formula [19]. Results from all replicates for the oil were subjected to probit analysis using PriProbit Program V1.6.3 (http://ars.usda.gov/Services/docs.htm?docid=11 284) to determine LC 50 values and their 95 % confidence intervals [17]. Samples for which the 95 % fiducial limits did not overlap were considered to be significantly different.

RESULTS
The yield of I. henryi essential oil was 0.23 % (v/w) while its density was determined to be 0.96 g/mL. A total of 25 components from the essential oil of I. henryi leaves were identified, accounting for 98.90 % of the total essential oil. The principal constituents of the essential oil of I. henryi leaves were safrole (54.09 %), myristicin (22.24 %), and 1, 8-cineole (5.43 %) ( Table 1).
The essential oil possessed strong larvicidal activity against the 4th instar larvae of A. albopictus with a LC 50 value of 35.43 μg/mL ( Table 2).
In the previous studies, one of the major constituent, safrole was found to possess larvicidal activity against two mosquitoes, A. aegypti and A. albopictus [21,22].  However, myristicin was shown to have contact and fumigant toxicity against several insect/mites and to exhibit strong synergistic activity because it has been demonstrated to possess strong inhibitory effects on many P450s, such as houseflies (Musca domestica) [23], hairy caterpillars (Spilarctia obliqua) [24], armyworms (Pseudaletia unipuncta) [25], navel orangeworm (Amyelois transitella) [26], and house dust mites (Dermatophagoides farinae, D. pteronyssinus) and mould mites (Tyrophagus putrescentiae) [27]. The isolation and identification of the bioactive compounds in the essential oil 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.
A literature survey has shown that there is no experimental data on toxicity of the essential oil of I. henryi leaves to human is available, to the best of our knowledge. Thus, to develop a practical application for the essential oil as novel insecticides, further research into the safety of the essential oil to humans is needed. The isolation and identification of the bioactive constituent compounds in the essential oil of I. henryi leaves are of utmost importance so that their potential application in controlling mosquitoes can be fully exploited. Additional studies on the development of formulations are also necessary to improve the efficacy and stability and to reduce cost. Moreover, field evaluation and further investigations on the effects of the essential oil on non-target organisms are necessary.

CONCLUSION
The essential oil of I. henryi leaves demonstrates some activity against Aedes albopictus mosquito larva but needs to be further evaluated for safety in humans and to develop formulations to improve the efficacy and stability as well as reducing cost.