Gas Chromatography-Mass Spectrometric Analysis and Insecticidal Activity of Essential Oil of Aerial Parts of Mallotus apelta (Lour.) Muell.-Arg. (Euphorbiaceae)

Purpose: To investigate the chemical composition and insecticidal activity of the essential oil of the aerial parts of Mallotus apelta against maize weevils, Sitophilus zeamais and booklice, Liposcelis bostrychophila. Methods: Water-distilled essential oil of M. apelta aerial parts was analyzed by gas chromatography/mass spectrometric (GC/MS) to determine its composition. Insecticidal activities of the essential oil were measured by using topical application/ impregnated filter paper and seal-spaced fumigation. Results: Thirty-six compounds, accounting for 97.75 % of the oil, were identified. The main compounds found were β -eudesmol (18.65 %), β -caryophyllene (9.83%), β -selinene (6.55 %), caryophyllene oxide (6.29 %), bornyl acetate (6.07%), γ -eudesmol (5.40 %) and α -selinene (5.06 %). The essential oil showed contact toxicity against adult maize weevils L. bostrychophila with LD 50 (lethal dosage, 50 %) value of 46.69 µ g/adult and 211.02 µ g/cm, respectively. The essential oil also exhibited fumigant toxicity against adult S. zeamais and L. bostrychophila with a LC 50 (median lethal concentration) value of 48.42 and 3.21 mg/l, respectively. Conclusion: The study indicates that the essential oil of M. apelta has the potential to be developed into a natural fumigant/insecticide for the control of stored product insects.

However, the constituents of the essential oil derived of M. apelta aerial parts have not been determined so far, to the best of our knowledge.Only the essential oil of M. apelta roots has been determined by GC and GC/MS [8].Moreover, a literature survey showed that insecticidal activities of the essential oil against grain storage insects were not measured.Thus, the present investigation consists of two parts: determination of chemical composition of the essential oil of M. apelta aerial parts; and evaluation of the essential oil as insecticide/fumigant against two grain storage insect pests, namely, maize weevil (Sitophilus zeamais Motsch.) and booklouse (Liposcelis bostrychophila Badonnel).

EXPERIMENTAL Plant material and essential oil extraction
Fresh aerial parts of M. apelta ( The booklice (L.bostrychophila) were obtained from laboratory cultures in the dark in incubators at 28-30 o C and 70-80 % RH and reared on a 1: 1:1 mixture, by mass, of milk powder, active yeast, and flour.All the containers housing insects and the Petri dishes used in experiments were made escape proof with a coating of polytetrafluoroethylene. Laboratory bioassays were done within one week after adult collections.

Gas chromatography-mass spectrometry
The essential oil was subjected to GC-MS analysis on an Agilent system consisting of a model 6890N gas chromatograph, a model 5973N mass selective detector (EIMS, electron energy, 70 eV), and an Agilent ChemStation data system.The GC column was an HP-5ms fused silica capillary with a 5 % phenylmethylpolysiloxane stationary phase, film thickness of 0.25 µm, a length of 30 m, and an internal diameter of 0.25 mm.
The GC settings were as follows: the initial oven temperature was held at 60 °C for 1 min and increased at 10 °C/min to 180 °C held for 1 min, and then increased at 20 °C/min to 280 °C and held for 15 min.The injector temperature was maintained at 270 °C.The sample (1 µl, diluted 100:1 in acetone) was injected, with a split ratio of 1:10.The carrier gas was helium at flow rate of 1.0 ml/min.Spectra were scanned from 20 to 550 m/z at 2 scans/s.Most constituents were identified by gas chromatography by comparison of their Kovats retention indices with those of 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 08 and Wiley 275 libraries or with mass spectra from literature [9].Component relative percentages were calculated based on GC peak areas without using correction factors.

Fumigant toxicity bioassay
Fumigant toxicity of M. apelta essential oil against maize weevils was determined by used the method of Liu and Ho [10].A serial dilution of the essential oil (5.0, 7.0, 9.0, 12.0, 17.0, and 22.0 %) was prepared in n-hexane.A Whatman filter paper (CAT no.1001020, diameter 2.0 cm) was placed on the underside of the screw cap of a glass vial (diameter 2.5 cm, height 5.5 cm, volume 24 ml).Ten microliters of an appropriate concentration of the essential oil was added to the filter paper.The solvent was allowed to evaporate for 15 s before the cap was placed tightly on the glass vial (with 10 unsexed insects) to form a sealed chamber.Preliminary experiments demonstrated that 15 s was sufficient for the evaporation of solvents.The vials were upright and the Fluon® coating restricted the insects to the lower portion of the vial to prevent them from the treated filter paper.n-Hexane was used as a control.They were incubated at 27 -29 o C and 70 -80 % RH for 24 h.Five replicates were carried out for all treatments and controls.The insects were considered dead if appendages did not move when probed with a camel brush.
The fumigant toxicity of the essential oil against L. bostrychophila was determined as described by Zhao et al [11].A filter paper strip (3.5 cm × 1.5 cm) treated with 10 µl of an appropriate concentration of the essential oil in acetone.The impregnated filter paper was then placed in the bottom cover of glass bottle of 250 ml.The insects, 10 adults in a small glass bottle (8 ml), were exposed for 24 h and each concentration with five replicates.Six concentrations (0.7, 1.0, 1.5, 2.0, 3.0 and 5.0 %) were used in all treatments and controls.Acetone was used as controls and dichlorvos was used as a positive control.Dichlorvos (99.9 %) was purchased from Aladdin-reagent Company (Shanghai).

Contact toxicity test (topical application)
Serial dilutions of the essential oil (5.0 -25.0 % v/w) were prepared in n-hexane.Aliquots of 0.5 µl of the dilutions were applied topically to the dorsal thorax of the weevils, using a Burkard Arnold microapplicator.Controls were determined using n-hexane.Both treated and control insects were then transferred to glass vials (10 insects per vial) with culture media and kept in incubators.The mortality of the weevils was observed after 24 h.

Contact toxicity test (treated filter paper)
The essential oil was diluted in acetone.The filter paper with 3.5 cm in diameter (Whatman) was treated with 150 µl of the solution.Then the filter paper after treated with solid glue (Glue Stick, Jong Ie Nara Co Ltd, Hong Kong) was placed in a Petri dish (3.5 cm diameter) and 10 booklice were put on the filter paper by using a hair brush.The plastic cover with holes was put and all the Petri dishes were kept in incubators at 27 -29 o C, 70 -80 % r.h. for 24 h.Acetone was used as controls and pyrethrum extract was used as a positive control.Six concentrations (1.0 -4.5 %) and five replicates of each concentration were used in all treatments and controls.Pyrethrum extract (25 % pyrethrin I and pyrethrin II) was purchased from Fluka Chemie (Buchs, Switzerland).

Data analysis
The observed mortality data were corrected for control mortality using Abbott's formula [12] and the results subjected to probit analysis using the PriProbit Program V1.6.3 to determine LC 50 or LD 50 values [13] at the 0.05 level of significance.
No death of insects was observed in the control under current concentration.The essential oil of M. apelta aerial parts possessed contact toxicity against S. zeamais adults with an LD 50 value of 46.69 µg/adult and L. bostrychophila with an LD 50 value of 211.02 µg/cm (Table 2).The essential oil of M. apelta also exhibited fumigant toxicity against S. zeamais adults and L. bostrychophila with LC 50 values of 48.42 mg/l and 3.21 mg/l, respectively (Table 2).
The essential oil exhibited contact toxicity against S. zeamais adults and L. bostrychophila.However, the essential oil of M. apelta demonstrated one-eleventh as acute toxic as the pyrethrum extract against S. zeamais (LD 50 = 4.29 µg/adult) [15] and L. bostrychophila (LD 50 = 18.99 µg/cm) [11].The essential oil of M. apelta also exhibited fumigant toxicity against S. zeamais adults and L. bostrychophila.The commercial grain fumigant, methyl bromide (MeBr) exhibited fumigant activity against S. zeamais adults with an LC 50 value of 0.67 mg/l thus the essential oil was 1/72 as toxic as MeBr to S. zeamais adults [10].
Moreover, the essential oil exhibited only 2400 times less toxicity than dichlorvos against the booklice because dichlorvos was reported to exhibit fumigant toxicity against L. bostrychophila with an LC 50 value of 1.35 × 10 -3 mg/l [11].However, compared with other essential oils in the previous studies that were tested using a similar bioassay, the essential oil of M. apelta aerial parts exhibited stronger or similar level of fumigant toxicity against S. zeamais adults, e.g., the essential oils of Aster ageratoides [13], Murraya exotica [14], Ostericum sieboldii [16] and several essential oils from Genus Artemisa [17,18].Moreover, the main constituent, βeudesmol, has been demonstrated to possess contact toxicity and ovicidal activity against diamondback moth, Plutella xylostella [19], and acute toxicity against the common vinegar fly, Drosophila melanogaster was also observed [20].Thus, the isolation and identification of the bioactive constituent compounds in the essential oil of M. apelta aerial parts are of utmost importance so that their potential application in controlling stored-product pests can be fully exploited.

CONCLUSION
Our findings suggest that the fumigant activity of the essential oil of M. apelta aerial parts is promising considering the currently used fumigants which are synthetic in origin.Since currently used fumigants are synthetic insecticides and the most effective fumigants are also highly toxic to humans and other non-target organisms, the essential oil of M. apelta aerial parts can play an important role in stored grain protection and also reduce the risks associated with synthetic insecticides.However, further studies on the safety of the essential oil in humans and also development of formulations are necessary to improve the efficacy and stability as well as reduce cost.

Table 1 :
Chemical constituents of essential oil of Mallotus apelta aerial parts