Chemical composition and toxic activity of essential oil of Caryopteris incana against Sitophilus zeamais

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INTRODUCTION
The maize weevil, Sitophilus zeamais (Motschulsky) is one of the most widespread and destructive primary insect pests of stored cereals (Liu and Ho, 1999).Infestations not only cause significant losses due to the consumption of grains; they also result in elevated temperature and moisture conditions that lead to an acelerated growth of molds, including toxigenic species (Magan et al., 2003).Fumigation plays a very important role in insect pest elimination in stored products not only because of their ability to kill a broad spectrum of pests but because of their easy penetration into the commodity while leaving minimal residues (Zettler and Arthur, 2000).The currently used synthetic fumigant is still the most effective means for the protection of stored food, feedstuffs and other agricultural commodities from insect infestation.However, repeated use of those synthetic *Corresponding author.E-mail: zhilongliu@cau.edu.cnTel/Fax: 86-10-62732800.
fumigants for decades has disrupted biological control by natural enemies and led to resurgence of stored-product insect pests, which sometimes resulted in the development of resistance, and had undesirable effects on nontarget organisms.Moreover, the use of methyl bromide (MeBr) will be prohibited in the near future because of its ozone depletion potential (United States Environmental Protection Agency, 1993).These problems have highlighted the need to develop new types of selective insectcontrol alternatives with fumigant action.Plant essential oils and their components have been shown to possess potential to be developed as new fumigants and they may have advantage over conventional fumigants in terms of low mammalian toxicity, rapid degradation and local availability (Isman, 2006(Isman, , 2008)).Essential oils derived from many plant species, so far have been eva-luated for fumigant toxicity against stored product insects (Rajendran and Srianjini, 2008).
Botanical pesticides have the advantage of providing novel modes of action against insects that can reduce the risk of cross-resistance as well as offering new leads for de-sign of target-specific molecules (Isman, 2006).During the screening program for new agrochemicals from Chinese medicinal herbs, the essential oil of Caryopteris incana (Thunb.ex Hout.)Miq.(Family: Verbenaceae) aerial parts was found to possess strong insecticidal toxicity against S. zeamais.

MATERIALS AND METHODS
S. zeamais were obtained from laboratory cultures maintained for the last 10 years in the dark in incubators at 27 to 29°C and 70 to 80% relative humidity.S. zeamais adults were reared on whole wheat at 12 to 13% moisture content.Unsexed adults of the maize weevils used in all the experiments was about 2 weeks old.
Ten kilograms of aerial parts of C. incana were purchased from Puning Chinese Medicinal herbs Market (Guangdong 515300, China).The species was identified, and the voucher specimens (BNU-Liuzhilong-Verb-09-302) were deposited at the Herbarium (BNU) of College of Life Sciences, Beijing Normal University.The herb was first ground to powder using a grinding mill (Retsch Muhle, Germany).Each 600 g portion of ground powder was mixed in 1,800 ml of distilled water and soaked for 3 h.The mixture was then boiled in a round-bottom flask, and steam distilled for 6 to 8 h.Essential oil from distillation was collected in a flask.Separation of the essential oil from the aqueous layer was done in a separatory funnel, using the non-polar solvent, n-hexane.The solvent was evaporated using a vacuum rotary evaporator (BUCHI Rotavapor R-124, Switzerland).The sample was dried over anhydrous Na2SO4 and kept in a refrigerator (4°C) for subsequent experiments.

of the essential oils
Components of the essential oil were separated and identified by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) Agilent 6890N gas chromatography hooked to Agilent 5973N mass selective detector.The same column and analysis conditions were used for both GC and GC/MS.They were equipped with a flame ionization detector and capillary column with HP-5MS (30 m x 0.25 mm x 0.25 µm).The 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 for 1 min, and then ramped at 20°C min −1 to 280°C for 15 min.The injector temperature was maintained at 270°C.The samples (1 µl) were injected neat, with a split ratio of 1:10.The carrier gas was helium at the flow rate of 1.0 ml min −1 Spectra were scanned from 20 to 550 m/z at 2 scans s -1 .Most constituents were identified by gas chromatography by comparison of their retention indices with Chu et al. 8477 those of the literature (Pu et al., 1984;Sun et al., 2004;Kim, 2008) or with those of authentic compounds available in our laboratories.The retention indices were determined in relation to a homologous series of n-alkanes (C8-C24) 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 (Adams, 2007).Component relative percentages were calculated based on GC peak areas without using correction factors.

Fumigant toxicity
A Whatman filter paper (diameter 2.0 cm, CAT No. 1001020) was placed on the underside of the screw cap of a glass vial (diameter 2.5 cm, height 5.5 cm, volume 24 ml).20 µl of 4.0 to 25.0% of the essential oil (V:V, 6 concentrations) was added to the filter paper.
The solvent was allowed to evaporate for 30 s before the cap was placed tightly on the glass vial (with 10 unsexed insects) to form a sealed chamber.Fluon (ICI America Inc) was used inside glass vial to prevent insects from the treated filter paper.n-Hexane was used as the controls.Six replicates were used in all the treatments and controls and they were incubated at 27 to 29°C and 70 to 80% relative humidity for 24 h.The insects were then transferred to clean vials with some culture media and kept in an incubator.Mortality of insects was observed daily until end-point mortality was reached one week after treatment.Results from all replicates were subjected to probit analysis using the PriProbit Program V1.6.3 to determine LC50 values (Sakuma, 1998).

Contact toxicity using topical application
The contact toxicity of essential oil against S. zeamais adults was measured as described by Liu and Ho (1999).A serial dilution of the essential oil (2.0 to 30.0%, 6 concentrations) was prepared in nhexane.Aliquots of 0.5 µl of the dilutions were applied topically to the dorsal thorax of the insects.Controls were determined using hexane.Six replicates were used in all the treatments and controls.Both treated and control insects were then transferred to glass vials (10 insects/vial) with culture media and kept in incubators.Mortality of insects was observed daily until end-point mortality was reached one week after treatment.The LD50 values were calculated by using probit analysis (Sakuma, 1998).
These results suggest that great variations in chemical composition of essential oil of C. incana aerial parts may be due to harvest time and local, climatic and seasonal factors as well as storage duration of the medicinal herbs.

Insecticidal activities
The essential oil of C. incana aerial parts showed contact toxicity against S. zeamais adults with an LD 50 value of 122.65 µg/adult (Table 2).However, the essential oil demonstrated weak acute toxicity against the weevil when compared with the control (pyrethrum extract, 25% pyrethrin I and pyrethrin II) because the pyrethrum extract had acute toxicity to the maize weevils with an LD 50 value of 4.3 µg/adult (Liu et al., 2010a).In previous studies, the two main components of the essential oil (estragole and linalool) demonstrated toxicity against several species of insects and mites (Sanchez-Ramos and Castanera, 2001;Sampson et al., 2005;Chang et al., 2009;Yang et al., 2009).
The earlier mentioned findings suggest that fumigant activity of the essential oil of C. incana aerial parts is quite promising by considering the currently used fumigants which are synthetic insecticides, and it shows potential to be developed as a possible natural fumigant for the control of stored product insects.However, for the practical application of the essential oil as novel fumigant, further studies on the safety of the essential oil to humans and on development of formulations are necessary to improve the efficacy and stability and to reduce cost.The isolation and identification of the bioactive compounds in the essential oil of C. incana aerial parts are of utmost importance so that their potential application in controlling stored-product pests can be fully exploited.Moreover, further studies on plant cultivation and essential oil standardization is necessary because chemical composition of the essential oil varies greatly with the plant population.

Table 1 .
Chemical constituents of the volatile oil from C. incana aerial parts.

Table 2 .
Insecticidal activity of C. incana essential oil against S. zeamais adults.
*Data from