GC-MS Analysis of Insecticidal Essential Oil of Aerial Parts of Echinops latifolius Tausch

The roots of Echinops latifolius Tausch (Asteraceae) have been used in the traditional medicine. However, no report on chemical composition and insecticidal activities of the essential oil of this plant exists. The aim of this research was to determine chemical composition and insecticidal activities of the essential oil of E. latifolius aerial parts against maize weevils (Sitophilus zeamais Motschulsky) for the first time. Essential oil of E. latifolius aerial parts at flowering stage was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 35 components of the essential oil of E. latifolius aerial parts were identified. The major compounds in the essential oil were 1,8-cineole (19.63%), (Z)-β-ocimene (18.44%), and β-pinene (15.56%) followed by β-myrcene (4.75%) and carvone (4.39%). The essential oil of E. latifolius possessed contact toxicity against S. zeamais with an LD 50 value of 36.40 μg/adult. The essential oil also exhibited fumigant toxicity against S. zeamais with an LC 50 value of 9.98mg/L.The study indicates that the essential oil of E. latifolius aerial parts has a potential for development into a natural insecticide/fumigant for control of insects in stored grains.


Introduction
Insects and fungi create serious quality problems in stored grains.The maize weevil (Sitophilus zeamais Motschulsky) is one of the major pests of stored grains and grain products in China, causing serious losses in stored products [1].The only way to eliminate pests completely from a food grain without leaving pesticide residues is fumigation [2].Currently, there are only two commonly used fumigants for stored food, methyl bromide (MeBr) and phosphine, which have led to the resurgence of pests.In addition to the resistance developed by the insect pests, chemical application has also exerted undesirable effects on nontarget organisms and fostered environmental and human health concerns [3].This necessitates the development of natural and safe products that are relatively less hazardous to mammalian health and the environment than existing conventional pesticides, as alternatives to nonselective synthetic pesticides to control the pests of medical and economic importance [4].Essential oils or their constituents have demonstrated to be potential sources of alternative compounds to currently used fumigants.Essential oils and their constituents have low toxicity to warm-blooded animals, high volatility, and toxicity to storedgrain insect pests [5,6].
During the screening program for new agrochemicals from Chinese medicinal herbs and wild plants, the essential oil of Echinops latifolius Tausch (Asteraceae) (syn.E. davuricus) aerial parts at flowering stage was found to possess strong insecticidal toxicity against a cosmopolitan pest of stored products, S. zeamais.Echinops is comprised of ca.120 species and distributed all over the world, mostly in the northern hemisphere [7].E. latifolius is a perennial growing to 0.5 m and distributed mainly in the north of China (e.g., Gansu, Hebei, Heilongjiang, Henan, Jilin, Liaoning, Ningxia, Shaanxi, Shandong, Shanxi, and Inner Mongolia) and Russia as well as Mongolia [7].The root of this plant is anti-inflammatory and galactagogue.It is used in the treatment of breast abscesses with inflammation, mastitis, lack of milk in nursing mothers, and distension of the breast for a long history and it has been recorded in Chinese Pharmacopoeia as one of the sources for Yuzhou Loulu [8].In previous studies, several triterpenoids, sesquiterpenoids, and thiophenes were isolated from E. latifolius and identified [9][10][11][12].However, a literature survey showed that there is no report on the volatile constituents and insecticidal activity of E. latifolius; thus we decided to investigate the chemical constituents and insecticidal activities of the essential oil of E. latifolius aerial parts against grain storage insects for the first time.

Essential Oil Extraction.
The sample was ground to a powder using a grinding mill (Retsch Mühle, Germany).Each 600 g portion of 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 6-8 h.Volatile 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 -hexane.The solvent was evaporated using rotary evaporator (BUCHI Rotavapor R-124, Switzerland).The sample was dried over anhydrous Na 2 SO 4 and kept in a refrigerator (4 ∘ C) for subsequent experiments.

Insects.
The maize weevils (S. zeamais) were obtained from laboratory cultures in the dark in incubators at 29-30 ∘ C and 70-80% relative humidity and were reared on whole wheat at 12-13% moisture content in glass jars (diameter 85 mm, height 130 mm).Unsexed adult weevils used in all the experiments were about one week old.All containers housing insects and the petri dishes used in experiments were made escape-proof with a coating of polytetrafluoroethylene (Fluon, Blades Biological, UK).

Gas Chromatography-Mass Spectrometry. The essential oil of E. latifolius 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% phenyl methylpolysiloxane stationary phase, film thickness of 0.25 m, 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 then heated at 180 ∘ C at a rate of 10 ∘ C/min, held for 1 min, and then heated to 280 ∘ C at 20 ∘ C/min and held for 15 min.The injector temperature was maintained at 270 ∘ C. The sample (1 L, 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 −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 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 -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 the literature [14].Component relative percentages were calculated based on normalization method without using correction factors.

Contact Toxicity by Topical Application.
Contact toxicity of the essential oil against S. zeamais adults was measured as described by Liu et al. [15].Range-finding studies were run to determine the appropriate testing concentrations of the essential oil of E. latifolius.A serial dilution of the essential oil (5.0%, 6.0%, 7.7%, 9.6%, 12.0%, and 15.0%) was prepared in -hexane.Aliquots of 0.5 L per insect were topically applied dorsally to the thorax of the weevils, using a Burkard Arnold microapplicator.Controls were determined using 0.5 L -hexane per insect.Ten insects were used for each concentration and control, and the experiment was replicated six times.Both the treated and control weevils were then transferred to glass vials (10 insects/vial) with culture media and kept in incubators at 29-30 ∘ C and 70-80% relative humidity.Mortality was observed after 24 h.The insects that did not present any movement when touched with a brush were considered as dead.
2.6.Fumigant Toxicity Bioassay.Range-finding studies were run to determine the appropriate testing concentrations of E. latifolius essential oil.The fumigant toxicity of E. latifolius essential oil was determined based on the method of Liu and Ho [1] with some modifications.A Whatman filter paper (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 the essential oil (5.39-20.00%,6 concentrations) 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.The vials were placed upright and the Fluon (ICI America Inc.) coating restricted the insects to the lower portion of the vial to prevent them from the treated filter paper.They were incubated at 27-29 ∘ C and 70-80% relative humidity for 24 h.Mortality of insects was observed.

Data Analysis.
The observed mortality data were corrected for control mortality using Abbott's formula and results from all replicates were subjected to probit analysis using the PriProbit Program V1.6.3 to determine LC 50 or LD 50 values [16].
The essential oil of E. latifolius aerial parts possessed fumigant toxicity against the maize weevils with an LC 50 value of 9.98 mg/L (Table 2).The commercial grain fumigant, methyl bromide (MeBr), was reported to have fumigant activity against S. zeamais adults with an LC 50 value of 0.67 mg/L  [1]; thus the essential oil was 15 times less toxic to S. zeamais adults compared with MeBr.However, compared with fumigant activity of the other essential oils in the literature using the same bioassay, the essential oil of E. latifolius exhibited stronger or the same level of fumigant toxicity against S. zeamais adults, for example, essential oils of S. multifida [28], Kadsura heteroclite [32], Murraya exotica [13], Ostericum grosseserratum [33], Saussurea nivea [34], and Illicium pachyphyllum [35] and several essential oils from genus Artemisia Moreover, (Z)--ocimene also exhibited fumigant toxicity against S. zeamais adults with a 24 h LC 50 value of 28.66 mg/L air [40].The above findings suggest that fumigant activity of the essential oil of E. latifolius aerial parts is quite promising.
Considering the currently used fumigants and synthetic insecticides, the oil shows potential to develop a possible new natural fumigant/insecticide for control of stored product insects.However, for the practical application of the essential oil as novel insecticide/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 costs.

Conclusion
The study indicates that the essential oil of E. latifolius aerial parts has a potential for development into a new natural insecticide/fumigant for control of insects in stored grains.However, further studies on the safety of the oil in humans as well as development studies are required to optimize the efficacy and stability of this extract and to reduce costs.

Table 1 :
Chemical constituents of essential oil derived fromEchinops latifolius aerial part.