Chemical Composition and Insecticidal Activity of Essential Oil of Artemisia frigida Willd (Compositae) against Two Grain Storage Insects

Purpose: To investigate the chemical composition and insecticidal activity of the essential oil of the aerial parts of Artemisia frigida against maize weevils (Sitophilus zeamais) and booklice (Liposcelis bostrychophila) ． Methods: Steam distillation of A. frigida aerial parts was carried out in a Clavenger apparatus to extract its volatile oil content. Gas chromatography/mass spectrometric (GC/MS) analyses (HP-5MS column) of the essential oil were performed and its contact toxicity was determined using topical application and filter paper impregnation technique while its fumigant toxicity was evaluated using sealed-space method. Results: A total of 32 components of the essential oil were identified. The principal compounds were cis-ρ-menth-2-en-1-ol (20.8%), 1,8-cineole (12.0%), borneol (10.2%), lavandulol (9.3%), camphor (6.9%), and bicyclogermacrene (5.5%). The oil exhibited contact toxicity against adult S. zeamais and L. bostrychophila with LC 50 value of 17.97 µg/adult and 254.38 µg/cm 2 , respectively. The essential oils also possessed fumigant toxicity against S. zeamais and L. bostrychophila with LC 50 value of 69.46 mg/L and 1.25 mg/L air. Conclusion: The study indicates that the essential oil of A. frigida has a potential to be developed to a natural fumigant/insecticide for the control of grain storage insects.


INTRODUCTION
The genus Artemisia belongs to the family Asteraceae (Compositae) and is a large, diverse genus of plants with about 380 species in the world, of which 186 species (82 endemic) are distributed in China [1]. Many species are rich in iso/chlorogenic acid, polyacetylenes, flavonoids, terpenoids, sesquiterpene lactones, and cyanogenic glycosides and are well-known medicinal plants [2]. Fringed sagebrush (Artemisia frigida Willd.) is a perennial semishrub distributed in the heavily grazed grasslands in Inner Mongolia, Qinghai, and Gansu Provinces in China [1]. The aerial part of A. frigida is a commonly used medical material in Mongolian folk medicine to treat joint swelling, renal heat, abnormal menstruation, and sore carbuncle, and is also one of the components of 'artificial holy water' [3]. Sesquiterpenoids, sesquiterpene lactone glycosides, coumarins and flavonoids have been reported from A. frigida [4][5][6]. The chemical composition of the essential oils derived from A. frigida aerial parts has also been determined [7][8][9].
During the screening program for new agrochemicals from Chinese medicinal herbs and wild plants, the essential oil of A. frigida aerial parts was found to possess insecticidal toxicity against maize weevils (Sitophilus zeamais Motsch.) and booklice (Liposcelis bostrychophila Badonnel) [10]. Essential oils of several Chinese Artemisia species were demonstrated to possess insecticidal activity to grain storage insects [11][12][13]. However, a literature survey shows that there is no report on insecticidal activity of the essential oil derived from A. frigida aerial parts. Thus, the objective of this study was to investigate the chemical constituents and insecticidal activity of the essential oil of A. frigida aerial parts against the two grain storage insects.

Extraction and isolation of essential oil
The samples were ground to powder using a grinding mill (Retsch Muhle, Germany). The powder (5 kg) was hydro-distilled for 6 h in a Clavenger apparatus. The oil was dried over anhydrous Na 2 SO 4 and kept in a refrigerator (4 o C) pending subsequent experiments.

Analysis of 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 o C and 300 o C, respectively. The components of the essential oils 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 (30m × 0.25mm × 0.25μm). 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. Retention index was 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 [14]. Relative proportions (%) of the oil components were calculated based on GC peak areas without using correction factors.

Rearing of insects
S. zeamais was obtained from laboratory cultures maintained for the last 10 years in the dark in incubators at 27-29 o C and 70 -80% relative humidity. Adult S. zeamais insects were reared on whole wheat at 12 -13% moisture content in glass jars (diameter 85 mm, height 130 mm). Unsexed adults of the insects 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).
Booklice (L. bostrychophila) were obtained from laboratory cultures in the dark in incubators at 28-30 o C and 70-80% r.h. 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 (Fluon®, Blades Biological, UK). Laboratory bioassays were done within one week after adult collections.

Fumigant toxicity test
The fumigant toxicity of the essential oil against S. zeamais was determined as described [15]. 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). Range-finding studies were run to determine the appropriate testing concentrations (2.0-40.0%, six concentrations). Ten microliters of essential oil solution 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 (containing 10 unsexed insects) to form a sealed chamber. Fluon (ICI America Inc) was used inside the glass vial to prevent insects from the treated filter paper. n-Hexane was used as controls and six replicates were used in all treatments and controls. They were incubated at 27 -29 o C and 70 -80% relative humidity for 24 h and mortality of insects was observed.
The fumigant toxicity of the essential oil against L. bostrychophila was determined as described [16]. A filter paper strip (3.5 cm×1.5 cm) treated with 10 μL of an appropriate concentration (0.80, 1.0, 1.8, 2.7, 4.0, and 6.0%) 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 all the treatments were replicated five times. Acetone was used as negative control and dichlorvos as positive control (purchased from Aladdin-reagent Company (Shanghai, China).

Contact toxicity test using topical application
The contact toxicity of the essential oil against S. zeamais was determined as described previously [15]. A serial dilution of the essential oil (6 concentrations, 2.6-13.3% v/w) was prepared in n-hexane. Aliquots of 0.5 µl of the dilutions were applied topically to the dorsal thorax of the insects. Controls were determined using nhexane. Six replicates were used in all 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 after 24 h. Pyrethrum extract (containing 25% pyrethrin I and pyrethrin II) was purchased from Fluka Chemie.

Contact toxicity test with treated filter paper
The contact toxicity of the essential oil against L. bostrychophila was determined as previously described [16]. 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 in 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, 1.2, 1.5, 1.8, 2.2, and 4.5%) and five replicates of each concentration were used in all treatments and controls.

Statistical analysis
The results from all replicates in fumigant and contact toxicity were subjected to Probit analysis [17] using PriProbit Program V1.6.3 to determine LD 50 and LC 50 values, respectively [18]. Samples for which the 95% fiducial limits did not overlap were considered to be significantly different.
The essential oil of A. frigida exhibited contact toxicity against S. zeamais and L. bostrychophila. However, compared with the positive control (Pyrethrum extract, LD 50 values of 4.3 μg/insect [19] and 19.0 µg/cm 2 , respectively), acute toxicity against the weevil and booklice was weak. The essential oil also showed fumigant toxicity against adult S. zeamais and L. bostrychophila. The commercial grain fumigant, methyl bromide (MeBr), is reported to possess fumigant activity against S. zeamais adults with an LC 50 value of0.67 mg/L [23]. However, compared with the fumigant activity of the other essential oils reported in the literature and which were tested using a similar bioassay, the essential oil obtained in the present study exhibited the same or stronger fumigant toxicity against maize weevils, e.g., the essential oils of A. vestita [11], A. eriopoda [13], A. lavandulaefolia and A. sieversiana [15], A. capillaris and A. mongolica [23], A. giraldii and A. subdigitata [12], and A. igniaria [24]. The essential oil of A. frigida aerial parts also possessed the same level of fumigant toxicity to the booklice as that of essential oils of Foeniculum vulgare [16], Illicium pachyphyllum fruits [25], and Curcuma wenyujin rhizomes [26].
The foregoing suggest that the fumigant activity of the essential oil of A. frigida has some promise as a possible natural fumigant/insecticide for the control of grain storage insects, especially as currently used fumigants are synthetic insecticides and the most effective fumigants are also highly toxic to humans and other non-target organisms [27]. However, to develop a practical application for the essential oil as novel fumigant/insecticide, further research into the safety of the essential oil in humans is needed. Additional studies on the development of formulations are also necessary to improve efficacy and stability as well as to reduce cost.

CONCLUSION
The essential oil of A. frigida aerial parts demonstrated some activity against maize weevil but needs to be further evaluated for safety in humans and to enhance its activity. The principal compounds found in the essential oil of A. frigida aerial parts harvested from Inner Mongolia were cis-ρ-menth-2-en-1-ol, 1,8-cineole, and borneol.