Chemical Composition and Insecticidal Activity of the Essential Oil of the Aerial Parts of Ostericum grosseserratum (Maxim) Kitag (Umbelliferae)

Purpose: To investigate the chemical composition and insecticidal activity of the essential oil of the aerial parts of Ostericum grosseserratum against the maize weevil, Sitophilus zeamais ． Methods: Steam distillation of the aerial parts of O. grosseserratum during the flowering stage was carried out using a Clavenger apparatus in order to obtain its volatile oil content. Gas chromatography/mass spectrometric (GC/MS) analyses (HP-5MS column) of the essential oil were performed and its contact and fumigant activity determined. Results: A total of 43 components of the essential oil were identified. The principal compounds were (d)-limonene (16.2 %), 4-terpineol (13.5 %), myristicin (11.3 %), γ -terpineol (8.3 %), β -pinene (5.1 %), β caryophyllene (4.6 %) and linalool (4.1 %). The oil exhibited contact toxicity against adult S. zeamais with lethal concentration (LC 50 ) value of 17.97 µg/adult. The essential oils also possessed fumigant toxicity against S. zeamais with LC 50 value of 13.70 mg/L air. Conclusion: The study indicates that the essential oil of O. grosseserratum has a potential to be developed into a natural fumigant/insecticide for the control of grain storage insects.


INTRODUCTION
The genus Ostericum belongs to the family Umbelliferae and is distinguished from the Genus Angelica by the presence of high concentrations of flavonoids in the leaf and mericarp of Ostericum [1].It comprises only of about 10 species in the world, of which seven are distributed in China [2].Ostericum grosseserratum (Maximowicz) Kitagawa is a herbaceous plant distributed mainly in some areas of China, Korea, Japan, Russia as well as Mongolia [2,3].The roots of O. grosseserratum have reputed medicinal value as a regional substitute for the traditional Chinese medicine, "Radix Angelicae Biseratae" (Angelica biserrata or A. pubescens) [2,3].This medicinal herb was used in traditional Chinese medicine as an analgesic and anti-inflammatory agent in the treatment of rheumatism and rheumatoid arthritis [4].
In a previous study, 8 constituent compounds (myristic acid, palmitic acid, stearic acid, octacosanoic acid, β-sitosterol, isoscopoletin, succinic acid, and β-sitosterol-β-D-glucoside) were isolated from the ethanol extract of O. grosseserratum roots [3].The chemical composition of the essential oils derived from O. grosseserratum roots has also been determined [5].During the screening program for new agrochemicals from Chinese medicinal herbs and wild plants, the essential oil of O. grosseserratum flowering aerial parts was found to possess insecticidal toxicity against the maize weevil, Sitophilus zeamais Motsch.
However, a literature survey shows that there is no report on the chemical composition and insecticidal activity of the essential oil derived from the aerial parts of O. grosseserratum.Thus, the objective of this study was to investigate the chemical constituents and insecticidal activity of the essential oil of O. grosseserratum aerial parts against grain storage insect.

EXPERIMENTAL Plant collection and identification
The aerial parts of O. grosseserratum were collected in August 2009 during the flowering stage from Xiaolongmen National Forest Park (39.48°N latitude and 115.25°E longitude, Mentougou District, Beijing 102300).The samples were air-dried and identified by Dr. Liu, (College of Life Sciences, Beijing Normal University, Beijing 100875, China) and a voucher specimen (no.ENTCAU-Umbelliferae-10003) was deposited at the Department of Entomology, China Agricultural University (Beijing 100193).

Extraction and isolation of essential oils
The flowering aerial parts were air-dried and first ground to powder using a grinding mill (Retsch Muhle, Germany).The powder was hydrodistilled for 6 h in a Clavenger apparatus.The oil was dried over anhydrous Na 2 SO 4 and kept in a refrigerator (4 °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 o C and 300 °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 °C for 1 min and ramped at 10 °C min −1 to 180 °C where it was held for 1 min, and then ramped at 20 °C min −1 to 280 °C and held there 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 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 [6].Relative percentages of the oil components were calculated based on GC peak areas without using correction factors.

Insects
S. zeamais was obtained from laboratory cultures maintained for the last 10 years in the dark in incubators at 27-29 °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).

Fumigant toxicity test
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.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 °C and 70 -80 % relative humidity for 24 h and mortality of insects was observed.

Contact toxicity test using topical application
The contact toxicity of the essential oil against adult S. zeamais was measured as described by Liu and Ho [10].Range-finding studies were run to determine the appropriate testing concentrations.A serial dilution of the essential oil 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 n-hexane.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 (25% pyrethrine I and pyrethrine II) was purchased from Fluka Chemie.

Statistical analysis
The results from all replicates in fumigant and contact toxicity were subjected to Probit analysis [7] using PriProbit Program V1.6.3 to determine LD 50 and LC 50 values, respectively [8].Samples for which the 95 % fiducial limits did not overlap were considered to be significantly different.
The essential oils of O. grosseserratum exhibited contact toxicity against S. zeamais adults.However, compared with the positive control (Pyrethrum extract, 25 % pyrethrine I and pyrethrine II) with an LC 50 value of 4.3 µg/insect [9], acute toxicity against the weevil was weak.The essential oil also showed fumigant toxicity against adult S. zeamais.The commercial grain fumigant, methyl bromide (MeBr), is reported to possess fumigant activity against S. zeamais adults with an LC 50 value of 0.67 mg/L [10].Thus, the essential oil is 20 times less toxic to adult S. zeamais than with MeBr.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 Artemisia lavandulaefolia (LC 50 = 11.2 mg/L) and A. sieversiana (LC 50 = 15.0 mg/L) [11], Illicium fragesii and I. simonsii (LC 50 = 11.36 mg/L and 14.95 mg/L, respectively) [12,13], and Kadsura heteroclita (LC 50 = 14.04 mg/L) [14] and Ostericum sieboldii (LC 50 = 20.92mg/L) [15].
The foregoing suggest that the fumigant activity of the essential oil of O. grosseserratum 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 [16].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 the flowering aerial parts of O. grosseserratum demonstrated some activity  [11]; **data from Liu and Ho [10]; *** methyl bromide.
against maize weevil but needs to be further evaluated for safety in humans and to enhance its activity.

Table 1 :
The main compounds of the essential oil of Ostericum grosseserratum