2.1.Collection, identification and extraction of plant , Ferulago angulate.
The plant of Ferulago angulate were collected in Chaharmahal and Bakhtiari Province in south-west of Iran (Fig.1) They were rapidly transported to the School of Public Health, Tehran University of Medical Sciences
2.2 Plantidentification: The plant was identified by experts in Department of Plant Sciences, Tehran University. (Fig.2)
2.3.Mosquitoes rearing
Susceptible strain of An. stephensi were reared and maintained at 28±2 °C and 65±5% relative humidity (RH) under a 16:8 (L: D) photoperiod. Under insectary situation .Guinea pigs is used as blood feeding female mosquitoes for maturing the eggs.
2.4.Repellency test
Females of An.stephensi were used for the repellency tests. 12 hour before starting the experiments, the sucrose solution was picked up from the cage . Various repellency tests including protection time, failure time, effective dose and killing effects of EOs were carried out according ASTM E951-9 against 5-8 old female An.stephensi.
2.5.Extraction of essential oil of plant
All the extraction was carried out at Faculty of Pharmacology, Tehran University of Medical Sciences. Essential oils (EOs) of native medicinal plant of Ferulago angulate, were hydrodistilled in a Clevenger-type apparatus for 4-6 h and dried over anhyrdrous sodium sulfate. The EOs were stored in the dark sealed vials at 4 °C until starting the repellency tests maximum after 2 days past of EO preparation.
2.6. Plant essential oils analysis
Chemical composition of plant was analyzed using an Agilent 7890–5975 gas chromatography mass spectrometer. With a HP- 5MS (5% Phenyl Methyl Silox) capillary column (30m×0.25mm, film thickness 0.25μm), split ratio, 1: 1, and using a flame ionization detector. The GC was programmed at 50 °C for 0.5 min and then increased at 5 °C/min to 280 °C, and finally held with an isothermal for 3min. The injector temperature was 280 °C. The flow rate of the carrier gas was 1ml/min. The identification of compounds was performed by comparing their retention times and mass spectra with mass spectra from Wiley library [19]
2.7.Plants essential oils analysis
Chemical composition of plant was analyzed using an Agilent 7890–5975 gas chromatography mass spectrometer. With a HP- 5MS (5% Phenyl Methyl Silox) capillary column (30m×0.25mm, film thickness 0.25μm), split ratio, 1: 1, and using a flame ionization detector. The GC was programmed at 50 °C for 0.5 min and then increased at 5 °C/min to 280 °C, and finally held with an isothermal for 3min. The injector temperature was 280 °C. The flow rate of the carrier gas was 1ml/min. The identification of compounds was performed by comparing their retention times and mass spectra with mass spectra from Wiley library.