Collection of plant leaves
Fresh mature leaves of Holoptelea integrifolia were randomly harvested from plants growing at outskirts of Debipur, Purba Bardhaman, West Bengal. Collected leaves were properly cleaned under tap water and then washed with distilled water to remove dust, debris and any kind of impurities on the leaf and soaked in paper towel.
Collection of mosquito larvae
Culex vishnui predominantly breed in rice field water. Egg rafts of Cx. vishnui were collected from rice fields surrounding Burdwan University Golapbag campus. The eggs were kept in plastic tray bearing volume of 12.6 x 10 x 6 inches3 in Mosquito and Microbiology Research Unit, The University of Burdwan. After hatching, larvae were fed with a mixture of dog biscuits and dried yeast powder in the ratio of 3:1.
Preparation of solvent extracts
Finely grounded shade dried leaves of 250 g were put into a Soxhlet apparatus for solvent extraction. Plant extracts were prepared using solvents of increasing polarity (non polar to polar) namely petroleum ether, ethyl acetate, acetone and absolute alcohol, applying successively (extraction period 72 hour in each solvent) with the same leaf sample. The extracts were collected separately and the Soxhlet apparatus was washed with 200 ml water and100 ml of similar solvent as an eluent after each solvent extraction procedure. Eluted material of each extract was concentrated below at 400 C temperatures to 100 ml of solution by evaporation in rotary evaporator. Resultant concentrated extract were kept in a deep freeze at -80 0 C (REVCO model No: ULT 790-3-V32) for 24 hour. The resulting freeze dried extract was lyophilized and the solid residue was weighed and then dissolved in suitable amount of sterilized distilled water to make the different graded concentrations. Total yield of ethyl acetate extract was 2.68g.
Bio assay with solvent extracts
The bioassay experiments were conducted on 3rd instars larvae of Cx. vishuni according to standard WHO procedure (1981)  with slight modification. Tween 20 was used as solubilizer for petroleum ether, ethyl acetate, acetone and absolute alcohol solvent extracts. The quantity of Tween 20 used to prepare the solution had been determined previously by tolerance experiments with Cx. vishuni larvae to find the non lethal concentrations. The stock solution of 500 ppm concentration of each extract was prepared. Different concentrations were obtained from this stock solution by addition of distilled water. Later those concentration of each extract was transferred to disposal plastic cups separated to carry out the tests, in which twenty five 3rd instars larvae of Cx. vishuni were placed with the help of disposable plastic pipette (WHO/VBC, 2005)  and a similar type of bio assay were conducted with only distilled water but without any of the solvent extract of the mature leaves as a control. The dead larvae were counted after every 24 hour up to 72 hour of exposure and percentage mortality was reported from the average of the three replicate taken.
The leaf extract was subjected to qualitative phytochemical analysis for determination of secondary metabolites using standard methods .
Column chromatographic analysis
Column chromatographic analysis was only done with the most effective extract which showed maximum mortality of the Cx. vishnui larvae in bioassay experiment. Dried 5 g sample to be analysed was transferred to the top of the prepared column. Solvent level was kept above the sample by adding the eluting solvent as necessary. Then the column was eluted with single and mixture of different ratios of organic solvents with increasing polarity like petroleum ether, petroleum ether: n- hexane , n- hexane, n- hexane ethyl acetate, ethyl acetate, ethyl acetate: chloroform, chloroform, chloroform: methanol, methanol, methanol: acetone, acetone, acetone: absolute alcohol and absolute alcohol were used as eluting solvent. The flow rate was 2 ml/ min. During the process of separation of the change for the eluting solvent to a more polar system, were previously determined by TLC. Several fractions were collected by combining the same fraction totalling 500 ml. Confirming homogeneity of compounds, the fractions with larvicidal activity against Cx. vishuni 3rd instar larvae was detected by bioassay.
Thin layered Chromatography (TLC) analysis
The bio active fractions were monitored by thin layer chromatography silica gel ʹGʹ (Merk, India) coated (0.5 mm thickness), using petroleum ether as mobile phase. TLC glass plates were placed in well lidded iodine chamber (21 x 21 x 9 cm) for 1 min to properly detect the bands. The plate was removed and the main band appeared on the subsequent plates with similar Rf (0.357) values were selected afterwards and mixed together and used as apparently purified compound. The Rf value was calculated using formula: Rf = Distance of spot centre from start point/Distance of solvent run from the start point.
IR and GC- MS analyses of bio active principle
The bioactive spots were scrapped from the glass plates and dissolved in absolute alcohol. The alcohol fractions were collected by discarding the silica G and filtered through Whatman No. 1 filter paper. For IR analysis, the sample was kept in vacuum desiccators over KOH pellets for 48 h, and then Infrared (IR) spectral analysis were done with 1 mg sample using potassium bromide (KBr) plates. The pellets were undergoing scanning in Jasco-Fourier Transformer Infrared Spectroscopy FTIR (FT/IR-42 Jasco) with a scanning period of 4 min/ sec and scanning speed of 2 mm sec-1. The purified fraction was analysed directly by GC on a Hewlett Packard (HP; Palo Alto CA, USA) model HP-6890 PLUS GLC and HP-3398a GC Chemstation instrument fitted with a column HP-5 (Capillary column of 0.32 mm in diameter and 30 m in length). The oven temperature programmed was initially 1500C (4 min) and then 2500C (4 min). The sample was introduced at 2500 C.
Bioassay with active principles
Bioassay experiments of active principles were conducted with active principles. Different concentrations of bioactive principles (5, 10 and 15 ppm) were applied on 3rd instar larvae of Cx. vishnui mosquitoes for the bioassay experiment according to standard protocol mentioned earlier.
Synthesis of silver nano particles
Ten gram of air dried and crushed leaves of H. integrifolia were weighed and put into three separate 500 ml beakers containing 100 ml double distilled water. Each beaker containing mixture of water and plant leaves was boiled for 10 minutes at 600C temperature. Then Whatman filter paper No: 42 were used for filtration of three separate extracts. The filtrates were treated with silver nitrate (AgNO3) solution for the reduction of Ag+ to Ag0.The strength of used silver nitrate aqueous solution was 10-3M AgNO3. The mixture was exposed to heat at 600C temperature and the colour changes take places within few minutes from colourless to reddish brown colour. The final nano- colloidal solution was centrifuged (twice) at 10,000 rpm for 15 min to isolate the pellet of synthesised nanoparticles from leaves of H. integrifolia for further use. After collecting the pellet from centrifuge tube was dried in vacuum desiccators for preparation of different concentrations of aqueous solution of nanoparticle for bio assay experiments.
Characterization of silver nano particles
Characterization of silver nanoparticle was conducted to determine shape and size of nanoparticles. Numbers of techniques were used for this study, including UV-visible spectroscopy, Scanning Electron Microscopy (SEM), Transmission electron microscopy (TEM), Fourior Transmission Infrared Spectroscopy (FTIR) and X-Ray Diffraction (XRD).
The formation of nanoparticles was verified by using UV-VIS due to surface plasmon resonance (SPR) absorption in the UV visible region. The nano particle surface plasmon resonance of the synthesised green nanoparticles in the centrifuged pellets was studied by UV-spectra analysis.
The transmission electron microscopy (TEM) image were obtained using Technai-20 Philips instrument operated at 200 kv and beam current of 104.1μA. Sample for this analysis were prepared by coating the aqueous AgNP on carbon coated copper grids (300 mesh size) by slow evaporation and then allowed to dry in vacuum at 250C for overnight.
Scanning electron microscope (SEM) analysis was employed to characterize the size, shape & morphologies of formed nano particles.
The FTIR study by using a FT IR spectrometer (Perkin Elmer Lx10-8873) with scanning range of 450-40000 cm-1 at the resolution of 4 cm-1 was used to analyse the vibration characteristics of chemical functional groups of the nano particles.
For the study of crystal structure, texture or orientation, X-ray-diffraction (XRD) studies were conducted using Siemens X-ray diffractometer (Japan), operated at 30 kv and 20 mA current with Cu Kα (I=1.54Å). Films of colloidal form AgNP were tested by drop coating on Si (III) substrates and data were recorded. The scanning range was selected between 10O and 80 O.
Bioassay of green silver nano particles
In case of bioassay of green nano particles synthesised from leaves of H. integrifolia, following concentrations were used i.e. 1.25, 2.5, 5, 7.5 and 10 ppm against all larval instars of Cx. vishnui.
Later those concentrations of nano particles were transferred to disposal plastic cups separated to carry out the tests, in which twenty five 1st to 4th instars larvae of Cx. vishuni were placed with the help of disposable plastic pipette (WHO/VBC, 2005)7 and similar type of bio assay were conducted. The dead larvae were counted after 24 hour of exposure and percentage mortality was reported from the average for the three replicates taken.
Toxicity test on non target organism
Active principle of ethyl acetate extract and silver nano particles of H. integrifolia leaves were tested against those organisms sharing the habitat of Cx. vishnui mosquito i.e. rice field. Some of the non-target organisms are natural predator of Cx. vishnui mosquitoes. It is very much essential to determine the effect of synthesised ethyl acetate and silver nano particles extract in laboratory condition on non target organisms to guess the probable effect that should occur after applying it in field conditions. Experiments were conducted on non-targets like Chironomus circumdatus, Daphnia sp, Diplonychus anulatum and Tadpole larvae.