The present experimental study was performed as follows:
Preparation of Plant extract by percolation method
The leaves of Duranta repens were collected from Serampore College campus. Leaves were air dried in shaded condition at room temperature for 7 days and after that powdered with a blender. The powder was preserved in glass jars for successive solution preparation. 3gm of powder was added in 50 ml of ethanol in brown flask bottle and kept for 3 days and shaken the bottle once in each of the 3 days at room temperature. At the 3rd day the mixture was filtered through whatman no. 1 filter paper. Then the yield % and the concentration of the stock solution were measured. The remaining stock solution was refrigerated at 4°C until the subsequent larvicidal bioassay. In present study the calculation of yield % is as follows-
Yield % = total amount of plant material before percolation - total amount of plant material after percolation × 100 / total amount of plant material before percolation.
Larvicidal efficacy test of plant extract on Culex larvae
Larvae of Culex mosquito were collected from surrounding areas of Serampore College (22045/ N, 88023/ E) Hooghly, in early morning with small glass jars and transferred to the laboratory of Department of Zoology, Serampore College. Larvae were placed in a tray containing water. Afterwards 50 ml of working solution was prepared from stock solution by following formula
V1S1 = V2S2
Larvicidal activity i.e % of larval mortality was calculated according to guidelines of WHO (2005) [14]. 25 larvae were released with the help of dropper into each tray. A control (ethanol only) tray was also included. Larvae were considered dead if they exhibited no sign of movement even after being touched with a glass rod. The % of mortality was recorded after 24 hrs of larvicidal exposure.
The % of larval mortality was corrected using Abbotts formula [15].
Corrected mortality % = % mortality introduced - % mortality in control X 100
100 - % mortality in control
Preparation of algae mixture
- At first collected algae were cleaned in water and filtered. 180 gm of the algae was taken in a mixer grinder with added water and beaten to make a bolus.
- Then algae bolus taken in a pan and boiled for 30 min on an oven. During this time period water was added for maintaining the liquid phase.
- 10gm of rice husk was added in pan which provided cementing property to the mixture.
- 10 gm of saw dust was added with the boiling mixture to provide thickness property to the mixture.
In this mixture formation the ratio were taken is 90:5:5 i.e in a 50 gm algae mixture there is 40 gm algae , 5 gm rice husk and 5 gm saw dust.
Development of an anti mosquito fast card
- Algae mixture was cooled, after cooling; ethanol extract of Duranta leaf was added in the mixture. The mixture was then stirred for proper mixing and kept for 30 minutes. In the present study 50:50 ratio was taken i.e 50 gm of algae mixture was added with 50 ml of ethanolic plant extract.
- After that mixture was taken in Deckle and mould tray and excess water was drained. Then the algal mould was pressed in a hydraulic pressing machine with temperature controller to provide the proper shape.
- After that the final prepared fast card was subjected to subsequent bioassays.
Test of efficacy of prepared fast card:
Bioassay was carried out in laboratory on adult Culex pipiens. 20 mosquitoes were released in two big bowls like jars by an aspirator. 1 X 1 cm2 diameter Duranta ethanolic algae card were burnt and suspended inside the first jar and 1x1 cm2 diameter commercial fast card was burnt in second jar and a thermocol piece was placed on each of the opening mouth of the jar. Cards were burnt slowly and smoke was released inside the jar. The process was allowed for 20 minutes, and then cards were taken out. Cotton mass soaked with sugar solution was placed inside the jar and the opening was covered by a small net and allowed for next 24 hr. After 2 hrs, cotton mass was removed and behavior of mosquitoes was recorded in a note book.
Evaluation of emitted gases by toxic indicator:
Emitted gases from Duranta fast card and commercially available fast card were identified and evaluated by toxic indicator of Model PPMTM gaZguard Tx. [Fig. 1]
For investigating anti mosquito repellent fast card activity, the prepared card was checked for its flammability, burning efficiency with respect to burning time and eventually its effective repellent activity.
From the produced smoke, emission of CO2, CO, and O3 were observed and recorded.
Characterization of plant materials:
GC-MS study
For GC-MS study the viscous dark mass of yielded plant product was dissolved in little quantity of chloroform and reduced to the volume of 2ml. The dissolved 5 µl sample diluted to 50 µl and has been filtered with the help of High-Performance Liquid Chromatography technique and the component analysis has been done by GC-MS (GC) using TR-WAX MS column (30mm×0.25mm ID ×0.25μM df, composed of PEG binding material, Polar column). Helium (99.99%) was used as carrier gas. An injection volume of one μl was used (split ratio of 10:1). The oven temperature was set from 60 °C (isothermal for two minutes), with a rise of 10°C per min, to 110°C, then 5°C per min to 260°C, ending with a 10 min isothermal condition at 260°C. Mass spectra have to be taken at 70 eV. Forty-nine minutes is the total running time for the study. Identification of the components was performed with the help of the datasheet of National Institute of Standards and Technology.
HPLC analysis:
Leaf extract of Duranta was further analyzed by a ‘Hitachi Chromaster quarternary gradient reverse phase HPLC system’ with 5160 pump, 5420 UV visible detecter for confirmation of the presence of Propionic acid. The extracts were filtered with syringe filter of 0.22micron (High Media). The mobile phase for this experiment was prepared by mixing of 50% HPLC grade Acetonitrile (Merck) and 50% HPLC grade water (Merck) followed by filtering the mixture with solvent filtration system (Borosil). The other parameters of both the pump and detectors are as below:
- Flow rate: 1ml/minute
- Run time: 20 minutes
- Absorbance range: 2 AU
- Wave length: 210 nm
Observation and result:
The present study on the plant extract has revealed the efficacy of Duranta repens as a potential mosquito larvicide. Table 1 depicts the impact of ethanolic extract of this plant on the mortality of third and fourth instar larvae of Culex sp after 24 hrs of larvicidal exposure. Ethanolic plant leaf extract at a concentration of 1000 ppm and 500 ppm shows 100% mortality of the studied mosquito larvae (Table1).
The present investigation also unfolds that the exposure of mosquitoes in Duranta extract- algae mixture card shows 100% knock down in first 20 minutes in comparison of 95% knock down by commercially available fast card. After 1 hour of exposure, the mosquitoes are treated with 10% of sugar solution on cotton wool and kept for 24 hrs at 27+ - 2 degree C and 80+-10% relative humidity. 70 % mosquitoes became died and 30 % showed knockdown. Contrarily commercially available fast card was responsible for 25 % mortality, 65% knockdown (Table 2) and the rest 10 % mosquitoes were active or in flying condition after this treatment with commercially available fast card.
During the burning of prepared card amount of some toxic gaseous products was considered. Result revealed that commercially available fast card discharged 2.20 ppm CO2, 1.72 ppm CO and 1.73 ppm O3. On the flip side, released amount of gases from the prepared organic fast card were 0.79 ppm, 1 ppm and 0.48 ppm respectively.
From the GC-MS analysis of the leaf extract the presence of 21 phytochemical constituents were revealed which are responsible for the larvicidal quality of the Duranta. The Propionic acid was the major bio active component of Duranta plant extract as it showed the highest area peak (49.70%) HPLC analysis of Duranta was performed. The crude extract and fractions from Duranta plants was investigated for the presence of Propionic acid (Fig 3).
Figure 3 also represented that the component which appeared in the peak number five and investigated at 3.260 minute showed high similarity with the standard compound at 3.487 minute that confirmed the presence of Propionic acid in the Duranta leaf extract. Further study of the larvicidal bioassay of the pure Propionic acid in ethanol solvent showed that this compound is highly effective as they caused 100%mortality of the fourth instar larvae in comparison to 13 % mortality caused by the solvent alone.