Journal of Mosquito Research 2015, Vol.5, No.10, 1-7
6
3.5 Larvicidal bioassay
Larvicidal bioassay were done according to the
protocal of WHO with slight modification (WHO,
2005). 30 larvae were put in plastic bowls containing
100 ml of test solutions of different concentrations of
crude extract (0.01- 0.05%) and different concentrations
of solvent extracts (1, 2, 4, and 8 ppm). Negative
c
ontrol experiments were set on 100 ml of tap water
only and ethanol treated control experiments were set
on 100 ml of tap water with 0.5 ml of ethanol (positive
control). Each set of experiment was replicated three
fold with three replicates of controls at laboratory
condition on separate three days. The percent
mortality was recorded after 24, 48 and 72 h of post
exposure cumulatively. Larvae were identified dead
when they could not move after touching the siphon or
cervical area with a fine brush.
3.6 Effect on Non Target Organisms
Non target organisms are animals which live in the
same place where larvae of mosquito grow. The effect
of ethyl acetate solvent extracts at LC
50
value of 3
rd
instar larvae of
Cx. quinquefasciatus
after 24 h of
exposure were observed upto 72 h on
Diplonychus
annulatum
,
Chironomus circumdatus
larvae
(insect)
and tadpole larvae of toad.
3.7 Qualitative Phytochemical analyses of Root
Extract of
A. reticulata
Aqueous and ethanolic root extracts (charcoal filtered)
of
A. reticulata
were tested for qualitative phytochemical
analyses following the standard protocal (Trease and
Evans, 1989) with slight modification.
3.7.1 Detection of Tannin and Phenolic Compounds
2 ml of aqueous extract were taken in a clean test tube
and was added 0.5 ml of ferric chloride solution. The
colour of solution change into blue green indicate the
presence of tannin and phenolic compounds.
3.7.2 Detection of Flavanoids
2 ml of aqueous extract was taken in a test tube and
was added few drops of NaOH solution. Intence
colour formation occur which become colourless on
addition of dilute HCL and indication of the presence
of flavanoids.
3.7.3 Detection of Alkaloids
2 ml of ethanolic extract were taken in a clean test
tube and added few drops of 2N HCL. Then mixed 1
or 2 drops of Mayer’s reagent [1.36 g of mercuric
chloride dissolve in 60 ml of H
2
O and then this
solution pour in potasium iodide solution (5 g potasium
iodide dissolve in 100 ml of water)]. Appearence
cream or pale yellow colour precipitation indicate the
presence of alkaloids.
3.7 4 Detection of Terpenoids (Salkowski test)
2 ml of ethanolic extract was taken in a test tube
and then added 2 ml of chloroform and 3 ml of
concentrated H
2
SO
4
carefully by the interior wall
of the test tube. A reddish brown colouration of the
interface was formed which indicate the presence
of terpenoids.
3.7.5 Detection of Steroids
2 ml of ethanolic extract was taken in a test tube and
then 5 ml of chloroform and 5 ml concentrated H
2
SO
4
was added carefully by the interior wall of the test
tube. The upper layer turns red and H
2
SO
4
layer
showed yellow with green fluorescence. This indicate
the presence of steroids.
3.7.6 Detection of Anthraquinones
2 ml of aqueous extract was added to 2 ml of 2N HCL
and NH
3
in a clean test tube
.
The appearence of pink
red was turned blue violet indicating the presence of
anthrocyanines.
3.7.7 Test of Saponins (Frothing test)
10 ml of aqueous extract were taken in a test tube and
was shaking vigorously. Persistence of frothing that is
the indication of the presence of saponines in the
sample.
3.8 Statistical analyses
MS EXCELL 2007 and the computer software STAT
PLUS 2009 (trial version) were used to calculate the
mean mortality percent, standard error, LC
50,
LC
90,
regression equation (Y= mortality, X= concentration),
coefficient of determination (R
2
) and ANOVA.
Acknowledgement
Authors are thankfull to Eminent Professor, Dr. A Mukhopadhyay,
Department of Botany, The University of Burdwan, Burdwan,
West Bengal, India for identification of the plant species. We
are also grateful to UGC DRS for providing financial supports.
References
Bernhard L., Bernhard P., and Magnussen P., 2003, Management of patient
with lymphoedema caused by filariasis in north- easternTanzania:
alternative approaches, Physiotherapy, 89: 743-749
