Comparative Studies on in- vitro Phytochemicals Analysis and Larvicidal Efficacy of Medicinal Plant Extracts against Culex quinquefasciatus

Int. J. Life. Sci. Scienti. Res., 2(6): 742-748 NOVEMBER- 2016
http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 742
Comparative Studies on in- vitro Phytochemicals
Analysis and Larvicidal Efficacy of Medicinal
Plant Extracts against Culex quinquefasciatus
S. Narendiran1
*, Janani. D2
, Keerthana. M2
, Nivethitha K. S2
, Nirmala Devi. S2
, Padmavathy. S2
, Supraja. T. S2
,
Sayeedur Rahman. H2
, Velvizhi. R2
, Swathi. N3
, Yasaswini. K. G3
1
Department of Biotechnology, Sree Sastha Institute of Engineering and Technology, Chembarambakkam, Chennai, India
2
Department of Biotechnology, VIT University, Vellore, India
3
Department of Biotechnology, Sathyabama University, Chennai, India
*
Address for Correspondence: S. Narendhiran, Research analyst, Food and Technology, Innovative Health Care India
Pvt Ltd, Chennai, India
Received: 17 September 2016/Revised: 04 October 2016/Accepted: 22 October 2016
ABSTRACT- Mosquitoes transmit human diseases, causing millions and millions of deaths every year Mosquito borne
diseases are one of the most serious public health problems in the developing countries. It can be controlled by using
repellent, causing larval mortality and the development of resistance to chemical insecticides resulting in rebounding
vectorial capacity. Plants may be alternative sources of mosquito control agents. Medicinal plants extracts of Vitex
negundo, Azadirachta indica and Eucalyptus tereticornis were tested for their larvicidal activity against Culex
quinquefasciatus. There are four different solvents were used (Petroleum-ether, Ethanol, Acetone and Hexane extract) for
the preparation of crude extracts from the plant leaves. The larval mortality of second and third instar larvae C.
quinquefasciatus after 24 hour to 48 hour of treatment were observed separately in control,100, 200,300,400 and 500
ppm concentrations of the leaf extract. The seven different solvent extract of Vitex negundo showed good larvicidal
activity.
Key-words- Larvicidal, Medicinal plant extracts, Phytochemicals Analysis, Culex quinquefasciatus
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INTRODUCTION
The mosquito is the principal vector of many of the vector
borne diseases affecting human beings and other animals.
Several mosquito species belonging to genera Anopheles,
Culex and Aedes are vectors for the pathogens of various
diseases like malaria, filariasis, Japanese encephalitis,
dengue fever, dengue hemorrhagic fever and yellow fever.
Repeated use of synthetic insecticides for mosquito control
has disrupted natural biological control systems and led to
resurgences in mosquito populations. It has also resulted in
the development of resistance, undesirable effects on
non-target organisms and fostered environmental and
human health concern, which initiated a search for alternat-
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DOI: 10.21276/ijlssr.2016.2.6.15
ive control measures. Plants are considered as a rich source
of bioactive chemicals and they may be an alternative
source of mosquito control agents One of the approaches
for control of these mosquito borne diseases is the
interruption of disease transmission by either killing,
preventing mosquitoes to bite human beings (by using
repellents) or by causing larval mortality in a large scale at
the breeding centers of the vectors. This study is concerned
with the using of such effective plant source against the
larval of Mosquito. Blood-feeding female mosquitoes are
responsible for the intolerable biting nuisance and
transmission of a large number of diseases such as malaria,
yellow fever, dengue, filariasis, chiken-gunya, and
encephalitis. They cause serious health problems to humans
and present obstacles to the socioeconomic development of
developing countries, particularly in the tropical region [1].
In addition to mortality, vector borne diseases cause
morbidity of millions of persons resulting in loss of
man-days and causing economic loss [2].
There are 300 species of mosquitoes belonging to 41
genera; all contained in the family Culicidae.
Research Article (Open access)

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 6
Aedes aegypti, a vector of dengue is now endemic and found to be widely distributed in the tropic and subtropics.
Synthetic chemical larvicides are parts of the world. But many of these chemicals are toxic to human, plant and animal
life and insecticides resistance can be problematic in maintaining control, especially with organophosphate and pyrethroid
larvicides [3]. Therefore, a more efficient approach to reduce the population of mosquitoes would be to target the larvae.
Mosquitoes are ecologically important components of the aquatic and terrestrial food chain, then they are the most
important group of insects in terms of public health importance, and thus, appropriate control programs are justified. Until
a few years ago, only the adults were sprayed, but now, it is well known that a more efficient way to reduce mosquito
populations is to target the larvae [3-4].
MATERIALS AND METHODS
The list of plant leaves included in this study was
1. Vitex negundo (Nochi)
2. Azadirachta indica (neem)
3. Eucalyptus tereticornis (Thailam)
Fig 1: Medicinal plants
COLLECTION OF MOSQUITO LARVAES
The mosquito larvae (Culex quinquefasciatus) were
collected from the Chembarambakkam Lake, situated near
Chennai-Bangalore highway Tiruvallur district of Tamil
Nadu, India. The larvae were collected in a container and
transferred to the laboratory immediately. From these
larvae, unwanted large size larvae and pupae were collected
and discarded from the remaining medium sized larvae
second and third instar larvae alone were collected for the
larvicidal bioassay. Feed is supplied to the mosquito larvae
for its growth.
Preparation of Medicinal Plant Extracts
10 grams of each plant leaves powder was taken separately
and 100ml of solvents (Ethanol, Petroleum ether, Hexane
and Acetone extract)was added and kept overnight in the
shaker. The extract is filtered again the solvent is added and
kept at room temperature in the shaker for 7 hours. In this
way the plant extract is collected and stored in the air tight
container for future use.
Test for Carbohydrates
1ml of different crude extract was dissolved in 10ml of
distilled water and filtered. The filtrate underwent Molish’s
test to confirm the presence of carbohydrates [5].
Molish’s Test
The filtrate was treated with 2ml of concentrated sulphuric
acid along the sides of the test tube. Appearance of violet
colour ring at the junction of the two liquid shows the
presence of carbohydrates.
Test of Glycosides
A small portion of different crude extract was hydrolyzed
with hydrochloric acid for few hours on water bath and the
hydrolysate was collected [5].
Fehling’s Test
2ml of extract was taken in a test tube. 1ml of Fehling’s A
solution and 1ml of Fehling’s B solution was added to the
extract, mixed well and boiled. Appearance of yellow or
red colour precipitate indicates the presence of glycosides
(reducing sugar).

Test for Proteins
A small portion of crude was dissolved in few ml of
distilled water and it was subjected to Xantho protein test to
confirm the presence of protein [5].
Xantho Test
Take 3ml of extract to which 1ml of concentrated nitric
acid was added. A white precipitate was obtained. The
solution was heated for 1 minute and cooled under the tap
water. 40% of NaOH was added to the solution.
Appearance of orange colour indicates the presence of
protein.
Test for Phenolic compounds and Tannins
A small portion of crude extract was dissolved in few ml of
distilled water and subjected to FeCl3 test [5].
FeCl3 Test
Few ml of extract 5% FeCl3 was added. Appearance of
violet colour indicates the presence of phenolic compounds
and tannins.
Test for Flavonoids
The crude extract was treated with concentrated sulphuric
acid. Appearance of yellowish orange colour indicates the
presence of anthocyanin, on further adding yellow turns to
orange which indicates the presence of flavones, on further
adding turns to crimson which indicates the presence of
flavonones [5].
Test for Terpenoids
2ml of crude extract was dissolved in 2ml of chloroform to
which 2ml of sulphuric acid was added and the heated for 2
minutes. Appearance of grayish colour indicates the
presence of terpenoids.
Test for Phlobatannins
2ml of extract was taken to which 2ml of 1% HCl was
added and boiled. Formation of red precipitate indicates the
presence of phlobatannins [5].
LARVICIDAL BIOASSAY
Initially 12.5 mg crude extract of petroleum ether solvent of
each solvent extract was taken and dissolved in 1ml of
acetone in an eppendorf. Then the dissolved crude extract
was mixed in container containing 50 larvae in 100ml of
water. Every 24 hours the mortality rate was noted and
reading was taken. The dead larvae were taken out at every
24 hours since it may leads to contamination of the water.
The readings were taken for 2 days (48hours) then 0.25%
(250ppm) concentration from each plant crude extract was
introduced into containers containing larvae. Similarly for
this reading wastaken for every 24 hours for 2 days. Then
0.50% concentration (500ppm) of crude extract was
introduced and reading was noted for every 24hrs for two
days [6].
Qualitative Analysis of Phytochemicals
The phyto constituents detected in the plant extract could
be responsible for their antioxidant and antimicrobial
activity.
Table 1: Qualitative analysis of phytochemicals in
various extracts of Vitex negundo
Phytochemicals/
Extracts
Ethanol
extract
Petroleum
ether
extract
Hexane
extract
Acetone
Extract
Alkaloids - + - +
Steroids + + + -
Triterpenoids + - - +
Flavanoids + + + +
Tannins - - - +
Phenols + + - -
Glycosides + - + +
Saponins - - - -
Phlobotannins + + + -
Anthraquinones + + - +
(+): Presence of phytochemicals
(-): Absence of phytochemicals
Table 2: Qualitative analysis of phytochemicals in
various extracts of Azadirachta indica
Phytochemicals/
Extracts
Ethanol
extract
Petroleum
ether
extract
Hexane
extract
Acetone
Extract
Alkaloids - + - +
Steroids + + + -
Triterpenoids + - - +
Flavanoids + + - +
Tannins + - + -
Phenols - + - -
Glycosides + - + +
Saponins - - + -
Anthraquinones + + - +

Table 3: Qualitative analysis of phytochemicals in various extracts of Eucalyptus tereticornis
Phytochemicals/Extracts Ethanol extract Petroleum ether
extract
Hexane extract Acetone Extract
Alkaloids - + - +
Steroids + + + -
Triterpenoids + + - -
Flavanoids + + + +
Tannins - - - +
Phenols + + - +
Glycosides + + + +
Saponins - + - +
Anthraquinones + - - +
RESULT AND DISCUSSION
Fig 2: Larvae mortality rate on Vitex negundo plant

Fig 3: Larvae mortality rate on Eucalyptus tereticornis plant
Fig 4: Larvae mortality rate on Azadirachta indica plant
Larvicidal activities of seven medicinal plant extracts were
studied with the control, 100, 200, 300, 400 and 500 ppm
concentrations. The present study assessed the role of
larvicidal activities of Medicinal plants extracts of Vitex
negundo and Azadirachta indica and Eucalyptus
tereticornis by Solvents Petroleum ether, Hexane, Acetone
and Ethanol against the larvae of malaria vector Culex
quinquefasciatus. The activity of crude extracts of the
medicinal plant is noted. The seven different solvents were
tested against Culex quinquefasciatus and larval mortality
percent was observed .through statistical analysis it was
analyzed that all plant extracts showed moderate toxic
effect on larval. However, the highest mortality rate was
found by the Eucalyptus tereticornis plant extracts.
CONCLUSION
An insecticide does will not cause high mortality on target
larvae but may serve medicinal plants extracts as suitable
alternatives to synthetic insecticides plant extract are more
safer, inexpensive, reduce dependence on imported
products (synthetic) and are readily available in many

region of the world. The findings of the present
investigation revealed that Eucalyptus tereticornis has good
larvicidal activity against Culex quinquefasciatus and
Azadirachta indica showed poor mortality against the
Culex quinquefasciatus larvae. These crude extracts can be
effectively used in the control of mosquitoes by replacing
the chemical pesticides which cause environmental
pollutions and other burdens. Statistically Vitex negundo
had high larvicidal efficacy. These Medicinal plants
extracts are the best alternate product against the control of
mosquito’s vector. It can be replace the chemical pesticides
which cause environmental pollutions and other health
problems.
REFERENCES
[1] Senthilkumar A, Kannathasan K, Venkatesalu V. Chemical
constituents and larvicidal property of the essential oil of
Blumeamollis (D. Don) Merr against Culex quinquefasciatus.
Parasitol Res 2008; 103: 959-962.
[2] Dhiman RC, Pahw S, Dhillon GP, Dash AP. Climate change
and threat of vector-borne diseases in India: are we
prepared? Parasitol Res 2010; 106(4): 763-773.
[3] Rahuman AA, Bagavan A, Kamaraj C, Saravanan E, Zahir
AA, Elango G. Efficacy of larvicidal botanical extracts
against Culex quinquefasciatus Say (Diptera: Culicidae).
Parasitol Res 2009; 104(6): 1365-1372.
[4] Govindarajan M, Jebanesan A, Pushpanathan T. Larvicidal
and ovicidal activity of Cassia fistula Linn. Leaf extract
against filarial and malarial vector mosquitoes. Parasitol Res
2008; 102(2): 289-292.
[5] Arun J, Pennarasi, Prasanna. M, SundaraMahalingam,
Narendhiran, Alagesan, Esaipriyan. Screening of
Phytochemical Constituents towards Antimicrobial Potency
of Hygrophilaschulli. A Journal of Biotechnology.
2231-3826, Special Issue 2013
[6] Maheswaran, R., Sathish. S and Ignacimuthu. S. 2008.
Larvicidal activity of Leucasaspera against the larvae of
Culex quinquefasciatus and Aedes aegypti. International
Journal of Integrative Biology, 2(3):214-217.
[7] Su T, Mulla MS. Ovicidal activity of neem products
(azadirachtin) against Culex tarsalis and Culex
quinquefasciatus (Diptera: Culicidae). J Am Mosq Control
Assoc 1998; 14:204-9.
[8] Sukumar K, Perich MJ, Boobar LR. Botanical derivatives in
mosquito control: a review. J Am Mosq Control Assoc
1991;7 : 210-37.
[9] Dhar R, Dawar H, Garg S,Basir SF, Talwar GP. Effect of
volatiles from Neem and other natural products on
gonotrophic cycle and oviposition of Anopheles stephensi
and An. culicifacies (Diptera: Culicidae). J Med Entomol
1996; 33:195-201.
[10]Su T, Mulla MS. Effects of Neem products containing
azadirachtin on blood feeding, fecundity, and survivorship of
Culex tarsalis and Culex quinquefasciatus (Diptera:
Culicidae). J Vector Ecol 1999; 24:202-15.
[11]Lucantoni L, Giusti F, Cristofaro M, Pasqualini L, Esposito
F, Lupetti P, et al. Effects ofneem extract on blood feeding,
oviposition and oocyte ultrastructure in Anopheles stephensi
Liston (Diptera: Culicidae). Tissue Cell 2006; 38: 361-71.
[12]Mulla MS, Su T. Activity and biological effects of
Neemproducts against arthropods of medical and veterinary
importance. J Am Mosq Control Assoc 1999; 15: 133-52.
[13]Boschitz C, Grunewald J. The effect of NeemAzal on
Aedesaegypti (Diptera: Culicidae).ApplParasitol 1994; 35:
251-6.
[14]Nathan SS, Kalaivani K, Murugan K. Effects of
Neemlimonoids on the malaria vector Anopheles stephensi
Liston (Diptera: Culicidae). Acta Trop 2005; 96: 47-55.
[15]World Health Organization. Instruction for determining
thesusceptibility or resistance of mosquito larvae to
insecticides.Geneva: WHO/VBC/81.807; 1981.
[16]Singh KRP, Patterson RS, LaBrecque GC, Razdan RK. Mass
rearing of Culex pipiensfatigans Wied. J Commun Dis
1975;7 : 31-53.
[17]Brown MD, Thomas D, Watson K, Kay BH. Laboratory and
field evaluation of efficacy of vectobac 12AS against Culex
sitiens (Diptera: Culicidae) larvae. J Am Mosq. Control
Assoc1998; 1: 183-5.
[18]Abbott WS. A method of computing the effectiveness of an
insecticide. J Econ Entomol 1925; 18: 265-7.
[19]Finney DJ. Probit analysis, 3rd ed. Cambridge: Cambridge
University Press; 1971.
[20]Sun C, Georghiou GP, Weiss K. Toxicity of Bacillus
thuringiensis var israelensis to mosquito larvae variously
resistant to conventional insecticides. Mosq News 1980;
40:614-8.
[21]Aly C, Mulla MS, Xu B, Schnetter W. Rate of ingestion by
mosquito larvae (Diptera: Culicidae) as a factor in the
effectiveness of a bacterial stomach toxin. J Med Entomol
1988; 25: 191-6.
[22]Aly C. Filtration rates of mosquito larvae in suspensions of
latex microspheres and yeast cells. Entomol Exp Appl 1988;
46: 55-61.
[23]Azmi MA, Naqvi SNH, Ahmad I, Tabassum R, Anbreen B.
Toxicity of Neem leaves extracts (NLX) “compare with
Malathion” (57 E.C.) against late 3rd instar larvae of Culex
fatigans (Wild Strain) by WHO method. Trop J Zool 1998;
22:213-8.
[24]Zebitz CPW. Potential of Neem seed kernel extracts in
mosquito control. Proceedings of the third International
Neem Conference (1986 Nairobi, Kenya) 1987. p. 555-73.
[25]El-Shazly MM, El-Sharnoubi ED. Toxicity of a Neem
(Azadirachta indica) insecticide to certain aquatic organisms.
J Egypt Soc Parasitol 2 000; 30: 221-31.
[26]Caboni P, Cabras M, Angioni A, Russo M, Cabras P.
Persistence of azadirachtin residues on olives after field
treatment. J Agric Food Chem 2002; 50: 3491-4.
[27]Caboni P, Sarais G, Angioni A, Garcia AJ, Lai F, Dedola F,
et al. Residues and persistence of Neem formulations on
strawberry after field treatment. J Agric Food Chem 2006;
54: 10026-32.
[28]Govindarajan M, Jebanesan A, Pushpanathan T, Samidurai
K. Studies on effect of Acalypha indica L. (Euphorbiaceae)
leaf extracts on the malarial vector, Anopheles stephensi
Liston (Diptera: Culicidae). Parasitol Res 2008; 103(3): 691-
695.

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How to cite this article:
Narendiran S, Janani D, Keerthana M, Nivethitha KS, Nirmala Devi S, Padmavathy S, Supraja TS, Sayeedur Rahman H, Velvizhi
R, Swathi N, Yasaswini KG: Comparative Studies on in-vitro Phytochemicals Analysis and Larvicidal Efficacy of
Medicinal Plant Extracts against Culex quinquefasciatus. Int. J. Life. Sci. Scienti. Res., 2016; 2(6): 742-748.
DOI:10.21276/ijlssr.2016.2.6.15
Source of Financial Support: Nil, Conflict of interest: Nil

Comparative Studies on in- vitro Phytochemicals Analysis and Larvicidal Efficacy of Medicinal Plant Extracts against Culex quinquefasciatus

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