Bincy Mol P.K., Shonima Govindan M. and Kunhi A.A.M. (2017) Phytochemical Analysis and Evaluation of Larvicidal Property of Leaf Extracts of Pogostemon quadrifolius against Culex quinquefasciatus. IOSR Journal of Biotechnology and Biochemistry, 3(6): 39-44. (ISSN: 2455-264X). DOI: 10.9790/264X-03063944

IOSR Journal of Biotechnology and Biochemistry (IOSR-JBB) ISSN: 2455-264X, Volume 3, Issue 6 (Nov.- Dec. 2017), PP 39-44 www.iosrjournals.org DOI: 10.9790/264X-03063944 www.iosrjournals.org 39 | Page 

Phytochemical Analysis and Evaluation of Larvicidal Property of Leaf Extracts of Pogostemonquadrifolius against Culex quinquefasciatus Bincy Mol P.K, ShonimaGovindan M* and A.A.M KunhiSIAS-Centre for Scientific Research (CSR), Safi Institute of Advanced Study Vazhayoor, Kerala, India

*Corresponding Author: shonima.g@gmail.com Abstract: The larvicidal potential of different solvent (Petroleum ether, chloroform, acetone, methanol, aqueous) extracts ofPogostemon quadrifoliusdried leaveswas tested against the 4th instar larvae of Culex quinquifasciatus.Larvicidal bioassaywas carried out using WHO standard method and the mortality was observed after 24 h exposure. The mortality data were subjected to probit analysis to determine the lethal concentration (LC50). The maximum larval mortality was detected in petroleum ether extract (LC50 0.112mg/ml) followed by acetone extract (LC50 0.0234 mg/ml). The phytochemical screening of the potent crude extracts revealed the presence of aikaloids, flavanoids, saponins and terpenoids. The petroleum ether extract was purified by column chromatography and the mobile phase used was designed from several experimental determination of TLC.All the recovered fractions were screened for larvicidal activity and the sixth fraction was found to be active. The active fraction was screened for bioactive phytochemical class by standard qualitative analysis and the presence of terpenoid was identified. This study provides first report on the mosquito larvicidal activity of the P.quadrifolius leaf extract against larvae of Cx. quinquefasciatus. Keywords: Culexquinquefasciatus, Pogostemonquadrifolius, Phytochemicals, Larvicidalactivity. ————————————————————————————————————————————— Date of Submission: 06-12-2017 Date of acceptance: 18-12-2017 ————————————————————————————————————————————— 

I. Introduction 

Over a third of the world population lives in areas at risk for epidemic transmission of mosquito-transmitted diseases like yellow fever, dengue hemorrhagic fever, and the emerging disease chikungunya. Mosquitoes are known to carry many infectious diseases from several different classes of microorganisms, including viruses and parasites(Nouret al., 2009). Repeated use of synthetic insecticides for mosquito control has disrupted naturalbiological control systems and led to resurgences in mosquitopopulations. It also resulted in the development of resistance,undesirable effects on non-target organisms and fosteredenvironmental and human health concern(Thomaset al., 2004).The WHO has estimated that there are 25 million cases of acute occupational pesticide poisoning in developing countries and that 20,000 deaths occur worldwide every year. One of the most effective alternative approaches under the biological control programme is to explore the land floral biodiversity and enter the field of using safer insecticides of botanical origin as a simple and sustainable method of mosquito control. The search for herbal preparations that do not produce any adverse effects in the non-target organisms and are easily biodegradable remains a top research issue for scientists associated with alternative vector control (Chowdhury et al., 2008).Many plant species are known to possess biological activity that is frequently assigned to the secondary metabolites. Several studies have identified and reported that plant extracts are effective against mosquitoes at various stages of development. The phytochemicals derived from plant sources possess a complex of chemicals with unique biological activity. The phytochemicals derived from plant resources can act as larvicides, insect growth regulators, repellents, and ovipositional attractants (Rawaniet al., 2010).Larval control can be an effective tool rather than mosquito repellent devices, which are now widely practiced. Due to the low mobility of larvae especially where the principal breeding habitats are manmade, larval control is very effective. 

The Lamiaceaeis one of the most diverse and widespread plant families in terms of ethnomedicine and its medicinal value is based on the volatile oils concentration (Sarac et al,2007).Some of the plants of this family are used as insect repellents because of their aromatic property. Some examples from this family include Pogostemon, Anisomeles, Colebrookea, Coleus, Hyptis, Leonotis, Leucas, Mentha, Ocimum, Oreganum and Salvia. With an ever increasing public interest and awareness about the environment in both developed and developing countries, positive public perception of natural insecticides is an added incentive for their Phytochemical Analysis and Evaluation of Larvicidal Property of Leaf Extracts of .. DOI: 10.9790/264X-03063944 www.iosrjournals.org 40 | Page 

development and use.In the present study the larvicidal activity of Pogostemon quadrifoliusplant extracts was investigated against the fourthinstar larvae of Cx.quinquefasciatus. 

II. Materials and methods 

Plant material Healthy leaves of Pogostemonquadrifolius was collected from in and around the campus of SAFI Institute of Advanced Study, Vazhayoor, Malappuram and the taxonomic identification was made byDr. Rajesh, Associate Professor, Zamorins, Guruvayurappan College, Calicut, Kerala. The leaves were washed, cleaned, air-dried at room temperature for two weeks and coarsely powdered. Test organism Mosquito larvae and eggs were collected regularly in the month of January- March. The collected larvae were transferred to clean plastic bags filled with water from the same breeding place and brought to the laboratory. The larvae hatched were identified, and recorded (Oguoma et al., 2010). Maintenance of mosquito larvae After hatching of the egg, the first instar larvae of Culex quinquefaciatus were transferred to an enamel tray of 30×25×5 cm3 containing well water. The larvae were fed on a diet of finely powdered biscuits and yeast in the ratio 3:1.The water in the tray was changed every day and dead larvae were removed (Gerber et al., 1994). Preparation of Plant extracts Leaves of Pogostemon quadrifolius were collected and washed thoroughly with tap water, dried in air and powdered. Extracts of the collected plant materials were taken by a modified method of Minjas and Sarda, 1986; Tandon and Sirohi, 2010. Powdered plant material (40g) was extracted sequentially using 400ml of different solvents of increasing polarity (petroleum ether, acetone, chloroform, methanol and distilled water) in shaking incubator for 12 h and the extracts were filtered through Whatman filter paper No. 4 and then concentrated at 40º C and stored at 4º C until testing for subsequent bioassays. Phytochemical screening Phytochemical screening was carried out using standard procedure (Harborne,1984). Larvicidal Bioassay The larvicidal activity of crude extracts of the selected plants was assessed by the protocol of WHOwith some modifications (Rahumannet al.,2000). For the bioassay 50-250ppm concentrations of the extracts were prepared using 1% of Dimethyl sulfoxide (DMSO) as an emulsifier. Controls were prepared by adding 1% DMSO in water. Twenty, 4th instar larvae of Culex quinquefaciatus were released into 250ml glass beaker containing 100ml water with each concentration independently. All experiments were conducted in six replicates along with control. Observation on mortality of the larvae was recorded after 24h of continuous exposure. After 24 hours exposures the dead larvae were counted and total was expressed as percentage of larval mortality for each concentration. When the Control mortality ranged from 5-20 per cent, the observed percentage mortality was corrected by Abbott’s formula(Abbott WS.,1925). Purification of larvicidal phytochemicals Thin Layer Chromatography of Petroleum Ether extract Mobile phases in Column chromatographic purification of the Petroleum ether extract was designed from several experimental determination of TLC. Chromatographic plate was prepared using silica gel under standard specifications. The mobile phase used for separation of the petroleum ether extract, was toluene: ethyl acetate solvents in 9:1. The plates were dried at room temperature and they were placed in iodine chamber for the development of chromatogram. Column chromatography of Petroleum Ether extract The potent crude extract was subjected to column chromatographic separation [Column length: 25cm, diameter: 2.5cm] using silica gel as stationary phase. 50mg extract mixed with 1.5ml petroleum ether, was applied on the column. Toluene: ethyl acetate was added as mobile phase and eluate was collected at a flow rate of 1ml/min. All fractions were monitored by thin layer chromatography until a single spot was obtained. The pure fractions were evaporated to dryness and were screened for their larvicidal activity. Phytochemical Analysis and Evaluation of Larvicidal Property of Leaf Extracts of .. DOI: 10.9790/264X-03063944 www.iosrjournals.org 41 | Page 

Statistical analysis Data were analyzed using one-way ANOVA. Significant differences between treatments were determined using Tukey’s test (P ≤ 0.01). LC50 values were calculated using probit analysis. 

III. Results