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ORIGINAL ARTICLE
Year : 2011  |  Volume : 134  |  Issue : 1  |  Page : 101-106

Larvicidal activity of medicinal plant extracts against Anopheles subpictus & Culex tritaeniorhynchus


Post Graduate & Research Department of Zoology, C. Abdul Hakeem College, Melvisharam, Vellore District, Tamil Nadu, India

Date of Submission17-Dec-2009
Date of Web Publication29-Jul-2011

Correspondence Address:
A Abdul Rahuman
Unit of Bioactive Natural Products, Post Graduate & Research Department of Zoology, C. Abdul Hakeem College, Melvisharam 632 509, Vellore District, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


PMID: 21808141

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   Abstract 

Background & objectives: Mosquitoes transmit serious human diseases, causing millions of deaths every year and the development of resistance to chemical insecticides resulting in rebounding vectorial capacity. Plants may be alternative sources of mosquito control agents. The present study assessed the role of larvicidal activities of hexane, chloroform, ethyl acetate, acetone, and methanol dried leaf and bark extracts of Annona squamosa L., Chrysanthemum indicum L., and Tridax procumbens L. against the fourth instar larvae of malaria vector, Anopheles subpictus Grassi and Japanese encephalitis vector, Culex tritaeniorhynchus Giles (Diptera: Culicidae).
Methods: Larvicidal activities of three medicinal plant extracts were studied in the range of 4.69 to 1000 mg/l in the laboratory bioassays against early 4 th instar larvae of An. subpictus and Cx. tritaeniorhynchus. The mortality data were subjected to probit analysis to determine the lethal concentrations (LC 50 and LC 90 ) to kill 50 and 90 per cent of the treated larvae of the respective species.
Results: All plant extracts showed moderate effects after 24 h of exposure; however, the highest toxic effect of bark methanol extract of A. squamosa, leaf ethyl acetate extract of C. indicum and leaf acetone extract of T. procumbens against the larvae of An. subpictus (LC 50 = 93.80, 39.98 and 51.57 mg/l) and bark methanol extract of A. squamosa, leaf methanol extract of C. indicum and leaf ethyl acetate extract of T. procumbens against the larvae of Cx. tritaeniorhynchus (LC 50 =104.94, 42.29 and 69.16 mg/l) respectively.
Interpretation & Conclusions: Our data suggest that the bark ethyl acetate and methanol extract of A. squamosa, leaf ethyl acetate and methanol extract of C. indicum, acetone and ethyl acetate extract of T. procumbens have the potential to be used as an ecofriendly approach for the control of the An. subpictus, and Cx. tritaeniorhynchus.

Keywords: Anopheles subpictus - Culex tritaeniorhynchus - medicinal plant extracts - larvicide


How to cite this article:
Kamaraj C, Bagavan A, Elango G, Zahir A A, Rajakumar G, Marimuthu S, Santhoshkumar T, Rahuman A A. Larvicidal activity of medicinal plant extracts against Anopheles subpictus & Culex tritaeniorhynchus. Indian J Med Res 2011;134:101-6

How to cite this URL:
Kamaraj C, Bagavan A, Elango G, Zahir A A, Rajakumar G, Marimuthu S, Santhoshkumar T, Rahuman A A. Larvicidal activity of medicinal plant extracts against Anopheles subpictus & Culex tritaeniorhynchus. Indian J Med Res [serial online] 2011 [cited 2019 Dec 14];134:101-6. Available from: http://www.ijmr.org.in/text.asp?2011/134/1/101/83362

Mosquitoes are the major vector for the transmission of malaria, dengue fever, yellow fever, filariasis, schistosomiasis and Japanese encephalitis (JE) [1] . In India, malaria is one of the most important causes of direct or indirect infant, child and adult mortality with approximately two to three million new cases arising every year. Anopheles subpictus Grassi is distributed throughout India, Afghanistan, Borneo, China, Malaysia, Philippines, Sri Lanka, Java and Indonesia. It is a dominant species in Haryana and Uttaranchal states [2] . Though it is a non-vector species, same infected specimens with malarial parasite have been reported from India, Indonesia and Java [3] . An. culicifacies is the main vector of malaria, and An. subpictus is a significant secondary vector in Sri Lanka [4] . An. subpictus is recognized as the secondary vector of malaria in South East Asia, with a large number of cases being reported from India. 2,400 million (about 40%) of the world's population [5],[6] . India contributes 77 per cent of the total malaria in Southeast Asia [7] . Cx. tritaeniorhynchus is a primary vector of Japanese encephalitis (JE) virus, with a distribution throughout Southeast Asia and South Asia. Keiser et al[8] have reported that global annual incidence and mortality estimates for JE are 30,000 to 50,000 and 10,000 respectively.

Annona squamosa , commonly known as custard apple is a native of West Indies and is cultivated throughout India, mainly for its edible fruit. Das et al[9] reported that the ethanol leaf extract of A. squamosa was found to have the most promising larvicidal activity against Cx. quinquefasciatus larvae. The larvicidal and mosquitocidal activities of ethanolic water mixture extract of A. squamosa and Centella asiaticai were effective against larvae of An. stephensi[10] . The leaves methanolic extract and the seeds petroleum ether extract of A. squamosa showed larvicidal activity when tested against An. stephensi, Cx. quinquefasciatus and Aedes aegypti[11] . Chrysanthemum indicum is a traditional herb used to treat various disorders, hypertension symptoms and several infectious diseases in Korean and Chinese medicine [12] . Beninger et al[13] reported that the leaves of C. morifolium extracted sequentially with hexane, ethyl acetate, and methanol were found to reduce the growth of Trichoplusia ni larvae. Tridax procumbens is commonly used in Indian traditional medicine as anticoagulant, antifungal and insect repellent, in bronchial catarrh, diarrhoea and dysentery [14] . The entire plant is used for the treatment of malaria, leishmaniasis, vaginitis, dysentery and gastrointestinal disorders [15] .

Though larvicides play a vital role in controlling mosquitoes in their breeding sites, these also show a negative impact in areas of beneficial and non-target organisms. In view of an increasing interest in developing plant origin insecticides as an alternative to chemical insecticide, this study was undertaken to assess the larvicidal potential of the extracts from the medicinal plants against two medically important species of malaria vector, An. subpictus and JE vector, Cx. tritaeniorhynchus.


   Material & Methods Top


Collection of plant materials : The bark of Annona squamosa L. (Annonaceae), leaf of Chrysanthemum indicum Linné (Compositae), Tridax procumbens Linn. (Asteraceae) were collected from Chitheri Hills, Dharmapuri District, Tamil Nadu, India in March 2009 and the taxonomic identification was made by Dr. C. Hema, Department of Botany, Arignar Anna Govt. Arts College for Women, Walajapet, Vellore, India. The voucher specimen was numbered and kept in the research laboratory of Unit of Bioactive Natural Products, Post Graduate & Research Department of Zoology, C. Abdul Hakeem College, Melvisharam, Vellore District, Tamil Nadu, India for further reference.

Preparation of plant extracts: The leaf and bark were dried for 7-10 days in the shade at the environmental temperatures (27-37 o C day time). The dried bark (600 g) and leaf (700 g) were powdered mechanically using commercial electrical stainless steel blender and extracted with hexane (2,200 ml, Fine Chemicals, Mumbai), chloroform (1,000 ml, Fine Chemicals, Mumbai), ethyl acetate (2,500 ml, Qualigens, Fine Chemicals, Mumbai, India), acetone (1,000 ml, Qualigens), and methanol (2,800 ml, Qualigens), in a soxhlet apparatus (boiling point range 60-80°C) for 6 h. The extracts were filtered through a Buchner funnel with Whatman number 1 filter paper. The extract was concentrated under reduced pressure 22 - 26 mm Hg at 45 o C and the residue obtained was stored at 4 o C. The residues were then made in to a 1 per cent stock solution with acetone (stock solution). From the stock solution, 1000-4.69 mg/l, dilutions were prepared with dechlorinated tap water. Polysorbate 80 (Qualigens) was used as an emulsifier at the concentration of 0.05 per cent in the final test solution [16],[17] .

Mosquito culture: An. subpictus and Cx. tritaeniorhynchus larvae were collected from rice field and stagnant water areas of Melvisharam and identified in Zonal Entomological Research Centre, Vellore, Tamil Nadu, to start the colony and larvae were kept in plastic and enamel trays containing tap water. They were maintained and all the experiments were carried out at 27 ± 2°C and 75-85 per cent relative humidity under 14:10 h light and dark cycles. Larvae were fed a diet of Brewer's yeast, dog biscuits and algae collected from ponds in a ratio of 3:1:1, respectively as per the method of Kamaraj et al[17] .

Larvicidal bioassay: During preliminary screening with the laboratory trial, the larvae of An. subpictus and Cx. tritaeniorhynchus were collected from the insect rearing cage and identified in Zonal Entomological Research Centre, Vellore. From the stock solution, 1000 mg/l extract was prepared with dechlorinated tap water. The larvicidal activity was assessed by the procedure of WHO [18] with some modification and as per the method of Rahuman et al[19] . For the bioassay test, larvae were taken in five batches of 20 in 249 ml of water and 1.0 ml of the desired plant extract concentration. The control was set up with acetone and polysorbate 80. The numbers of dead larvae were counted after 24 h of exposure and the percentage of mortality was reported from the average of five replicates. The experimental media in which 100 per cent mortality of larvae occurs alone were selected for dose response bioassay. Hundred per cent mortality was observed in bark methanol extract of A. squamosa at concentration of (500, 250, 125, 62.5, 31.25, 15.63 and 7 mg/l), leaf methanol (300, 150, 75, 37.5, 18.75, 9.38 and 4.69 mg/l) and ethyl acetate (500, 250, 125, 62.5, 31.25, 15.63 and 7 mg/l) extract of C. indicum, leaf acetone (800, 400, 200, 100, 50, 25 and 12.5 mg/l) and ethyl acetate (600, 300, 150, 75, 37.5, 18.8 and 9.4 mg/l) extract of T. procumbens tested against An. subpictus and Cx. tritaeniorhynchus [Table 1] .
Table 1: Larvicidal activity of different concentrations against An. subpictus and Cx. tritaeniorhynchus (mg/l)

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Dose-response bioassay: From the stock solution, different concentrations ranging from 4.69-1000 mg/l were prepared. Based on the preliminary screening results 100 per cent mortality of larvae extracts alone were tested at different concentrations. Crude different solvents of leaf, and bark extracts prepared from A. squamosa, C. indicum and T. procumbens was subjected to dose response bioassay against An. subpictus, and Cx. tritaeniorhynchus respectively. The numbers of dead parasite and mosquito larvae were counted after 24 h of exposure, and the percentage mortality was reported from the average of five replicates. However, at the end of 24 h, the selected test samples turned out to be equal in their toxic potential.

Statistical analysis: The average larval mortality data were subjected to probit analysis for calculating LC 50, LC 90 , and other statistics at 95 per cent fiducial limits of upper confidence limit and lower confidence limit, and Chi-square values were calculated by using the software developed by Reddy et al[20] . P<0.05 was considered to be statistically significant.


   Results & Discussion Top


The activity of crude plant extracts is often attributed to the complex mixture of active compounds. In the preliminary screening, potential larvicidal activity of plants, different solvents crude extracts of three plants was noted ([Table 2], [Figure 1] A, B and C). Five different solvents were tested against An. subpictus and Cx. tritaniorhynchus and 100 per cent larval mortality was observed in the bark methanol extract of A. squamosa, leaf ethyl acetate and methanol extract of C. indicum and leaf acetone and ethyl acetate extract of T. procumbens [Table 2].
Figure 1: Graph showing the larvicidal activity of (A) Annona squamosa bark (B) Chrysanthemum indicum leaf and (C) Tridax procumbens leaf crude extracts against the fourth instar larvae of An. subpictus and Cx. tritaeniorhynchus at 1000 mg/l. Values are mean SD of 5 replicates.

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Table 2: Preliminary screenings of different solvent extracts against fourth instar larvae of An. subpictus and Cx. tritaeniorhynchus at1000 mg/l

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All plant extracts showed moderate toxic effect on An. subpictus and Cx. tritaeniorhynchus after 24 h of exposure at 1000 mg/l; however, the highest mortality was found in bark methanol extract of A. squamosa, leaf ethyl acetate extract of C. indicum and leaf acetone extract of T. procumbens against the larvae of An. subpictus (LC 50 =93.80, 39.98, and 51.57 mg/l; LC 90 =524.90, 145.70, and 226.56 mg/l); and bark methanol extract of A. squamosa, leaf methanol extract of C. indicum and leaf ethyl acetate of T. procumbens against the larvae of Cx. tritaeniorhynchus (LC 50 =104.94, 42.29 and 69.16 mg/l; LC 90 =443.79, 172.34, and 287.21 mg/l) respectively. The data obtained were analyzed using Chi-squared test, comparing experimental and control groups, with a significance level established at P<0.05. [Table 3]).
Table 3: LC50, LC90, and other statistical analysis of different solvent plant extracts against, fourth instar larvae of An. subpictus and Cx. tritaeniorhynchus

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Crude extracts of leaves or bark of these plants have been tested earlier by several investigators [21],[22],[23],[24],[25],[26],[27],[28],[29] and larvicidal and antifeedent activities have been shown against An. subpictus and Cx. tritaeniorhynchus, and also Leishmania maxicone[30] . C. fuscatum and C. myconis flowers powders have been tested for the antifeeding and growth regulatory activity against Spodoptera littoralis[28] . The leaves essential oils of T. procumbens are more effective repellents activity at 6 per cent concentration against An. stephens[29] and leaves methanol extracts and purified compound (35)-16,17-didehydrofalcerinol (1) showed effective antileishmanial activity against promastigotes of Leishmania mexicana[30] .

In conclusion, our findings showed that leaf and bark extract of A. squamosa, C. indicum and T. procumbens can be developed as ecofriendly larvicides. Also our results open the possibility for further investigations of the efficacy of larvicidal properties of natural product extracts.


   Acknowledgment Top


The authors acknowledge C. Abdul Hakeem College Management, Dr S. Mohammed Yousuff, Principal, Dr K. Abdul Subhan, Associate Professor and Head, Zoology Department, and Dr Sait Sahul Hameed, Associate Professor in Zoology for their help and suggestions.

 
   References Top

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2.Nagpal BN, Sharma VP. Indian anophelines. New Delhi: Oxford & IBH Publishing Co. Pvt. Ltd.; 1995. p. 1-416.  Back to cited text no. 2
    
3.Kirti JS, Kaur J. Variations in ornamentation of wings and palpi of Anopheles (Cellia) subpictus Grassi collected from northwest India. J Vector Borne Dis 2004; 41 : 37-41.   Back to cited text no. 3
    
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8.Keiser J, Maltese MF, Erlanger TE, Bos R, Tanner M, Singer BH, et al. Effect of irrigated rice agriculture on Japanese encephalitis, including challenges and opportunities integrated vector management. Acta Trop 2005; 95 : 40-57.  Back to cited text no. 8
    
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13.Beninger CW, Abou-Zaid MM, Kistner AL, Hallett RH, Iqbal MJ, Grodzinski B, et al. A flavanone and two phenolic acids from Chrysanthemum morifolium with phytotoxic and insect growth regulating activity. J Chem Ecol 2004; 30 : 589-606.  Back to cited text no. 13
    
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15.Caceres A, Cano O, Samayoa B, Aguilar L. Plants used in Guatemala for the treatment of gastrointestinal disorders. 1. Screening of 84 plants against enterobacteria. J Ethnopharmacol 1990; 30 : 55-73.  Back to cited text no. 15
    
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23.Kamaraj C, Abdul Rahman A, Bagavan A, Abduz Zahir A, Elango G, Kandan P, et al. Larvicidal efficacy of medicinal plant extracts against Anopheles stephensi and Culex quinquefasciatus (Diptera: Culicidae). Trop Biomed 2010; 27: 211-9.  Back to cited text no. 23
    
24.Bagavan A, Kamaraj C, Abdul Rahuman A, Elango G, Abduz Zahir A, Pandiyan G. Evaluation of larvicidal and nymphicidal potential of plant extracts against Anopheles subpictus Grassi, Culex tritaeniorhynchus Giles and Aphis gossypii Glover Parasitol Res 2009; 104:1109-17.  Back to cited text no. 24
    
25.Elango G, Rahuman AA, Kamaraj C, Bagavan A, Zahir AA. Screening for feeding deterrent activity of herbal extracts against the larvae of malaria vector Anopheles subpictus Grassi. Parasitol Res. In press 2011.  Back to cited text no. 25
    
26.Kamaraj C, Kaushik NK, Mohanakrishnan D, Elango G, Bagavan A, Zahir AA, et al . Antiplasmodial potential of medicinal plant extracts from Malaiyur and Javadhu hills of South India. Parasitol Res. In press 2011.  Back to cited text no. 26
    
27.Saxena RC, Dixit OP, Sukumaran P. Laboratory assessment of indigenous plant extracts for anti-juvenile hormone activity in Culex quinquefasciatus. Indian J Med Res 1992; 95 : 204-6.  Back to cited text no. 27
    
28.Haouas D, Ben Halima Kamel M, Harzallah FS, Ben Hammouda MH. Bioactivities of seven chrysanthemum species flowers powder on Spodoptera littoralis (Boiduval) larvae. Commun Agric Appl Biol Sci 2005; 70 : 799-807.  Back to cited text no. 28
    
29.Rajkumar S. Jebanesan A. Repellent activity of selected plant essential oils against the malarial fever mosquito Anopheles stephensi. Trop Biomed 2007; 2 : 71-5.  Back to cited text no. 29
    
30.Martin-Quintal Z, Moo-Puc R, Gonzalez-Salazar F, Chan-Bacab MJ, Torres-Tapia LW, Peraza-Sanchez SR. In vitro activity of Tridax procumbens against promastigotes of Leishmania mexicana. J Ethnopharmacol 2009; 122 : 463-7.  Back to cited text no. 30
    


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  [Table 1], [Table 2], [Table 3]



 

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