ORIGINAL PAPER

Laboratory evaluation of Indian medicinal plants as repellentsagainst malaria, dengue, and filariasis vector mosquitoes

Marimuthu Govindarajan & Rajamohan Sivakumar

Received: 3 November 2014 /Accepted: 6 November 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Mosquito-borne diseases have an economic im-pact, including loss in commercial and labor outputs, particu-larly in countries with tropical and subtropical climates; how-ever, no part of the world is free from vector-borne diseases.Mosquitoes are the carriers of severe and well-known illnessessuch as malaria, arboviral encephalitis, dengue fever,chikungunya fever, West Nile virus, and yellow fever. Thesediseases produce significant morbidity and mortality inhumans and livestock around the world. In view of the recent-ly increased interest in developing plant origin insecticides asan alternative to chemical insecticides, in the present study, therepellent activity of crude hexane, ethyl acetate, benzene,chloroform, and methanol extracts of leaf of Erythrina indicaand root of Asparagus racemosus were assayed for theirrepellency against three important vector mosquitoes, viz.,Anopheles stephensi , Aedes aegypt i , and Culexquinquefasciatus. The crude extract was applied on a mem-brane used for membrane feeding of unfed mosquitoes in a 1-ft cage. About 50 unfed 3–4-day-old laboratory-reared patho-gen-free strains of A. stephensi , A. aegypti , andC. quinquefasciatus were introduced in a 1-ft cage fitted witha membrane with blood for feeding with temperature main-tained at 37 °C through circulating water bath maintained at40–45 °C. Three concentrations (1.0, 2.0, and 5.0 mg/cm2) ofthe crude extracts were evaluated. Repellents in E. indicaafforded longer protection time against A. stephensi,A. aegypti, andC. quinquefasciatus than those inA. racemosusat 5.0 mg/cm2 concentration, and the mean complete protec-tion time ranged from 120 to 210 min with the differentextracts tested. In this observation, these two plant crude

extracts gave protection against mosquito bites; also, the re-pellent activity is dependent on the strength of the plantextracts. These results suggest that the leaf extract of E. indicaand root extract of A. racemosus have the potential to be usedas an ideal eco-friendly approach for the control of mosqui-toes. This is the first report on the mosquito repellent activityof the reported A. racemosus and E. indica plants.

Keywords Repellent activity .Erythrina indica . Asparagusracemosus . Mosquitoes

Introduction

Mosquitoes are vectors of several diseases affecting humansand domestic animals worldwide (WHO 2012). Mosquito is aserious insect to public health, which transmits several dan-gerous diseases such as dengue, filariasis, malaria, yellowfever, and Japanese encephalitis. Mosquitoes also cause aller-gic responses that include local skin and systemic reactionssuch as angioedema in humans. Anopheles stephensi Liston isthe major humanmalarial mosquito vector prevalent in severalcountries including the Middle East and South Asia (Bianet al. 2013), which harbors and transmits the malarial proto-zoan parasite Plasmodium falciparum (Corby-Harris et al.2010). Malaria is a deadly disease that resulted in 207 millioncases and about 627,000 deaths in 2012 (WHO 2013). Aedesaegypti is responsible for spreading dengue and chikungunya.Dengue is prevalent throughout the tropics and subtropics.The World Health Organization estimates that around 2.5billion people are at risk of dengue. Infections have dramati-cally increased in recent decades due to increased urbaniza-tion, trade, and travel. No effective drug or vaccine is availableso far. The only solution is to prevent the disease-carryingmosquito from breeding and biting humans. Dengue is themost important mosquito-spread viral disease and a major

M. Govindarajan (*) :R. SivakumarUnit of Vector Control, Phytochemistry and Nanotechnology,Department of Zoology, Annamalai University, Annamalainagar,Tamilnadu 608 002, Indiae-mail: drgovindzoo@yahoo.com

Parasitol ResDOI 10.1007/s00436-014-4222-0

international public health concern. It is a self-limiting diseasefound in tropical and subtropical regions around the world,predominantly in urban and semi-urban areas. Dengue feveror dengue hemorrhagic fever is caused by dengue virus, whichbelongs to the genus Flavivirus and family Flaviviridae, andincludes serotypes 1, 2, 3, and 4 (Den-1, Den-2, Den-3, andDen-4) (WHO 2010).

Culex quinquefasciatus acts as a vector for filariasis inIndia. Human filariasis is a major public health hazard andremains a challenging socioeconomic problem in many of thetropical countries (Govindarajan 2011). Lymphatic filariasiscaused by Wuchereria bancrofti and transmitted by mosquitoC. quinquefasciatus is found to be more endemic in the Indiansubcontinent. It is reported that C. quinquefasciatus infectsmore than 100 million individuals worldwide annually(Govindarajan et al. 2011). W. bancrofti is the most predom-inant filarial nematode, which is usually characterized byprogressive debilitating swelling at the extremities, scrotum,or breast (elephantiasis) in an infected individual(Kannathasan et al. 2007). These diseases are transmitted tohuman beings through mosquito bite only. Since there is noeffective vaccine available for the control of these diseases,prevention of mosquito bites is one of the main strategies tocontrol or minimize the incidence of these diseases. The use ofinsect repellents can provide a practical and economicalmeans of preventing mosquito-borne diseases. It is importantnot only for local people in disease risk areas, especially intropical countries, but also for travelers who are vulnerable todiseases (Tawatsin et al. 2006). Thus, the risk of being infectedwith a mosquito-borne disease is caused by the risk of beingbitten by the infected mosquito. Generally, mosquito bites arealso very itchy and painful sometimes. The continuousscratching over the skin will irritate it even more and makeyou more desperate.

Repellency is known to play an important role inpreventing the vector-borne diseases by reducing man–vectorcontact. Synthetic chemicals and insecticides used for controlof vectors are causing irreversible damage to the ecosystem, assome of them are non-degradable in nature. Some repellentsof synthetic origin may cause skin irritation and affect thedermis (Das et al. 2000). Majority of commercial repellentsare prepared by using chemicals like allethrin, N,N-diethyl-m-toluamide (DEET), dimethyl phthalate (DMP), and N,N-diethyl mendelic acid amide (DEM). It has been reported thatthese chemical repellents are not safe for public use (Ronaldet al. 1985). Because of unpleasant smell, oily feeling to someusers, and potential toxicity, some prefer to use natural insectrepellent products (Robbins and Cherniack 1986). Repellentsof plant origin do not pose hazards of toxicity to human anddomestic animals and are easily biodegradable. Natural prod-ucts are safe for human when compared to that of syntheticcompounds (Sharma and Ansari 1994). Therefore, it is thehour to launch extensive search to explore eco-friendly

biological materials for control of insect pests. The searchfor new strategies or natural products to control destructiveinsects and vectors of diseases is desirable due to the prevalentoccurrence of vector resistance to synthetic insecticides andthe problem of toxic non-biodegradable residues contaminat-ing the environment and the undesirable effects on non-targetorganisms (Jantan et al. 2005). It has been shown that the useof plant extracts as mosquito control agents can be effective,and it has been shown to minimize the impact that mostpesticidal compounds impose on the environment (Fatopeet al. 1993). Botanical insecticides have long been touted asattractive alternatives to synthetic chemical insecticides forvector management because botanicals reputedly pose littlethreat to the environment or to human health. The body ofscientific literature documenting bioactivity of plant deriva-tives to arthropod insects continues to expand, yet only ahandful of botanicals are currently used in agriculture in theindustrialized world, and there are few prospects forcommercial development of new botanical products.

Dhivya and Manimegalai (2013) have studied the repellentefficacy of the flower extracts of Calotropis gigantea againstC. quinquefasciatus mosquito and screened the bioactivecompounds present in the flower extract. The ethanolic extractof leaves of Datura stramonium was evaluated for larvicidaland mosquito repellent activities against A. aegypti,A. stephensi, and C. quinquefasciatus (Swathi et al. 2012).Trongtokit et al. (2005) have assessed repellent activity of 38Thai essential oils and found that an effective time ofrepellency strongly depended on the concentrations,experiment designs, and mosquito species. Govindarajan(2009) reported that the leaf methanol, benzene, and acetoneextracts of Cassia fistula were studied for the larvicidal,ovicidal, and repellent activities against A. aegypti. Petroleumether extracts of the leaves of Vitex negundo were consideredas a promising repellent against Culex tritaeniorhynchus(Karunamoorthi et al. 2008). The repellent activity of theessential oils of Thymus and Mentha species againstOchlerotatus caspius were evaluated by Koc et al. (2012).Repellent activity of essential oils derived from 10 Thai nativeplants belonging to three families was evaluated against fe-male A. aegypti and C. quinquefasciatus (Phukerd andSoonwera 2014). Karunamoorthi et al. (2014) evaluated therepellency of Ethiopian ethnomedicinal plant Juniperusprocera against the Afro-tropical malarial vector Anophelesarabiensis, respectively.

The essential oil (EO) extracted from fresh leaves ofHyptissuaveolens and its main constituents were evaluated for larvi-cidal and repellent activity against the Asian tiger mosquitoAedes albopictus (Conti et al. 2012). Mullai et al. (2008) havereported that the leaf extract of Citrullus vulgaris with differ-ent solvents, viz., benzene, petroleum ether, ethyl acetate, andmethanol, were tested for larvicidal, ovicidal, repellent, andinsect growth regulatory activities against A. stephensi.

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Choochote et al. (2004) studied the crude seed extract ofcelery Apium graveolens for repellent activity againstA. aegypti. It showed repellency against A. aegypti. Jantanand Zaki (1998) reported that the four essential oils of Litseaelliptica, Cinnamomum mollissimum, Cymbopogon nardus,and Pogostemom cablin, respectively, for their repellencyeffect against A. aegypti. The essential oils extracted fromZingiber officinalewas evaluated for repellent activity againstthe three cockroach species Periplaneta americana, Blattellagermanica, and Neostylopyga rhombifolia under laboratoryconditions (Thavara et al. 2007). The efficacy of DEET (N,N-diethyl-3-methylbenzamide) in providing a long-lasting pro-tection against many mosquito species has been documentedin several studies (Klun et al. 2006).

Govindarajan et al. (2011) evaluated the ovicidal and re-pellent activities of methanol leaf extract of Ervatamiacoronaria and Caesalpinia pulcherrima against A. stephensi,A. aegypti, and C. quinquefasciatus.

Erythrina indica belongs to the Family Fabaceae and isalso known as Indian coral tree, tropical coral tree, Tiger’sclaw, Moochy wood tree, variegated coral tree, sunshine tree,coral bean, and Kalyana murungai (Tamil). E. indica is acompact shrub with knobby stems. It possesses dense clustersof deep crimson flowers that spread broadly open. E. indica isa medium-sized, spiny, deciduous tree normally growing to 6–9 m tall. Young stems and branches are thickly armed withstout conical spines up to 8-mm long, which fall off after 2–4 years rarely; a few spines persist and are retained with thecorky bark. Bark is smooth and green when young, exfoliatingin papery flakes, becoming thick, corky, and deeply fissuredwith age. Leaves are trifoliate, alternate, bright emerald green,petioles are long about 6–15 cm, rachis 5–30-cm long, andprickly, and leaflets are smooth, shiny, and broader than long,8–20 by 5–15 cm, ovate to acuminate with an obtusely point-ed end. Leaf petiole and rachis are spiny, respectively (Yadavaand Reddy 1999). Asparagus racemosus (Shatavari) is anarmed climbing under shrub with woody terete stems andrecurved and rarely straight spines. Young stems very delicate,brittle, and smooth; leaves reduced tominute chaffy scales andspines; cladodes triquetrous, curved in tufts or two to six.Flowers are white, fragrant in fascicles or racemes on thenaked nodes of the main shoots or in the axils of the thorns.Fruits are subglobose pulpy berries, purplish black when ripe.Seeds are three to six, globose or angled having brittle andhard testa. The tuberous succulent roots are 30 cm to a meteror more in length, fascicled at the stem base and smoothtapering at both ends. Root contains saponin, water-solubleconstituents 52.1/2 %, moisture 1 %, glucose 7 %, and ashfrom dried root 4 %. The roots of A. racemosus are fleshy,whitish brown in color, lightly sweet in taste, emollient,cooling, nervine tonic, and possesses rejuvenating, carmina-tive, and aphrodisiac properties. Different scientific studieshave proved its efficacy in a number of physical and mental

ailments, respectively (Nadkarni 1976). As far as our literaturesurvey could ascertain, no information was available on therepellent activities of the experimental plant species givenhere against A. stephensi, A. aegypti, and C. quinquefasciatus.Therefore, the aim of this study was to investigate the mos-quito repellent activity of the different solvent extracts ofE. indica and A. racemosus. This is the first report on themosquito repellent activity of different solvent extracts ofselected plants.

Materials and methods

Collection of plants

The healthy root of A. racemosus (Fig. 1) and leaves ofE. indica (Fig. 2) were collected from Tamil Nadu, India.These were authenticated by a plant taxonomist from theDepar tmen t o f Botany, Annamala i Unive r s i ty,Annamalainagar. A voucher specimen was deposited at theDepartment of Zoology, Annamalai University.

Extraction

The healthy roots and leaves were washed with sterile distilledwater, shade-dried, and finely ground. The finely ground rootand leaf powder (500 g/solvent) was extracted with fivedifferent solvents, viz., hexane, benzene, chloroform, ethylacetate, and methanol, using a Soxhlet extraction apparatus,and the extraction was continued till visibly no further extrac-tion is possible. The solvents from the extracts were removedusing a rotary vacuum evaporator to collect the crude extract

Asparagus racemosus plantFig. 1 Asparagus racemosus plant

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and stored at 4 °C. Standard stock solutions were prepared at1 % by dissolving the residues in ethanol. From this stocksolution, different concentrations were prepared and thesesolutions were used for repellent bioassay.

Test organisms

The laboratory-bred pathogen-free strains of mosquitoes werereared in the vector control laboratory, Department of Zoolo-gy, Annamalai University. The larvae were fed on dog biscuits

and yeast powder in a 3:1 ratio. At the time of adult feeding,these mosquitoes were 3–4 days old after emergences (main-tained on raisins and water) and were starved for 12 h beforefeeding. Each time, 500 mosquitoes per cage were fed onblood using a feeding unit fitted with parafilm as membranefor 4 h. A. aegypti feeding was done from 12 noon to 4.00P.M.

and A. stephensi andC. quinquefasciatuswere fed during 6.00to 10.00P.M. A membrane feeder with the bottom end fittedwith parafilm was placed with 2.0 ml of the blood sample(obtained from a slaughterhouse by collecting in a heparinizedvial and stored at 4 °C) and kept over a netted cage ofmosquitoes. The blood was stirred continuously using anautomated stirring device, and a constant temperature of37 °C was maintained using a water jacket circulating system.After feeding, the fully engorged females were separated andmaintained on raisins. Mosquitoes were held at 28±2 °C, 70–85 % relative humidity, with a photoperiod of 12 h light and12 h dark.

Repellent activity

The crude extract was made up to known concentration solu-tion in ethanol. The crude extract was applied on a membraneused for membrane feeding of unfedmosquitoes in a 1-ft cage.About 50 unfed 3–4-day-old laboratory-reared pathogen-freestrains of C. quinquefasciatus, A. aegypti, and A. stephensiwas introduced in a 1-ft cage fitted with a membrane withblood for feeding with temperature maintained at 37 °Cthrough circulating water bath maintained at 40–45 °C. The

Erythrina indica plant

Fig. 2 Erythrina indica plant

Table 1 Repellency of different solvent root extracts of Asparagus racemosus against Anopheles stephensi

Solvent Concentration (mg/cm2) Repellency%±SD

Time of post application (min)

15 30 60 90 120 150 180 210

Methanol 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 86.4±1.67 73.2±1.09

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.4±1.67 85.2±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 92.8±1.09

Ethyl acetate 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 87.6±0.89 71.2±1.09 69.6±0.89

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 91.2±1.09 82.4±1.67 72.4±0.89

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 93.6±1.69 81.6±1.67

Chloroform 1.0 100±0.0 100±0.0 100±0.0 100±0.0 93.6±0.89 80.4±0.89 67.2±1.09 61.6±1.67

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 88.8±1.09 74.4±0.89 68.4±0.89

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.4±1.67 76.4±0.89

Benzene 1.0 100±0.0 100±0.0 100±0.0 100±0.0 88.8±1.09 78.4±1.67 61.6±1.67 52.4±0.89

2.0 100±0.0 100±0.0 100±0.0 100±0.0 92.8±1.09 82.4±1.67 69.6±0.89 58.4±1.67

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 91.6±1.67 84.8±1.09 68.4±1.67

Hexane 1.0 100±0.0 100±0.0 100±0.0 92.8±1.09 83.6±1.67 70.8±1.09 53.6±0.89 43.6±1.67

2.0 100±0.0 100±0.0 100±0.0 100±0.0 91.6±1.62 76.4±1.67 62.4±1.67 52.8±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 88.8±1.09 80.4±0.89 61.6±1.67

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time taken for the first feeding in the cage containing themembrane treated with repellent needs to be observed at30-min intervals, and each observation was made for 60 s.The experiment was repeated at this application rate forfive times to confirm reproducible results. The time taken

for feeding is considered as the protection time (in hours).Each test included one membrane feeding unit as controlwithout applying any repellent. The testing period was0600–1400 h for A. aegypti and 1800–0200 h forA. stephensi and C. quinquefasciatus.

Table 2 Repellency of different solvent root extracts of Asparagus racemosus against Aedes aegypti

Solvent Concentration (mg/cm2) Repellency%±SD

Time of post application (min)

15 30 60 90 120 150 180 210

Methanol 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 92.4±0.89 81.6±1.67 69.6±0.89

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 87.6±0.89 77.2±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.4±1.67

Ethyl acetate 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 84.4±1.67 65.6±0.89 62.4±1.67

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 89.2±1.09 76.4±0.89 68.4±1.67

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.4±0.89 75.6±0.89

Chloroform 1.0 100±0.0 100±0.0 100±0.0 100±0.0 90.8±1.09 78.4±0.89 60.8±1.09 58.8±1.09

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 83.6±1.67 69.2±1.09 62.8±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 88.8±1.09 71.2±1.09

Benzene 1.0 100±0.0 100±0.0 100±0.0 92.8±1.09 82.4±1.67 72.4±0.89 54.4±1.67 48.4±0.89

2.0 100±0.0 100±0.0 100±0.0 100±0.0 90.8±1.09 76.4±1.67 62.4±1.67 54.4±1.67

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 86.8±1.09 80.4±0.89 64.4±1.67

Hexane 1.0 100±0.0 100±0.0 100±0.0 90.8±1.09 75.6±0.89 68.4±1.67 49.2±1.09 39.2±1.09

2.0 100±0.0 100±0.0 100±0.0 100±0.0 84.8±1.09 71.2±1.09 57.6±1.67 49.2±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 82.4±1.67 75.2±1.09 58.8±1.09

Table 3 Repellency of different solvent root extracts of Asparagus racemosus against Culex quinquefasciatus

Solvent Concentration (mg/cm2) Repellency%±SD

Time of post application (min)

15 30 60 90 120 150 180 210

Methanol 1.0 100±0.0 100±0.0 100±0.0 100±0.0 90.4±0.89 83.6±1.67 71.2±1.09 63.2±1.09

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 89.2±1.09 76.4±0.89 67.2±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 92.4±0.89 86.8±1.09

Ethyl acetate 1.0 100±0.0 100±0.0 100±0.0 100±0.0 87.2±1.09 78.4±0.89 62.4±1.67 58.4±1.67

2.0 100±0.0 100±0.0 100±0.0 100±0.0 90.8±1.09 82.4±1.67 71.6±0.89 60.4±1.67

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 91.6±1.67 85.6±1.67 71.2±1.09

Chloroform 1.0 100±0.0 100±0.0 100±0.0 91.2±1.09 84.8±1.09 71.2±1.09 56.8±1.09 48.8±1.09

2.0 100±0.0 100±0.0 100±0.0 100±0.0 89.6±1.67 76.4±0.89 63.6±0.89 56.8±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 87.6±0.89 79.6±0.89 63.6±0.89

Benzene 1.0 100±0.0 100±0.0 100±0.0 85.2±1.09 79.6±0.89 62.4±1.67 48.4±1.67 39.2±1.09

2.0 100±0.0 100±0.0 100±0.0 92.4±0.89 83.6±1.67 69.2±1.09 58.8±1.09 49.2±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 90.8±1.09 81.6±1.67 72.8±1.09 58.8±1.09

Hexane 1.0 100±0.0 100±0.0 100±0.0 80.4±0.89 67.6±0.89 56.4±1.67 42.4±1.67 32.8±1.09

2.0 100±0.0 100±0.0 100±0.0 88.4±0.89 76.4±0.89 63.2±1.09 53.2±1.09 38.4±1.67

5.0 100±0.0 100±0.0 100±0.0 100±0.0 85.2±1.09 76.4±1.67 64.4±1.67 52.4±1.67

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If more than 4 h is taken at 2 mg/cm2 application, theplant extract was considered to exhibit promising repel-lency. If the protection time is <1–2 h at 2 mg/cm2

application rate, the plant extract may be discarded forrepellence testing.

The percentage of repellency was calculated by the follow-ing formula:

% Repellency ¼ Ta– Tbð Þ=Ta½ � � 100

where Ta is the number of mosquitoes in the control groupand Tb is the number of mosquitoes in the treated group.

Results

The results of the repellent activity of different solvent extractsof A. racemosus against A. stephensi, A. aegypti, andC. quinquefasciatus under laboratory conditions are given inTables 1, 2, and 3 (Fig. 3). When the repellent activity of threedifferent concentrations was compared (1.0, 2.5, and 5.0 mg/cm2) on membrane, the results revealed that the protectionobserved at 5.0 mg/cm2 concentration was more than the othertwo concentrations. Among the tested solvents, methanolextract provided significant repellency against A. stephensi,A. aegypti, and C. quinquefasciatus. The higher concentrationof 5.0 mg/cm2 provides 100 % protection for 180 min againstA. stephensi and A. aegypti. Against C. quinquefasciatus,

1.0 (mg/cm2)2.0 (mg/cm2)5.0 (mg/cm2)

1.0 (mg/cm2)2.0 (mg/cm2)5.0 (mg/cm2)

1.0 (mg/cm2)2.0 (mg/cm2)5.0 (mg/cm2)

Fig. 3 Repellent efficiency ofAsparagus racemosus againstthree important vectormosquitoesat various concentrations

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methanol extract provided 150 min of protection. Data of therepellent activity of E. indica against three species of mosqui-toes are presented in Tables 4, 5, and 6 (Fig. 4). Among thefive solvent tested for repellent activity against A. stephensi,A. aegypti, and C. quinquefasciatus, the methanol extract

afforded the maximum protection for A. stephensi followedby A. aegypti and C. quinquefasciatus. The higher concentra-tion of 5.0 mg/cm2 provides 100 % protection for 210 minaga in s t A. s t ephens i and A. aegyp t i . Aga in s tC. quinquefasciatus, methanol extract provided 180 min of

Table 4 Repellency of different solvent leaf extracts of Erythrina indica against Anopheles stephensi

Solvent Concentration (mg/cm2) Repellency%±SD

Time of post application (min)

30 60 90 120 150 180 210 240

Methanol 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 92.8±1.09 85.2±1.09 74.4±1.67

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.4±1.67 82.4±1.67

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 93.2±1.09

Ethyl acetate 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.8±1.09 77.6±0.89 60.4±0.89

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 93.6±0.89 81.6±1.67 72.8±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 91.2±1.09 86.8±1.09

Chloroform 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 82.4±1.67 64.4±1.67 52.4±1.67

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.8±1.09 72.4±1.67 68.4±1.67

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 84.4±0.89 75.6±0.89

Benzene 1.0 100±0.0 100±0.0 100±0.0 100±0.0 91.2±1.09 76.4±0.89 57.6±0.89 48.4±0.89

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 81.2±1.09 62.4±1.67 52.4±0.89

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 92.8±1.09 74.4±0.89 62.8±1.09

Hexane 1.0 100±0.0 100±0.0 100±0.0 92.4±1.67 82.4±1.67 64.4±1.67 48.4±1.67 39.2±1.09

2.0 100±0.0 100±0.0 100±0.0 100±0.0 90.4±1.67 72.4±0.89 58.8±1.09 46.4±1.67

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 86.4±0.89 65.2±1.09 54.4±1.67

Table 5 Repellency of different solvent leaf extracts of Erythrina indica against Aedes aegypti

Solvent Concentration (mg/cm2) Repellency%±SD

Time of post application (min)

30 60 90 120 150 180 210 240

Methanol 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 86.4±0.89 76.4±0.89 66.4±1.67

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 81.2±1.09 74.4±1.67

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 87.6±0.89

Ethyl acetate 1.0 100±0.0 100±0.0 100±0.0 100±0.0 92.8±1.09 80.4±0.89 62.4±1.67 56.4±1.67

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.8±1.09 71.6±0.89 63.6±1.67

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 86.8±1.09 72.4±0.89

Chloroform 1.0 100±0.0 100±0.0 100±0.0 100±0.0 90.4±0.89 71.2±1.09 52.4±0.89 46.8±1.09

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 83.6±1.67 62.4±1.67 52.4±0.89

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 79.6±1.67 65.2±1.09

Benzene 1.0 100±0.0 100±0.0 100±0.0 100±0.0 87.6±0.89 63.2±1.09 45.2±1.09 39.2±1.09

2.0 100±0.0 100±0.0 100±0.0 100±0.0 92.4±0.89 73.2±1.09 56.8±1.09 48.4±0.89

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 89.2±1.09 66.8±1.09 57.6±0.89

Hexane 1.0 100±0.0 100±0.0 100±0.0 88.8±1.09 78.4±1.67 58.4±1.67 42.4±0.89 33.6±0.89

2.0 100±0.0 100±0.0 100±0.0 100±0.0 86.8±1.09 66.8±1.09 48.4±1.67 39.2±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 82.4±1.67 61.6±1.67 49.2±1.09

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protection. Among the two plants tested for repellentactivity against three species of mosquitoes, E. indicaafforded the maximum protection against all three mos-quitoes. The repellent activity is dependent on thestrength of the plant extracts. The tested plant crudeextracts have exerted promising repellency against vec-tor mosquitoes.

Discussion

Vector control is facing a threat due to the emergence ofresistance of vector mosquitoes to conventional syntheticinsecticides, warranting countermeasures such as the develop-ment of novel insecticides. Crude extracts from plants havebeen used as insecticides in many countries for centuries.Crude plant extracts often consist of complex mixtures ofactive compounds. Advances of using complete mixturemay act synergistically, and they may show greater overallbioactivity compared to the individual constituents. Ourresults showed that the crude hexane, benzene, chloroform,ethyl acetate, and methanol solvent extracts of E. indica andA. racemosus were effective against three important vectormosqui toes , viz . , A. stephensi , A. aegypt i , andC. quinquefasciatus. This result is also comparable to earlierreports of Panneerselvam and Murugan (2013) who observedthe repellent activity of A. stephensi the hexane, ethyl acetate,benzene, aqueous, and methanol extract of Andrographispaniculata, Cassia occidentalis, and Euphorbia hirta plants

at three different concentrations of 1.0, 3.0, and 6.0 mg/cm2. Five different concentrations, 5, 10, 15, 20, and25 % (volume by volume), were prepared from eachextract stock. Topical application of the extract concen-trations on human volunteers revealed that 20 and 25 %repelled mosquitoes 2 and 5 h, respectively. The methanolextract of Ervatamia coronaria was found to be morerepellent than Caesalpinia pulcherrima extract. A higherconcentration of 5.0 mg/cm2 provided 100 % protectionup to 150, 180, and 210 min against C. quinquefasciatus,A. aegypti, and A. stephensi, respectively (Govindarajanet al. 2011). Karunamoorthi et al. (2008) have also report-ed that the leaves of Echinops sp. (92.47 %), Ostostegiaintegrifolia (90.10 %), and Olea europaea (79.78 %) werealso effective and efficient to drive away mosquitoes, andthe roots of Silene macroserene (93.61 %), leaves ofEchinops sp. (92.47 %), O. integrifolia (90.10 %), andO. europaea (79.78 %) exhibited a significant repellencyby direct burning. Park et al. (2005) found that monoter-penes from the Lamiaceae could be used to repel mosqui-toes of the genus Culex. Similarly, octacosane derivedfrom Moschosma polystachyum was effective in repellingCulex. Even though anethole is sometimes an ingredientof commercial repellents, it is used to improve the aromaof the products and not for its repellent properties. On theother hand, application of Tagetes minuta at 90 % con-centration deterred A. aegypti from biting, consistent withprevious reports indicating a moderate repellence ofT. minuta against mosquitoes (Moore et al. 2004).

Table 6 Repellency of different solvent leaf extracts of Erythrina indica against Culex quinquefasciatus

Solvent Concentration (mg/cm2) Repellency%±SD

Time of post application (min)

30 60 90 120 150 180 210 240

Methanol 1.0 100±0.0 100±0.0 100±0.0 100±0.0 93.6±1.67 81.6±1.67 68.4±0.89 58.8±1.09

2.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.4±1.67 75.6±0.89 69.6±0.89

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 92.8±1.09 80.4±0.89

Ethyl acetate 1.0 100±0.0 100±0.0 100±0.0 92.8±1.09 86.8±1.09 75.6±0.89 58.4±1.67 52.4±1.62

2.0 100±0.0 100±0.0 100±0.0 100±0.0 91.2±1.09 87.2±1.09 69.6±0.89 58.8±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 93.6±1.67 81.6±1.67 67.2±1.09

Chloroform 1.0 100±0.0 100±0.0 100±0.0 92.4±0.89 80.4±0.89 67.6±0.89 45.6±0.89 39.2±1.09

2.0 100±0.0 100±0.0 100±0.0 100±0.0 91.6±1.67 78.4±0.89 56.4±1.67 49.2±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.4±0.89 72.8±1.09 60.4±1.67

Benzene 1.0 100±0.0 100±0.0 100±0.0 92.4±0.89 75.6±0.89 55.6±0.89 39.2±1.09 33.6±0.89

2.0 100±0.0 100±0.0 100±0.0 90.8±1.09 82.4±1.67 69.6±1.67 47.2±1.09 42.8±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 91.2±1.09 82.8±1.09 60.4±0.89 53.2±1.09

Hexane 1.0 100±0.0 100±0.0 100±0.0 85.2±1.09 71.6±0.89 49.2±1.09 33.6±0.89 29.6±1.67

2.0 100±0.0 100±0.0 100±0.0 87.6±0.89 75.6±0.89 62.4±1.67 42.8±1.09 32.8±1.09

5.0 100±0.0 100±0.0 100±0.0 100±0.0 89.6±1.67 77.2±1.09 58.4±1.67 42.8±1.09

Parasitol Res

The seed acetone extract of Tribulus terrestris showedstrong repellent activity against Anopheles culicifacies spe-cies, 100 % repellency in 1 and 6 h; A. stephensi 100 %repellency in 0, 4, and 6 h; and C. quinquefasciatus 100 %repellency in 0, 2, and 4 h, at 10 % concentration, respectively(Singh and Saratchandra 2005). Choi et al. (2002) tested therepellent activity of Lavandula officinalis and Rosmarinusofficinalis EOs against Culex pipiens pallens, showing aneffective repellent effect mainly to adult mosquitoes due toα-terpinene, carvacrol, and thymol. Tawatsin et al. (2001)have reported repellent activity against A. aegypti, Anophelesdirus, and C. quinquefasciatus, which is due to 5 % vanillin,

which has been added to the essential oil of Curcumalonga. The repellent effects and the result showed thatt h e n i g h t - b i t i n g m o s q u i t o e s A . d i r u s a n dC. quinquefasciatus and A. albopictus were more sensi-tive to all the essential oils (repellency 4.5–8 h) than wasA. aegypti (repellency 0.3–2.8 h), whereas DEET andIR3535 provided excellent repellency against A. aegypti,A. albopictus, A. dirus, and C. quinquefasciatus (repel-lency 6.7–8 h). Neem products are good mosquito repel-lents showing 90 to 100 % protection against malariavectors and about 70 % against C. quinquefasciatus(Ansari and Razdan 1994).

1.0 (mg/cm2)2.0 (mg/cm2)5.0 (mg/cm2)

1.0 (mg/cm2)2.0 (mg/cm2)5.0 (mg/cm2)

1.0 (mg/cm2)2.0 (mg/cm2)5.0 (mg/cm2)

Fig. 4 Repellent efficiency ofErythrina indica against threeimportant vector mosquitoes atvarious concentrations

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Amer and Mehlhorn (2006) have reported that the fivemost effective oils were those of Litsea (Litsea cubeba),Cajeput (Melaleuca leucadendron), Niaouli (Melaleucaquinquenervia), Violet (Viola odorata), and Catnip (Nepetacataria), which induced a protection time of 8 h at the max-imum repellency against A. aegypti, A. stephensi, andC. quinquefasciatus. The repellent activity, compared to aprevious report at the dose of 25.0 mg/mat, of thymol provid-ed complete repellency, whereas Trachyspermum ammi seedoil could achieve a repellency of 45.0 %, and the repellentdoses (RD50) observed were 25.02 and 11.63 mg/mat forT. ammi seed oil and thymol, respectively, against A. stephensiadults (Pandey et al. 2009). The leaf methanol, benzene, andacetone extract of C. fistulawere studied for the larvicidal andrepellent activity against A. aegypti, and the mortality wasobserved in 24 h LC50 concentration of the extracts at 10.69,18.27, and 23.95 mg/l; the crude extract of C. fistula showssignificant repellency against A. aegypti (Govindarajan 2009).Khanna et al. (2011) have reported that the larvicidal crudeleaf extract of Gymnema sylvestre showed the highest mortal-ity at the concentration of 1000 ppm against the larvae ofAnopheles subpictus (LC50=166.28 ppm) and against thelarvae of C. quinquefasciatus (LC50=186.55 ppm), and themaximum efficacy was observed in gymnemagenol com-pound isolated frompetroleum ether leaf extract ofG. sylvestrewith LC50 values against the larvae of A. subpictus at22.99 ppm and against C. quinquefasciatus at 15.92 ppm,respectively. Prophiro et al. (2012) reported that the suscepti-bility of larvae was determined under three different temper-atures, 15, 20, and 30 °C, with lethal concentrations forCopaifera sp. ranging from LC50 of 47 mg/l to LC90 of91 mg/l and for Carapa guianensis LC50 of 136 mg/l toLC90 of 551 mg/l.

Xue et al. (2006) pointed out the oviposition deterrenteffectiveness (76–100 % repellency) against A. albopictus of21 commercial insect repellent products (at 0.1 % concentra-tion), including 12 botanical, six DEET-based, and three syn-thetic organics. Xue et al. (2003) have reported the oviposi-tional deterrent effects of DEET and several repellent com-pounds, such as AI3-37220, AI3-35765, AI3-54995, and AI3-55051, against A. albopictus under laboratory and fieldconditions. Autran et al. (2009) have reported that the essentialoil from leaves and stems of Piper marginatum exhibited anoviposition deterrent effect against A. aegypti at 50 and100 ppm in that significantly lower numbers of eggs(<50%) were laid in glass vessels containing the test solutionscompared with the control solution. The oviposition deterrentproperties against A. stephensi have been observed for variousplant extracts including the methanol extract of Pelargoniumcitrosa, which exhibited 56 and 92% inhibition of ovipositionat 1 and 4 ppm, respectively (Jeyabalan et al. 2003). Repellentingredients from Lantana camera flowers were isolated andtheir repellency was tested against Aedes mosquito. One

application of this fraction gave 100 % protection for 2 hand may protect 75.8 % at 7 h (Dua et al. 2003). To reducenumbers of mosquitoes indoors at night and mosquito bitingactivity, the following plant products were used: the freshsmoldering Hyptis suaveolens (85.4 % repellency), smoke ofthe bark of Daniellia oliveri (74.7 %), smoke of theinfructescence of Elaeis guineensis (72.2 %), smoke of theseed capsules of Parkia biglobosa, smoke of the leaves ofA. indica (76.0 %) and Eucalyptus sp. (69.0 %), freshOcimumcanum (63.6 %), and fresh Senna occidentalis (29.4 %)(Palsson and Jaenson 1992). In conclusion, an attempt hasbeen made to evaluate the role of medicinal plant extracts fortheir repellent bioassay against A. stephensi, A. aegypti, andC. quinquefasciatus. Compared with earlier reports, our re-sults revealed that the experimental plant extracts were effec-tive in controlling three mosquito species. From these results,it was concluded that the plant leaf and root extracts ofE. indica and A. racemosus exhibit repellent activity againstan important vector mosquito. The results reported in thisstudy open the possibility for further investigations of theefficacy of repellent property of natural product extracts.Plants can provide safer alternatives for modern deadly poi-sonous synthetic chemicals.

Acknowledgments The authors are grateful to the Indian Council ofMedical Research (ICMR ref. letter no. 5/8-7(246)/2012-ECD-II), NewDelhi, India for providing financial assistance and to Dr. N. Indra,Professor and Head of the Department of Zoology, Annamalai Universityfor the laboratory facilities provided. The authors would also like toacknowledge the cooperation of staff members of the VCRC (ICMR),Pondicherry.

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