Adulticidal and repellent properties of Cassia tora Linn. (Family: Caesalpinaceae) against Culex...

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Parasitology ResearchFounded as Zeitschrift fürParasitenkunde ISSN 0932-0113 Parasitol ResDOI 10.1007/s00436-012-3042-3

Adulticidal and repellent properties ofCassia tora Linn. (Family: Caesalpinaceae)against Culex quinquefasciatus, Aedesaegypti, and Anopheles stephensi

Duraisamy Amerasan, KadarkaraiMurugan, Kalimuthu Kovendan,Palanisamy Mahesh Kumar, ChellasamyPanneerselvam, Jayapal Subramaniam,et al.

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ORIGINAL PAPER

Adulticidal and repellent properties of Cassia tora Linn.(Family: Caesalpinaceae) against Culex quinquefasciatus,Aedes aegypti, and Anopheles stephensi

Duraisamy Amerasan & Kadarkarai Murugan &

Kalimuthu Kovendan & Palanisamy Mahesh Kumar &

Chellasamy Panneerselvam & Jayapal Subramaniam &

Samuel John William & Jiang-Shiou Hwang

Received: 20 June 2012 /Accepted: 5 July 2012# Springer-Verlag 2012

Abstract Mosquitoes have developed resistance to varioussynthetic insecticides, making its control increasingly diffi-cult. Insecticides of botanical origin may serve as suitablealternative biocontrol techniques in the future. The adultici-dal and repellent activities of crude hexane, chloroform,benzene, acetone, and methanol extracts of the leaf of Cas-sia tora were assayed for their toxicity against three impor-tant vector mosquitoes, viz., Culex quinquefasciatus, Aedesaegypti, and Anopheles stephensi. The adult mortality wasobserved after 24 h of exposure. All extracts showed mod-erate adulticidal effects; however, the highest adult mortalityobserved was found in methanol extract. The LC50 and LC90

values of C. tora leaf extracts against adulticidal activity of(hexane, chloroform benzene, acetone, and methanol) C.quinquefasciatus, A. aegypti, and A. stephensi were thefollowing: C. quinquefasciatus LC50 values were 338.81,315.73, 296.13, 279.23, and 261.03 ppm and LC90 valueswere 575.77, 539.31, 513.99, 497.06, and 476.03 ppm; A.aegypti LC50 values were 329.82, 307.31, 287.15, 269.57,and 252.03 ppm and LC90 values were 563.24, 528.33,

496.92, 477.61, and 448.05 ppm; and A. stephensi LC50

values were 317.28, 300.30, 277.51, 263.35, and251.43 ppm and LC90 values were 538.22, 512.90, 483.78,461.08, and 430.70 ppm, respectively. The results of therepellent activity of hexane, chloroform, benzene, acetone,and methanol extracts of C. tora plant at three differentconcentrations of 1.0, 2.5, and 5.0 mg/cm2 were appliedon skin of forearm in man and exposed against adult femalemosquitoes. In this observation, this plant crude extractsgave protection against mosquito bites without any allergicreaction to the test person, and also, the repellent activity isdependent on the strength of the plant extracts. These resultssuggest that the leaf solvent plant extracts have the potentialto be used as an ideal eco-friendly approach for the controlof mosquitoes. This is the first report on mosquito adultici-dal and repellent activities of the reported C. tora againstmosquito vectors from Southern India.

Introduction

Mosquito-borne diseases such as malaria, filariasis, dengue,and viral encephalitis contribute to a larger proportion ofhealth problems of developing countries. Repeated use ofsynthetic insecticides for mosquito control has disruptednatural biological control systems and led to resurgencesin mosquito populations. It also resulted in the developmentof resistance, undesirable effects on nontarget organisms,and fostered environmental and human health concern(Thomas et al. 2004).

Anopheles stephensi transmits malaria in the plains ofrural and urban areas of India and other parts of the world.In 1993, it was estimated that 2,020 million people in some90 countries or territories were at risk of malaria infection

D. Amerasan :K. Murugan :K. Kovendan (*) :P. Mahesh Kumar :C. Panneerselvam : J. SubramaniamDivision of Entomology, Department of Zoology,School of Life Sciences, Bharathiar University,Coimbatore 641 046, Tamil Nadu, Indiae-mail: [email protected]

S. John WilliamP.G. Research & Department of Advanced Zoologyand Biotechnology, Loyola College,Nungambakkam,Chennai 600 034, Tamil Nadu, India

J.-S. HwangInstitute of Marine Biology, National Taiwan Ocean University,Keelung 202-24, Taiwan

Parasitol ResDOI 10.1007/s00436-012-3042-3

Author's personal copy

and that some 300–500 million people would contact ma-laria each year, with 1.5–2.7 million deaths annually, includ-ing one child every 30 s (WHO 1999). A. stephensi isrecognized as a major vector for urban malaria in India.This species prefers to breed in small synthetic water col-lections and is responsible for frequent outbreaks of malaria,particularly at construction sites in urban areas (Mittal et al.2005). Aedes aegypti, the primary carrier for viruses thatcause dengue fever, dengue haemorrhagic fever, chikungu-nya fever, and yellow fever is widespread over large areas ofthe tropics and subtropics (Service 1983; Yang et al. 2009).According to WHO (2009) about two-fifths of the world'spopulation are now at risk of dengue, and the only way toprevent dengue virus transmission is to combat the diseasecarrying mosquitoes. In India, a total of 28,292 cases and110 deaths were reported because of dengue in 2010 (Na-tional Vector Borne Disease Control Programme 2011a).Culex quinquefasciatus, a vector of lymphatic filariasis, iswidely distributed in tropical zones with around 120 millionpeople infected worldwide and 44 million people havingcommon chronic manifestation (Bernhard et al. 2003). De-spite its debilitating effects, lymphatic filariasis is given avery low control priority (Ramaiah et al. 2000).

Seyoum et al. (2002a) studied the repellency of livepotted plants against Anopheles gambiae from human baitsin semifield experimental huts. Ocimum americanum(Labiatae), Lantana camara (Verbenaceae), and Lippia uck-ambensis (Verbenaceae) repelled at an average of 39.7 %,32.4 %, and 33.3 % of the mosquitoes, respectively. Thecombination of O. americanum with either L. camara or L.uckambensis repelled 31.6 % and 45.2 % of mosquitoes,respectively. The ethanol extract of Curcuma aeruginosa,Curcuma aromatica, and Curcuma xanthorrhiza were testedfor repellent activity against Aedes togoi, Armigeres subal-batus, C. quinquefasciatus, and C. tritaeniorhynchus (Pita-sawat et al. 2003). Choochote et al. (2004) studied the crudeseed extract of celery, Apium graveolens, for repellent ac-tivity against A. aegypti. It showed repellency against A.aegypti adult females with ED50 and ED95 values of 2.03and 28.12 mg/cm2, respectively. It also provided bitingprotection time of 3 h when applied at a concentration of25 %. Topical application of the ethanol-extracted A. grave-olens did not induce dermal irritation.

Yang et al. (2004) studied the repellent activity of meth-anol extracts from 23 aromatic medicinal plant species and asteam distillate against female blood-starved A. aegypti. At adose of 0.1 mg/cm2, the repellency extracts of Cinnamomumcassia root bark (91 %), Nardostachys chinensis rhizome(81 %), Paeonia suffruticosa root bark (80 %), and Cinna-momum camphora steam distillate (94 %) were compared toDEET (N,N-diethyl-m-toluamide) and lasted for l h. Rela-tively short duration of repellency was observed in P. suf-fruticosa root bark extract and C. camphora steam distillate.

Dua et al. (2003) evaluated the repellent properties of dif-ferent fractions isolated from L. camara flowers by usingsteam distillation, solvent partition, and chromatographicmethods against Aedes mosquitoes. Maximum protectiontime of one fraction eluted by chloroform from a silica gelcolumn was 3.45 h. One application of this fraction gave100 % protection for 2 h and protected 75.8 % at 7 h againstthe bites of Aedes mosquitoes. The chemicals derived fromplants have been projected as weapons in future mosquitocontrol programs as they are shown to function as generaltoxicant, growth and reproductive inhibitors, repellents, andoviposition-deterrent (Sukumar et al. 1991).

Cassia tora (Family: Cesalpinaceae), commonly knownas Tagarai sedi in Tamil, is a very common Indian herbhaving various medicinal properties for the treatment ofdifferent kinds of disease, viz. antifungal, wound healing,and antidiabetic agents, respectively (Nadkarni 1982;Christopher 2005). These herbs have been reported for theirusefulness in the form of decoctions, infusions, and tincturesin traditional system of medicines for treating skin diseaseslike psoriasis, leprosy, etc. (Horvath and Ferenc 1992; Zahraet al. 2000; Cordova et al. 2002; Harrison and Dorothy2003). Plants such as Calendula officinalis, C. tora, andMomordica charantia are well known herbs used in ayuverdictraditional medicine for their effectiveness against a widerange of diseases including skin infections due to the advan-tage of the diversity of secondary metabolites responsible fortheir antibacterial activity. Moreover, the number of immunecompromised patients is on the increase due to great progressin the field of intensive care, organ transplantation, and AIDS.HIV-positive patients have developed resistance to treatmentwith existing antibiotics (Silver and Bostian 1993).This indi-cates that aqueous extracts are better as compared to otherextracts. Literature also reveals the use of aqueous paste ofseeds of C. tora, M. charantia seed juice (prepared inwater), and Calendula flower water decoctions in variousskin conditions associated with bacterial infections (Brownand Dattner 1998; Grover and Yadav 2004). Manyresearchers have reported on the effectiveness of plantextract against mosquito studies (Kalyanasundaram andDas 1985; Kovendan et al. 2012a, b, c, d, e; MaheshKumar et al. 2012a).

TaxonomyKingdom: PlantaePhylum: TracheophytaSubphylum: EuphyllophytinaClass: SpermatopsidaSubclass: RosidaeSuperorder: RosanaeOrder: FabalesFamily: CaesalpinaceaeSubfamily: CaesalpinioideaeTribe: Cassieae

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Genus: CassiaSpecies: toraBotanical name: Cassia tora L. Zipcodezoo (2012)Trongtokit et al. (2005) have assessed repellent activ-

ity of 38 Thai essential oils and found that an effectivetime of repellency strongly depended on the concentra-tions, experiment designs, and mosquito species. Mooreet al. (2007) demonstrated efficacy of low-cost repellentcontaining PMD and lemongrass oil (with fixatives) forup to 6 h, which was more effective than DEET, againstAnopheles darlingi in Peru. However, prolonged protec-tion effects have been reported from 20 % of the oilfrom turmeric, citronella, and hairy basil, especially withthe addition of 5 % vanillin as fixative in which theyeach can repel three species of mosquitoes for up to 6 h(Jaenson et al. 2006). The present investigation aims toassess the adulticidal and repellent properties of the plantextract against medically important mosquitoes of filarial,dengue, malaria vector, C. quinquefasciatus, A. aegypti,and A. stephensi as target species of the vector controlprogrammes.

Materials and methods

Collection of larvae and maintenance

The larvae of A. stephensi, A. aegypti, and C. quinquefas-ciatus were collected from the National Centre for DiseaseControl field station of Mettupalayam, Tamil Nadu using an“O”-type brush. The mosquito larvae were fed with grounddog biscuits and yeast at 3:1 ratio. The feeding was contin-ued until the larvae transformed into the pupal stage.

Maintenance of pupae and adults

The pupae were collected from the culture trays and trans-ferred to plastic containers (12×12 cm) containing 500 mLof water with the help of a dipper. The plastic jars were keptin a 90×90×90 cm mosquito cage for adult emergence.Mosquito larvae were maintained at 27±2 °C, 75–85 %relative humidity, under a photoperiod of 14:10 (light/dark).A 10 % sugar solution was provided for a period of 3 daysbefore blood feeding.

Blood feeding of adult mosquito vectors

The adult female mosquitoes were allowed to feed on theblood of a rabbit (a rabbit per day, exposed on the dorsalside) for 2 days to ensure adequate blood feeding for 5 days.After blood feeding, enamel trays with water from theculture trays were placed in the cage as ovipositionsubstrates.

Collection of plants and preparation of plant extracts

The C. tora plants were collected in and around Marutha-malai hills, Coimbatore in Tamil Nadu, India. The plantswere identified by a taxonomist in the Department of Bot-any, Bharathiar University, Coimbatore. The fresh leaves ofC. tora were washed thoroughly in tap water and shade-dried at room temperature (28±2 °C) for 5 to 10 days. Theair-dried materials were powdered separately using commer-cial electrical blender. From the plants, 500 g powder wasmacerated with 1.5 L organic solvents of hexane, chloro-form, benzene, acetone, and methanol sequentially for aperiod of 72 h each and filtered. The yield of the C. toracrude extracts by hexane, chloroform, benzene, acetone andmethanol were 13.67, 12.89, 11.75, 14.36 and 15.23 g,respectively. The extracts were concentrated at reduced tem-perature on a rotary vacuum evaporator and stored at atemperature of 4 °C. One gram of the plant residue wasdissolved in 100 mL of acetone (stock solution) consideredas 1 % stock solution. From this stock solution, concentra-tions were prepared ranging from 180, 260, 340, 420, and500 ppm, respectively.

Repellent bioassay

The stock solutions of the extracts were diluted with ace-tone, polysorbate 80, and distilled water to obtain test sol-utions of 1.0, 2.5, and 5.0 mg/cm2 (hexane, chloroform,benzene, acetone, and methanol) prepared separately. Forrepellent experiment, 50 laboratory reared blood-starvedadult female mosquitoes that were between 3 and 10 daysold were placed into separate laboratory cages (45×45×40 cm). Before each test, the forearm and hand of a humansubject were washed with unscented neutral soap, thorough-ly rinsed, and allowed to dry for 10 min before extractapplication. The different plant extracts being tested wereapplied from the elbow to the fingertips. The arm was leftundisturbed. An arm treated with acetone and polysorbate80 served as control. The control and treated arms wereintroduced simultaneously into the cage. The number ofbites was counted over 15 min, every 30 min, and from1800 h to 0600 h. Protection time was recorded as the timeelapsed between repellent application and the observationperiod immediately preceding that in which a confirmed bitewas obtained. If no bites were confirmed at 210 min, testswere discontinued and protection time was recorded as210 min. An attempt of the mosquito to insert its styletswas considered a bite. No mosquito attempted to bite thecontrol arm during the observation period; that trial wasdiscarded, and the test was repeated with a new batch ofmosquitoes to ensure that lack of bites was due to repellenceand not to mosquitoes not being predisposed to get a bloodmeal at the time. The experiments were conducted five times

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in separate cages and in each replicate different volunteerswere used to nullify any effect of skin differences on repel-lency. It was observed that there was no skin irritation from

the plant extract. The percentage protection was calculatedby using the following formula (Fradin and Day 2002;Venkatachalam and Jebanesan 2001):

Protection ¼ No:ofbitesreceivedbycontrolarmf g � No:ofbitesreceivedbytreatedarmf gð ÞNo:ofbitesreceivedbycontrolarmð Þ � 100

Adulticidal bioassay

Sugar-fed adult female mosquitoes (5 to 6 days old) wereused. The C. tora leaf extract were diluted with acetoneto make different concentrations. The diluted plantextracts were impregnated on filter papers (140×

120 mm). A blank paper consisting of only ethanol wasused as control. The papers were left to dry at roomtemperature to let the ethanol evaporate overnight. Im-pregnated papers were prepared fresh prior to testing.The bioassay was conducted in an experimental kit con-sisting of two cylindrical plastic tubes both measuring

Table 1 Adulticidal activity of different solvent extracts of Cassia tora against Culex quinquefasciatus

Name of the extract Concentration (ppm) % mortality±SD LC50 (ppm) (LFL–UFL) LC90 (ppm) (LFL–UFL) χ2

Hexane Control 0.0±0.0 338.81 (316.61–360.91) 575.77 (531.52–639.94) 1.17*

180 20.0±1.63

260 30.3±1.69

340 54.3±1.88

420 66.6±1.24

500 80.0±1.41

Chloroform Control 0.0±0.0 315.73 (293.67–336.42) 539.31 (500.83–593.84) 0.98*

180 22.6±1.24

260 34.6±1.88

340 59.0±1.63

420 71.0±0.81

500 85.6±1.69

Benzene Control 0.0±0.0 296.13 (273.34–316.58) 513.99 (478.48–563.79) 0.65*

180 26.6±0.94

260 38.6±1.24

340 60.0±1.63

420 78.0±0.81

500 88.3±1.69

Acetone Control 0.0±0.0 279.23 (254.95–300.18) 497.06 (462.85–544.97) 1.83*

180 30.6±1.69

260 40.0±0.81

340 66.3±0.94

420 80.3±1.24

500 90.0±1.41

Methanol Control 0.0±0.0 261.03 (235.07–282.59) 476.03 (443.54–521.43) 0.53*

180 33.3±0.94

260 47.6±1.24

340 68.0±1.63

420 82.0±0.81

500 93.3±0.94

LFL lower fiducidal limits, UFL upper fiducidal limits, χ2 Chi square value*P<0.05 (statistical significance)

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125×44 mm following the method of WHO (1981). Onetube served to expose the mosquitoes to the plant extractand another tube was used to hold the mosquitoes beforeand after the exposure periods. The impregnated paperswere rolled and placed in the exposure tube. Each tubewas closed at one end with a 16-mesh wire screen.Sucrose-fed and blood-starved mosquitoes (20) were re-leased into the tube, and the mortality effects of theextracts were observed every 10 min for a 3-h exposureperiod. At the end of 1, 2, and 3 h exposure periods, themosquitoes were placed in the holding tube. Cotton padssoaked in 10 % sugar solution with vitamin B complexwas placed in the tube during the holding period for24 h. Mortality of the mosquitoes was recorded after24 h. The above procedure was carried out in triplicatefor plant extract of each concentration.

Statistical analysis

The average adult mortality data were subjected to probitanalysis for calculating LC50, LC90, and other statistics at95 % fiducidal limits of upper fiducidal limit and lowerfiducidal limit, and Chi-square values were calculated usingthe SPSS Statistical software package 16.0 version wasused. Results with P<0.05 were considered to be statistical-ly significant.

Results

The results of the adulticidal activity of hexane, chloroform,benzene, acetone, and methanol extracts of C. tora againstthe adult of three important vector mosquitoes, viz., C.

Table 2 Adulticidal activity of different solvent extracts of Cassia tora against Aedes aegypti


Hexane Control 0.0±0.0 329.82 (307.58–351.42) 563.24 (520.90–624.22) 0.86*

180 21.0±1.72

260 32.3±1.59

340 55.7±1.65

420 68.5±1.43

500 81.9±1.19


180 24.3±1.20

260 35.7±1.76

340 61.0±1.29

420 72.5±0.98

500 87.3±1.55

Benzene Control 0.0±0.0 287.15 (264.52–307.18) 496.92 (463.80–542.82) 0.97*

180 28.2±0.95

260 39.7±1.25

340 62.3±1.37

420 80.1±0.89

500 90.6±1.47

Acetone Control 0.0±0.0 269.57 (245.50–290.09) 477.61 (445.94–521.42) 2.35*

180 32.8±1.96

260 41.7±1.66

340 68.2±0.91

420 81.7±1.29

500 93.2±1.17


180 35.6±1.65

260 48.3±1.73

340 70.5±1.39

420 85.2±1.47

500 96.7±1.58


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quinquefasciatus, A. aegypti, and A. stephensi, are presentedin Tables 1, 2, and 3 (Fig. 1). Among the three vectorstested, the highest adulticidal activity was observed in thehighest mortality followed by A. stephensi, A. aegypti, andC. quinquefasciatus. At higher concentrations, the adultshowed restless movement for some time with abnormalwagging and died. The rates of mortality were directlyproportional to concentration. The LC50 and LC90 valuesof C. tora leaf extracts against adulticidal activity of (hex-ane, chloroform benzene, acetone, and methanol) C. quin-quefasciatus, A. aegypti, and A. stephensi were thefollowing: C. quinquefasciatus LC50 values were 338.81,315.73, 296.13, 279.23, and 261.03 ppm and LC90 valueswere 575.77, 539.31, 513.99, 497.06, and 476.03 ppm; A.aegypti LC50 values were 329.82, 307.31, 287.15, 269.57,and 252.03 ppm and LC90 values were 563.24, 528.33,

496.92, 477.61, and 448.05 ppm; and A. stephensi LC50

values were 317.28, 300.30, 277.51, 263.35, and251.43 ppm and LC90 values were 538.22, 512.90, 483.78,461.08, and 430.70 ppm, respectively.

In the present observation, the results from the skin-repellent activity of hexane, chloroform benzene, acetone,and methanol extracts of C. tora against blood-starved adultfemale of C. quinquefasciatus, A. aegypti, and A. stephensiare given in Tables 4, 5 and 6. The hexane, chloroformbenzene, acetone, and methanol extracts of C. tora showsignificant repellency against C. quinquefasciatus, A.aegypti, and A. stephensi. In this observation, these plantcrude extracts gave protection against mosquito bites with-out any allergic reaction to the test person, and also, therepellent activity is dependent on the strength of the plantextracts. The highest repellency of 210 min was observed in

Table 3 Adulticidal activity of different solvent extracts of Cassia tora against Anopheles stephensi


Hexane Control 0.0±0.0 317.28 (295.57–337.77) 538.22 (500.19–591.95) 0.93*

180 22.3±1.24

260 34.6±1.69

340 57.9±1.05

420 70.1±1.16

500 86.5±1.41


180 25.4±0.94

260 36.7±1.05

340 62.0±1.44

420 74.2±1.09

500 89.8±1.62

Benzene Control 0.0±0.0 277.51 (254.44–297.56) 483.78 (451.92–527.76) 1.23*

180 30.3±1.69

260 42.6±1.14

340 63.3±1.24

420 81.2±1.63

500 93.0±1.41

Acetone Control 0.0±0.0 263.35 (239.89–283.29) 461.08 (431.68–501.23) 1.61*

180 33.0±1.55

260 44.6±1.68

340 68.3±1.86

420 84.8±1.16

500 94.7±1.05


180 35.3±1.10

260 48.9±1.40

340 70.6±1.21

420 86.2±1.94

500 99.5±1.88


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methanol extract of C. tora against A. stephensi followed byA. aegypti and C. quinquefasciatus. The results clearly showthat repellent activity was dose-dependent.

Discussion

Mosquito control represents an important strategy for pre-vention of disease transmission and epidemic outbreaks.

However, a high level of insecticide resistance has devel-oped through chemical control of the vector and peststhreatening the control strategies. To overcome these prob-lems, it is necessary to search for alternative methods ofvector control. The failure of chemical insecticides to con-trol the insect and growing public concern for safe food anda healthy environment have catalyzed the search for moreenvironmentally benign control methods for the manage-ment of the vectors. Biopesticides provide an alternative tosynthetic pesticides because of their generally low environ-mental pollution, low toxicity to humans, and other advan-tages (Liu et al. 2000).

The effects of the tested extract, adult emergence, andadulticidal activity of the mosquitoes are remarkably greaterthan those reported for other plant extracts in the literature.For example, at the highest concentration, 50 % inhibitionof the emergence of the adult mosquitoes was observed bythe use of the ethyl acetate fractions of Calophyllum ino-phyllum seed and leaf, Solanum suratense, and Samaderaindica leaf extracts and the petrol ether fraction of Rhino-canthus nasutus leaf extract on C. quinquefasciatus, A.stephensi, and A. aegypti (Muthukrishnan and Puspalatha2001). Similarly 88 % of the adult mortality was observedby the use of Pelargonium citrosa leaf extracts at 2 %concentration against A. stephensi (Jeyabalan et al. 2003).A similar result was obtained in the root extract of Valerianajatamansi which exhibited adulticidal activity of 90 % lethalconcentration against adult A. stephensi, A. culicifacies, A.aegypti, A. albopictus, and C. quinquefasciatus and were0.14, 0.16, 0.09, 0.08, and 0.17 and 0.24, 0.34, 0.25, 0.21,and 0.28 mg/cm2, respectively (Dua et al. 2008).

Nathan et al. (2005) considered pure limonoids of neemseed, testing for biological, larvicidal, pupicidal, adulticidal,and antiovipositional activities of A. stephensi, and larvalmortality was dose-dependent with the highest dose of1 ppm azadirachtin evoking almost 100 % mortality, affect-ing pupicidal and adulticidal activities and significantlydecreased fecundity and longevity of A. stephensi. The high-est adulticidal effect was established from Piper sarmento-sum, followed by Piper ribesoides, and Piper longum, withLD50 values of 0.14, 0.15, and 0.26 μg/female, respectively(Choochote et al. 2006). This result is also comparable toearlier reports of Dua et al. (2010) who observed that theadulticidal activity of the essential oil of L. camara wasevaluated against different mosquito species on 0.208 mg/cm2

impregnated papers, and the KDT50 and KDT90 values of theessential oil were 20, 18, 15, 12, and 14 min and 35, 28, 25, 18,and 23 min against A. aegypti, C. quinquefasciatus, A. culicif-acies, A. fluvialitis, and A. stephensi with percent mortality of93.3 %, 95.2 %, 100 %, 100 %, and 100 %, respectively. Adultmortality was found in methanol extract of A. paniculataagainst the adults of C. quinquefasciatus and A. aegypti withthe LC50 and LC90 values were 149.81 and 172.37 ppm, and

329.

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Hexane Chloroform Benzene Acetone Methanol

Aedes aegypti

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LC90

338.

81

575.

77

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513.

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Culex quinquefasciatus

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Fig 1 Graph of the adulticidal activity of crude plant extracts againstmosquito vectors of the LC50 and LC90 values of Anopheles stephensi(a), Aedes aegypti (b), and Culex quinquefasciatus (c)

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288.12 and 321.01 ppm, respectively (Govindarajan andSivakumar 2012). In the present results, the LC50 and LC90

values of C. tora leaf extracts against adulticidal activity of(hexane, chloroform benzene, acetone, and methanol) C. quin-quefasciatus, A. aegypti, and A. stephensi were the following:C. quinquefasciatus LC50 values were 338.81, 315.73, 296.13,279.23, and 261.03 ppm and LC90 values were 575.77, 539.31,513.99, 497.06, and 476.03 ppm; A. aegypti LC50 values were329.82, 307.31, 287.15, 269.57, and 252.03 ppm and LC90

values were 563.24, 528.33, 496.92, 477.61, and 448.05 ppm;and A. stephensi LC50 values were 317.28, 300.30, 277.51,263.35, and 251.43 ppm and LC90 values were 538.22, 512.90,483.78, 461.08, and 430.70 ppm, respectively.

A large number of synthetic chemical have been testedfor their repellent activity against mosquitoes. However, theprohibitive retail cost of proprietary formulations of chem-icals like DEET restricts their usage by the poor in countriessuch as India. Hence, the search for a safer, better, andcheaper repellent is an ongoing effort. Since cost is animportant factor, investigation on the use of local plantsas repellents is strongly recommended (Curtis 1990). Theessential oil of Tagetes minuta, providing a repellency of90 % protection for 2 h against A. stephensi, C. quinque-fasciatus, and A. aegypti was reported by Tyagi et al. (1994).Venkatachalam and Jebanesan (2001) have also reported thatthe repellent activity of methanol extract of Ferronia elephan-tum leaves against A. aegypti activity at 1.0 and 2.5 mg/cm2

concentrations gave 100 % protection up to 2.14±0.16 h and4.00±0.24 h, respectively, and the total percentage protection

was 45.8 % at 1.0 mg/cm2 and 59.0 % at 2.5 mg/cm2 for 10 h.It was found that a CO2 extract of the seeds of the Mediterra-nean plant Vitex agnus castus can be used as a spray to keepaway insects, especially Ixodes ricinus and Rhipicephalussanguineus ticks, from animals and humans for at least 6 h.In addition, mosquitoes, biting flies, and fleas are also repelledfor about 6 h (Mehlhorn et al. 2005). Larvicidal, ovicidal, andrepellent activities of the leaf extract of C. maxima plantsagainst C. quinquefasciatus (Mullai and Jebanesan 2007)have also been observed.

There are few published studies using essential oils orplant derivatives for repellency against mosquitoes. Manydifferent techniques have been used to measure mosquitorepellency. Besides different techniques, there are differen-ces in biting pressure in a mosquito population which isanother factor affecting repellency testing (Barnard et al.1998). R. officinalis was effective in terms of repellencetime against A. aegypti in concentrations as low as 12.5 %.These results are consistent with reports of insect repellentproperties of rosemary, including C. pipiens pallens (Choi etal. 2002). The mosquito sensitivity to repellents variesamong Aedes, Anopheles, and Culex mosquitoes (Amerand Mehlhorn 2006). A large number of plant extracts havebeen reported to have mosquitocidal or repellent activityagainst mosquito vectors, but very few plant products haveshown practical utility for mosquito control. Some of theplants that have been tested against mosquito larvae in Indiaare Cleome viscosa, Ocimum basilicum, Vitex negundo,Delonix regia, Oligo chaetaramosa, Azadirachta indica,

Table 4 Repellency of different solvent extracts of Cassia tora against Culex quinquefacistus

Solvents Concentration (mg/cm2) % of repellency±SD

Time post-application of repellent (min)

15 30 60 90 120 150 180 210

Hexane 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 64.6±1.3 63.4±1.3 51.2±1.5

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 71.2±1.5 66.8±1.1 60.6±1.2

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 89.7±1.0 71.9±1.5 68.7±1.7

Chloroform 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 69.5±1.3 65.6±1.0 55.4±1.3

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 79.4±1.8 71.7±1.2 63.1±1.2

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 92.1±1.2 79.4±1.7 74.8±1.9

Benzene 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 72.8±1.9 67.5±0.4 59.7±0.7

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 84.6±1.1 76.2±1.9 65.2±1.5

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 94.7±1.7 83.8±0.8 69.3±0.9

Acetone 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 78.3±1.4 73.4±1.2 63.5±1.2

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 87.5±1.6 80.7±1.6 70.9±0.4

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 96.8±1.4 89.1±1.0 81.2±2.1

Methanol 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 85.9±1.2 78.9±1.1 69.1±1.6

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 91.6±1.0 87.3±1.7 73.8±1.5

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 98.5±1.5 93.5±1.3 86.3±1.9

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Quassia amara, Anacardium occidentale, Thevetianeriifolia, etc. Natural products are preferred because of theirbiodegradability and lesser toxicity compared to the synthet-ic ones (Datta et al. 2010).

Benzene and methanol extracts of Artemisia vulgarishave been reported to have repellent activity against A.

aegypti 17 (Yit et al. 1985). Neem products are good mos-quito repellents showing 90 to 100 % protection againstmalaria vectors and about 70 % against C. quinquefasciatus(Sharma and Ansari 1994). One controlled study evaluatedthe efficacy of a cream formulation containing 5 % neem oilagainst C. quinguefasciatus and A. culicifacies. Four to five

Table 6 Repellency of different solvent extracts of Cassia tora against Anopheles stephensi



15 30 60 90 120 150 180 210

Hexane 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 71.2±1.4 63.4±1.3 56.5±1.3

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 75.8±1.7 67.6±1.2 60.8±1.9

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 98.3±1.1 78.8±1.4 75.9±1.4

Chloroform 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 76.6±1.5 66.3±1.0 61.5±1.0

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 82.4±1.8 73.7±1.2 66.3±1.2

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±1.5 82.4±1.7 79.8±1.4

Benzene 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 81.2±1.3 70.2±0.4 66.7±0.6

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 86.5±1.1 75.9±1.0 70.2±1.0

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.7 85.8±0.8 83.3±0.8

Acetone 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 88.6±1.4 74.5±1.1 70.4±1.1

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 92.5±1.6 79.9±0.6 74.8±0.6

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±1.9 89.6±2.0 85.3±2.0

Methanol 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 93.7±1.0 82.3±1.9 73.2±1.3

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 97.9±1.1 88.8±1.7 77.5±1.4

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±1.3 96.2±1.6 89.6±1.5

Table 5 Repellency of different solvent extracts of Cassia tora against Aedes aegypti



15 30 60 90 120 150 180 210

Hexane 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 68.3±1.6 65.5±1.2 54.6±1.1

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 73.8±1.4 69.8±1.5 63.9±1.3

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 93.5±1.9 74.6±1.3 70.2±1.0

Chloroform 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 73.6±1.3 68.4±1.0 59.8±1.9

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 79.4±1.2 73.7±1.1 64.5±1.2

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 96.8±1.1 81.3±1.6 76.3±1.5

Benzene 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 78.3±1.5 71.9±1.9 62.8±1.7

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 85.6±1.6 78.1±1.7 68.7±1.3

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 98.9±1.3 86.8±1.8 82.3±1.6

Acetone 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 87.6±1.7 75.3±1.1 66.5±1.5

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 89.4±1.4 84.6±1.5 72.9±0.9

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±1.1 92.5±2.0 83.3±2.4

Methanol 1.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 90.7±1.9 81.5±1.3 70.4±1.0

2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 94.3±1.3 92.4±1.7 76.8±1.5

5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±1.5 94.8±1.8 87.6±1.3

Parasitol Res


grams of the cream was applied to the exposed skin areas ofhuman volunteers in Ghaziabad, India in the summermonths of May/June and the monsoon months of August/September. Neem cream was found to offer 82 % protectionagainst Culex bites and 100 % protection against Anophelesbites, as compared to untreated controls (Nagpal et al.2001). The ethanolic extracts of the orange peel (C. sinensis)was tested for the toxicity effect on the larvae of the yellowfever mosquito A. aegypti (Amusan et al. 2005).

Ansari et al. (2000) reported that the peppermint oil gave94.1% protection for 6 h, while mylol oil gave 95% protectionfor 7.2 h. They also reported that mylol oil and peppermint oilgave 100 % protection for 11 h against A. annularis. Mylol oilgave 95.4 % protection for 8.7 h for the Anopheline species,whereas the peppermint oil gave 86.3 % protection for 8 h.Phukan and Kalita (2005) showed that Litsea salicifoliarecorded 70 % and 50 % repellency for 3 and 4 h, respectively,againstA. aegypti, but they failed to showmuch activity againstC. quinquefasciatus. Mullai et al. (2008) have also reported thatthe skin repellent test at 1.0, 2.5, and 5.0 mg/cm2 concentra-tions gave the mean complete protection time range from119.17 to 387.83 min against A. stephensi with the benzene,petroleum ether, ethyl acetate, and methanol extracts ofCitrullus vulgaris tested. Our results showed that hexane,chloroform, benzene, acetone, and methanol extracts ofC. tora have significant repellent activity against C. quinque-fasciatus, A. aegypti, and A. stephensi mosquitoes. The high-est repellency of 210 min was observed in methanol extract ofC. tora against A. stephensi followed by A. aegypti andC. quinquefasciatus.

Das et al. (2000) evaluated the repellent properties of Zan-thoxylum armatum, Zanthoxylum alatum, C. aromatica, andAcalypha indica against mosquitoes in two bases of mustard oiland coconut oil. The active components, dymalol, nymania-3,and triterpenes, isolated from the extract of Dysoxylum mala-baricum act as an oviposition repellent and/or deterrent to A.stephensi (Govindachari et al. 1999). Certain natural productshave been investigated for repellent activity against mosqui-toes. Z. armatum, DC. syn. Z. alatum Roxb. (Rutaceae); Aza-dirachta indica (Maliaceae) and C. aromatica (Zingiberaceae)were among them and have been reported to possess repellentproperties against mosquitoes (Das et al. 2000). The repellentactivity of constituents identified in Foeniculum vulgare fruitagainst A. aegypti was studied by Hyoung et al. (2002).Rajkumar and Jebanesan (2002) reported that knocking downand killing effect of Solanum aerianthum exhibited repellentactivity against C. quinquefasciatus. The larvicidal, ovicidal,and repellent activities of crude benzene and ethyl acetateextracts of leaf of Ervatamia coronaria and Caesalpinia pul-cherrimawere assayed for their toxicity against three importantvector mosquitos (Govindarajan et al. 2011).

In conclusion, an attempt has been made to evaluate therole of C. tora leaf extracts for their adulticidal and repellent

bioassay against C. quinquefasciatus, A. aegypti, and A.stephensi as target species. The results reported in this studyopen the possibility for further investigations of the efficacyof adulticidal and repellent properties of natural productextracts. These are environmentally safe and eco-friendlyapproaches for the vector control programs.

Acknowledgments We thank Dr. K. Sasikala, Professor and Head,Department of Zoology, Bharathiar University for the laboratory facil-ities provided. The authors are grateful to Mr. N. Muthukrishnan,technician, and Mr. A. Anbarasan, lab assistant, National Centre forDiseases Control (NCDC), Mettupalayam, Tamil Nadu for their help inmosquito collection and for the mosquito samples provided for thepresent work.

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