Parasitol Res (2008) 102:867873 DOI 10.

1007/s00436-007-0839-6

ORIGINAL PAPER

Larvicidal activity of some Euphorbiaceae plant extracts against Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae)
A. Abdul Rahuman & Geetha Gopalakrishnan & P. Venkatesan & Kannappan Geetha

Received: 16 November 2007 / Accepted: 30 November 2007 / Published online: 29 December 2007 # Springer-Verlag 2007

Abstract Larvicidal activity of ethyl acetate, butanol, and petroleum ether extracts of five species of Euphorbiaceae plants, Jatropha curcas, Pedilanthus tithymaloides, Phyllanthus amarus, Euphorbia hirta, and Euphorbia tirucalli, were tested against the early fourth instar larvae of Aedes aegypti L. and Culex quinquefasciatus (Say). The larval mortality was observed after 24 h of exposure. All extracts showed low larvicidal effects; however, the highest larval mortality was found in petroleum ether extract. The LC50 value of petroleum ether extracts of J. curcas, P. tithymaloides, P. amarus, E. hirta, and E. tirucalli were 8.79, 55.26, 90.92, 272.36, and 4.25 ppm, respectively, against A. aegypti and 11.34, 76.61, 113.40, 424.94, and 5.52 ppm, respectively, against C quinquefasciatus. Of the various ratios tested, the petroleum ether extracts of J. curcas and E. tirucalli were observed to be more efficient than the other plant extracts. It is, therefore, suggested that E. tirucalli can be applied as an ideal potential larvicide against A.
A. A. Rahuman (*) Unit of Bioactive Natural Products, Department of Zoology, C. Abdul Hakeem College, Melvisharam 632 509, India e-mail: abdulrahuman6@hotmail.com G. Gopalakrishnan Center for Natural Products, SPIC Science Foundation, 64, Mount Road, Chennai 600 032, India P. Venkatesan Department of Zoology, Loyola College, Chennai 600 032, India K. Geetha Department of Chemistry, Muthurangam Government Arts College, Vellore 632 002, India

aegypti and C. quinquefasciatus. This is an ideal ecofriendly approach for the control of the dengue vector, A. aegypti, and the lymphatic filariasis vector, C. quinquefasciatus.

Introduction Mosquito-transmitted diseases remain a major cause of the loss of human life worldwide with more than 700 million people suffering from these diseases annually (Taubes 1997). Mosquito-borne diseases have an economic impact, including loss in commercial and labor outputs, particularly in countries with tropical and subtropical climates; however, no part of the world is free from vector-borne diseases (Fradin and Day 2002). Aedes aegypti, a vector of dengue is widely distributed in tropical and subtropical zones. Dengue fever incidence has increased fourfold since 1970 and nearly half the worlds population is now at risk. In 1990, almost 30% of the world population, 1.5 billion people, lived in regions where the estimated risk of dengue transmission was greater than 50% (Hales et al. 2002). An outbreak of chikungunya virus infection emerged in the southwest Indian Ocean islands in 2005, spread out to India, and resulted in an ongoing outbreak that has involved >1.5 million patients, including travelers who have visited these areas (Taubitz et al. 2007). Culex quinquefasciatus, a vector of lymphatic filariasis, is widely distributed in tropical zones with around 120 million people infected worldwide and 44 million people having common chronic manifestation (Bernhard et al. 2003). Despite its debilitating effects, lymphatic filariasis is given a very low control priority (Ramaiah et al. 2000). A number of workers have used plant products in the control of various mosquito species. The petroleum ether extracts of Rhinacanthus nasutus, Derris elliptica, Trigonostemon reidioides, Homalomena aromatica, Stemona tuberose, and

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Acorus calamus (Komalamisra et al. 2005); Piper nigrum (Rasheed et al. 2005; Siddiqui et al. 2004); Chenopodium album and Sonchus oleraceus (Sharma et al. 2006); Argemone mexicana (Sakthivadivel and Thilagavathy 2003); Solanum xanthocarpum (Mohan et al. 2005); Ajuga remota (Sharma et al. 2004); Thymus capitatus (Mansour et al. 2000); Vitex negundo, Nerium oleander, and Syzygium jambolanum (Pushpalatha and Muthukrishnan 1995); Murraya koenigii, Coriandrum sativum, Ferula asafetida, and Trigonella foenum (Harve and Kamath 2004); and Eichhornia crassipes, Ageratum conyzoides, Cleome icosandra, Tagetes erectes, and Tridax procumbens (Saxena et al. 1992) have been tested against the larvae of A. aegypti and C. quinquefasciatus. The genus Euphorbia (Euphorbiaceae) is chemically defined by the occurrence of a large number of polyfunctional diterpenoids with the tigliane (phorbol), ingenane, and daphnane skeletons (Evans and Soper 1978); lectins; and lysozymes with recognized biological properties (Wititsuwannakul et al. 1998; OKeefe 2001). Most of these are skin irritants and toxic; in addition, many of them are skin tumor promoters. Nonirritant polyfunctional macrocyclic diterpenoids with the lathyrane and jatrophane skeletons have also been isolated from the Euphorbia species. Jatropha curcas is a shrub small tree that grows up to 15 ft high. It is known as Jungli erand in Hindi and Katalamanakku by the local people. It is used in traditional medicine for fevers, venereal diseases, dysentery (Iwu 1993), wounds (Diallo et al. 2002), and inhibits Staphylococcus aureus and Escherichia coli (Adamu et al. 2005); the seeds of J. curcas are a valuable source of biodiesel in Asian countries (Heller 1996). The extracts showed nematicidal, fungicidal (Sharma and Trivedi 2002), antifeedant (Meshram et al. 1996), molluscicidal (Liu et al. 1997), and abortifacient activities (Goonasekara et al. 1995) against white flies (B. tabaci), fourth instar mosquito (Ochlerototatus triseriatus) larvae, and neonates of H. zea and H. virescens (Georges et al. 2007), and exhibited insecticidal activities against moths, butterflies, aphids, bugs, beetles, flies, and cockroaches (Wink et al. 1997). Pedilanthus tithymaloides (L.) Poit. is a shrub with smooth green fleshy stems, which grow up to 6 ft tall with milky sap. The local people call it Ladakaa in Oriya and Kandai kalli in Tamil, which grows profusely in marginal wasteland in northern and eastern India without any agricultural management. It is reported to have an antiprotozoa (Luize et al. 2003), mitogenic (Seshagirirao 1995), antiinflammatory (Dhar et al. 1988; Abreu et al. 2006), and antimicrobial (Vidotti et al. 2006) activities minimizing nematode damage to mushrooms (Rao et al. 1991). Phyllanthus amarus Schum. & Thonn. is an annual, glabrous herb that grows to between 30 and 60 cm. Known as Kilanelli by local people, it is found in tropical areas, in subtropical regions, and is usually quite scattered in its

distribution (Unander et al. 1990). Fresh leaves are ground and mixed with a cup of cow or goats milk and taken internally to cure jaundice. The aerial parts of the herb have been widely used in folk medicine in India and other tropical countries for the treatment of various diseases and disorders, such as jaundice, diarrhea, ringworm, ulcers, malaria, genitourinary infections, hemorrhoids, and gonorrhea (Unander et al. 1991). The extracts were found to possess antiviral property against hepatitis B virus (HBV) (Thyagarajan et al. 1988; Yeh et al. 1993) and antitumor, anticarcinogenic, and antiinflammatory properties (Kiemer et al. 2003; Rajeshkumar et al. 2002). Euphorbia hirta L. is a medicinal, rhizomatous herb distributed in Southern Western Ghats of India and Northern East Coast of Tamil Nadu. The local people know the plant as Amman pachcharsi. The plant has been reported to contain quercitrin (Glvez et al. 1993) and polyphenols (Chen 1991). The extracts were reported as anthelmintic (Iwu 1993), repellent, antifeedant and controlling Plutella xylostella (Wei et al. 2005, 2004) and Rotylenchulus reniformis (Inserra et al. 1989), antimicrobial (Adamu et al. 2005), antibacterial, and against worms (Parekh and Chanda 2007). Euphorbia tirucalli is a small shrub erect tree, 36 m high with thin, cylindrical, and spreading branches locally known as Thirugu kalli or Kodikalli; pencil tree in English; Khaadi Siju in Oriya. It is typically used as fence plants as stray animals and cattle do not eat them. It is reported to control Lymnaea acuminata (Tiwari and Singh 2005), activate the EpsteinBarr virus (MacNeil et al. 2003), larvicidal (Yadav et al. 2002), antibacterial (Parekh and Chanda 2007), anticancer, heal wounds, and toxic against Artemia salinai (Brasileiro et al. 2006). Synthetic insecticides have created a number of ecological problems, such as the development of resistant insect strains, ecological imbalance, and harm to mammals. Hence, there is a constant need for developing biologically active plant materials as larvicides, which are expected to reduce the hazards to human and other organisms by minimizing the accumulation of harmful residues in the environment. Natural products are generally preferred because of their less harmful nature to nontarget organisms and due to their innate biodegradability. As far as our literature survey could ascertain, no information was available on the larvicidal activities of the Phyllanthus species given here. Therefore, the aim of this study was to investigate the mosquito larvicidal activity of the petroleum ether extracts of five plant species from India. The present study was an attempt to assess the larvicidal activity of leaf and stem bark extracts against A. aegypti and C. quinquefasciatus. This is the first report on the mosquito larvicidal activity of the petroleum ether extract of Euphorbiaceae plants.

Parasitol Res (2008) 102:867873

Materials

869

and methods

Mosquito culture A. aegypti and C. quinquefasciatus colonies were maintained in our insectary (454540 cm) at 272C and 80 2% RH with a photoperiod of 14:10-h light and dark cycles as per the procedure of Sharma and Saxena (1994). The egg strips were obtained from Zonal Entomological Research Centre, Vellore (12 55 48 N, 79 7 48 E) to start the colony. The strips were immersed in dechlorinated tap water for hatching. Larvae were fed with a diet of finely ground brewer yeast and dog biscuits (3:1). The adults were given a blood meal from a pigeon (Reuben 1987). Glass Petri dishes with 50 ml of tap water lined with filter paper was kept inside the cage for oviposition. Plant materials The leaf of J. curcas L., P. tithymaloides (L.) Poit., P. amarus Schum.& Thonn. (syn. P. niruri auct. non L.), and E. hirta L., and stem bark of E. tirucalli L. were collected from Javadhu Hills, Tiruvannamalai district (12 36 10 N, 078 53 07 E, altitude 705 m), Tamil Nadu in August 2004 and was authenticated by Dr. B. Annadurai, Department of Plant Biology and Biotechnology, C. Abdul Hakeem College, Melvisharam, Vellore, India. The voucher specimens have been deposited in the zoology laboratory. Preparation of plant extracts The dried leaf (350 g) and the stem bark (400 g) were powdered mechanically using a commercial electrical stainless steel blender and extracted with ethyl acetate (Qualigens), butanol (Qualigens), petroleum ether (6080C, Qualigens) in a soxhlet apparatus (1,700 ml) separately until exhaustion (Irungu and Mwangi 1995). The extract was concentrated under reduced pressure 2226 mg Hg at 45C, and the residue obtained was stored at 4C. Larvicidal bioassay One gram of crude extract was first dissolved in 100 ml of petroleum ether (stock solution). From the stock solution, different concentrations ranging from 0.94 to 600 ppm were prepared with dechlorinated tap water. Polysorbate 80 (Qualigens) was used as an emulsifier at the concentration of 0.05%. Experiments were conducted for 24 h at room temperature (282C). The larvicidal activity was assessed by the procedure of the WHO (1996) with some modifications and as per the method of Rahuman et al. (2000). For bioassay test, larvae were taken in 5 batches of 20 in 249 ml of water and 1.0 ml of the desired plant extract

concentration. The control was set up with petroleum ether and polysorbate 80. The numbers of dead larvae were counted after 24 h of exposure, and the percentage mortality was reported from the average of five replicates. Statistical analysis The average larval mortality data were subjected to probit analysis for calculating LC50, LC90, and other statistics at 95% fiducial limits of the upper confidence limit (UCL) and lower confidence limit (LCL), and chi-square values were calculated by using the software developed by Reddy et al. (1992).

Results and discussion The ethyl acetate and butanol extracts showed very low larvicidal effects; however, the highest larval mortality was found in the petroleum ether extract. The larvicidal activity of the petroleum ether extracts of five species of Euphorbiaceae plants are noted and presented in Table 1 and Fig. 1. Among the extracts tested, the extracts of J. curcas and E. tirucalli were highly effective against the larvae of A. aegypti (LC50 = 8.79 and 4.25 ppm) and against C. quinquefasciatus (LC50 = 11.34 and5.52 ppm). The LC90 values were 35.39, 256.77, 384.19, 703.76, and 13.14 ppm against A. aegypti and 46.52, 307.07, 465.28, 1,314.01, and 25.67 ppm against C. quinquefasciatus for J. curcas, P. tithymaloides, P. amarus, E. hirta, and E. tirucalli, respectively. Chi-square value was significant at the P<0.05 level on A. aegypti and C. quinquefasciatus larvae. Yadav et al. (2002) have reported the methanolic, chloroform, and ether extracts of E. tirucalli latex and stem bark were evaluated for larvicidal activity against laboratory-reared larvae of C. quinquefasciatus. In our observation, the stem bark petroleum ether extract of E. tirucalli possessed higher activity than the other solvent extracts. The larvicidal activity of the essential oil aqueous solutions of the stalks and leaves of Croton argyrophylloides, Croton nepetaefolius, Croton sonderianus, and Croton zehntneri showed 100% mortality at 50 ml against A. aegypti (Lima et al. 2006); the oil from Cinnamomum camphora, Boswellia carteri, Anethum graveolens, and Myrtus communis showed 100% mortality at 50 ppm within 3 h against the third instar larvae of A. aegypti (Amer and Mehlhorn 2006). The LC50 values after a 12-h contact with 13 selected oils observed against the third instar larvae of A. aegypti ranged from 1 to 101.4 ppm and the LC50 values for C. quinquefasciatus larvae ranged between 1 and 50.2 ppm (Amer and Mehlhorn 2006). Morais et al. (2006) also reported that the main components methyleugenol and alpha-copaene for C. nepetaefolius (LC50 of 84 ppm); alpha-pinene and beta-pinene for C. argyrophyloides (LC50

870 Table 1 Larvicidal activity of petroleum ether extracts against A. aegypti L. and C. quinquefasciatus Say Name of the plants Jatropha curcas L. Pedilanthus tithymaloides (L.) Poit. Phyllanthus amarus Schum. & Thonn. Euphorbia hirta L. Euphorbia tirucalli L. Mosquito species Aedes aegypti L. Culex quinquefasciatus Aedes aegypti L. Culex quinquefasciatus Aedes aegypti L. Culex quinquefasciatus Aedes aegypti L. Culex quinquefasciatus Aedes aegypti L. Culex quinquefasciatus LC50 (ppm) (UCLLCL) 8.79 (9.927.59) 11.34 (12.859.82) 55.26 (62.8347.68) 76.61 (90.8662.35) 90.92 (115.6080.23) 113.40 (128.5498.26) 272.36 (312.44231.77) 424.94 (473.74376.13) 4.25 (4.733.76) 5.52 (6.284.768)

Parasitol Res (2008) 102:867873

LC90 (ppm) (UCLLCL) 35.39 (42.9427.27) 46.52 (56.9136.12) 256.77 (316.61196.93) 307.07 (402.72211.42) 384.19 (489.82258.55) 465.28 (569.18361.38) 703.76 (874.98532.54) 1314.01 (1551.121076.81) 13.14 (15.5110.76) 25.67 (31.6619.69)

c 2 (df=5) 3.51 7.06 8.54 3.47 5.15 7.26 4.16 2.75 3.46 8.53

Say Say Say Say Say

The control has nil mortality. Values were significant at the P<0.05 level. LC50: lethal concentration that kills 50% of the exposed larvae, LC90: lethal concentration that kills 90% of the exposed larvae, UCL: upper confidence limit (95% fiducial limit), LCL: lower confidence limit (95% fiducial limit), 2 : chi-square, df: degrees of freedom

of 102 ppm); and alpha-pinene, beta-phelandrene, and transcaryophyllene for C. sonderianus (LC50 of 104 ppm) and Croton zenhtneri exhibited higher larvicidal activity with an LC50 of 28 ppm against A. aegypti. The toxicity of Euphorbia milii molluscicidal latex and niclosamide showed toxic affect to Anopheles albitarsis, A. aegypti, and Aedes fluviatilis larvae (Filho and Paumgartten 2000). Earlier authors reported that the petroleum ether extract of Rhinacanthus nasutus possessed larvicidal effects with LC50
Fig. 1 Graph showing the LC50 and LC90 values of mosquito vectors a A. aegypti and b C. quinquefasciatus
Doses(ppm)

values between 3.9 and 11.5 mg/l and Derris elliptica showed LC50 values between 11.2 and 18.84 mg/l against A. aegypti, C. quinquefasciatus, Anopheles dirus, and Mansonia uniformis (Komalamisra et al. 2005). The same extracts of A. mexicana, J. curcus, P. extensa, and W. sornnifera showed acute toxicity causing 100% mortality at 1,000, 500, and 250 ppm, respectively, against C. quinquefasciatus larvae (Karmegam et al. 1997). Solanum xanthocarpum fruits petroleum ether extract was observed as the most toxic with
LC50 LC90
703.76
384.19
256.77

1000 800 600 400

8.79

a
2000

J.curcas

P.tithymaloides

P.amarus

E.hirta

E.tirucalli

LC50

LC90

1500
Doses(ppm)

1000
465.28

46.52

76.61

113.4

500
11.34

307.07

424.94

1314.01

4.25

J.curcas

P.tithymaloides

P.amarus

E.hirta

E.tirucalli

5.52

25.67

13.14

200

35.39

55.26

90.92

272.36

Parasitol Res (2008) 102:867873

871 Amer A, Mehlhorn H (2006) Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae). Parasitol Res 99:466472 Bernhard L, Bernhard P, Magnussen P (2003) Management of patients with lymphoedema caused by filariasis in North-eastern Tanzania: alternative approaches. Physiotherapy 89:743749 Bigi MF, Torkomian VL, de Groote ST, Hebling MJ, Bueno OC, Pagnocca FC, Fernandes JB, Vieira PC, da Silva MF (2004) Activity of Ricinus communis (Euphorbiaceae) and ricinine against the leafcutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae) and the symbiotic fungus Leucoagaricus gongylophorus. Pest Manag Sci 60(9):933938 Brasileiro BG, Pizziolo VR, Raslan DS, Jamal CM, Silveira D (2006) Antimicrobial and cytotoxic activities screening of some Brazilian medicinal plants used in Governador Valadares district. RBCF Rev Bras Cienc Farm 42(2):195202 Chen L (1991) Polyphenols from leaves of Euphorbia hirta L. Zhongguo Zhongyao Zazhi 16(1):3839 Civelek HS, Weintraub PG (2004) Effects of two plant extracts on larval leafminer Liriomyza trifolii (Diptera: Agromyzidae) in tomatoes. J Econ Entomol 97(5):15816 Dhar SN, Ray SM, Roy A, Dutta SK (1988) Oral anti-inflammatory activity of pedilanthaina new proteolytic enzyme from Pedilanthus tithymaloides. Indian J Pharm Sci 50:281283 Diallo D, Sogn C, Samake FB, Paulsen BS, Michaelsen TE, Keita A (2002) Wound healing plants in Mali, the Bamako region. An ethnobotanical survey and complement fixation of water extracts from selected plants. Pharm Biol 40:117128 Evans FJ, Soper CJ (1978) The tigliane, daphnane and ingenane diterpenes, their chemistry, distribution and biological activities. A review. Lloydia 41:193233 Filho ECO, Paumgartten FJ (2000) Toxicity of Euphorbia milii latex and niclosamide to snails and nontarget aquatic species. Ecotoxicol Environ Saf 46(3):342350 Fradin MS, Day JF (2002) Comparative efficacy of insect repellents against mosquitoes bites. N Engl J Med 347:1318 Glvez J, Crespo ME, Jimnez J, Surez A, Zarzuelo A (1993) Antidiarrhoeic activity of quercitrin in mice and rats. J Pharm Pharmacol 45(2):157159 Georges K, Jayaprakasam B, Dalavoy SS, Nair MG (2007) Pestmanaging activities of plant extracts and anthraquinones from Cassia nigricans from Burkina Faso. Bioresour Technol (in press) Goonasekara MM, Gunawardhana VK, Jayaseana K, Mohammed SG, Balasubramaniam S (1995) Pregnancy terminating effect of Jatropha curcas in rats. J. Ethnopharmacol 47:117123 Hales S, Wet ND, Maindonald J, Woodward A (2002) Potential effect of population and climate changes on global distribution of dengue fever: an empirical model. Lancet 360:830834 Harve G, Kamath V (2004) Larvicidal activity of plant extracts used alone and in combination with known synthetic larvicidal agents against Aedes aegypti. Indian J Exp Biol 42(12):12161219 Heller J (1996) Promoting the conservation and use of under utilized and neglected crops. 1. Physic nut: Jatropha curcas L. International Plant Genetic Resources Institute, Rome Inserra RN, Dunn RAM, Sorley R, Langdow KR, Richmer AY (1989) Weed hosts of Rotylenchulus reniformis in ornamental nurseries of S. Florida. Weed Abstr 39:444 Irungu LW, Mwangi RW (1995) Effects of a biologically active fraction from Melia volkensii on Culex quinquefasciatus. Insect Sci Appl 16:159162 Iwu MM (1993) Handbook of African medicinal plants. CRC, Boca Raton, FL, pp 2433 Karmegam N, Sakthivadivel M, Anuradha V, Daniel T (1997) Indigenous-plant extracts as larvicidal agents against Culex quinquefasciatus Say. Bioresour Technol 59(23):137140

LC50 values of 62.62 ppm after 24 h and 59.45 ppm after 48 h of exposure period against the larvae of C. quinquefasciatus (Mohan et al. 2005). In the present study, P. tithymaloides, P. amarus, and E. hirta exhibited a moderate larvicidal activity against A. aegypti (LC50 =55.26, 90.92, and 272.36 ppm) and C. quinquefasciatus (LC50 =76.61, 113.40, and 424.94 ppm). Our results showed that the extracts of J. curcas and E. tirucalli were highly effective against the larvae of A. aegypti (LC50 =8.79 and 4.25 ppm) and against C. quinquefasciatus (LC50 =11.34 and 5.52 ppm). Euphorbiaceae species are commonly used in traditional medicine and has been reported to possess various biological activities. Aqueous extracts of Euphorbia myrsinites L. was tested for their insecticidal activity against the leafminer Liriomyza trifolii (Civelek and Weintraub 2004), the ethanolic extract of aerial and root parts of P. amarus showed insecticidal activity against stored grain pest Tribolium castaneum (Khanna et al. 2003), aqueous leaf extract of Ricinus communis L. showed excellent insecticidal activity against Callosobruchus chinensis L. (Upasani et al. 2003) and against the bug Eurystylus oldi (Ratnadass et al. 2001). Bigi et al. (2004) tested the isolated compounds, fatty acids, and ricinine from leaf extracts of R. communis L. against the leaf-cutting ant Atta sexdens rubropilosa Forel and its symbiotic fungus Leucoagaricus gongylophorus (Singer) Mller. In conclusion, an attempt has been made to evaluate the role of plant extracts in mosquito larvicidal activity. The results reported in this study open the possibility of further investigations on the efficacy of the larvicidal properties of natural product extracts. The isolation and purification of the crude extract of J. curcas and E. tirucalli are in progress.
Acknowledgements The authors are grateful to the C. Abdul Hakeem College Management, Prof. U. Peer, Principal and Dr. Ahmed Najib, HOD of the Zoology Department for their help and suggestions. We wish to thank the Principal and the HOD of the Zoology Department, Loyola College, Chennai for providing the necessary facilities for our experimental work. We are thankful to Dr. S. Narasimhan, Associate Director, SPIC Science Foundation, Chennai for his help and encouragement. AR is indebted to the University Grants Commission, New Delhi for the award of fellowship.

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