Adulticidal and larvicidal efficacy of some medicinal plant extracts against tick, fluke and mosquitoes

Veterinary Parasitology 166 (2009) 286–292

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Adulticidal and larvicidal efficacy of some medicinal plant extracts against tick, fluke and mosquitoes A. Bagavan, C. Kamaraj, G. Elango, A. Abduz Zahir, A. Abdul Rahuman * Unit of Bioactive Natural Products, P.G. & Research Department of Zoology, C. Abdul Hakeem College, Melvisharam - 632 509, Vellore District, Tamil Nadu, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 May 2009 Received in revised form 6 August 2009 Accepted 15 September 2009

The adulticidal and larvicidal effect of indigenous plant extracts were investigated against the adult cattle tick Haemaphysalis bispinosa Neumann, 1897 (Acarina: Ixodidae), sheep fluke Paramphistomum cervi Zeder, 1790 (Digenea: Paramphistomatidae), fourth instar larvae of malaria vector, Anopheles subpictus Grassi and Japanese encephalitis vector, Culex tritaeniorhynchus Giles (Diptera: Culicidae). The aim of this study was to evaluate the toxic effect of leaf hexane, chloroform, ethyl acetate, acetone and methanol extracts of Annona squamosa L., Centella asiatica (L.) Urban, Gloriosa superba L., Mukia maderaspatensis (L.) M.Roem, Pergularia daemia (Forsk.) Chiov. and Phyllanthus emblica L. were exposed to different concentrations. All plant extracts showed moderate toxic effect on parasites after 24 h of exposure; however, the highest mortality was found in leaf hexane extract of A. squamosa, methanol extracts of G. superba and P. emblica against H. bispinosa (LC50 = 145.39, 225.57 and 256.08 ppm); methanol extracts of C. asiatica, G. superba, P. daemia and P. emblica against P. cervi (LC50 = 77.61, 60.16, 59.61, and 60.60 ppm); acetone, ethyl acetate extracts of A. squamosa, methanol extract of C. asiatica, acetone extracts of G. superba, ethyl acetate, hexane and methanol extracts of P. daemia against A. subpictus (LC50 = 17.48, 18.60, 26.62, 18.43, 34.06, 13.63, and 50.39 ppm); and chloroform, ethyl acetate extracts of A. squamosa, ethyl acetate extract of P. daemia, ethyl acetate and methanol extracts of P. emblica against C. tritaeniorhynchus (LC50 = 63.81, 60.01, 31.94, 69.09, and 54.82 ppm), respectively. These results demonstrate that methanol extracts of C. asiatica, G. superba, P. daemia and P. emblica extracts may serve as parasites control even in their crude form. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Medicinal plant extracts Haemaphysalis bispinosa Paramphistomum cervi Anopheles subpictus Culex tritaeniorhynchus Control

1. Introduction Ticks are currently considered to be second only to mosquitoes as vectors of human infectious diseases in the world. It has been reported that 80% of 1200 million cattle are at risk for ticks and tick-borne diseases causing a global annual loss of US$ 7000 million. About 40% cattle have been found positive for Haemaphysalis bispinosa infestation and detected on 7.6% of examined cattle, 55.4% goats, and 13.2% pigs (van den Broek et al., 2003; Wall, 2007; Ghosh et al., 2007).

* Corresponding author. Tel.: +91 9442310155; fax: +91 04172269487. E-mail address: [email protected] (A.A. Rahuman). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.09.007

Helminthiasis is one of the most important groups of parasitic diseases in India resulting in heavy production losses in livestock. Development of resistance to most of the commercially available anthelmintics became a severe problem worldwide (Waller, 2003). There are 350–500 million clinical cases of malaria per year with about one million deaths. In India around two million malaria cases are being reported annually (Kumar et al., 2007). Japanese encephalitis is the leading cause of viral encephalitis in Asia with 50,000 cases reported annually. Evaluation of the activities of medicinal plants claimed for parasitic property is getting attention these days (Alawa et al., 2003; Gathuma et al., 2004; Iqbal et al., 2004; Pessoa et al., 2002). Extracts or essential oils from plants may be alternative sources of acaricidal, anthelmintic and insecti-

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cidal control agents, since they constitute a rich source of bioactive compounds that are biodegradable into nontoxic products and potentially suitable for use in control of parasites. Annona squamosa, commonly known as custard apple is a native of West Indies and is cultivated throughout India, mainly for its edible fruit. The compound C37 trihydroxy adjacent bistetrahydrofuran acetogenin isolated from ethyl acetate seeds extract of A. squamosa showed anthelmintic activity against Haemonchus contortus (Souza et al., 2008) and the annonaceous acetogenins compounds isolated from the seeds aqueous and organic extracts have been reported to have potent pesticidal and parasiticidal activities (Pardhasaradhi et al., 2005). The larvicidal and mosquitocidal activities of ethanolic water mixture extract of A. squamosa and Centella asiatica (Senthilkumar et al., 2009); the leaves methanolic extract (Jaswanth et al., 2002) and the seeds petroleum ether extract showed larvicidal activity were tested against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti. The ethyl acetate extract of C. asiatica showed antihelmintic properties and antifilarial effects (Chakraborty et al., 1996). The larvicidal activity of crude acetone, hexane, ethyl acetate, methanol, and petroleum ether extracts of the leaf of C. asiatica was assayed for their toxicity against the early fourth instar larvae of C. quinquefasciatus (Rahuman et al., 2008b). The methanol extract of the rhizomes of Gloriosa superba and its subsequent fractions in different solvent systems were screened for antibacterial and antifungal activities (Khan et al., 2008). Pergularia daemia leaf petroleum ether crude extract showed toxic effect against fourth instar larvae of C. quinquefasciatus, A. stephensi and A. aegypti (Sakthivadivel and Danial, 2008). Zahir et al. (2009) have reported that the antiparasitic activities of acetone, chloroform, ethyl acetate, hexane, and methanol dried leaf, flower, and seed extracts of Achyranthes aspera, Anisomeles malabarica, G. superba, Psidium guajava, Ricinus communis, and Solanum trilobatum were tested against the larvae of cattle tick Rhipicephalus microplus, sheep parasite Paramphistomum cervi and fourth instar larvae of Anopheles subpictus and Culex tritaeniorhynchus. The acaricidal activity of oleoresinous extract (oleoresin) from Copaifera reticulata was investigated against R. microplus larvae were exposed to filter paper envelopes impregnated with different oleoresin concentrations (Fernandes and Freitas, 2007). The leaf acetone, chloroform, ethyl acetate, hexane, and methanol extracts of Aegle marmelos, Andrographis lineata, Andrographis paniculata, Cocculus hirsutus, Eclipta prostrata, and Tagetes erecta (Elango et al., 2009); extracts of leaf, flower and seed of Cassia auriculata, Leucas aspera, Rhinacanthus nasutus, Solanum torvum and Vitex negundo (Kamaraj et al., 2009) and the leaf extracts of Citrus sinensis, Ocimum canum, O. sanctum and R. nasutus (Bagavan et al., 2009) were tested against the fourth instar larvae of A. subpictus, C. tritaeniorhynchus and feeding deterrence to nymphs of Aphis gossypii. The aim of this study was to investigate the parasitic activities of the different solvent extracts of six plant species from Tamil Nadu, India. This is the first report on R.

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microplus, P. cervi, A. subpictus and C. tritaeniorhynchus activity of the solvent extracts of selected plants. 2. Materials and methods 2.1. Collection of plant materials The leaf of A. squamosa L. (Annonaceae), C. asiatica (L.) Urban (Umbelliferae) (syn. Hydrocotyle asiatica L.), G. superba L. (Colchicaceae), Mukia maderaspatensis (L.) M.Roem (Cucurbitaceae), P. daemia (Forsk.) Chiov. (Asclepiadaceae) and Phyllanthus emblica L. (Euphorbiaceae) (syn. Emblica officinalis Gaertn) were collected from Javadhu Hills, Tiruvannamalai district (128360 10N, 0788530 07E, altitude 705 m) and Chitheri Hills, Dharmapuri district (118530 28N, 0788300 26E, altitude 959), Tamil Nadu, India in July 2007 and the taxonomic identification was made by Dr. B. Annadurai, Department of Plant Biology and Biotechnology, C. Abdul Hakeem College, Melvisharam, India. The voucher specimen was numbered and kept in our research laboratory for further reference. 2.2. Preparation of plant extracts The leaves were dried for 7–10 days in the shade at the environmental temperatures (27–37 8C day time). The dried leaves were powdered mechanically using commercial electrical stainless steel blender and the powdered leaves (700 g) were extracted with hexane (1600 ml, Fine), chloroform (1700 ml, Fine), ethyl acetate (2400 ml, Qualigens), acetone (1400 ml, Qualigens) and methanol (2800 ml, Qualigens) in a soxhlet apparatus (boiling point range 60–80 8C) for 8 h. The extract was concentrated under reduced pressure 22–26 mm Hg at 45 8C and the residue obtained was stored at 4 8C. One gram of crude extract was first dissolved in 100 ml of acetone (stock solution). The control was set up with acetone and polysorbate 80 (Qualigens). From the stock solution, 2500 and 1000 ppm were prepared with dechlorinated tap water. Polysorbate 80 was used as an emulsifier at the concentration of 0.05% in the final test solution. 2.3. Parasite collection and bioassay The attached adult of H. bispinosa Neumann, 1897 (Acarina: Ixodidae) were collected from inside the ears and very rarely were found elsewhere on the body of cattle. The parasites were identified by Dr. A. Sangaran, Department of Veterinary Parasitology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu. The applied method in the present study to verify the acaricidal activity of different solvent plant extracts against adult of H. bispinosa was developed as per the method of FAO (2004), incorporating slight modifications to improve practicality and efficiency of tested materials (Fernandes, 2001; Fernandes et al., 2005). From the stock solution, 2500 ppm was prepared and a series of filter paper envelopes (Whatman filter paper No. 1, 125 mm dia.) with micropores were treated with each concentration of extracts previously listed. Each envelope

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was treated with 3 ml solution uniformly distributed with a pipette on internal surfaces. Five envelopes were impregnated with each tested solution. The control papers were impregnated with acetone, polysorbate 80 and distilled water only. The opening of the envelopes (treated and inoculated with adult ticks) was folded (10 mm) and re-sealed with metallic clip, with its identification mark (tested solution and concentration) on the outside. The packets are placed in the BOD incubator at a temperature of 28–30 8C and 80–90% RH for 24 h. The envelopes were opened 24 h after exposure and recorded the number of live and mortality parasites were recorded (Fernandes and Freitas, 2007; Zahir et al., 2009). The experimental media, in which 100% mortality of adult occurs alone, were selected for dose response bioassay. Adult Paramphistomum cervi (Zeder 1790) (Sey, 1982) were collected in 0.9% phosphate-buffered saline (pH 7– 7.2) from the rumen of infected sheep killed for consumption at the local slaughterhouses and identified. One gram of crude extract was first dissolved in 100 ml of acetone (stock solution). The anthelmintic assay was carried as per the method of Tandon et al. (1997) with necessary modifications. From the stock solution, 2500 ppm was prepared with 100 ml PBS solution and 1.0 ml of the desired plant extract concentration. The adult P. cervi parasites were incubated at 37  2 8C in media containing no extract (control) or crude extract in PBS supplemented with 0.5% dimethylsulfoxide (Qualigens). Five replicates were used for each concentration. The time required for complete paralysis and death of the parasite were recorded. After being removed from the experimental medium and dipped in slightly warm water and on gentle stimulation, the paralyzed parasite confirmed motility. The numbers of dead parasite were counted after 24 h of incubation at 37 8C of exposure, and the percentage mortality was reported from the average of five replicates. The experimental media, in which 100% mortality of parasite occurs alone, were selected for dose response bioassay. 2.4. Insect rearing and bioassay A. subpictus and C. tritaeniorhynchus larvae were collected from the rice field and stagnant water area of Melvisharam (128560 2300 N, 798140 2300 E) and identified by Dr. V. Rajagopal, Senior Entomologist, Zonal Entomological Research Centre, Vellore (128550 4800 N, 79870 4800 E), Tamil Nadu, to start the colony, and the larvae were kept in plastic and enamel trays containing tap water. They were maintained and reared in the laboratory as per the method of Kamaraj et al. (2009). During preliminary screening with the laboratory trial, the larvae of A. subpictus and C. tritaeniorhynchus were collected from the insect rearing cage and identified in Zonal Entomological Research Centre, Vellore. From the stock solution, 1000 ppm was prepared with dechlorinated tap water. The larvicidal activity was assessed by the procedure of WHO (1996) with some modification and as per the method of Rahuman et al. (2000). For the bioassay test, larvae were taken in five batches of 20 in 249 ml of water and 1.0 ml of the desired plant extract concentration. The control was set up with

acetone and polysorbate 80. The numbers of dead larvae were counted after 24 h of exposure, and the percentage mortality was reported from the average of five replicates. The experimental media, in which 100% mortality of larvae occurs alone, were selected for dose response bioassay. 2.5. Dose response bioassay From the stock solution, different concentrations ranging from 10.94 to 2500 ppm for parasites and 2.34 to 1000 ppm for mosquito were prepared. Based on the preliminary screening results, different crude solvent extracts prepared from the leaf of A. squamosa, C. asiatica, G. superba, M. maderaspatensis, P. daemia and P. emblica were subjected to dose response bioassay against H. bispinosa, P. cervi, A. subpictus, and C. tritaeniorhynchus, respectively. The numbers of dead parasite and mosquito larvae were counted after 24 h of exposure, and the percentage mortality was reported from the average of five replicates. However, at the end of 24 h, the selected test samples turned out to be equal in their toxic potential. 2.6. Statistical analysis The average parasite and larval mortality data were subjected to Probit analysis for calculating LC50, LC90, and other statistics at 95% fiducial limits of upper confidence limit and lower confidence limit, and chi-square values were calculated by using the software developed by Reddy et al. (1992). Results with p < 0.05 were considered to be statistically significant. 3. Results Larvicidal activities of different crude solvent extracts of six plants are noted and presented in Table 1. All plant extracts showed moderate toxic effect on parasites after 24 h of exposure; however, the highest mortality was found in leaf hexane extract of A. squamosa, acetone and methanol extracts of G. superba, methanol extracts of P. daemia and P. emblica against H. bispinosa (LC50 = 145.39, 419.83, 225.57, 294.46, and 256.08 ppm; LC90 = 689.08, 1579.56, 852.34, 1404.61 and 1025.60 ppm), ethyl acetate and methanol extracts of A. squamosa, methanol extracts of C. asiatica, G. superba, M. maderaspatensis, P. daemia and P. emblica against P. cervi (LC50 = 269.44, 197.67, 77.61, 60.16, 242.53, 59.61, and 60.60 ppm; LC90 = 974.77, 886.76, 308.76, 250.36, 1190.39, 423.98, and 287.48 ppm), acetone, chloroform, ethyl acetate and methanol extracts of A. squamosa, methanol extract of C. asiatica, acetone and methanol extracts of G. superba, ethyl acetate, hexane and methanol extracts of P. daemia against A. subpictus (LC50 = 17.47, 76.04, 18.60, 119.93, 26.62, 18.43,64.87, 34.06, 13.63, and 50.39 ppm; LC90 = 195.86, 320.91, 75.19, 451.25, 111.40, 63.20, 257.82, 125.21, 57.23 and 230.58 ppm) and acetone, chloroform, ethyl acetate and methanol extracts of A. squamosa, acetone and methanol extracts of G. superba, chloroform, ethyl acetate, methanol extract of P. daemia, ethyl acetate and methanol extracts of P. emblica against C. tritaeniorhynchus (LC50 = 106.41, 63.81, 60.01, 78.21, 87.25,

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Table 1 Parasitic activity of crude plant extract against the adult of Haemaphysalis bispinosa, Paramphistomum cervi at 2500 ppm and fourth instar larvae of Anopheles subpictus and Culex tritaeniorhynchus at 1000 ppm. % Mortalitya  SD

Botanical name/family (herbarium numbers) vernacular names

Species

Hexane

Chloroform

Ethyl acetate

Acetone

Methanol

Annona squamosa L./Annonaceae (ZD/AS/045-08) Pangiee/Sita

H. bispinosa P. cervi A. subpictus C. tritaeniorhynchus

100  0.000 85  2.449 88  1.291 77  4.159

74  2.864 62  3.647 100  0.000 100  0.000

57  4.037 100  0.000 100  0.000 100  0.000

80  3.316 73  2.408 100  0.000 100  0.000

79  1.924 100  0.000 100  0.000 100  0.000

Centella asiatica (L.) Urban/ Umbelliferae (ZD/CS/036-08)Vallarai

H. bispinosa P. cervi A. subpictus C. tritaeniorhynchus

72  2.302 80  2.236 75  3.873 51  1.924

76  1.924 52  2.702 33  1.923 65  2.550

89  2.302 61  3.962 52  3.209 74  3.033

72  2.702 82  1.817 33  2.408 51  3.701

48  4.856 100  0.000 100  0.000 84  1.924

Gloriosa superba L./ Liliaceae (ZD/GS/073-08) Nabhikkodi

H. bispinosa P. cervi A. subpictus C. tritaeniorhynchus

85  1.414 82  1.817 78  3.507 81  2.387

77  2.702 88  1.817 76  2.775 68  3.209

79  1.789 62  3.209 62  3.209 83  1.817

100  0.000 61  2.387 100  0.000 100  0.000

100  0.000 100  0.000 100  0.000 100  0.000

Mukia maderaspatensis (L.) M.Roem/ Cucurbitaceae (ZD/GS/073-08) Musumusukkai

H. bispinosa P. cervi A. subpictus C. tritaeniorhynchus

62  1.817 86  3.162 73  2.881 37  2.702

79  1.924 76  3.114 41  2.490 56  5.630

71  2.774 45  2.739 34  1.789 60  4.528

65  1.581 57  3.647 32  3.782 55  4.000

83  3.391 100  0.000 31  2.280 62  2.302

Pergularia daemia (Forssk.) Chiov./ Asclepiadaceae (ZD/PD/063-08) Vaeliparuththi

H. bispinosa P. cervi A. subpictus C. tritaeniorhynchus

86  2.588 59  2.168 100  0.000 89  1.095

67  2.881 60  2.236 90  1.581 100  0.000

76  3.033 71  3.633 100  0.000 100  0.000

79  3.114 86  2.387 83  1.949 66  3.421

100  0.000 100  0.000 100  0.000 100  0.000

Phyllanthus emblica L./ Euphorbiaceae (ZD/PE/088-08) Kattunelli

H. bispinosa P. cervi A. subpictus C. tritaeniorhynchus

90  1.414 77  3.302 53  3.578 74  3.768

58  2.074 76  1.924 32  3.507 80  3.316

79  1.924 74  2.588 78  2.078 100  0.000

88  1.140 81  2.168 45  4.000 67  5.459

100  0.000 100  0.000 75  4.359 100  0.000

Control—nil mortality. a Mean value of five replicates.

92.43, 124.73, 31.94, 76.64,69.09, and 54.82 ppm; LC90 = 360.76, 191.51, 224.14, 276.58, 313.15, 375.28, 731.98, 114.91, 235.93, 311.25, and 199.89 ppm), respectively (Table 2 and Fig. 1).

4. Discussion This has been observed earlier the potential of acaricidal activity of Matricaria chamomile flowers’ extract

Fig. 1. Graph showing the LC50 and LC90 values of crude leaf extracts against the adult of H. bispinosa, P. cervi, larvae of A. subpictus, and C. tritaeniorhynchus.

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Table 2 LC50, LC90, and other statistical analysis of different solvent plant extracts against the adult of Haemaphysalis bispinosa, Paramphistomum cervi, and fourth instar larvae of Anopheles subpictus and Culex tritaeniorhynchus. Name of the plants

Solvents

Species

LC50  SE (ppm) (UCL–LCL)

Annona squamosa

Acetone

A. subpictus C. tritaeniorhynchus A. subpictus C. tritaeniorhynchus P. cervi A. subpictus C. tritaeniorhynchus H. bispinosa P. cervi A. subpictus C. tritaeniorhynchus

17.48  1.623(20.66–14.30) 106.41  6.51(119.16–93.65) 76.04  5.15(86.14–65.94) 63.81  3.66(70.99–56.64) 269.44  16.96(302.68–236.20) 18.60  1.33(22.20–16.01) 60.01  3.83(67.52–52.50) 145.39  10.27(165.52–125.27) 197.67  13.76(224.64–170.71) 119.93  8.44(136.48–103.39) 78.21  4.88(87.77–68.65)

Chloroform Ethyl acetate

Hexane Methanol

195.86  21.32(237.64–154.08) 360.76  35.08(429.51–292.01) 320.91  35.12(389.76–252.07) 191.51  17.13(225.08–157.93) 974.77  98.35(1,167.53–782.01) 75.19  8.16(91.17–59.20) 224.14  22.39(268.03–180.24) 689.08  84.14(854.00–524.16) 886.76  101.00(1,084.72–688.80) 451.25  51.53(552.24–350.26) 276.58  28.58(332.61–220.56)

x2 (df = 4) 3.19 15.06 4.49 15.85 4.99 2.60 4.89 10.94 1.17 6.91 9.42

Centella asiatica

Methanol

P. cervi A. subpictus

Gloriosa superba

Acetone

Methanol

H. bispinosa A. subpictus C. tritaeniorhynchus H. bispinosa P. cervi A. subpictus C. tritaeniorhynchus

419.83  27.05(472.85–366.83) 18.43  1.13(20.65–16.21) 87.25  5.42(97.88–76.63) 225.57  14.43(253.84–197.29) 60.16  4.02(68.03–52.28) 64.87  4.53(73.75–55.98) 92.43  6.12(104.42–80.43)

1,579.56  168.49(1,909.80–1,249.32) 63.20  6.37(75.68–50.72) 313.15  30.04(372.02–254.28) 852.34  87.00(1,022.87–681.80) 250.36  26.16(301.64–199.07) 257.82  27.10(310.73–204.90) 375.28  38.84(451.41–299.15)

Mukia maderaspatensis

Methanol

P. cervi

242.53  17.55(276.92–208.14)

1,190.39  142.42(1,469.54–911.24)

Pergularia daemia

Chloroform Ethyl acetate

C. tritaeniorhynchus A. subpictus C. tritaeniorhynchus A. subpictus H. bispinosa P. cervi A. subpictus C. tritaeniorhynchus C. tritaeniorhynchus

124.73  9.60(143.56–105.91) 34.06  2.17(38.31–29.80) 31.94  2.08(36.02–27.86) 13.63  0.92(15.44–11.82) 294.46  20.85(335.33–253.59) 59.61  5.33(70.05–49.17) 50.39  3.59(57.43–43.36) 76.64  4.64(85.74–67.55) 69.09  4.72(78.35–59.84)

731.98  100.78(929.52–534.45) 125.21  12.68(150.07–100.35) 114.91  11.56(137.56–92.25) 57.23  67.11(69.21–45.26) 1,404.61  166.31(1,730.27–1,078.64) 423.98  59.32(540.25–307.72) 230.58  25.49(280.54–180.63) 235.93  23.91(302.79–209.08) 311.25  34.17(378.22–244.27)

13.64 11.32 2.65 6.17 9.43 6.93 3.47 11.59 6.64

H. bispinosa P. cervi C. tritaeniorhynchus

256.08  16.85(289.11–223.04) 60.60  4.38(69.71–52.02) 54.82  3.48(61.65–47.99)

1,025.60  110.85(1,242.87–808.33) 287.48  33.14(352.44–222.52) 199.89  20.61(240.29–159.49)

5.48 5.07 11.96

Hexane Methanol

Phyllanthus emblica

Ethyl acetate Methanol

77.61  5.13(87.67–67.55) 26.62  1.79(30.13–23.11)

LC90  SE (ppm) (UCL–LCL)

308.76  31.60(370.69–246.83) 111.40  12.17(135.25–87.55)

2.39 4.47 15.28 13.74 5.15 5.77 5.81 17.93 4.06 8.32

Control—nil mortality. Significant at 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; LCL: lower confidence limit; x2—Chi-square; df: degree of freedom.

was studied against engorged Rhipicephalus annulatus showed the mortality rate ranged from 6.67% to 26.7%, whereas no mortality was recorded for non-treated control group (Pirali-Kheirabadi and Razzaghi-Abyaneh, 2007). Fernandes and Freitas (2007) have reported that the oleoresinous extract (oleoresin) from C. reticulata tested against R. (Boophilus) microplus larvae showed LC50 and LC99 values were 1579 and 3491 ppm, respectively. The chemotherapeutic value of both the extracts is also evident from an earlier study, wherein the aqueous extracts from certain medicinal plants, including Butea monosperma, Embelia ribes, and Roltlesia tinctoria reportedly influenced drastic decrease in the activities of both acid phosphatase and alkaline phosphatase in the trematode, P. cervi (Chopra et al., 1991). Zahir et al. (2009) have reported that the highest parasite mortality was found in the ethyl acetate extract of A. aspera, leaf methanol extract of A. malabarica, flower methanol extract of G. superba, and leaf methanol extract of R. communis against the larvae of R. microplus (LC50 = 265.33, 95.97, 153.73, and 181.49 ppm; LC90 = 1130.18, 393.88, 1794.25, and 1829.94 ppm); leaf acetone and chloroform of A. malabarica, flower acetone extract of G. superba, and leaf chloroform and methanol of R. communis against the adult of P. cervi (LC50 = 108.07,

106.69, 157.61, 69.44, and 168.24 ppm; LC90 = 521.77, 463.94, 747.02, 256.52, and 809.45 ppm); leaf ethyl acetate extract of A. aspera, leaf chloroform extract of A. malabarica, flower methanol of G. superba, and leaf methanol extract of R. communis against the larvae of A. subpictus (LC50 = 48.83, 135.36, 106.77, and 102.71 ppm; LC90 = 225.36, 527.24, 471.90, and 483.04 ppm); and leaf ethyl acetate extract of A. aspera, leaf chloroform extract of A. malabarica, flower methanol extract of G. superba, and leaf methanol extract of R. communis against the larvae of C. tritaeniorhynchus (LC50 = 68.27, 95.98, 59.51, and 93.94 ppm; LC90 = 306.88, 393.83, 278.99, and 413.27 ppm), respectively. The extracts from the Piper longum (fruits), along with Azadirachta indica (bark), Butea frondosa (seed), and Nigella sativa (seeds), produced broad spectrum anthelmintic action against roundworms (H. contortus and Oesophagostomum columbianum) and flukes (P. cervi) in calves (Raje et al., 2003). In vitro treatment of the parasites with the crude extract of Flemingia vestita (50 mg/ml) in PBS revealed complete immobilization of the trematode and cestode in about 43 and 20 min, respectively, and appears to be effective against Paramphistomum sp. since within a considerably short interval, complete inactivation and irreversible immobilization was observed in the treated

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worms, indicating a lethal effect on the parasite (Tandon et al., 1997). Oxyclozanide and rafoxanide at 10(3) M inhibited enzyme activity by 100% in homogenates Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum while hexachlorophene at 10(3) M also caused 100% inhibition in homogenates from F. gagantica and P. explanatum but only 65% of malate oxidation in F. buski homogenates (Probert et al., 1981). Bagavan et al. (2009) have reported that peel chloroform extract of C. sinensis, leaf ethyl acetate extracts of O. canum and O. sanctum and leaf chloroform extract of R. nasutus against the larvae of A. subpictus (LC50 = 58.25, 88.15, 21.67 and 40.46 ppm; LC90 = 298.31, 528.70, 98.34 and 267.20 ppm), peel methanol extract of C. sinensis, leaf methanol extract of O. canum, ethyl acetate extracts of O. sanctum and R. nasutus against the larvae of C. tritaeniorhynchus (LC50 = 38.15, 72.40, 109.12 and 39.32 ppm; LC90 = 184.67, 268.93, 646.62 and 176.39 ppm), respectively. This has been observed earlier by Kamaraj et al. (2009) that the highest larval mortality was found in leaf petroleum ether, flower methanol extracts of C. auriculata, flower methanol extracts of L. aspera and R. nasutus, leaf and seed methanol extracts of S. torvum and leaf hexane extract of V. negundo against the larvae of A. subpictus (LC50 = 44.21, 44.69, 53.16, 41.07, 35.32, 28.90 and 44.40 ppm; LC90 = 187.31, 188.29, 233.18, 142.66, 151.60, 121.05 and 192.11 ppm, respectively) and against the larvae of C. tritaeniorhynchus (LC50 = 69.83, 51.29, 81.24, 71.79, 44.42, 84.47 and 65.35 ppm; LC90 = 335.26, 245.63, 300.45, 361.83, 185.09, 351.41 and 302.42 ppm, respectively). Alkaloids isolated from A. squamosa have shown larvicidal growth-regulating and chemosterilant activities against A. stephensi at concentrations of 50– 200 ppm, the larvae, pupae and adults produced about a 52–92% decrease in the laboratory experiment (Saxena et al., 1993). Earlier authors reported that the larvicidal activity of crude acetone, ethyl acetate, hexane, methanol, and petroleum ether extracts of the leaf of C. asiatica and Mukia scabrella showed percent mortality of 36  4.6, 24  3.63, 28  3.28, 06  1.78, 46  3.89 and 00  0.00 00  0.00 08  1.6733 18  2.9664 00  0.00 at 1000 ppm, respectively against the early fourth instar larvae of C. quinquefasciatus (Rahuman et al., 2008b). The LC50 value of petroleum ether extracts of Jatropha curcas, Pedilanthus tithymaloides, Phyllanthus amarus, Euphorbia hirta, and Euphorbia 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 (Rahuman et al., 2008a). Kamaraj et al. (2008a) have reported that the peel methanol extract of C. sinensis, leaf and flower ethyl acetate extracts of O. canum against the larvae of A. stephensi (LC50 = 95.74,101.53,28.96; LC90 = 303.20,492.43 and 168.05 ppm), respectively. The highest larval mortality was found in methanol extract of O. canum, R. nasutus and acetone extract of O. sanctum against the larvae of A. aegypti (LC50 = 99.42, 94.43 and 81.56 ppm) and against C. quinquefasciatus (LC50 = 44.54, 73.40 and 38.30 ppm), respectively (Kamaraj et al., 2008b). A bioassay-guided fractionation of A. aspera led to the separation and identification of a saponin as a potential mosquito larvicidal

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compound, with LC50 value of 18.20 and 27.24 ppm against A. aegypti and C. quinquefasciatus, respectively (Bagavan et al., 2008). Amer and Mehlhorn (2006) reported that Lippia citriodora essential oil having LC50 value of 101.4 ppm against third instar larvae of A. stephensi. In conclusion, an attempt has been made to evaluate the adulticidal and larvicidal activity of plant extracts against H. bispinosa, P. cervi, A. subpictus and C. tritaeniorhynchus. The results reported here open the possibility of further investigations of efficacy on their larvicidal properties of natural product extracts. The isolation and purification of crude ethyl acetate and methanol extracts of A. squamosa, methanol extract of C. asiatica, acetone extracts of G. superba, ethyl acetate, hexane, methanol extracts of P. daemia and methanol extracts of P. emblica are in progress. Acknowledgments The authors are grateful to C. Abdul Hakeem College Management, Dr. S. Mohammed Yousuff, Principal, Dr. Ahmed Najib, HOD of Zoology Department, and Dr. Sait Sahul Hameed, Reader in Zoology, for their help and suggestion. The authors wish to thank Dr. A. Sangaran, Department of Parasitology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India for identification of parasites. References Alawa, C.B., Adamu, A.M., Gefu, J.O., Ajansui, O.J., Abdu, P.A., Chiezey, N.P., Alawa, J.N., Bowman, D.D., 2003. In vitro screening of two Nigerian medicinal plants (Vernonia amiygdalina and Annona senegalensis) for anthelmintic activity. Vet. Parasitol. 113, 73–81. Bagavan, A., Kamaraj, C., Rahuman, A.A., Elango, G., Zahir, A.A., Pandiyan, G., 2009. Evaluation of larvicidal and nymphicidal potential of plant extracts against Anopheles subpictus Grassi, Culex tritaeniorhynchus Giles and Aphis gossypii Glover. Parasitol. Res. 104 (5), 1109– 1117. Bagavan, A., Rahuman, A.A., Kamaraj, C., Geetha, K., 2008. Larvicidal activity of saponin from Achyranthes aspera against Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae). Parasitol. Res. 103 (1), 223–229. Chakraborty, T., Babu, S.P.S., Sukul, N.C., Babu, S.P.S., 1996. Preliminary evidence of antifilarial effect of Centella asiatica on Canine dirofilariasis. Fitoterapia 67 (2), 110–112. Chopra, A.K., Sharma, M.K., Upadhyay, V.P., 1991. Effects of ayurvedic anthelmintics on phosphatase activity of Paramphistomum cervi. Indian J. Parasitol. 431, 65–69. Elango, G., Rahuman, A.A., Bagavan, A., Kamaraj, C., Zahir, A.A., Venkatesan, C., 2009. Laboratory study on larvicidal activity of indigenous plant extracts against Anopheles subpictus and Culex tritaeniorhynchus. Parasitol. Res. 104, 1381–1388. FAO (Food and Agriculture Organization of the United Nations), 2004. Module 1. Ticks: acaricide resistance: diagnosis management and prevention. In: Guidelines Resistance Management and Integrated Parasite Control in Ruminants, FAO Animal Production and Health Division, Rome. Fernandes, F.F., 2001. Toxicological effects and resistance to pyretroids in Boophilus microplus from Goia’s., Brasil. Arq. Bras. Med. Vet. Zoot. 53, 548–552. Fernandes, F.F., Freitas, E.P.S., 2007. Acaricidal activity of an oleoresinous extract from Copaifera reticulata Leguminosae: Caesalpinioideae against larvae of the southern cattle tick, Rhipicephalus Boophilus microplus Acari: Ixodidae. Vet. Parasitol. 1471-2, 150–154. Fernandes, F.F., Freitas, E.P.S., Costa, A.C., Silva, I.G., 2005. Larvicidal potential of Sapindus saponaria to control the cattle tick Boophilus microplus. Pesq. Agropec. Bras. 40, 1243–1245. Gathuma, J.M., Mbaria, J.M., Wanyama, J., Kaburia, H.F., Mpoke, L., Mwangi, J.N., 2004. Efficacy of Myrsine africana, Albizia anthelmintica and Hilderbrantia sepalosa herbal remedies in Samburu district, Kenya. J. Ethnopharmacol. 91, 7–12.

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