ORIGINAL PAPER

Efficacy of plant-mediated synthesized silver nanoparticlesagainst hematophagous parasites

Chidambaram Jayaseelan & Abdul Abdul Rahuman & Govindasamy Rajakumar &

Thirunavukkarasu Santhoshkumar & Arivarasan Vishnu Kirthi &Sampath Marimuthu & Asokan Bagavan & Chinnaperumal Kamaraj &Abdul Abduz Zahir & Gandhi Elango & Kanayairam Velayutham &

Kokati Venkata Bhaskara Rao & Loganathan Karthik & Sankariah Raveendran

Received: 1 May 2011 /Accepted: 17 May 2011 /Published online: 4 June 2011# Springer-Verlag 2011

Abstract The purpose of the present study was toinvestigate the acaricidal and larvicidal activity against thelarvae of Haemaphysalis bispinosa Neumann (Acarina:Ixodidae) and larvae of hematophagous fly Hippoboscamaculata Leach (Diptera: Hippoboscidae) and against thefourth-instar larvae of malaria vector, Anopheles stephensiListon, Japanese encephalitis vector, Culex tritaeniorhyn-chus Giles (Diptera: Culicidae) of synthesized silver nano-particles (AgNPs) utilizing aqueous leaf extract from Musaparadisiaca L. (Musaceae). The color of the extractchanged to light brown within an hour, and later it changedto dark brown during the 30-min incubation period. AgNPsresults were recorded from UV–vis spectrum at 426 nm;Fourier transform infrared (FTIR) analysis confirmed thatthe bioreduction of Ag+ ions to silver nanoparticles are due

to the reduction by capping material of plant extract, X-raydiffraction (XRD) patterns clearly illustrates that the nano-particles formed in the present synthesis are crystalline innature and scanning electron microscopy (SEM) supportthe biosynthesis and characterization of AgNPs with rod inshape and size of 60–150 nm. After reaction, the XRDpattern of AgNPs showed diffraction peaks at 2θ=34.37°,38.01°, 44.17°, 66.34° and 77.29° assigned to the (100),(111), (102), (110) and (120) planes, respectively, of a facedcentre cubic (fcc) lattice of silver were obtained. Forelectron microscopic studies, a 25 μl sample was sputter-coated on copper stub, and the images of nanoparticleswere studied using scanning electron microscopy. The spotEDX analysis showed the complete chemical compositionof the synthesized AgNPs. The parasite larvae wereexposed to varying concentrations of aqueous extract ofM. paradisiaca and synthesized AgNPs for 24 h. In thepresent study, the percent mortality of aqueous extract of M.paradisiaca were 82, 71, 46, 29, 11 and 78, 66, 38, 31and 16observed in the concentrations of 50, 40, 30, 20, 10 mg/l for24 h against the larvae of H. bispinosa and Hip. maculata,respectively. The maximum efficacy was observed in theaqueous extract of M. paradisiaca against the H. bispinosa,Hip. maculata, and the larvae of A. stephensi, C. tritaenio-rhynchus with LC50 values of 28.96, 31.02, 26.32, and20.10 mg/lm, respectively (r2=0.990, 0.968, 0.974, and0.979, respectively). The synthesized AgNPs of M. para-disiaca showed the LC50 and r2 values against H. bispinosa,(1.87 mg/l; 0.963), Hip. maculata (2.02 mg/l; 0.976), andlarvae of A. stephensi (1.39; 0.900 mg/l), against C.tritaeniorhynchus (1.63 mg/l; 0.951), respectively. The χ2

values were significant at p<0.05 level.

C. Jayaseelan :A. A. Rahuman (*) :G. Rajakumar :T. Santhoshkumar :A. V. Kirthi : S. Marimuthu :A. Bagavan :C. Kamaraj :A. A. Zahir :G. Elango :K. VelayuthamUnit of Nanotechnology and Bioactive Natural Products,Post Graduate and Research Department of Zoology,C. Abdul Hakeem College,Melvisharam- 632 509,Vellore District, Tamil Nadu, Indiae-mail: abdulrahuman6@hotmail.com

K. V. B. Rao : L. KarthikEnvironmental Biotechnology Division,School of Bio Sciences and Technology, VIT University,Vellore, Tamil Nadu, India

S. RaveendranPost Graduate and Research Department of Zoology,Khadir Mohideen College,Adirampattinam,614 701, Thanjavur District, Tamil Nadu, India

Parasitol Res (2012) 111:921–933DOI 10.1007/s00436-011-2473-6

Introduction

Ectoparasites of livestock cause great economic lossbeing pests as well as vectors of various diseases. Untilnow, several chemicals including hydrocarbons andorganophosphates were commonly used. Tick productionlosses largely result from meat quantity loss caused bytick worry, and the death of cattle from tick fever and thereduction in meat quantity associated with tick infesta-tion were estimated (Sing et al. 1983). About 40% ofcattle have been found positive for Haemaphysalisbispinosa infestation, and it was detected on 7.6% ofexamined cattle, 55.4% goats, and 13.2% pigs (van denBroek et al. 2003; Wall 2007; Ghosh et al. 2007).Hippobosca maculata Leach is a serious pest of equinesin India and is also cosmopolitan in distribution (Parasharet al. 1991). These flies are adapted to a more or lesscontinuous existence on the bodies of their host. Blood-sucking flies cause damage to livestock through bothdirect damage and as vectors of viral and bacterialinfections. Among haematophagous flies, Hippoboscidaeare well known to infest horses and cattle in different partsof the world (Soulsby 1982).

Malaria is one of the most serious health threats, evennow, at national and international levels. Every year, about300–500 million people are estimated to be affected bymalaria, and it further threatens 2.4 billion (about 40%) ofthe world’s population (WHO 2005). Japanese encephalitis(JE) is a disease caused by an arbovirus that is mainlytransmitted by the bite of infected Culex tritaeniorhynchusmosquitoes. The annual incidence and mortality estimatesfor JE are 30,000–50,000 and 10,000, respectively (Solomon2004). JE results in thousands of deaths annually. In additionto mortality, vector-borne diseases (VBDs) cause morbidityof millions of persons resulting in loss of man–dayscausing economic loss (Dhiman et al. 2010).

Insecticides that can be used in control are increasinglybecoming limited. C. tritaeniorhynchus was found suscep-tible to permethrin and resistant to DDT, dieldrin, fenitro-thion and propoxur (Bansal and Singh 1995) and resistantto organophosphorous insecticides (Watanabe et al. 1991).Most of the insecticides available in the market aresynthetic chemical products which apart from their prohib-itively high costs persistent applications have unintendedimplications including the production of resistant strains ofmosquitoes, ecological imbalance and elimination of non-target organisms in the environment (Anyaele and Amusan2003). Extracts or essential oils from plants may bealternative sources of mosquito larval control agents, sincethey constitute a rich source of bioactive compounds thatare biodegradable into non-toxic products and potentiallysuitable for use in control of mosquito larvae. In fact, manyresearchers have reported on the effectiveness of plant

extracts or essential oils against mosquito larvae (Amer andMehlhorn 2006a, b; Rahuman et al. 2008a, b).

Nanoparticles play an indispensable role in drug deliv-ery, diagnostics, imaging, sensing, gene delivery, artificialimplants, and tissue engineering (Morones et al. 2005). Thebiosynthesis of nanoparticles is advantageous over chemi-cal and physical methods because it is a cost-effective andenvironment-friendly method, where it is not necessary touse high pressure, energy, temperature, and toxic chemicals(Goodsell 2004). AgNPs may be released into the environ-ment from discharges at the point of production, fromerosion of engineered materials in household products(antibacterial coatings and silver-impregnated water filters),and from washing or disposal of silver-containing products(Benn and Westerhoff 2008). Using plants for nanoparticlesynthesis can be advantageous over other biologicalprocesses because it eliminates the elaborate process ofmaintaining cell cultures and can also be suitably scaled upfor large-scale nanoparticle synthesis (Shankar et al. 2004).Synthesis of nanoparticles using microorganisms or plantscan potentially eliminate this problem by making thenanoparticles more biocompatible. Nanoparticles, generallyconsidered as particles with sizes of up to 100 nm, exhibitcompletely new or improved properties compared to thelarger particles of the bulk material that they are composedof, based on specific characteristics such as size, distribu-tion, and morphology (Willems and van den Wildenberg2005). In recent years, the biosynthesis method using plantextracts has received more attention than chemical andphysical methods, and even more than the use of microbes,for the nanoscale metal synthesis due to the absence of anyrequirement to maintain an aseptic environment. Nano-particles have attracted considerable attention because oftheir various applications. AgNPs are reported to possessanti-viral (Rogers et al. 2008), anti-bacterial (Sathishkumaret al. 2009) and anti-fungal properties (Panacek et al. 2009).Use of plant extract for the synthesis of nanoparticles couldbe advantageous over other environmentally benign bio-logical processes because it eliminates the elaborate processof maintaining cell cultures. Recently, green silver nano-particles have been synthesized using various naturalproducts like Nelumbo nucifera (Santhoshkumar et al.2011) and Pongamia pinnata (Rajesh et al. 2010).

Musa paradisiaca (Banana peel) contained largeamounts of dopamine and L-dopa, catecholamines with asignificant antioxidant activity. Tetracyclic triterpenoidswere isolated from the methanolic extract of fresh leavesof Azadirachta indica, showing mortality on the fourthinstar larvae of Anopheles stephensi (Siddiqui et al. 2003).An acute toxicity test confirmed that exposure to AgNPscould significantly increase the metal burden in gill tissueand in the whole body of adult female zebrafish (Griffitt etal. 2008). It is well known that silver in various chemical

922 Parasitol Res (2012) 111:921–933

forms exhibit strong toxicity to a wide range of micro-organisms (Liau et al. 1997).

Materials and methods

Materials

The peel of M. paradisiaca L. (Musaceae) was collected inChitheri Hills, Dharmapuri district (11°53′28″N, 078°30′26″E, altitude 959 m), Tamil Nadu, India. Taxonomic identifi-cation was done by Dr. C. Hema from the Department ofBotany, Arignar Anna Government Arts College for Women,Walajapet, Vellore, India. The voucher specimen wasnumbered and kept in our research laboratory for furtherreference. Silver nitrate was obtained from Qualigens FineChemicals (Mumbai, India).

Collection of H. bispinosa and Hip. maculata

The larvae of H. bispinosa Neumann (Acarina: Ixodidae)and adult fly of Hip. maculata Leach (Diptera: Hippo-boscidae) were collected from inside the ears and very rarelyfound elsewhere on the body of cattle. The parasites wereidentified by Department of Veterinary Parasitology, MadrasVeterinary College, Tamil Nadu Veterinary and AnimalSciences University, Chennai, Tamil Nadu.

Bioassay of H. bispinosa and Hip. maculata

The newly attached larvae of H. bispinosa and Hip.maculata were collected from the softer skin inside thethigh, flanks, abdomen, brisket, and forelegs of naturallyinfested cattle. The method used in this study to verify theacaricidal activity of synthesized AgNPs against the larvaeof H. bispinosa and Hip. maculata were developed as perthe method of FAO (2004), incorporating slight modifica-tions to improve practicality and efficiency of testedmaterials (Fernandes et al. 2005). First, 500 mg ofsynthesized AgNPs were dissolved in 1000 ml of distilledwater (stock solution). From the stock solution, 100 mg/l wasprepared and a series of filter paper envelopes (Whatmanfilter paper no. 1, 125 mm diameter) with microporeswere treated with each concentration of extract of M.paradisiaca. The synthesized particles were impregnatedwith 20 mg/l, of which 3 ml solution of the stock wasuniformly distributed with a pipette on internal surfaces.Five envelopes were impregnated with each testedsolution. Control papers were impregnated with distilledwater only. The opening of the envelopes (treated andinoculated with larval ticks) were folded (10 mm) and re-sealed with a metallic clip, with its identification mark(tested solution and concentration) on the outside. The

packets were placed in the BOD incubator at thetemperature of 28–30°C and 80–90% RH for 24 h. Theenvelopes were opened 24 h after exposure, and the number oflive and mortality larvae were recorded (Fernandes and Freitas2007). Death of parasites were confirmed when there wascessation of motility or waggling of the appendages ontouching with a needle. The experimental media, in which100% mortality of larvae occurs alone, were selected fordose response bioassay.

Mosquito culture

A. stephensi and C. tritaeniorhynchus larvae were collectedfrom rice field and stagnant water area of Melvisharam(12°56′23″N, 79°14′23″E) and identified in Zonal Entomo-logical Research Centre, Vellore (12°55′48″ N, 79°7′48″E),Tamil Nadu, India. To start the colony, the collected larvaewere kept in plastic and enamel trays containing tap water.They were maintained and reared in the laboratory as perthe method described by Kamaraj et al. (2009).

Larvicidal bioassay

During preliminary screening with the laboratory trial, thelarvae of A. stephensi and C. tritaeniorhynchus werecollected from the insect-rearing cage and identified inZonal Entomological Research Centre, Vellore. One gramof aqueous leaf extract was first dissolved in 100 ml ofdistilled water for bioassay test of plant extract (stocksolution). From the stock solution, 100 mg/l was preparedwith dechlorinated tap water for bioassay test of plantextract. The larvicidal activity was assessed following theprocedure of WHO (1996) with some modification and asper the method described by Rahuman et al. (2000). Forbioassay test, larvae were taken in five batches of 20 in249 ml of water and 1.0 ml of aqueous plant extractconcentration. Control was set up with dechlorinated tapwater. The numbers of dead larvae were counted after 24 hof exposure, and the percentage of mortality was reportedfrom the average of five replicates. The experimental mediain which 100% mortality of larvae occurs alone wereselected for dose–response bioassay.

Synthesized AgNPs toxicity test was performed byplacing 20 mosquito larvae into 200 ml sterilized doubledistilled water with nanoparticles into a 250-ml beaker(Borosil). The nanoparticle solutions were diluted usingdouble distilled water as a solvent according to the desiredconcentrations (5.0, 4.0, 3.0, 2.0 and1.0 mg/l). Each testincluded a set control groups (silver nitrate and distilledwater) with five replicates for each individual concentra-tion. Mortality was assessed after 24 h to determine theacute toxicities on fourth instar larvae of A. stephensi andC. tritaeniorhynchus.

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Twenty mosquito larvae were placed into 250 mL glassbeakers (Borosil) and set in an environmental chamber at25°C with a 16:8 h light/dark cycle. Each beaker containingmosquito larvae distilled water was spiked with stocksolutions of AgNPs in order to achieve target nominalconcentrations of 5.0, 4.0, 3.0, 2.0 and1.0 mg/l with a finalvolume of 200 ml. A negative control (1 mM silver nitratesolution) was used in all experiments, and all conditionswere tested in five replicates. In order to compare themortality of synthesized AgNPs to that of dissolved Agreleased and the 20 mosquito larvae were exposed to arange of dissolved Ag concentrations so as to cover therange released from all doses of AgNPs. To avoid settlingof particles especially at higher doses, all treatmentsolutions were sonicated for an additional 5 min prior toaddition of the mosquito larvae. Since this additionalsonication appeared to significantly decrease the settling ofparticles.

Dose–response bioassay

Based on the preliminary screening results, crude peelextract of M. paradisiaca and synthesized AgNPs weresubjected to dose–response bioassay for larvicidal activityagainst the larvae of H. bispinosa, Hip. maculata, A.stephensi and C. tritaeniorhynchus. Different concentra-tions ranging from 5.0 to 25.0 mg/l (for aqueous peel plantextract) and 1.0 to 5.0 mg/l (for synthesized AgNPs andsilver nitrate) were prepared for larvicidal activity ofparasites. The numbers of dead larvae were counted after24 h of exposure, and the percentage of mortality wasreported from the average of five replicates. However, atthe end of 24 h, the selected test samples turned out to beequal in their toxic potential.

Preparations of M. paradisiaca aqueous peel extract

The peel was finely cut into small pieces and the aqueousextract was prepared by mixing 10 g of fresh peel pieceswith 100 ml double distilled water (boiled and cooleddistilled water) with constant stirring on a magnetic stirrer(Minjas and Sarda 1986). The suspension of fresh peel inwater was left for 3 h, filtered through Whatman no. 1 filterpaper, and the filtrate was stored in amber colored air tightbottle at 10°C and used within a week.

Synthesis of silver nanoparticles using M. paradisiaca peelextract

M. paradisiaca plant peel broth solution was prepared bytaking 10 g of thoroughly washed and finely cut peels in a300 ml Erlenmeyer flask along with 100 ml sterilizeddouble distilled water and then boiling the mixture for

5 min before finally decanting it. The extract was filteredwith Whatman filter paper no. 1 and stored at −15°C andwas within 1 week. The filtrate was treated with aqueous1 mM AgNO3 solution and incubated at room temperature.80 ml aqueous solution of 1 mM of AgNO3 was reducedusing 20 ml peel extract at room temperature for 10 min,resulting in a brown-yellow solution indicating the formationof AgNPs. The nanoparticle solution was diluted 20 timeswith double-distilled water to avoid errors due to high opticaldensity of the solution. The bioreduction of AgNPs ions insolution was monitored by periodic sampling of aliquots(1.0 ml) of aqueous component and measuring UV–visspectra of the solution (Parashar et al. 2009).

Characterization of silver nanoparticles

Silver nanoparticles formed in the M. paradisiaca peelfiltrate were monitored under UV–visible spectrophotome-ter. The bioreduction of the Ag+ ions in solutions wasmonitored by periodic sampling of aliquots (1 ml) of theaqueous component after 20 times dilution and measuringthe UV–vis spectra of the solution. UV–vis spectra of thesealiquots were monitored as a function of time of reaction ona Schimadzu 1601 spectrophotometer in the 300- 700-nmrange operated at a resolution of 1 nm. Furthermore, thereaction mixture was subjected to centrifugation at 60,000× g for 40 min; resulting pellet was dissolved in deionizedwater and filtered through a Millipore filter (0.45 μm). Thepurified pellets were then dried and the powders subjectedto Fourier transform infrared (FTIR) spectroscopy measure-ment. Characterization involved FTIR analysis of the driedpowder of AgNPs, by scanning it in the range 350–3,000 cm−1 at a resolution of 4 cm−1. These measurementswere carried out on a Perkin–Elmer Spectrum Oneinstrument in the diffuse reflectance mode at a resolutionof 4 cm−1 in KBr pellets and the pellets were mixed withKBr powder and pelletized after drying properly. Thepellets were later subjected to FTIR spectroscopy measure-ment. The FTIR spectra of peel extracts taken before andafter synthesis of AgNPs were analyzed and then examinedfor possible functional groups for the formation of AgNPs.An aliquot of this filtrate containing AgNPs was used forX-ray diffraction (XRD) and FTIR analysis. Elementalanalysis was carried out under energy dispersive X-rayspectroscopy (EDX). For XRD studies, dried nanoparticleswere coated on XRD grid, and the spectra were recorded byusing Phillips PW 1830 instrument operating at a voltage of40 kV and a current of 30 mA with Cu Kα1 radiation. Forelectron microscopic studies, a 25 μl sample was sputtercoated on copper stub, and the images of nanoparticleswere studied using scanning electron microscopy (SEM;JEOL, Model JFC-1600). EDX was carried out to deter-mine the chemical composition of the synthesized AgNPs.

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Data analysis

The average larval mortality data were subjected to probitanalysis for calculating LC50 and other statistical data at95% fiducial limits of upper confidence limit and lowerconfidence limit, and χ2 values were calculated using thesoftware developed by Reddy et al. (1992). Results with p<0.05 were considered statistically significant. Mean percentlarval mortality data were subjected to analysis of varianceand compared with Duncan’s multiple range tests todetermine any differences between plant species and withinspecies and concentration (SPSS 2007). Prior to analysis,mortality in treatments was corrected for that in controlsusing Abbott formula (Abbott 1925).

Results

In the present study, the percent mortality of aqueousextract of M. paradisiaca were 82, 71, 46, 29, 11 and 78,66, 38, 31 and 16 observed in the concentrations of 50, 40,30, 20, 10 mg/l for 24 h against the larva of H. bispinosa

and Hip. maculata, respectively. The synthesized AgNPs ofM. paradisiaca was tested in the concentrations of 5, 4, 3, 2and 1 mg/l and the percent mortality were 100, 83, 77, 48,28 and 100, 87, 71, 46 and 19 against H. bispinosa andHip. maculata, respectively. The LC50 were in aqueousextract and synthesized AgNPs were in H. bispinosa 28.96and 1.87 mg/l (r2=0.990 and 0.963) and in Hip. maculata31.02 and 2.02 mg/l (r2=0.968 and 0.976) (Table 1). Thisproves that concentration plays important role in acaricidalactivity. The control (distilled water) showed nil mortalityin the concurrent assay. χ2 value was significant at p≤0.05level.

Larvicidal activity of synthesized AgNPs

Results suggested that the mortality effects of synthesizedAgNPs of M. paradisiaca were 55, 96, 100% and 39, 83,100% at 6, 12, and 24 h, respectively, against A. stephensiand C. tritaeniorhynchus. The larvicidal percent mortalityof aqueous extract of M. paradisiaca were 91, 79, 46, 33,17 and 95, 88, 66, 47 and 26 observed in the concentrationsof 50, 40, 30, 20, 10 mg/l for 24 h against the fourth instar

Table 1 Acaricidal activity of aqueous and synthesized silver nanoparticles using Musa paradiciaca leaf extract against Haemaphysalis bispinosaand Hippobosca maculata

Extracts Species Concentrations (mg/l) Percenta mortality± SD LC50 (mg/l) (LCL–UCL) Slope r2 χ2 (df=4)

Plant aqueous H. bispinosa 50 82±1.271 28.96 (26.28−31.94) 29 0.990 1.25540 71±1.534

30 46±0.359

20 29±0.624

10 11±0.739

Hip. maculata 50 78±0.346 31.02 (27.72−34.72) 31 0.968 2.84740 66±0.878

30 38±0.676

20 31±1.765

10 16±0.912

SynthesizedAgNPs

H. bispinosa 05 100±0.000 1.87 (1.62−2.16) 48 0.963 8.44704 83±0.347

03 77±0.558

02 48±1.743

01 28±0.547

Hip. maculata 05 100±0.000 2.02 (1.81-2.26) 46 0.976 3.71904 87±0.788

03 71±1.045

02 46±0.874

01 19±0.678

Control−nil mortality. Significant at p<0.05 level

LC50 lethal concentration that kills 50% of the exposed larvae, UCL upper confidence limit, LCL lower confidence limit, r2 regression coefficientχ2 chi-square, df degree of freedomaMean value of five replicates

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larva of A. stephensi and C. tritaeniorhynchus, respectively.Synthesized AgNPs of M. paradisiaca was tested inconcentrations of 5, 4, 3, 2 and 1 mg/l and the percentmortality were 100, 100, 88, 62, 39 and 100, 96, 79, 56and 33 against A. stephensi and C. tritaeniorhynchus,respectively. In the present study, the toxic effect wasstudied on fourth instar larvae after 24 h of exposure;however, the mortality of aqueous crude peel extract of M.paradisiaca, against the larvae of A. stephensi and C.tritaeniorhynchus (LC50=26.32 and 20.10 mg/l; r2=0.974and 0.979 mg/l) (Table 2). The synthesized silver nano-particles of M. paradisiaca acted against the larvae of A.stephensi and C. tritaeniorhynchus (LC50=1.39 and1.63 mg/l; r2=0.900 and 0.951) (Table 2). The χ2 valuewas significant at p≤0.05 level. Not more than 4%mortality was observed in controls, indicating that theaqueous extract of M. paradisiaca did not contribute to theoverall mortalities during the bioassay. All tested com-ponents showing lethal effect and mortality were posi-tively dose-dependent against the immature stages ofmosquitoes. 88% mortality was observed at 25mg/l plant extract and 5 mg/l of control as silver showsnil mortality.

Characterization of silver nanoparticles

The change in color was noted by visual observation in theM. paradisiaca peel extract when it was incubated withAgNO3 solution. M. paradisiaca peel extract withoutAgNO3 did not show any change in color (Fig. 1a). The

Table 2 Larvicidal activity of aqueous and synthesized silver nanoparticles using M. paradiciaca leaf extract against fourth instar larvae ofAnopheles stephensi and Culex tritaeniorhynchus

Extracts Species Concentrations(mg/l)

Percenta mortality± SD LC50 (mg/l)(LCL–UCL)

Slope r2 χ2 (df=4)

Plant aqueous A. stephensi 50 91±0.474 26.32 (23.7–29.24) 33 0.974 4.13640 79±1.643

30 46±0.975

20 33±0.624

10 17±0.647

C. tritaeniorhynchus 50 95±0.346 20.10 (17.42–23.18) 47 0.979 0.81740 88±0.578

30 66±0.909

20 47±1.032

10 26±0.912

Synthesized AgNPs A. stephensi 05 100±0.000 1.39 (1.13–1.71) 62 0.900 4.26804 100±0.000

03 88±0.917

02 62±1.032

01 39±0.819

C. tritaeniorhynchus 05 100±0.000 1.63 (1.37–1.94) 56 0.951 2.40804 96±0.934

03 79±1.681

02 56±0.548

01 33±0.875

Control−nil mortality. Significant at p<0.05 level

LC50 lethal concentration that kills 50% of the exposed larvae, UCL upper confidence limit, LCL lower confidence limit, r2 regression coefficient,χ2 chi-square, df degree of freedomaMean value of five replicates

Fig. 1 a Change in color after adding AgNO3 before reaction andafter reaction time of 30 min. b UV–vis spectra of aqueous silvernitrate with M. paradisiaca peel extract at different time intervals

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color of the extract changed to light brown within an hourand then later changed to dark brown during the 30 minincubation period. No significant change occurred after30 min. The absorption spectrum of M. paradisiaca peelextract at different wavelengths ranging from 300 to700 nm revealed a peak at 420 nm (Fig. 1b). FTIR analysisof the purified nanoparticles showed the presence of bandsdue to C=O group (1,629 cm−1), C–C and C–N stretching(1,384 cm−1), O–H stretch (1,082 cm−1), aromatic C–H outof plane deformation (821 cm−1) and N–H wag (759 cm−1)(Fig. 2). Figure 3 shows the XRD pattern of AgNPsobtained in this study. After the reaction, diffraction peaksat 2θ=34.37°, 38.01°, 44.17°, 66.34° and 77.29° assignedto the (100), (111), (102), (110) and (120) planes of a facedcentre cubic (fcc) lattice of silver were obtained. SEM

micrographs of the synthesized AgNPs of M. paradisiacamagnified at ×5,000 and ×20,000, times its size wasmeasured at 50 to 150 nm are shown in Fig. 4a and b.EDX proves the chemical purity of the synthesized AgNPs(Fig. 4c).

Discussion

The reduction of silver ions and formation of stablenanoparticles occurring within 4 h of the reaction was alsoachieved with Camellia sinensis extract (Vilchis-Nestora etal. 2009). In basil plant, the plasmon intensity at thereaction time of 11 h is near to that obtained at 15 h,implying the completion of the reaction. AgNPs wereobserved to be stable in solution and showed very littleaggregation (Ahmad et al. 2010). When metal nanoparticlesform in solution, they must be stabilized against the van derWaals forces of attraction, which may otherwise causecoagulation. Physisorbed surfactant and polymers maycause steric or electrostatic barriers or purely electrostaticbarriers around the particle surface and may therebyprovide stabilization (Mulvaney 1996). A few unassignedpeaks were also noticed in the vicinity of the characteristicpeaks (Sathishkumar et al. 2009). These sharp Bragg peaksmight have resulted due to the capping agent stabilizing thenanoparticle. The XRD pattern of pure silver ions is knownto display peaks at 2θ=7.9°, 11.4°, 17.8°, 30.38° and 44°(Gong et al. 2007). Therefore, XRD results also suggestthat crystallization of the bioorganic phase occurs on thesurface of the AgNPs. Silver nanoparticles were synthe-

Fig. 2 FTIR spectrum of silver nanoparticles synthesized by M.paradisiaca peel extract

Fig. 3 XRD pattern of AgNPs synthesized using M. paradisiaca peel extract

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sized using leaf extract of Acalypha indica; from the SEMimage, the size of the control silver nitrate obtained wasmore than 1000 nm, whereas synthesized silver nano-particles measured 20–30 nm in size (Krishnaraj et al.2010).

There is a need for an inexpensive nanoparticle toxicitymodel, using a delivery apparatus simple enough for benchtop use yet capable of delivering a wide range of nano-particles, and an organismal model that is inexpensive,experimentally powerful and relevant to human health(Schneider 2000). Nanoparticles have properties differentfrom bulk materials due to their increased surface area,affecting their ability to penetrate the skin, pass through cell

membranes, cross the blood brain barrier, be absorbed intolymphatic channels, and reach bone marrow, lymph nodes,heart, lungs and the central nervous system (Oberdörster etal. 2004, 2005). Malondialdehyde level, an end product oflipid peroxidation was significantly higher while antioxi-dant glutathione content was significantly lower in AgNPsexposed organisms (Ahamed et al. 2010). Drosophilamelanogaster was exposed to coated and uncoated AgNPsin a chronic toxicity assay, and tested for Heat shockproteins (Hsp 70) protein expression, and Western blotsprobed with anti-Hsp70 antibody demonstrated that Hsp70protein expression was higher in silver nanoparticleexposed flies as compared to the negative control (Posgai

Fig. 4 Image of scanning electron microscopic observation of synthesized silver nanoparticles: a lower magnification b higher magnification, cEDX, showing the chemical composition

928 Parasitol Res (2012) 111:921–933

et al. 2009). The toxic effects of well characterizedpolysaccharide coated 10 nm AgNPs on heat shock stress,oxidative stress, DNA damage and apoptosis in Drosophilamelanogaster. The third instar larvae of D. melanogasterwere fed a diet of standard cornmeal media mixed withAgNPs at the concentrations of 50 and 100 μg/ml for 24and 48 h. AgNPs up regulated the expression of heat shockprotein 70 and induced oxidative stress in D. melanogaster.Nanofibrous silk membranes with fiber diameters of 460±40 nm were electrospun from an aqueous Bombyx morifibroin solution, and incorporating silver nanoparticles wereprepared by dipping the membranes in aqueous silvernitrate (AgNO3) solution (0.5 or 1.0 wt.%) followed byphotoreduction (Kang et al. 2007). In Daphnia magnaAgNPs accumulation reached as high as 22.9 mg Ag/g drywt at the highest AgNPs concentration tested (500 μg/l),and it was extremely sensitive to free Ag ion (Ag+, addedas AgNO3), with a measured 48 h 50% lethal concentrationof 2.51 μg/l (Zhao and Wang 2010).

Ecotoxicity study was determined by AgNPs in 48 heffective concentration 50 (EC50) values for D. magna withsuspensions of 60 nm and 300 nm AgNPs, and were foundto be 1.0 (95% confidence interval (CI)=0.1–1.3) and 1.4(95% CI=0.3–2.1) μg Ag/l, respectively, and for Oryziaslatipes for 96 h; LC50 values of 60 and 300 nm AgNPsuspensions were 28 (95% CI=23–34) and 67 (95% CI=45–108) μg Ag/l, respectively, and there was no acutetoxicity with the AgNPs suspensions not containing Ag+

(Kim et al. 2010). AgNPs also induced chromosomalaberrations and aneuploidy, which showed that silvernanoparticles were cytotoxic and genotoxic to fish cells(Wise et al. 2010). Earthworms (Eisenia fetida) wereexposed to AgNO3, (94.21 mg kg−1) and AgNPs(727.6 mg kg−1 with similar size ranges coated with eitherpolyvinylpyrrolidone (hydrophilic) or oleic acid (amphi-philic) 773.3 mg kg−1during a standard sub-chronicreproduction toxicity test (Shoults-Wilson et al. 2010).The use of nanoparticulate silver, copper, and their oxideswill be considered in relation to their effects on bacterialpopulations. Silver nanoparticles formed exhibited goodantibiotic activity against both Gram-positive and Gram-negative pathogens and Candida albicans, suggesting theirbroad spectrum antimicrobial activity (Kumar et al. 2010).The toxicity of AgNPs to natural aquatic bacterial assemb-lages appears to be concentration dependent for concen-trations between 0 and 5 μM (Dasari and Hwang 2010).Mechanisms of toxicity are still poorly understood althoughit seems clear that in some cases, nanoscale specificproperties may cause bio-uptake and toxicity over andabove that caused by the dissolved Ag ion (Fabrega et al.2011). Kamaraj et al. (2010) reported that the leaf methanolof Rhinacanthus nasutus, leaf and seed methanol ofSolanum torvum, and seed acetone of Terminalia chebula

against the adult of H. bispinosa (LC50=333.15, 328.98,312.28, and 186.46 ppm; LC90=1,056.07, 955.39, 946.63,and 590.76 ppm), the leaf methanol of Cassia auriculata,the leaf and flower methanol of R. nasutus, the leaf ethylacetate of S. torvum against the Hip. maculata (LC50=303.36, 177.21, 204.58, and 211.41 ppm; LC90=939.90,539.39, 599.43, and 651.90 ppm). Bagavan et al. (2009)reported the leaf hexane extract of Annona squamosa andmethanol extracts of Gloriosa superba and Phyllanthusemblica against H. bispinosa with LC50=145.39, 225.57,and 256.08 ppm, respectively. The marine actinobacterialextracts showed moderate to high larvicidal effects after24 h of exposure at 1,000 ppm, and the highest larvalmortality was found in the extract of Streptomycesgedanensis (LC50=108.08 ppm) against the larvae of C.gelidus and (LC50=146.24 ppm) and against the larvae ofC. tritaeniorhynchus (Karthik et al. 2011). SynthesizedAgNPs using Tinospora cordifolia extract tested againstthe larvae of A. subpictus (LC50=6.43 mg/l) and againstthe larvae of C. quinquefasciatus (LC50=6.96 mg/l)(Jayaseelan et al. 2011). Larvicidal studies were carriedout against C. quinquefasciatus, and results were com-pared with bulk permethrin. The LC50 of nanopermethrinand bulk permethrin to C. quinquefasciatus were 0.117and 0.715 mg/l, respectively (Anjali et al. 2010). Sakulkuet al. (2009) have reported the low release rate ofnanoemulsion with a large droplet size that resulted inprolonged mosquito repellant activity compared to thenanoemulsion with small droplet size.

Results of a comparative study of larvicidal andadulticidal activity of different synthesized nanoparticlesof acaricides and insecticides are shown in Table 3.Larvicidal activity of synthesized AgNPs utilizing anaqueous extract from Eclipta prostrata, a member ofAsteraceae, was investigated against the fourth instar larvaeof filariasis and malaria, and the efficacy was observed incrude aqueous, and synthesized AgNPs against C. quinque-fasciatus (LC50=27.49 and 4.56 mg/l; LC90=70.38 and13.14 mg/l) and against A. subpictus (LC50=27.85 and5.14 mg/l; LC90=71.45 and 25.68 mg/l), respectively(Rajakumar and Rahuman 2011). AgNPs synthesized byfilamentous fungus Cochliobolus lunatus and its larvicidalactivity was tested in various concentrations (10, 5, 2.5,1.25, 0.625, and 0.3125 ppm) against second, third, andfourth instar larvae of Aedes aegypti (LC50 1.29, 1.48, and1.58; LC90 3.08, 3.33, and 3.41 ppm) and against A.stephensi (LC50 1.17, 1.30, and 1.41; LC90 2.99, 3.13, and3.29 ppm) (Salunkhe et al. 2011). The larvicidal effect ofaqueous crude leaf extracts, silver nitrate solution andsynthesized AgNPs of Mimosa pudica showed that thehighest mortality was found in synthesized AgNPs againstthe larvae of A. subpictus (LC50=8.89, 11.82, and 0.69 ppm)and against the larvae of C. quinquefasciatus (LC50=9.51,

Parasitol Res (2012) 111:921–933 929

Tab

le3

Com

parativ

estud

yof

larvicidal

andadulticidal

activ

ityof

differentsynthesizednano

particles

Sou

rce

Com

mon

name

Parts

used

Treated

samples

Particle

size

(nm)

Biological

activ

ityTarget

organism

sLC50

Reference

Nelum

bonu

cifera

Lotus

leaf

AgNPs

25–8

0Larvicidal

A.subp

ictus

0.69

ppm

Santhoshk

umar

etal.20

11C.qu

inqu

efasciatus

1.10

ppm

Mimosapu

dica

Sensitiv

ePlant

leaf

AgNPs

25–6

0Larvicidal

A.subp

ictus

13.90mg/l

Marim

uthu

etal.20

11C.qu

inqu

efasciatus

11.73mg/l

R.microplus

8.98

mg/l

Tino

sporacordifo

liaGud

uchi

leaf

AgNPs

55–8

0Larvicidal

A.subp

ictus

6.43

mg/l

Jayaseelan

etal.20

11C.qu

inqu

efasciatus

6.96

mg/l

Adu

lticidal

P.hu

man

uscapitis

12.46mg/l

Eclipta

prostrata

False

daisy

leaf

AgNPs

35–6

0Larvicidal

A.subp

ictus

5.14

mg/l

Rajakum

arandRahum

an20

11C.qu

inqu

efasciatus

4.56

mg/l

Citron

ella

oil(Cym

bopo

gonna

rdus)

−−

Nanoemulsion

120–20

0Repellent

A.aegypti

−Sakulku

etal.20

09

Lag

enidium

giga

nteum

−SiNPswith

Nacl

−Larvicidal

Mosqu

itoes

−Vandergheyn

stet

al.20

06

Lag

enidium

giga

nteum

−−

SiNPs

−Larvicidal

Mosqu

itoes

0.5mg/cm

2Vandergheyn

stet

al.20

07

Cochliobo

lusluna

tus

−−

AgNPs

3–21

Larvicidal

A.aegypti

1.58

mg/l

Salun

kheet

al.20

11A.stephensi

1.41

mg/l

Polym

ethacrylate

−−

AgNPs

−Larvicidal

A.aegypti

5pp

mSap-Lam

etal.20

10

Citron

ella

oil,Hairy

basiloil,andVetiver

oil

−−

Nanoemulsion

150–12

20Repellent

A.aegypti

−Nuchu

chua

etal.20

09

Zincnitrate

Zincnitrate

−ZnO

NPs

60–1

20Larvicidal

A.subp

ictus

11.14mg/l

Kirthiet

al.20

11C.qu

inqu

efasciatus

12.39mg/l

R.microplus

29.14mg/l

Adu

lticidal

P.hu

man

uscapitis

11.80mg/l

Cu(II)acetatehy

drate

Cu(II)acetatehy

drate

−CuNPs

35–8

0Larvicidal

A.subp

ictus

23.47mg/l

Ram

yadevi

etal.20

11C.qu

inqu

efasciatus

15.24mg/l

R.microplus

14.14mg/l

Permethrin

Permethrin

−Permethrin

NPs

151±27

Larvicidal

C.qu

inqu

efasciatus

0.117mg/l

Anjaliet

al.20

10

LC50lethal

concentrationthat

kills

50%

oftheexpo

sedlarvae

930 Parasitol Res (2012) 111:921–933

13.65, and 1.10 ppm) (Marimuthu et al. 2010). Thesynthesized zinc oxide nanoparticles showed the LC50 andr2 values against Rhipicephalus microplus (13.41 mg/l;0.982), Pediculus humanus capitis (11.80; 0.966 mg/l), andthe larvae of A. subpictus (3.19; 0.945 mg/l) and C.quinquefasciatus (4.87; 0.970 mg/l), respectively (Kirthi etal. 2011). The highest mortality was found in methanol,aqueous, and synthesized AgNPs, which used Nelumbonucifera plant extract against the larvae of A. subpictus(LC50=8.89, 11.82, and 0.69 ppm; LC90=28.65, 36.06, and2.15 ppm) and against the larvae of C. quinquefasciatus(LC50=9.51, 13.65, and 1.10 ppm; LC90=28.13, 35.83, and3.59 ppm) (Santhoshkumar et al. 2011).

All our experiments have largely validated our premisethat nanoparticles of various kinds can be put to use to asnovel agents of defense against old and common problemsthat plague mankind. The present study demonstrated thatuse of a natural, low-cost biological reducing agent; M.paradisiaca peel extracts (aqueous) can produce metalnanostructures, through efficient green nanochemistrymethodology, avoiding the presence of hazardous and toxicsolvents and waste; furthermore, the nanostructures showedexcellent acaricidal and larvicidal activity.

Acknowledgements The authors are grateful to C. Abdul Hakeemof the College Management; Dr. S. Mohammed Yousuff, Principal;and Dr. K. Abdul Subhan, HOD of Zoology Department for providingus the facilities to carry out this work.

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