Layout 1 [Journal of Entomological and Acarological Research 2013; 45:e11] [page 57] Green synthesis of silver nanoparticles using Cadaba indica lam leaf extract and its larvicidal and pupicidal activity against Anopheles stephensi and Culex quinquefasciatus K. Kalimuthu,1,2 C. Panneerselvam,1 K. Murugan,1 J.-S. Hwang2 1Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore, India; 2Institute of Marine Biology, National Taiwan Ocean University, Taiwan Abstract Green nanoparticle synthesis was achieved using environmentally acceptable plant extracts and eco-friendly reducing and capping agents. In the present study, activity of silver nanoparticles (AgNPs) synthesized using Cadaba indica lam plant against Anopheles stephen- si and Culex quinquefasciatus was determined. A range of concentra- tions of synthesized AgNPs (3.125, 6.25, 12.5, 25, 50 ppm) and crude extract (50, 100, 150, 200, 250 ppm) were tested against A. stephensi and C. quinquefasciatus. The synthesized AgNPs from C. indica lam were much more toxic than crude extract in both mosquito species. The cured extract high mortality values were 50% lethal concentration (LC50)=88.22, 90.84 ppm; 90% lethal concentration (LC90)=172.94, 178.55 ppm, and the AgNPs high mortality values were LC50=3.90, 4.39 ppm; LC90=19.04, 17.35 ppm against A. stephensi and C. quinquefascia- tus, respectively. The results recorded from ultraviolet-visible spec- trophotometer, scanning electron microscopy, energy dispersive X-ray and Fourier transformed infrared support the biosynthesis and charac- terization of silver nanoparticles. These results suggest that the leaf cured extracts of C. indica lam and green synthesis of silver nanopar- ticles have the potential to be used as an ideal eco-friendly approach for the control of A. stephensi and C. quinquefasciatus. Introduction Vector-borne diseases transmitted by blood-feeding mosquitoes, such as dengue hemorrhagic fever, Japanese encephalitis, malaria, and filariasis, are increasing in prevalence worldwide, particularly in tropical and subtropical zones. Malaria now is responsible for illness in more than an estimated 300 million people, resulting in one million deaths per year (WHO, 2007). Culex quinquefasciatus is a vector of lymphatic filariasis, which affects 120 million people worldwide, and approximately 400 million people are at risk of contracting filariasis, resulting in an annual economic loss of 1.5 billion dollars (WHO, 2002). Lymphatic filariasis is a serious public health problem in India, constituting one third of the infected population in the world (WHO et al., 1997). Mosquito-borne diseases are endemic to India due to favor- able ecological conditions for the vectors, their close contact with humans, and their reproductive biology. In rubber plantations, the rich organic content, stagnant water, low light levels and protected condi- tions in the coconut shells used in rubber production favors intense breeding (Sumodan, 2003). Mosquito control is improving in many areas, but there are significant challenges, including increasing resistance to insecticides and a lack of alternative, cost-effective, and safe insecticides. This increase in insecticide resistance requires the development of strategies for prolonging the use of highly effective vector control compounds. The use of combinations of multiple insec- ticides and phytochemicals is one such strategy that may be suitable for mosquito control. Attempts to develop novel materials as mosquito larvicides are still necessary. With the progress of nanotechnology research, many laboratories around the world have investigated silver nanoparticles (AgNPs) production. The development of green processes for the synthesis of nanoparti- cales is evolving into an important brach of nanotechnology. Nanoparticles play an indispensable role in drug delivery, diagnostics, imaging, sensing, gene delivery, artificial implants, and tissue engi- neering (Morones & Elechigerra, 2005). The development of a reliable green process for the synthesis of silver nanoparticles is an important aspect of current nanotechnology research. Biological methods for nanoparticle synthesis using microorganisms, enzymes, and plants or plant extracts have been suggested as possible eco-friendly alterna- tives to chemical and physical methods (Mohanpuria et al., 2008). Recently, green silver nanoparticles have been synthesized using var- ious natural products such as Nelumbo nucifera (Santhoshkumar et Correspondence: Kandasamy Kalimuthu, Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore - 641 046, India. Tel.: +91.770.875.5603. E-mail: biokalimuthu@yahoo.in Key words: Cadaba indica lam, silver nanoparticles, Anopheles stephensn, Culex quinquefasciatus. Acknowledgments: the authors are grateful to Dr. K. Sasikala, Professor and Head, Department of Zoology, Bharathiar University for the laboratory facil- ities providing for this experiment. Received for publication: 12 December 2012. Revision received: 31 May 2013. Accepted for publication: 7 June 2013. ©Copyright K. Kalimuthu et al., 2013 Licensee PAGEPress, Italy Journal of Entomological and Acarological Research 2013; 45:e11 doi:10.4081/jear.2013.e11 This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 3.0) which permits any noncom- mercial use, distribution, and reproduction in any medium, provided the orig- inal author(s) and source are credited. Journal of Entomological and Acarological Research 2012; volume 44:eJournal of Entomological and Acarological Research 2013; volume 45:e11 Jear_2013_2:Hrev_master 16/09/13 13.56 Pagina 57 No n- co mm er cia l =3.90, 4.39 No n- co mm er cia l =3.90, 4.39 C. quinquefascia- No n- co mm er cia l C. quinquefascia- respectively. The results recorded from ultraviolet-visible spec- No n- co mm er cia l respectively. The results recorded from ultraviolet-visible spec- trophotometer, scanning electron microscopy, energy dispersive X-ray No n- co mm er cia l trophotometer, scanning electron microscopy, energy dispersive X-ray and Fourier transformed infrared support the biosynthesis and charac- No n- co mm er cia l and Fourier transformed infrared support the biosynthesis and charac- terization of silver nanoparticles. These results suggest that the leaf No n- co mm er cia l terization of silver nanoparticles. These results suggest that the leaf tropical and subtropical zones. Malaria now is responsible for illness in No n- co mm er cia l tropical and subtropical zones. Malaria now is responsible for illness inmore than an estimated 300 million people, resulting in one million No n- co mm er cia l more than an estimated 300 million people, resulting in one milliondeaths per year (WHO, 2007). No n- co mm er cia l deaths per year (WHO, 2007). lymphatic filariasis, which affects 120 million people worldwide, and No n- co mm er cia l lymphatic filariasis, which affects 120 million people worldwide, and No n- co mm er cia l approximately 400 million people are at risk of contracting filariasis, No n- co mm er cia l approximately 400 million people are at risk of contracting filariasis, No n- co mm er cia l Correspondence: Kandasamy Kalimuthu, Division of Entomology, No n- co mm er cia l Correspondence: Kandasamy Kalimuthu, Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, No n- co mm er cia l Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore - 641 046, India. Tel.: +91.770.875.5603. No n- co mm er cia l Coimbatore - 641 046, India. Tel.: +91.770.875.5603. lam, silver nanoparticles, No n- co mm er cia l lam, silver nanoparticles, us e Vector-borne diseases transmitted by blood-feeding mosquitoes, us e Vector-borne diseases transmitted by blood-feeding mosquitoes,such as dengue hemorrhagic fever, Japanese encephalitis, malaria, us e such as dengue hemorrhagic fever, Japanese encephalitis, malaria, and filariasis, are increasing in prevalence worldwide, particularly inus e and filariasis, are increasing in prevalence worldwide, particularly in tropical and subtropical zones. Malaria now is responsible for illness inus e tropical and subtropical zones. Malaria now is responsible for illness in more than an estimated 300 million people, resulting in one millionus e more than an estimated 300 million people, resulting in one million on ly and on ly and C. quinquefasciatus on ly C. quinquefasciatus on ly Vector-borne diseases transmitted by blood-feeding mosquitoes, on ly Vector-borne diseases transmitted by blood-feeding mosquitoes, [page 58] [Journal of Entomological and Acarological Research 2013; 45:e11] al., 2011), Pongamia pinnata (Rajesh et al., 2010), Azadirachta indica (Tripathi et al., 2009), Glycine max (Vivekanandhan et al., 2009), Cinnamon zeylanicum (Sathishkumar et al., 2009), and Camellia sinensis (Begum et al., 2009). In recent studies, potential mosquito lar- vicidal activity of synthesized AgNPs from plant extracts as well as physical methods is well documented (Marimuthu et al., 2011; Thirunavukkarasu et al., 2010; Sap-Iam et al., 2010). Plants are rich sources of bioactive organic chemicals and offer an advantage over synthetic pesticides, as these are less toxic, less prone to development of resistance, and easily biodegradable. India can uti- lize its rich supply of herbs for such purposes, as plant extracts are not only potentially insecticides, but also can act as effective antimicrobial, antifungal, anti- parasitic and anti-malarial agents. Plant materials not only offer effective mosquito control agents, but also promise to be environmentally safer. Therefore, an alternative approach for mosquito control is the use of natural products of plant origin. The botanical insecticides are generally pest-specific, readily biodegradable, and usu- ally lack toxicity to higher animals (Bowers, 1992). In traditional med- icine systems, different parts of the plant have been described to be useful against a variety of diseases. The leaves of Cadaba indica lam plant are rich in lactones, steroids, flavonoids, alkaloids, reducing sug- ars and tannins (Peach & Tracy 1955; Rastogi & Mehrotra, 1991). C. indica lam leaf extract is used on boils; its leaf juice is used as eye drops. Against cattle fever, a decoction of fresh leaves, pepper and gar- lic is administered orally (Reddy et al., 2007). However, the activity of the ethanol extract of the leaves was found to be most effective against bacteria and fungi (Selvamani & Latha, 2005). The leaf and flower liq- uid extract mixed with castor oil and turmeric is taken as a remedy for menorrhagia, syphilis, and as a purgative (Alagesaboopathi, 2009). Pathak et al. (2000) reported that the steam-distilled whole plant oil extract of Tagetes minuta gave 100% mortality against larvae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti at doses lower than 100 ppm. Volatile oil extracted from the peel of citrus fruits has also shown toxic effects on mosquito larvae as well as adults (Ezeonu et al., 2001). In the present study, we report on the synthesis of silver nanoparti- cles, reducing the silver ions present in the solution of silver nitrate by C. indica lam leaf extract, and its efficacy against A. stephensi and C. quinquefasciatus. Materials and methods Materials The Cadaba indica lam plants were collected in and around Kaveri river bank, Namakkal District, in Tamilnadu, India, and identified by the taxonomist, Department of Botany, Bharathiar University, Coimbatore, India. The voucher specimen was numbered and kept in the authors’ research laboratory for further reference. Silver nitrate (AgNO3) was purchased from Precision Scientific Co., Coimbatore, India. Mosquito rearing The eggs of A. stephensi and C. quinquefasciatus were collected from the National Centre for Disease Control field station of Mettupalayam, Tamil Nadu, India. These were brought to the laboratory and trans- ferred (in approximately the same aliquot numbers of eggs) into 18 cm L×13 cm W×4 cm D enamel trays containing 500 mL of water, where they were allowed to hatch. Mosquito larvae were reared (and adult mosquitoes held) at 27±2°C and 75-85% relative humidity in a 14:10 (L/D) photoperiod. The larvae were fed 5-g ground dog biscuit and brewer’s yeast daily in a 3:1 ratio. The pupae were collected and trans- ferred into plastic containers with 500 mL of water. The container was placed inside a screened cage (90 cm L×90 cm H×90 W) to retain emerging adults, for which 10% sucrose in water solution (v/v) was made available. On day 5 post-emergence, the mosquitoes were provid- ed access to a rabbit host for blood feeding. The shaved dorsal side of the rabbit was positioned on the top of the mosquito cage in contact with the cage screen (using a cloth sling to hold the rabbit) and held in this position overnight. Glass Petri dishes lined with filter paper and containing 50 mL of water were subsequently placed inside the cage for oviposition by female mosquitoes. Synthesis of silver nanoparticles Leaves were washed with distilled water and dried for 2 days at room temperature. A plant leaf broth was prepared by placing 10 g of the leaves (finely cut) in a 300-mL flask with 100 mL of sterile distilled water. This mixture was boiled for 5 min, decanted, stored at −4°C, and used in our tests within 1 week. The filtrate was treated with aqueous 1 mM AgNO3 solution in an Erlenmeyer flask and incubated at room temperature. As a result, in a brown-yellow solution indicating the for- mation of AgNPs, it was found that aqueous silver ions can be reduced by aqueous extract of the plant parts to generate extremely stable silver nanoparticles in water. Characterization of silver nanoparticles The presence of synthesized silver nanoparticles was confirmed by sampling the reaction mixture at regular intervals and the absorption maxima was scanned by ultraviolet-visible (UV-vis) spectra at the wavelengths of 350-600 nm in a UV-3600 Shimadzu spectrophotometer at 1 nm resolution. Further, the reaction mixture was subjected to cen- trifugation at 15,000 rpm for 20 min; the resulting pellet was dissolved in deionized water and filtered through a millipore filter (0.45 μm). An aliquot of this filtrate containing silver nanoparticles was used for scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) studies. Thin films of the sample were prepared on a carbon- coated copper grid by dropping a small amount of the sample on the grid; extra solution was removed using a blotting paper, and the films on the SEM grid were allowed to dry by placing it under a mercury lamp for 5 min. The surface groups of the nanoparticles were qualitatively confirmed by using Fourier transformed infrared spectroscopy (FTIR) (Stuart, 2002), with spectra recorded by a Perkin-Elmer Spectrum 2000 FTIR spectrophotometer. Larval/pupal toxicity test Twenty-five larvae (instars I-IV) or pupae were placed in 249 mL of dechlorinated water in a 500-mL glass beaker, and 1 mL of the desired concentration of silver nanoparticles was added; 0.5 mg of larval food was provided for each test concentration. Tests of each concentration against each instar and the pupae were replicated three times. In each case, the control comprised 25 larvae or pupae in 250 mL of distilled water. Control mortality was corrected by using Abbott’s formula (Abbott, 1925), and percent mortality was calculat- ed as follows: Percent mortality= Number of dead larvae/pupae ¥100 Number of larvae/pupae introduced Statistical analysis Average larval mortality data were subjected to probit analysis for calculating 50% and 90% lethal concentration (LC50 and LC90) values, using the method of Finney (1971). SPSS software, ver. 9.0 (StataCorp., College Station, TX, USA), was used. Results were considered to be sta- tistically significant at P<0.05. Article Jear_2013_2:Hrev_master 16/09/13 13.56 Pagina 58 No n- co mm er cia l lower than 100 ppm. Volatile oil extracted from the peel of citrus fruits No n- co mm er cia l lower than 100 ppm. Volatile oil extracted from the peel of citrus fruits has also shown toxic effects on mosquito larvae as well as adults No n- co mm er cia l has also shown toxic effects on mosquito larvae as well as adults In the present study, we report on the synthesis of silver nanoparti- No n- co mm er cia l In the present study, we report on the synthesis of silver nanoparti- cles, reducing the silver ions present in the solution of silver nitrate by No n- co mm er cia l cles, reducing the silver ions present in the solution of silver nitrate by A. stephensi No n- co mm er cia l A. stephensi and No n- co mm er cia l and No n- co mm er cia l lam plants were collected in and around KaveriN on -co mm er cia l lam plants were collected in and around Kaveri wavelengths of 350-600 nm in a UV-3600 Shimadzu spectrophotometer No n- co mm er cia l wavelengths of 350-600 nm in a UV-3600 Shimadzu spectrophotometerat 1 nm resolution. Further, the reaction mixture was subjected to cen- No n- co mm er cia l at 1 nm resolution. Further, the reaction mixture was subjected to cen-trifugation at 15,000 rpm for 20 min; the resulting pellet was dissolved No n- co mm er cia l trifugation at 15,000 rpm for 20 min; the resulting pellet was dissolved in deionized water and filtered through a millipore filter (0.45 No n- co mm er cia l in deionized water and filtered through a millipore filter (0.45 aliquot of this filtrate containing silver nanoparticles was used for No n- co mm er cia l aliquot of this filtrate containing silver nanoparticles was used for scanning electron microscopy (SEM) and energy dispersive X-ray No n- co mm er cia l scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) studies. Thin films of the sample were prepared on a carbon- No n- co mm er cia l (EDX) studies. Thin films of the sample were prepared on a carbon- us e Characterization of silver nanoparticles us e Characterization of silver nanoparticles The presence of synthesized silver nanoparticles was confirmed by us e The presence of synthesized silver nanoparticles was confirmed bysampling the reaction mixture at regular intervals and the absorption us e sampling the reaction mixture at regular intervals and the absorption us e maxima was scanned by ultraviolet-visible (UV-vis) spectra at theus e maxima was scanned by ultraviolet-visible (UV-vis) spectra at the wavelengths of 350-600 nm in a UV-3600 Shimadzu spectrophotometerus e wavelengths of 350-600 nm in a UV-3600 Shimadzu spectrophotometer at 1 nm resolution. Further, the reaction mixture was subjected to cen-us e at 1 nm resolution. Further, the reaction mixture was subjected to cen- on ly temperature. As a result, in a brown-yellow solution indicating the for- on ly temperature. As a result, in a brown-yellow solution indicating the for- mation of AgNPs, it was found that aqueous silver ions can be reduced on ly mation of AgNPs, it was found that aqueous silver ions can be reduced by aqueous extract of the plant parts to generate extremely stable silver on lyby aqueous extract of the plant parts to generate extremely stable silver Characterization of silver nanoparticleson ly Characterization of silver nanoparticles The presence of synthesized silver nanoparticles was confirmed by on ly The presence of synthesized silver nanoparticles was confirmed by Results and discussion Several approaches have been employed to obtain better biosynthe- sis of nanoparticles, which is preferable to chemical and physical meth- ods, as it is a cost-effective and environmentally friendly method, and does not require the use of high pressure, energy, temperature, or toxic chemicals (Sinha et al., 2009; Goodsell, 2004). In the present study, the larvicidal and pupicidal effects of ethanol leaf extracts and synthesized AgNPs of C. indica lam were noted; the highest mortality was found in synthesized AgNps against larvae and pupae of A. stephensi (LC50=3.90, 4.67, 10.20, 15.41, 25.27 ppm and LC90=19.04, 27.06, 47.72, 61.07, 78.32 mg/L) and C. quinquefasciatus (LC50=4.39, 5.07, 8.21, 15.44, 23.83 mg/L and LC90=17.35, 20, 35.76, 58.37, 75.33 ppm), respectively (Table 1 and Figure 1). The highest mortality was found against the larvae and pupae of A. stephensi (LC50=88.22, 107.34, 136.98, 169.04, 270.68 mg/L and LC90=172.94, 209.09, 284.08, 314.46, 474.85 ppm) and C. quinque- fasciatus (LC50=90.84, 115.37, 145.18, 172.92, 288.86 mg/L and LC90=178.55, 211.37, 277.29, 311.29, 525.13 ppm), respectively (Table 2 and Figure 2). Chi-square values were significant at the P≤0.05 level. Fifty-percent hydroethanolic extracts of Bonninghausenia albiflora whole plant, Calotropis procera root, Citrus maxima flower, Acorus cala- mus rhizome, and Weidelia chinensis whole plant showed acaricidal efficacy ranging from 4% to 35% within 24 h of application on Rhipicephalus (Boophilus) microplus. Rhizome extract of A. calamus revealed that a 79.31% correlation with log concentration in probit mor- tality could be assigned to the concentration of the extract, and the regression line of the extract showed the LC85 as 11.26% (Ghosh et al., 2011). Chandran et al. (2006) synthesized silver nanoparticles by using Aloe vera extract at 24 h of incubation. Previous authors reported that the methanol extract of Cassia fistula exhibited LC50 values of 17.97 and 20.57 mg/L for A. stephensi and C. quinquefasciatus, respectively (Govindarajan et al., 2008). A 23% mortality was noted against first- instar larvae of A. stephensi by treatment of A. ilicifolius extract at 20 ppm; this increased to 89% at 100 ppm (Kovendan & Murugan, 2011). Mosquitocidal properties of Calotropis gigantea leaf extract and bacter- ial insecticidal properties of Bacillus thuringiensis against these mos- quito vectors have been reported by Kovendan et al. (2012). Reduction of silver ions in the aqueous solution during reaction with the ingredients present in plant leaf extract was observed by UV-visible spectroscopy. The color change was noted by visual observation in the C. indica lam leaf extracts when incubated with AgNO3 solution. C. indica lam leaf extract without AgNO3 did not show any change in color (Figure 3). The color of the extract changed to light brown within an hour, and later changed to dark brown during a 1 h incubation period, after which no significant change occurred. Appearance of the yellow- ish brown color was an indication of formation of colloidal silver nanoparticles in the medium. The brown color could be due to the exci- [Journal of Entomological and Acarological Research 2013; 45:e11] [page 59] Article Table 1. Larvicidal activity of C. indica lam crude leaf ethnolic extract against larva and pupa of A. stephensi and C. quinquefasciatus. Species Life Percentage of larval and pupal mortality Slope LC50(LC90) 95% confidence limit X2 stages Concentration of EEC (ppm) LCL UCL (df=3) (instars) 50 100 150 200 250 LC50(LC90) LC50(LC90) A. stephensi I 30.0±2.4 56.2±2.3 80.0±2.0 95.8±2.2 100±1.2 0.359 88.22 (172.94) 77.06 (160.36) 97.89 (189.33) 1.383 II 25.6±3.6 42.2±2.4 71.8±2.6 88.4±3.0 96.2±1.0 0.374 107.34 (209.09) 95.61 (194.30) 117.80 (228.84) 1.051 III 21.2±3.3 33.8±3.0 62.8±2.2 70.4±2.2 81.2±1.6 0.313 136.98 (284.08) 122.60 (258.08) 150.50 (321.54) 3.890 IV 13.8±2.2 24.2±3.6 49.6±2.8 61.6±3.8 73.4±1.2 0.313 169.04 (314.46) 155.65 (285.28) 183.56 (356.63) 2.571 Pupa 8.0±1.0 15.2±3.4 22.8±2.0 30.2±3.0 46.4±2.8 0.183 270.68 (474.85) 241.07 (402.53) 320.08 (605.86) 0.522 C. quinquefasciatus I 28.2±2.2 54.8±2.0 81.2±2.0 92.6±2.2 100±1.0 0.362 90.84 (178.55) 79.50 (165.69) 100.69 (195.26) 1.731 II 21.0±2.4 40.8±2.2 66.4±1.6 85.6±2.8 97.8±1.2 0.396 115.37 (211.37) 104.70 (197.44) 125.20 (230.68) 1.107 III 16.8±3.0 30.2±2.8 58.6±2.2 70.0±3.0 82.40±1.4 0.342 145.18 (277.29) 132.51 (254.27) 157.58 (309.39) 2.607 IV 11.8±2.8 22.6±3.4 45.2±2.8 64.6±2.2 71.8±1.0 0.324 172.92 (311.29) 160.05 (283.70) 187.01 (350.82) 2.921 Pupa 8.8±2.1 15.4±2.2 24.6±3.2 31.4±3.2 40.6±2.6 0.159 288.86 (525.13) 251.76 (432.37) 357.53 (709.61) 0.387 LC50, 50% lethal concentration; LC90, 90% lethal concentration; EEC, ethanolic extract of Cadaba indica lam; Control nil mortality; LCL, lower confidence limit; UCL, upper confidence limit; df, degrees of freedom. Each value is the mean±SD of five replicates. Table 2. Larvicidal activity of synthesized silver nanoparticles using C. indica lam leaf extract against larvae and pupa of A. stephensi and C. quinquefasciatus. Species Life Percentage of larval and pupal mortality Slope LC50(LC90) 95% confidence limit X2 stages Concentration of AgNPs (ppm) LCL UCL (df=3) (instars) 3.125 6.25 12.5 25 50 LC50(LC90) LC50(LC90) A. stephensi I 42.0±2.8 61.2±3.0 80.8±1.0 94.6±1.1 100±1.2 1.068 3.90 (19.04) 1.42 (16.48) 5.72 (23.00) 3.378 II 40.8±2.4 55.6±3.8 71.4±2.4 88.4±1.4 98.8±1.0 1.128 4.67 (27.06) 1.43 (23.32) 7.13 (32.80) 3.376 III 35.2±2.0 42.6±3.5 58.2±2.5 75.0±1.8 88.4±1.4 1.099 10.20 (47.72) 6.04 (40.95) 13.68 (58.18) 4.909 IV 29.8±3.4 40.0±2.6 51.2±2.2 66.4±2.4 80.2±1.0 1.010 15.41 (61.07) 10.97 (51.50) 19.51 (76.74) 4.895 Pupa 21.8±3.6 33.4±2.8 42.6±2.6 55.8±2.6 68.8±1.3 0.918 25.27 (78.32) 13.80 (53.53) 45.48 (190.03) 6.095 C. quinquefasciatus I 44.2±3.0 53.2±2.4 85.8±1.0 96.2±1.0 100±1.0 1.099 4.39 (17.35) 2.37 (15.12) 5.93 (20.74) 5.051 II 40.0±2.4 55.2±2.1 77.8±2.1 94.2±1.0 100±1.0 1.158 5.07 (20.00) 2.89 (17.40) 6.76 (23.96) 1.843 III 36.8±1.6 43.2±2.0 63.4±2.7 82.4±2.0 95.4±1.4 1.227 8.21 (35.76) 4.99 (31.11) 10.92 (42.62) 4.851 IV 30.4±3.4 36.4±2.0 51.6±3.5 68.4±1.8 81.2±1.2 1.063 15.44 (58.37) 3.93 (42.23) 25.07 (110.23) 5.801 Pupa 27.2±3.0 30.4±2.4 42.6±3.3 56.4±2.7 71.4±1.0 0.942 23.83 (75.33) 19.26 (62.54) 29.18 (97.33) 2.888 LC50, 50% lethal concentration; LC90, 90% lethal concentration; AgNPs, silver nanoparticles; Control nil mortality; LCL, lower confidence limit; UCL, upper confidence limit; df, degrees of freedom. Each value is the mean±SD of five replicates. Jear_2013_2:Hrev_master 16/09/13 13.56 Pagina 59 No n- co mm er cia l lam crude leaf ethnolic extract against larva and pupa of No n- co mm er cia l lam crude leaf ethnolic extract against larva and pupa of No n- co mm er cia l Species Life Percentage of larval and pupal mortality Slope LC No n- co mm er cia l Species Life Percentage of larval and pupal mortality Slope LC No n- co mm er cia l Concentration of EEC (ppm) No n- co mm er cia l Concentration of EEC (ppm) No n- co mm er cia l (instars) 50 100 150 200 250 No n- co mm er cia l (instars) 50 100 150 200 250 I 30.0±2.4 56.2±2.3 80.0±2.0 95.8±2.2 100±1.2 0.359 88.22 (172.94) 77.06 (160.36) 97.89 (189.33) 1.383 No n- co mm er cia l I 30.0±2.4 56.2±2.3 80.0±2.0 95.8±2.2 100±1.2 0.359 88.22 (172.94) 77.06 (160.36) 97.89 (189.33) 1.383 II 25.6±3.6 42.2±2.4 71.8±2.6 88.4±3.0 96.2±1.0 0.374 107.34 (209.09) 95.61 (194.30) 117.80 (228.84) 1.051 No n- co mm er cia l II 25.6±3.6 42.2±2.4 71.8±2.6 88.4±3.0 96.2±1.0 0.374 107.34 (209.09) 95.61 (194.30) 117.80 (228.84) 1.051 III 21.2±3.3 33.8±3.0 62.8±2.2 70.4±2.2 81.2±1.6 0.313 136.98 (284.08) 122.60 (258.08) 150.50 (321.54) 3.890 No n- co mm er cia l III 21.2±3.3 33.8±3.0 62.8±2.2 70.4±2.2 81.2±1.6 0.313 136.98 (284.08) 122.60 (258.08) 150.50 (321.54) 3.890 No n- co mm er cia l IV 13.8±2.2 24.2±3.6 49.6±2.8 61.6±3.8 73.4±1.2 0.313 169.04 (314.46) 155.65 (285.28) 183.56 (356.63) 2.571 No n- co mm er cia l IV 13.8±2.2 24.2±3.6 49.6±2.8 61.6±3.8 73.4±1.2 0.313 169.04 (314.46) 155.65 (285.28) 183.56 (356.63) 2.571 Pupa 8.0±1.0 15.2±3.4 22.8±2.0 30.2±3.0 46.4±2.8 0.183 270.68 (474.85) 241.07 (402.53) 320.08 (605.86) 0.522 No n- co mm er cia l Pupa 8.0±1.0 15.2±3.4 22.8±2.0 30.2±3.0 46.4±2.8 0.183 270.68 (474.85) 241.07 (402.53) 320.08 (605.86) 0.522 No n- co mm er cia l I 28.2±2.2 54.8±2.0 81.2±2.0 92.6±2.2 100±1.0 0.362 90.84 (178.55) 79.50 (165.69) 100.69 (195.26) 1.731 No n- co mm er cia l I 28.2±2.2 54.8±2.0 81.2±2.0 92.6±2.2 100±1.0 0.362 90.84 (178.55) 79.50 (165.69) 100.69 (195.26) 1.731 No n- co mm er cia l II 21.0±2.4 40.8±2.2 66.4±1.6 85.6±2.8 97.8±1.2 0.396 115.37 (211.37) 104.70 (197.44) 125.20 (230.68) 1.107 No n- co mm er cia l II 21.0±2.4 40.8±2.2 66.4±1.6 85.6±2.8 97.8±1.2 0.396 115.37 (211.37) 104.70 (197.44) 125.20 (230.68) 1.107 No n- co mm er cia l III 16.8±3.0 30.2±2.8 58.6±2.2 70.0±3.0 82.40±1.4 0.342 145.18 (277.29) 132.51 (254.27) 157.58 (309.39) 2.607 No n- co mm er cia l III 16.8±3.0 30.2±2.8 58.6±2.2 70.0±3.0 82.40±1.4 0.342 145.18 (277.29) 132.51 (254.27) 157.58 (309.39) 2.607 No n- co mm er cia l IV 11.8±2.8 22.6±3.4 45.2±2.8 64.6±2.2 71.8±1.0 0.324 172.92 (311.29) 160.05 (283.70) 187.01 (350.82) 2.921 No n- co mm er cia l IV 11.8±2.8 22.6±3.4 45.2±2.8 64.6±2.2 71.8±1.0 0.324 172.92 (311.29) 160.05 (283.70) 187.01 (350.82) 2.921 No n- co mm er cia l No n- co mm er cia l No n- co mm er cia l No n- co mm er cia l No n- co mm er cia l Pupa 8.8±2.1 15.4±2.2 24.6±3.2 31.4±3.2 40.6±2.6 0.159 288.86 (525.13) 251.76 (432.37) 357.53 (709.61) 0.387 No n- co mm er cia l Pupa 8.8±2.1 15.4±2.2 24.6±3.2 31.4±3.2 40.6±2.6 0.159 288.86 (525.13) 251.76 (432.37) 357.53 (709.61) 0.387 , 90% lethal concentration; EEC, ethanolic extract of No n- co mm er cia l , 90% lethal concentration; EEC, ethanolic extract of us e nanoparticles in the medium. The brown color could be due to the exci- us e nanoparticles in the medium. The brown color could be due to the exci- lam crude leaf ethnolic extract against larva and pupa of us e lam crude leaf ethnolic extract against larva and pupa of on ly leaf extract without AgNO on ly leaf extract without AgNO (Figure 3). The color of the extract changed to light brown within an on ly (Figure 3). The color of the extract changed to light brown within an hour, and later changed to dark brown during a 1 h incubation period, on lyhour, and later changed to dark brown during a 1 h incubation period,after which no significant change occurred. Appearance of the yellow- on lyafter which no significant change occurred. Appearance of the yellow- ish brown color was an indication of formation of colloidal silveron ly ish brown color was an indication of formation of colloidal silver nanoparticles in the medium. The brown color could be due to the exci-on ly nanoparticles in the medium. The brown color could be due to the exci- [page 60] [Journal of Entomological and Acarological Research 2013; 45:e11] tation of surface plasmon vibrations, typical of silver nanoparticles (Ahmad et al., 2003; Krishnaraj et al., 2010). The dark brown color of the silver colloid is attributable to surface plasmon resonance arising from the group of free conduction electrons induced by an interacting electromagnetic field (Song & Kim, 2008). The strong surface plasmon resonance band appears at the range of 420-480 nm and the broaden- ing peak indicates that the particles are monodispersed (Figure 4). These color changes arise because of the excitation of surface plasmon vibrations in the silver nanoparticles (Mulvaney, 1996). SEM (JEOL-MODEL 6390) image showing high density Ag nanopar- ticles synthesized by C. indica lam plant extracts further confirmed the presence of Ag nanoparticles (Figure 5). It was shown that relatively spherical and uniform Ag nanoparticles were formed with a diameter of 30-60 nm. The SEM image of silver nanoparticles synthesized by plant extracts were assembled on the surface due to interactions such as hydrogen bonding and electrostatic interactions between the bio- organic capping molecules bound to the Ag nanoparticles. It was found that relatively spherical and uniform silver nanoparticles were formed. The nanoparticles were not in direct contact, even within the aggre- gates, indicating stabilization of the nanoparticles by a capping agent (Song & Kim, 2008). Silver nanoparticles have been characterized using SEM by various investigators (Durán et al., 2005; Balaji et al., 2009). Silver nanoparticles were synthesized using leaf extracts of Acalypha indica; from the SEM image, the size of the control silver nitrate obtained was more than 1000 nm, whereas synthesized silver nanoparticles measured 20-30 nm in size (Krishnaraj et al., 2010). Tian et al. (2007) reported that numerous flavonoids, including quercetin or quercetin 3-O-glycosides, were isolated from lotus leaves that were used for silver nanoparticle synthesis. The element analysis of the sil- ver nanoparticles was performed using EDX on the SEM. Figure 6 Article Figure 1. Larvicidal activity of C. indica lam crude leaf ethnolic extract against larva and pupa of A. stephensi and C. quinquefas- ciatus. Figure 2. Larvicidal activity of syn- thesized silver nanoparticles using C. indica lam leaf extract against larvae and pupa of A. stephensi and C. quinquefasciatus. Jear_2013_2:Hrev_master 16/09/13 13.56 Pagina 60 No n- co mm er cia l No n- co mm er cia l u se us e us e o nlyon ly shows the EDX spectrum of AgNPs synthesized at 25ºC and 80ºC; strong signals from the silver atoms in the nanoparticles were observed, and signals from calcium, potassium, oxygen, sodium, magnesium, sul- phur, Ag and chloro were also recorded. The results indicate that the reaction product was composed of higher level Ag nanoparticles. The AgNPs produced by C. indica lam leaf extract were distinct and scattered in distribution. The Fourier transformed infrared spectra of AgNPs exhibited prominent peaks at 3453; 3288; 1790; 1638; 1384; 1114; 1077; 371; 360 cm−1 (Figure 7). The sharp absorption peak at 1638 cm−1 was assigned to C=O stretching vibration in the carbonyl compounds, which may be characterized by the presence of a high con- tent of terpenoids and flavonoids. The peaks at 1077 cm−1 correspond to C–N stretching vibration of aliphatic amines or alcohols/phenols, representing the presence of polyphenols. The absorption bands at 1088 cm−1 in the fingerprint region indicate several modes such as C–H deformation or C–O or C–C stretching, pertaining to carbohy- drates. The bands at 1383 to 1431 cm−1 were assigned to scissoring modes of methylene tails, CH3 R. A broad intense band at 3402 cm−1 in both the spectra can be assigned to the N–H stretching frequency aris- ing from the peptide linkages present in the proteins of the extract (Mukherjee et al., 2008). Conclusions The present study of green synthesis shows that the environmental- ly benign and renewable source of C. indica lam is used as an effective reducing agent for the synthesis of AgNPs. This biological reduction of silver nanoparticles would be a boon for the development of a clean, nontoxic, and environmentally acceptable green approach to produc- tion of AgNPs, involving organisms extending even to higher plants. The AgNPs did not exhibit any noticeable effects on C. indica lam expo- sure at their LC50 and LC90 values against larvae of A. stephensi and C. [Journal of Entomological and Acarological Research 2013; 45:e11] [page 61] Article Figure 4. Ultraviolet-visible spectra of aqueous silver nitrate with C. indica lam leaf extract at different time intervals. Figure 3. Photographs showing change in color after adding silver nitrate (AgNO3) before reaction and after reaction time of 30 min. Figure 5. Image of scanning electron microscopic observation of synthesized silver nanoparticles. A) Lower magnification (0.5 µm); B) Higher magnification (1 µm). Jear_2013_2:Hrev_master 16/09/13 13.56 Pagina 61 No n- co mm er cia l No n- co mm er cia l No n- co mm er cia l No n- co mm er cia l No n- co mm er cia l No n- co mm er cia l No n- co mm er cia l Figure 3. Photographs showing change in color after adding silver No n- co mm er cia l Figure 3. Photographs showing change in color after adding silver No n- co mm er cia l ) before reaction and after reaction time of 30 min. No n- co mm er cia l ) before reaction and after reaction time of 30 min. No n- co mm er cia l u se us e o nlyon ly [page 62] [Journal of Entomological and Acarological Research 2013; 45:e11] Article Figure 6. Energy dispersive X-ray spectra recorded form a film, after formation of silver nanoparticles with different X-ray emission peaks labeled. Cl, chloro; K, potassium; Ca, calcium; O, oxygen; Na, sodium; Mg, magnesium; S, sulphur; Ag, silver. Figure 7. Fourier transformed infrared spectroscopy spectrum of silver nanoparticle synthesized by reacting silver nitrate with C. indi- ca lam leaf extract. Jear_2013_2:Hrev_master 16/09/13 13.56 Pagina 62 No n- co mm er cia l No n- co mm er cia l No n- co mm er cia l No n- co mm er cia l Figure 6. Energy dispersive X-ray spectra recorded form a film, after formation of silver nanoparticles with different X-ray emission No n- co mm er cia l Figure 6. Energy dispersive X-ray spectra recorded form a film, after formation of silver nanoparticles with different X-ray emission No n- co mm er cia l peaks labeled. Cl, chloro; K, potassium; Ca, calcium; O, oxygen; Na, sodium; Mg, magnesium; S, sulphur; Ag, silver. No n- co mm er cia l peaks labeled. 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TRIPATHI A., CHANDRASEKARAN N., RAICHUR A.M., MUKHERJEE A., [Journal of Entomological and Acarological Research 2013; 45:e11] [page 63] Article Jear_2013_2:Hrev_master 16/09/13 13.56 Pagina 63 No n- co mm er cia l CHANDRAN S.P., CHAUDHARY M., PASRICHA R., AHMAD A., SASTRY No n- co mm er cia l CHANDRAN S.P., CHAUDHARY M., PASRICHA R., AHMAD A., SASTRY M., 2006 - Synthesis of gold nanotriangles and silver nanoparticles No n- co mm er cia l M., 2006 - Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract. - Biotechnol. Prog. 22: 577-583. No n- co mm er cia l using Aloe vera plant extract. - Biotechnol. Prog. 22: 577-583. DURÁN N., MARCATO P.D., ALVES O.L., SOUZA G.I., ESPOSITO E., 2005 No n- co mm er cia l DURÁN N., MARCATO P.D., ALVES O.L., SOUZA G.I., ESPOSITO E., 2005 - Mechanistic aspects of biosynthesis of silver nanoparticles by sev- No n- co mm er cia l - Mechanistic aspects of biosynthesis of silver nanoparticles by sev- eral Fusarium oxysporum strains. - J. Nanobiotechnol. 13: 3-8. No n- co mm er cia l eral Fusarium oxysporum strains. - J. Nanobiotechnol. 13: 3-8. EZEONU F.C., CHIDUME G.I., UDEDI SC., 2001- Insecticidal properties No n- co mm er cia l EZEONU F.C., CHIDUME G.I., UDEDI SC., 2001- Insecticidal properties of volatile extracts of orange peels. - Bioresource. Technol. 76: 273. No n- co mm er cia l of volatile extracts of orange peels. - Bioresource. Technol. 76: 273. 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