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Spectral and HRTEM analyses of Annona muricata leaf extract mediated silver nanoparticles and its Larvicidal efficacy against three mosquito vectors Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti
Journal of Photochemistry & Photobiology, B: Biology 153 (2015) 184–190
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Spectral and HRTEM analyses of Annona muricata leaf extract mediated silver nanoparticles and its Larvicidal efficacy against three mosquito vectors Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti Shanthi Bhupathi Santhosh, Chinnasamy Ragavendran, Devarajan Natarajan ⁎ Natural Drug Research Laboratory, Department of Biotechnology, School of Biosciences, Periyar University, Periyar Palkalai Nagar, Salem 636 011, Tamil Nadu, India
a r t i c l e
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Article history: Received 2 April 2015 Received in revised form 15 August 2015 Accepted 16 September 2015 Available online 21 September 2015 Keywords: Silver nanoparticles Annona muricata larvicidal activity Anopheles stephensi Culex quinquefasciatus Aedes aegypti
a b s t r a c t Mosquitoes transmit various diseases which mainly affect the human beings and every year cause millions of deaths globally. Currently available chemical and synthetic mosquitocidal agents pose severe side effects, pollute the environment vigorously, and become resistance. There is an urgent need to identify and develop the cost effective, compatible and eco-friendly product for mosquito control. The present study was aimed to find out the larvicidal potential of aqueous crude extract and green synthesized silver nanoparticles (AgNPs) from Annona muricata leaves were tested against fourth instar larvae of three important mosquitoes i.e. Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti using different concentrations of AgNPs (10, 20, 30, 40 and 50 ppm) and the aqueous leaf extract (100, 200, 300, 400, and 500 ppm) for 24 and 48 h. The maximum mortality was noticed in AgNPs than aqueous leaf extract of A. muricata against tested mosquitoes with least LC50 values of 37.70, 31.29, and 20.65 ppm (24 h) and 546.7, 516.2, and 618.4 ppm (48 h), respectively. All tested concentrations of AgNps exhibited 100% mortality in A. aegypti larvae at 48 hour observation. In addition, the plant mediated AgNPs were characterized by UV–vis spectrum, Fourier transform infrared spectroscopy, particle size analyser, X-ray diffraction, high resonance transmission electron microscopy, and energy-dispersive X-ray spectroscopy analysis for confirmation of nanoparticle synthesis. Based on the findings of the study suggests that the use of A. muricata plant mediated AgNPs can act as an alternate insecticidal agents for controlling target mosquitoes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Mosquitoes play an important role in transmitting diseases such as malaria, dengue fever/dengue hemorrhagic fever (DHF), chikungunya, Japanese encephalitis, Yellow fever, Lymphatic filariasis, West Nile virus infection, and Leishmaniasis [1]. Mosquito-transmitted diseases kill nearly one million people every year [2]. Anopheles stephensi is solely responsible for the transmission of malaria in urban regions of India [3]. Among 53 anopheline species present in India, nine are vectors of malaria. In Southeast Asia, 100 million malaria cases occur every year and 70% of these are reported from India [4]. Culex quinquefasciatus is an important mosquito vector of lymphatic filariasis, which is a widely distributed tropical disease with more than 120 million people infected worldwide, and nearly 44 million people have common chronic manifestation [5]. Aedes aegypti is a vector of dengue that carries the arbovirus solely responsible for dengue diseases. Recently, the occurrence of dengue has increased dramatically throughout the world [6]. Around 80 million people were infected with dengue and reported the global ⁎ Corresponding author. Tel.: +91 9443857440, +91 0427 23457125; fax: +91 0427 2345124 (office). E-mail addresses: [email protected], [email protected] (D. Natarajan).
http://dx.doi.org/10.1016/j.jphotobiol.2015.09.018 1011-1344/© 2015 Elsevier B.V. All rights reserved.
attack rate was 4% per year [7]. The chemical insecticides namely permethrin, dieldrin, fenitrothion, and propoxur that were widely used to control mosquitoes are often harmful to other beneficial organisms including human beings [8]. Moreover, the control of vector borne disease is difficult due to the increased resistance of mosquito populations to synthetic insecticides. Therefore, there is a need for an alternative pest control strategies, especially the effective, environmentally friendly and low-cost ones [9–11]. Nanoparticles play a major role in drug delivery, diagnosis, imaging, sensing, gene delivery, artificial implants, gene targeting, and tissue engineering [12]. In recent years, the biosynthetic method using plant extracts has received maximum attention than physiochemical methods because this method of nanoparticle synthesis can cause cytotoxicity effect to humans. Synthesis of nanoparticles using plants can potentially eliminate this problem by making the nanoparticles more biocompatible. The silver nanoparticles are reported to possess antiviral, antibacterial [13], larvicidal [14], anticancer [15], and antifungal activity [16]. Recently, silver nanoparticles have been synthesized using various medicinal plants like Murraya koenigii [2], Nerium oleander [17], Chomelia asiatica [18], and Cipadessa baccifera [19]. One such plant, Annona muricata (L.) a small, upright evergreen tree belonging to the family Annonaceae, has been selected for this study. The biological
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properties of plant are reported as having anti-viral [20,21], anti-cancer [22], antidiabetic [23], anti-fungal [24], anti-tubercular [25], and larvicidal activities [26]. Hence, the present study was aimed for the synthesis, characterization, and mosquito larvicidal potential of silver nanoparticles from A. muricata leaves. 2. Materials and Methods 2.1. Collection of Plant and Preparation of Extract The A. muricata (Fig. 1) plant leaves were collected from the Salem District, Tamil Nadu, India. The leaves were washed several times with tap-water to remove the dust particles and shade-dried at room temperature (28 ± 2 °C) for 15 to 20 days. The dried plant leaves were cut into small pieces and powdered mechanically using an electrical stainless steel blender. The extract was used for the reduction of silver nitrate to silver nanoparticles by placing 5 g of leaf powder in 250 ml glass beaker along with 100 ml of sterile double distilled water. The mixture was boiled for 30 min until the color of the aqueous solution changes from watery to light yellow using a magnetic stirrer. The extract was cooled at room temperature and obtained extract was filtered through a Whatman No. 1 filter paper. The extract was stored at −4 °C in a refrigerator until further use. 2.2. Mosquito Culture Laboratory culture of mosquito larvae A. stephensi and C. quinquefasciatus was obtained from the National Centre for Disease Control (NCDC), Mettupalayam, Tamil Nadu, India. The wild A. aegypti larvae were collected from Salem region, Tamil Nadu, India. All the larvae were kept in plastic trays containing tap water and were maintained in the laboratory. All the experiments were carried out at 27 ± 2 °C and 75–85% relative humidity under 14:10 light/dark photoperiod cycles. Larvae were fed with dog biscuit and yeast powder in the ratio of 3:1. The cultures were maintained and reared in the laboratory. 2.3. Synthesis of Silver Nanoparticles The silver nitrate (AgNO3) aqueous solution (1 mM) was freshly prepared with Milli Q water and used for the synthesis of silver nanoparticles. 10 ml of aqueous extract was added to 80 ml of 1 mM AgNO3 solution in an Erlenmeyer flask and the solution was kept under stirring condition for 1 h and the temperature was fixed at 60 °C for synthesis of
Fig. 2. UV–vis absorption spectra of silver nanoparticles synthesized using A. muricata leaf extract.
silver nanoparticles. Formation of AgNPs (Fig. 2) indicated by the yellow to brown color formation of the solution suggests that the aqueous silver ions can be reduced by aqueous extract of plants to develop stable silver nanoparticles [27]. 2.4. Characterization of the Synthesized AgNPs 2.4.1. UV–visible Spectra Analysis The bioreduction of AgNO3 solution was monitored using a UV– visible spectrometer, at the wavelength of 200–700 nm. The absorption spectra of synthesized NPs concentrations were measured at different time intervals of 0 min, 10 min, 30 min, and 1 h (Schimadzu UV Spectrophotometer, model UV-1800). 2.4.2. Fourier Transform Infrared (FTIR) Spectroscopy For FTIR analysis, the sample was prepared as per the modified method of Vivek et al., [28] the colloidal solution which contains the silver nanoparticles was centrifuged at 8000 rpm for 10 min and the pellet was collected. Then, the supernatant was again centrifuged at 8000 rpm for 10 min and the obtained pellet was washed with deionized water and allowed to dry. In the functional groups of the synthesized AgNPs, the dried AgNPs were analyzed in the mid IR region of 400–4000 cm−1 by KBr pellet technique. 2.4.3. X-ray Diffraction (XRD) Pattern The dried powder containing silver nanoparticles was subjected to XRD analysis for confirming the crystalline nature of AgNPs. The average crystalline structure was calculated using Debye Scherer equation: d = 0.9 λ / β cos θ where, β is full width at half maxima (FWHM), θ is the diffracted angle and λ is wavelength of the X-ray. The spectrum was recorded in an Advance power X-ray diffractometer, Brucker, Germany model D8. 2.4.4. EDX and HRTEM Analyses Energy-dispersive X-ray spectroscopy (EDX) was used to analyze the purity and chemical composition of AgNPs and HRTEM (high resonance transmission electron microscopy — Hitachi H-7100 using an accelerating voltage of 120 kv and water as solvent) was used to magnify the lattice arrangements of atoms and shape of the AgNPs.
Fig. 1. Annona muricata plant.
2.4.5. Particle Size Analysis The average particle size distribution of silver nanoparticles was analyzed in the particle size analyser system (Zeta sizer, Malvern
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and 50 ppm). Each test was conducted in three replicates along with control group (distilled water). Mortality was assessed after 24 and 48 h to determine the acute toxicities on fourth instar larvae of mosquitoes. 2.5.1. Dose–response Bioassay Based on the preliminary screening results, aqueous plant extract and synthesized AgNPs were subjected to dose–response bioassay for larvicidal activity against the fourth instar larvae of three mosquitoes. Different concentrations of aqueous extract (100–500 ppm) and AgNPs (10–50 ppm) were prepared. The numbers of dead larvae were counted after 24 and 48 h of exposure. The LC50 and LC90 values were calculated from the average of three replicates.
Fig. 3. Nanoparticle synthesis of A. muricata leaf extract and its change after adding AgNO3.
Instruments Ltd., USA). The average size distribution of AgNPs were noted based on their intensity, number weightage and volume respectively.
2.5.2. Statistical Analysis The average larval mortality data were subjected to probit analysis for calculating LC50, LC90 statistics at 95% confidence limits of upper confidence limit (UCL), and lower confidence limit (LCL) values, and chi-square test was calculated using the SPSS (Statistical Package of Social Sciences) software package 16.0 version. Results with p b 0.05 were considered to be significant. 3. Results and Discussion
2.5. Larvicidal Activity 3.1. UV–visible Spectra Analysis Larvicidal bioassay of extracts was carried out according to WHO protocols [29] with some modifications. Twenty-five numbers of fourth instar larvae were introduced into a 500 ml glass beaker containing 249 ml of dechlorinated water and the desired concentrations of aqueous plant extract. The control was set up with dechlorinated tap water. After 24 and 48 h of exposures, the number of dead larvae was counted and the LC50 and LC90 values were calculated. Synthesized AgNP leaf extract toxicity tests were conducted using a multi-concentration test, consists of a control and different concentrations of AgNP leaf extract. Each test was performed by placing 25 mosquito larvae into 250 ml of sterilized double distilled water with silver nanoparticles into a glass beaker. Nanoparticle solution was diluted using double distilled water as a solvent according to the desired concentrations (10, 20, 30, 40,
UV–visible spectra of the mixture of AgNPs–AgNO3 solution were recorded against time of reaction. UV–visible spectroscopy is a widely used technique for structural characterization of silver nanoparticles [30]. Generally, the results of the UV–visible absorption showed increasing color intensity with increased time intervals and this might be due to the production of the nanoparticles [31]. The color of the solution that gradually turned from light yellow into brown in 1 h, indicates the formation of AgNPs. The appearance of the brown color was due to the excitation of the surface plasmon vibrations. The formation of AgNPs was monitored by UV–visible spectroscopy in the 200–800 nm range. Typically, the synthesized AgNPs are having λmax values which are in the visible range of 400–500 nm (Fig. 3).
Fig. 4. FTIR spectrum of silver nanoparticles synthesized by A. muricata leaf extract.
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nanoparticles with controllable size and uniform shape can be easily obtained with the aqueous reduction method. The mean size of silver nanoparticles was calculated using the Debye–Scherrer's equation by determining the width of the 111 Bragg's reflections. The crystalline size was calculated from the width of the XRD peaks and assumed that they are free from non-uniform strains, by using the Scherrer formula. D = kλ / βcosθ, where D is the crystallite size, k is the Scherrer coefficient (0.94), λ is the wavelength of X-rays (CuKα) (1.5406 Å), β is the full width half maxima (FWHM) (value was 0.72574), and θ Bragg's angle (half of 2θ) = 16.20°. The average crystalline size of the synthesized AgNPs was approximately 30 nm, which is significantly related with HRTEM results.
Fig. 5. XRD pattern of AgNPs synthesized using A. muricata.
3.2. Fourier Transform Infrared (FTIR) Spectroscopy Analysis FTIR analysis was carried out to identify the possible interactions between silver and bioactive molecules, which may be responsible for synthesis and stabilization (capping) of silver nanoparticles. The prominent peaks in the FTIR results show the corresponding values in the amide group (N–H stretching—3416 cm − 1 ), alkane group (CH stretching—2921 cm− 1), alkene group (C = C − 1619 cm− 1), anhydrides group (C–O − 1075 cm− 1), and chloride group (C–X − 616 cm− 1) respectively (Fig. 4). Asmathunisha et al. [32] reported that the observed peaks of amide and aromatic rings are considered as functional groups of flavonoids, triterpenoids, and polyphenols. The linkage of metal ions and the amide group (containing enzymes) of plant biomolecules is responsible for the reduction, synthesis, and stabilization of the metal ions as well as the amine containing organics (polyphenols) having good potential reducing agents in the synthesis of silver nanoparticles [32−35].
3.3.1. HRTEM Imaging and EDX Analyses The size, shape, and morphology of the silver nanoparticles were studied by the transmission electron microscopy images. The grid used in the HRTEM was prepared by placing a drop of the bioreduced diluted solution on a carbon-coated copper grid and further dried it under a lamp. The HRTEM images confirmed that the biosynthesized silver nanoparticles were in the size of 30–45 nm (Fig. 6). The shape of the nanoparticles was analyzed as spherical and few of the AgNPs were agglomerated. The energy dispersive spectrum revealed that the clear identification of the elemental composition is present in the synthesized nanoparticles, which suggests the presence of silver as the ingredient element. Silver nanoparticles show an optical absorption peak at 3 keV due to the surface plasmon resonance [36]. However, other element signals along with silver nanoparticles were recorded because of the sample placed in a carbon coated copper grid (Fig. 7). 3.4. Particle Size Analysis The size distribution analysis of the capped silver nanoconjugates strongly confirmed that the particles were well dispersed. The average size distribution of silver nanoparticles (based on intensity, volume and number weightage in colloidal solution) was found to be 101.1 nm (Fig. 8).
3.3. X-ray Diffraction Analysis
3.5. Larvicidal Activity
The strong and narrow diffraction peaks of nanoparticles indicate that the product has well crystallized. The XRD peak intensities at 38.33, 44.28, 64.42, and 77.60 can be indexed to the 111, 200, 220, and 311 Bragg's reflections of cubic structure of silver (Fig. 5). The broadening of Bragg's peaks indicates that the formation of silver
Larvicidal activity of different concentrations of aqueous leaf extract and synthesized silver nanoparticles of A. muricata was tested against fourth instar larvae of three mosquitoes in 24 and 48 h intervals. The LC50 values of 24 and 48 h of aqueous extract are 546.74, 516.25, and 618.43, and 458.21, 442.38, and 349.13 ppm. The aqueous leaf extract
Fig. 6. Transmission electron microscopy image of synthesized silver nanoparticles (AgNPs) from A. muricata (scale bar corresponds to 50 nm) leaves.
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Fig. 7. EDX showing the chemical constituents of the synthesized nanoparticles.
LC90 values of 24 and 48 h are 937.06, 936.64, and 1240.62 and 852.04, 807.21, and 703.73 ppm (Table 1) respectively. Previously, several plant crude extracts have been reported as potential larvicidal agents against target mosquitoes namely Annona squamosa [37], Murraya koengii
[2,38], Cedrus deodora, and Nicotiana tobacum [39] with least LC50 and LC 90 values and support the outcome of present study. Nanoparticles opened a modern era in biological sciences and have been used specifically as gene carriers, tissue engineering, and
Fig. 8. The particle size distribution histogram of AgNPs shows size distribution of AgNPs.
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Table 1 Larvicidal activity of A. muricata aqueous leaf extract against fourth instar of A. stephensi, C. quinquefasciatus, and A. aegypti of 24 h and 48 h observation. Test insects
Concentrations (ppm)
Aqueous leaf extract (24 h) LC50 (LCL–UCL)
A. stephensi
C. quinquefasciatus
A. aegypti
100 200 300 400 500 100 200 300 400 500 100 200 300 400 500
Aqueous leaf extract (48 h) x2
LC90 (LCL–UCL)
df
LC50 (LCL–UCL)
LC90 (LCL–UCL)
x2
546.7 (482.9–650.3)
937.0 (795.4–1185.3)
5.25
458.2 (408.7–530.4)
852.0 (733.0–1049.5)
11.21
16
516.2 (454.5–614.8)
936.6 (791.8–1190.4)
9.25
442.3 (397.9–504.5)
807.2 (702.8–974.2)
8.88
16
618.4 (510.0–846.4)
1240.6 (967.1–1856.5)
16.87
349.1 (313.4–391.8)
703.7 (620.8–829.7)
16.91
16
Control—nil mortality, UCL—upper confidence limit, LCL—lower confidence limit, x2—Chi-square test, df—degree of freedom, significant at p b 0.05 level.
Table 2 Larvicidal activity of A. muricata synthesized AgNPs against fourth instar of A. stephensi, C. quinquefasciatus, and A. aegypti of 24 h and 48 h observation. Test insects
A. stephensi
C. quinquefasciatus
A. aegypti
Concentrations (ppm)
10 20 30 40 50 10 20 30 40 50 10 20 30 40 50
AgNPs (24 h)
AgNPs (48 h)
df
LC50 (LCL–UCL)
LC90 (LCL–UCL)
x2
LC50 (LCL–UCL)
LC90 (LCL–UCL)
x2
37.70 (33.92–42.47)
73.67 (64.22–87.50)
14.61
25.47 (22.63–28.29)
53.18 (48.27–59.93)
19.31
16
31.29 (28.43–34.40)
59.54 (53.89–67.48)
12.00
21.10 (17.41–24.52)
44.34 (39.29–51.79)
26.70
16
20.65 (17.89–23.23)
45.58 (41.57–50.92)
25.41
7.41 (5.71–9.22)
20.11 (18.27–23.25)
16.20
16
Control—nil mortality, UCL—upper confidence limit, LCL—lower confidence limit, x2—Chi-square test, df—degree of freedom, significant at p b 0.05 level.
insecticides. The mortality effect of silver AgNPs on mosquito larvae may be enabled due to the small size of the particles, which allows passage through the insect cuticle and into individual cells where they interfere with molting and many other physiological processes. About 1 μg/ml concentration of AgNPs inhibited cell growth by b30%,
whereas at 5 μg/mL, cell growth was inhibited by N60% [40]. The present results, show that synthesized AgNPs had shown an excellent activity against fourth instar larvae of A. stephensi, C. quinquefasciatus, and A. aegypti with low LC50 and LC90 values (Table 2). The LC50 values of 24 and 48 h of AgNPs are 37.70, 31.29, and 20.65 and 25.47, 21.10, and 7.41 ppm. The AgNP LC90 values of 24 and 48 h are 73.67, 59.54, and 45.58 and 53.18, 44.34, and 20.11 ppm respectively. During 48 h observation, 100% mortality was observed in A. aegypti at all concentrations of extracts (Fig. 9). Similar kind of work has been done by several researchers and reported the plant mediated silver nanoparticles have better larvicidal activity against selected mosquitoes viz. Pedilanthus tithymaloides [27], Rhipicephalus microplus, Pediculus humanus capitis [41], Chrysosporium tropicum [42], Tinospora cordifolia [43], and Heliotropium indicum [44] which supports the findings of present investigation. 4. Conclusion
Fig. 9. Mortality of A. aegypti mosquito larvae after 24 h exposure of AgNPs.
This study reports a green, eco-friendly, low cost, nontoxic and single step synthesis of AgNPs using A. muricata leaf extract. The physical properties of synthesized AgNPs were characterized using relevant techniques like HRTEM and XRD reveals AgNPs are in spherical shape with an average size of 22 nm. The outcome of this study confirms that the plant mediated AgNPs are having an excellent larvicidal potency against selected mosquitoes A. stephensi, C. quinquefasciatus
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and A. aegypti. We strongly recommend that the synthesized AgNPs can be used to control the mosquito vector especially A. aegypti due to great outcome of 100% mortality within 48 h observation. Acknowledgments We thank the Department of Biotechnology, Periyar University, Salem, India for the laboratory facilities provided. Authors are thankful to DST Unit of Nanoscience, IIT Madras for HRTEM and EDX characterization. The authors would also like to thank Department of Physics, Periyar University for the XRD and FTIR analyses. References [1] K. Veerakumar, M. Govindan, M. Rajeswary, Green synthesis of silver nanoparticles using Sida acuta (Malvaceae) leaf extract against Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti (Diptera: Culicidae), Parasitol. Res. 112 (2013) 4073–4085. [2] A. Suganya, K. Murugan, K. Kovendan, P. Mahesh Kumar, J. Shiou Hwang, Green synthesis of silver nanoparticles using Murraya koenigii leaf extract against Anopheles stephensi and Aedes aegypti, Parasitol. Res. 112 (2013) 1385–1397. [3] S.J. Rahman, S.K. Sharma, Rajagopal, Manual on Entomological Surveillance of Vector Borne Diseases, NICD, New Delhi, 1989. [4] World Health Organization, First Meeting of the Regional Technical Advisory Group on Malaria, Manesar, Haryana, India, SEA-MAL, 2392004 1–38. [5] L. Bernhard, P. Bernhard, P. Magnussen, Management of patients with lymphoedema caused by filariasis in North-eastern Tanzania: alternative approaches, Physiology 89 (2003) 743–749. [6] C.D. Patil, H.P. Borase, S.V. Patil, R.B. Salunkhe, B.K. Salunke, Larvicidal activity of silver nanoparticles synthesized using Pergularia daemia plant latex against Aedes aegypti and Anopheles stephensi and nontarget fish Poecillia reticulata, Parasitol. Res. 111 (2012) 555–562. [7] C. Pancharoen, W. Kulwichit, T. Tantawichien, U. Thisyakorn, C. Thisyakorn, Dengue infection: a global concern, J. Med. Assoc. Thail. 85 (2002) 25–33. [8] A. Amer, H. Mehlhorn, Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae), Parasitol. Res. 99 (2006) 466–472. [9] K. Hargreaves, L.L. Koekemoer, B.D. Brooke, R.H. Hunt, J. Mthembu, M. Coetzee, Anopheles funestus resistant to pyrethroid insecticides in South Africa, Med. Vet. Entomol. 14 (2000) 181–189. [10] H. Ranson, L. Rossiter, F. Ortelli, B. Jensen, X. Wang, C.W. Roth, F.H. Collins, J. Hemingway, Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector, Anopheles gambiae, Biochem. J. 359 (2001) 295–304. [11] A. Gericke, J.M. Govere, D.N. Durrheim, Insecticide susceptibility in the South African malaria mosquito Anopheles arabiensis (Diptera: Culicidae), S. Afr. J. Sci. 98 (2002) 205–208. [12] J.R. Morones, J.L. Elechiguerra, A. Camacho, K. Holt, J.B. Kouri, J.T. Ramfrez, M.J. Yacaman, The bactericidal effect of silver nanoparticles, Nanotechnology 16 (2005) 2346–2353. [13] C. Krishnaraj, E.G. Jagan, S. Rajasekar, P. Selvakumar, P.T. Kalaichelvan, N. Mohan, Synthesis of silver nanoparticles using Acalypha indica leaf extracts and its antibacterial activity against water borne pathogens, Colloids Surf. B: Biointerfaces 76 (2010) 50–56. [14] N. Soni, S. Prakash, Silver nanoparticles: a possibility for malarial and filarial vector control technology, Parasitol. Res. 113 (2014) 4015–4022. [15] B. Venkatesan, V. Subramanian, A. Tumala, E. Vellaichamy, Rapid synthesis of biocompatible silver nanoparticles using aqueous extract of Rosa damascene petals and evalution of their anticancer activity, Asian Pac. J. Trop. Med. 7 (Suppl. 1) (2014) 294–300. [16] N. Soni, S. Prakash, Antimicrobial and mosquitocidal activity of microbial synthesized silver nanoparticles, Parasitol. Res 114 (3) (2015) 1023–1030. [17] M. Roni, K. Murugan, C. Panneerselvam, J. Subramanian, J.S. Hwang, Evaluation of leaf aqueous extract and synthesized silver nanoparticles using Nerium oleander against Anopheles stephensi (Diptera: Culicidae), Parasitol. Res. 112 (2013) 981–990. [18] U. Muthukumaran, M. Govindarajan, M. Rajeswary, Mosquito larvicidal potential of silver nanoparticles synthesized using chomelia asiatica (Rubiaceae) against Anopheles stephensi, Aedes aegypti, and Culex quinquefaciatus (Diptera: Culicidae), Parasitol. Res 114 (3) (2015) 989–999. [19] G. Ramkumar, S. Karthi, R. Muthusamy, D. Natarajan, M.S. Shivakumar, Adulticidal and smoke toxicity of Cipadessa baccifera (Roth) plant extracts against Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus, Parasitol. Res. 114 (2015) 167–173. [20] P. Padma, N.P. Pramod, S.P. Thyagarajan, R.L. Khosa, Effect of the extract of Annona muricata and Petunia nyctaginiflora on herpes simplex virus, J. Ethnopharmacol. 61 (1998) 81–83.
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jpb
Spectral and HRTEM analyses of Annona muricata leaf extract mediated silver nanoparticles and its Larvicidal efficacy against three mosquito vectors Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti Shanthi Bhupathi Santhosh, Chinnasamy Ragavendran, Devarajan Natarajan ⁎ Natural Drug Research Laboratory, Department of Biotechnology, School of Biosciences, Periyar University, Periyar Palkalai Nagar, Salem 636 011, Tamil Nadu, India
a r t i c l e
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Article history: Received 2 April 2015 Received in revised form 15 August 2015 Accepted 16 September 2015 Available online 21 September 2015 Keywords: Silver nanoparticles Annona muricata larvicidal activity Anopheles stephensi Culex quinquefasciatus Aedes aegypti
a b s t r a c t Mosquitoes transmit various diseases which mainly affect the human beings and every year cause millions of deaths globally. Currently available chemical and synthetic mosquitocidal agents pose severe side effects, pollute the environment vigorously, and become resistance. There is an urgent need to identify and develop the cost effective, compatible and eco-friendly product for mosquito control. The present study was aimed to find out the larvicidal potential of aqueous crude extract and green synthesized silver nanoparticles (AgNPs) from Annona muricata leaves were tested against fourth instar larvae of three important mosquitoes i.e. Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti using different concentrations of AgNPs (10, 20, 30, 40 and 50 ppm) and the aqueous leaf extract (100, 200, 300, 400, and 500 ppm) for 24 and 48 h. The maximum mortality was noticed in AgNPs than aqueous leaf extract of A. muricata against tested mosquitoes with least LC50 values of 37.70, 31.29, and 20.65 ppm (24 h) and 546.7, 516.2, and 618.4 ppm (48 h), respectively. All tested concentrations of AgNps exhibited 100% mortality in A. aegypti larvae at 48 hour observation. In addition, the plant mediated AgNPs were characterized by UV–vis spectrum, Fourier transform infrared spectroscopy, particle size analyser, X-ray diffraction, high resonance transmission electron microscopy, and energy-dispersive X-ray spectroscopy analysis for confirmation of nanoparticle synthesis. Based on the findings of the study suggests that the use of A. muricata plant mediated AgNPs can act as an alternate insecticidal agents for controlling target mosquitoes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Mosquitoes play an important role in transmitting diseases such as malaria, dengue fever/dengue hemorrhagic fever (DHF), chikungunya, Japanese encephalitis, Yellow fever, Lymphatic filariasis, West Nile virus infection, and Leishmaniasis [1]. Mosquito-transmitted diseases kill nearly one million people every year [2]. Anopheles stephensi is solely responsible for the transmission of malaria in urban regions of India [3]. Among 53 anopheline species present in India, nine are vectors of malaria. In Southeast Asia, 100 million malaria cases occur every year and 70% of these are reported from India [4]. Culex quinquefasciatus is an important mosquito vector of lymphatic filariasis, which is a widely distributed tropical disease with more than 120 million people infected worldwide, and nearly 44 million people have common chronic manifestation [5]. Aedes aegypti is a vector of dengue that carries the arbovirus solely responsible for dengue diseases. Recently, the occurrence of dengue has increased dramatically throughout the world [6]. Around 80 million people were infected with dengue and reported the global ⁎ Corresponding author. Tel.: +91 9443857440, +91 0427 23457125; fax: +91 0427 2345124 (office). E-mail addresses: [email protected], [email protected] (D. Natarajan).
http://dx.doi.org/10.1016/j.jphotobiol.2015.09.018 1011-1344/© 2015 Elsevier B.V. All rights reserved.
attack rate was 4% per year [7]. The chemical insecticides namely permethrin, dieldrin, fenitrothion, and propoxur that were widely used to control mosquitoes are often harmful to other beneficial organisms including human beings [8]. Moreover, the control of vector borne disease is difficult due to the increased resistance of mosquito populations to synthetic insecticides. Therefore, there is a need for an alternative pest control strategies, especially the effective, environmentally friendly and low-cost ones [9–11]. Nanoparticles play a major role in drug delivery, diagnosis, imaging, sensing, gene delivery, artificial implants, gene targeting, and tissue engineering [12]. In recent years, the biosynthetic method using plant extracts has received maximum attention than physiochemical methods because this method of nanoparticle synthesis can cause cytotoxicity effect to humans. Synthesis of nanoparticles using plants can potentially eliminate this problem by making the nanoparticles more biocompatible. The silver nanoparticles are reported to possess antiviral, antibacterial [13], larvicidal [14], anticancer [15], and antifungal activity [16]. Recently, silver nanoparticles have been synthesized using various medicinal plants like Murraya koenigii [2], Nerium oleander [17], Chomelia asiatica [18], and Cipadessa baccifera [19]. One such plant, Annona muricata (L.) a small, upright evergreen tree belonging to the family Annonaceae, has been selected for this study. The biological
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properties of plant are reported as having anti-viral [20,21], anti-cancer [22], antidiabetic [23], anti-fungal [24], anti-tubercular [25], and larvicidal activities [26]. Hence, the present study was aimed for the synthesis, characterization, and mosquito larvicidal potential of silver nanoparticles from A. muricata leaves. 2. Materials and Methods 2.1. Collection of Plant and Preparation of Extract The A. muricata (Fig. 1) plant leaves were collected from the Salem District, Tamil Nadu, India. The leaves were washed several times with tap-water to remove the dust particles and shade-dried at room temperature (28 ± 2 °C) for 15 to 20 days. The dried plant leaves were cut into small pieces and powdered mechanically using an electrical stainless steel blender. The extract was used for the reduction of silver nitrate to silver nanoparticles by placing 5 g of leaf powder in 250 ml glass beaker along with 100 ml of sterile double distilled water. The mixture was boiled for 30 min until the color of the aqueous solution changes from watery to light yellow using a magnetic stirrer. The extract was cooled at room temperature and obtained extract was filtered through a Whatman No. 1 filter paper. The extract was stored at −4 °C in a refrigerator until further use. 2.2. Mosquito Culture Laboratory culture of mosquito larvae A. stephensi and C. quinquefasciatus was obtained from the National Centre for Disease Control (NCDC), Mettupalayam, Tamil Nadu, India. The wild A. aegypti larvae were collected from Salem region, Tamil Nadu, India. All the larvae were kept in plastic trays containing tap water and were maintained in the laboratory. All the experiments were carried out at 27 ± 2 °C and 75–85% relative humidity under 14:10 light/dark photoperiod cycles. Larvae were fed with dog biscuit and yeast powder in the ratio of 3:1. The cultures were maintained and reared in the laboratory. 2.3. Synthesis of Silver Nanoparticles The silver nitrate (AgNO3) aqueous solution (1 mM) was freshly prepared with Milli Q water and used for the synthesis of silver nanoparticles. 10 ml of aqueous extract was added to 80 ml of 1 mM AgNO3 solution in an Erlenmeyer flask and the solution was kept under stirring condition for 1 h and the temperature was fixed at 60 °C for synthesis of
Fig. 2. UV–vis absorption spectra of silver nanoparticles synthesized using A. muricata leaf extract.
silver nanoparticles. Formation of AgNPs (Fig. 2) indicated by the yellow to brown color formation of the solution suggests that the aqueous silver ions can be reduced by aqueous extract of plants to develop stable silver nanoparticles [27]. 2.4. Characterization of the Synthesized AgNPs 2.4.1. UV–visible Spectra Analysis The bioreduction of AgNO3 solution was monitored using a UV– visible spectrometer, at the wavelength of 200–700 nm. The absorption spectra of synthesized NPs concentrations were measured at different time intervals of 0 min, 10 min, 30 min, and 1 h (Schimadzu UV Spectrophotometer, model UV-1800). 2.4.2. Fourier Transform Infrared (FTIR) Spectroscopy For FTIR analysis, the sample was prepared as per the modified method of Vivek et al., [28] the colloidal solution which contains the silver nanoparticles was centrifuged at 8000 rpm for 10 min and the pellet was collected. Then, the supernatant was again centrifuged at 8000 rpm for 10 min and the obtained pellet was washed with deionized water and allowed to dry. In the functional groups of the synthesized AgNPs, the dried AgNPs were analyzed in the mid IR region of 400–4000 cm−1 by KBr pellet technique. 2.4.3. X-ray Diffraction (XRD) Pattern The dried powder containing silver nanoparticles was subjected to XRD analysis for confirming the crystalline nature of AgNPs. The average crystalline structure was calculated using Debye Scherer equation: d = 0.9 λ / β cos θ where, β is full width at half maxima (FWHM), θ is the diffracted angle and λ is wavelength of the X-ray. The spectrum was recorded in an Advance power X-ray diffractometer, Brucker, Germany model D8. 2.4.4. EDX and HRTEM Analyses Energy-dispersive X-ray spectroscopy (EDX) was used to analyze the purity and chemical composition of AgNPs and HRTEM (high resonance transmission electron microscopy — Hitachi H-7100 using an accelerating voltage of 120 kv and water as solvent) was used to magnify the lattice arrangements of atoms and shape of the AgNPs.
Fig. 1. Annona muricata plant.
2.4.5. Particle Size Analysis The average particle size distribution of silver nanoparticles was analyzed in the particle size analyser system (Zeta sizer, Malvern
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and 50 ppm). Each test was conducted in three replicates along with control group (distilled water). Mortality was assessed after 24 and 48 h to determine the acute toxicities on fourth instar larvae of mosquitoes. 2.5.1. Dose–response Bioassay Based on the preliminary screening results, aqueous plant extract and synthesized AgNPs were subjected to dose–response bioassay for larvicidal activity against the fourth instar larvae of three mosquitoes. Different concentrations of aqueous extract (100–500 ppm) and AgNPs (10–50 ppm) were prepared. The numbers of dead larvae were counted after 24 and 48 h of exposure. The LC50 and LC90 values were calculated from the average of three replicates.
Fig. 3. Nanoparticle synthesis of A. muricata leaf extract and its change after adding AgNO3.
Instruments Ltd., USA). The average size distribution of AgNPs were noted based on their intensity, number weightage and volume respectively.
2.5.2. Statistical Analysis The average larval mortality data were subjected to probit analysis for calculating LC50, LC90 statistics at 95% confidence limits of upper confidence limit (UCL), and lower confidence limit (LCL) values, and chi-square test was calculated using the SPSS (Statistical Package of Social Sciences) software package 16.0 version. Results with p b 0.05 were considered to be significant. 3. Results and Discussion
2.5. Larvicidal Activity 3.1. UV–visible Spectra Analysis Larvicidal bioassay of extracts was carried out according to WHO protocols [29] with some modifications. Twenty-five numbers of fourth instar larvae were introduced into a 500 ml glass beaker containing 249 ml of dechlorinated water and the desired concentrations of aqueous plant extract. The control was set up with dechlorinated tap water. After 24 and 48 h of exposures, the number of dead larvae was counted and the LC50 and LC90 values were calculated. Synthesized AgNP leaf extract toxicity tests were conducted using a multi-concentration test, consists of a control and different concentrations of AgNP leaf extract. Each test was performed by placing 25 mosquito larvae into 250 ml of sterilized double distilled water with silver nanoparticles into a glass beaker. Nanoparticle solution was diluted using double distilled water as a solvent according to the desired concentrations (10, 20, 30, 40,
UV–visible spectra of the mixture of AgNPs–AgNO3 solution were recorded against time of reaction. UV–visible spectroscopy is a widely used technique for structural characterization of silver nanoparticles [30]. Generally, the results of the UV–visible absorption showed increasing color intensity with increased time intervals and this might be due to the production of the nanoparticles [31]. The color of the solution that gradually turned from light yellow into brown in 1 h, indicates the formation of AgNPs. The appearance of the brown color was due to the excitation of the surface plasmon vibrations. The formation of AgNPs was monitored by UV–visible spectroscopy in the 200–800 nm range. Typically, the synthesized AgNPs are having λmax values which are in the visible range of 400–500 nm (Fig. 3).
Fig. 4. FTIR spectrum of silver nanoparticles synthesized by A. muricata leaf extract.
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nanoparticles with controllable size and uniform shape can be easily obtained with the aqueous reduction method. The mean size of silver nanoparticles was calculated using the Debye–Scherrer's equation by determining the width of the 111 Bragg's reflections. The crystalline size was calculated from the width of the XRD peaks and assumed that they are free from non-uniform strains, by using the Scherrer formula. D = kλ / βcosθ, where D is the crystallite size, k is the Scherrer coefficient (0.94), λ is the wavelength of X-rays (CuKα) (1.5406 Å), β is the full width half maxima (FWHM) (value was 0.72574), and θ Bragg's angle (half of 2θ) = 16.20°. The average crystalline size of the synthesized AgNPs was approximately 30 nm, which is significantly related with HRTEM results.
Fig. 5. XRD pattern of AgNPs synthesized using A. muricata.
3.2. Fourier Transform Infrared (FTIR) Spectroscopy Analysis FTIR analysis was carried out to identify the possible interactions between silver and bioactive molecules, which may be responsible for synthesis and stabilization (capping) of silver nanoparticles. The prominent peaks in the FTIR results show the corresponding values in the amide group (N–H stretching—3416 cm − 1 ), alkane group (CH stretching—2921 cm− 1), alkene group (C = C − 1619 cm− 1), anhydrides group (C–O − 1075 cm− 1), and chloride group (C–X − 616 cm− 1) respectively (Fig. 4). Asmathunisha et al. [32] reported that the observed peaks of amide and aromatic rings are considered as functional groups of flavonoids, triterpenoids, and polyphenols. The linkage of metal ions and the amide group (containing enzymes) of plant biomolecules is responsible for the reduction, synthesis, and stabilization of the metal ions as well as the amine containing organics (polyphenols) having good potential reducing agents in the synthesis of silver nanoparticles [32−35].
3.3.1. HRTEM Imaging and EDX Analyses The size, shape, and morphology of the silver nanoparticles were studied by the transmission electron microscopy images. The grid used in the HRTEM was prepared by placing a drop of the bioreduced diluted solution on a carbon-coated copper grid and further dried it under a lamp. The HRTEM images confirmed that the biosynthesized silver nanoparticles were in the size of 30–45 nm (Fig. 6). The shape of the nanoparticles was analyzed as spherical and few of the AgNPs were agglomerated. The energy dispersive spectrum revealed that the clear identification of the elemental composition is present in the synthesized nanoparticles, which suggests the presence of silver as the ingredient element. Silver nanoparticles show an optical absorption peak at 3 keV due to the surface plasmon resonance [36]. However, other element signals along with silver nanoparticles were recorded because of the sample placed in a carbon coated copper grid (Fig. 7). 3.4. Particle Size Analysis The size distribution analysis of the capped silver nanoconjugates strongly confirmed that the particles were well dispersed. The average size distribution of silver nanoparticles (based on intensity, volume and number weightage in colloidal solution) was found to be 101.1 nm (Fig. 8).
3.3. X-ray Diffraction Analysis
3.5. Larvicidal Activity
The strong and narrow diffraction peaks of nanoparticles indicate that the product has well crystallized. The XRD peak intensities at 38.33, 44.28, 64.42, and 77.60 can be indexed to the 111, 200, 220, and 311 Bragg's reflections of cubic structure of silver (Fig. 5). The broadening of Bragg's peaks indicates that the formation of silver
Larvicidal activity of different concentrations of aqueous leaf extract and synthesized silver nanoparticles of A. muricata was tested against fourth instar larvae of three mosquitoes in 24 and 48 h intervals. The LC50 values of 24 and 48 h of aqueous extract are 546.74, 516.25, and 618.43, and 458.21, 442.38, and 349.13 ppm. The aqueous leaf extract
Fig. 6. Transmission electron microscopy image of synthesized silver nanoparticles (AgNPs) from A. muricata (scale bar corresponds to 50 nm) leaves.
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Fig. 7. EDX showing the chemical constituents of the synthesized nanoparticles.
LC90 values of 24 and 48 h are 937.06, 936.64, and 1240.62 and 852.04, 807.21, and 703.73 ppm (Table 1) respectively. Previously, several plant crude extracts have been reported as potential larvicidal agents against target mosquitoes namely Annona squamosa [37], Murraya koengii
[2,38], Cedrus deodora, and Nicotiana tobacum [39] with least LC50 and LC 90 values and support the outcome of present study. Nanoparticles opened a modern era in biological sciences and have been used specifically as gene carriers, tissue engineering, and
Fig. 8. The particle size distribution histogram of AgNPs shows size distribution of AgNPs.
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Table 1 Larvicidal activity of A. muricata aqueous leaf extract against fourth instar of A. stephensi, C. quinquefasciatus, and A. aegypti of 24 h and 48 h observation. Test insects
Concentrations (ppm)
Aqueous leaf extract (24 h) LC50 (LCL–UCL)
A. stephensi
C. quinquefasciatus
A. aegypti
100 200 300 400 500 100 200 300 400 500 100 200 300 400 500
Aqueous leaf extract (48 h) x2
LC90 (LCL–UCL)
df
LC50 (LCL–UCL)
LC90 (LCL–UCL)
x2
546.7 (482.9–650.3)
937.0 (795.4–1185.3)
5.25
458.2 (408.7–530.4)
852.0 (733.0–1049.5)
11.21
16
516.2 (454.5–614.8)
936.6 (791.8–1190.4)
9.25
442.3 (397.9–504.5)
807.2 (702.8–974.2)
8.88
16
618.4 (510.0–846.4)
1240.6 (967.1–1856.5)
16.87
349.1 (313.4–391.8)
703.7 (620.8–829.7)
16.91
16
Control—nil mortality, UCL—upper confidence limit, LCL—lower confidence limit, x2—Chi-square test, df—degree of freedom, significant at p b 0.05 level.
Table 2 Larvicidal activity of A. muricata synthesized AgNPs against fourth instar of A. stephensi, C. quinquefasciatus, and A. aegypti of 24 h and 48 h observation. Test insects
A. stephensi
C. quinquefasciatus
A. aegypti
Concentrations (ppm)
10 20 30 40 50 10 20 30 40 50 10 20 30 40 50
AgNPs (24 h)
AgNPs (48 h)
df
LC50 (LCL–UCL)
LC90 (LCL–UCL)
x2
LC50 (LCL–UCL)
LC90 (LCL–UCL)
x2
37.70 (33.92–42.47)
73.67 (64.22–87.50)
14.61
25.47 (22.63–28.29)
53.18 (48.27–59.93)
19.31
16
31.29 (28.43–34.40)
59.54 (53.89–67.48)
12.00
21.10 (17.41–24.52)
44.34 (39.29–51.79)
26.70
16
20.65 (17.89–23.23)
45.58 (41.57–50.92)
25.41
7.41 (5.71–9.22)
20.11 (18.27–23.25)
16.20
16
Control—nil mortality, UCL—upper confidence limit, LCL—lower confidence limit, x2—Chi-square test, df—degree of freedom, significant at p b 0.05 level.
insecticides. The mortality effect of silver AgNPs on mosquito larvae may be enabled due to the small size of the particles, which allows passage through the insect cuticle and into individual cells where they interfere with molting and many other physiological processes. About 1 μg/ml concentration of AgNPs inhibited cell growth by b30%,
whereas at 5 μg/mL, cell growth was inhibited by N60% [40]. The present results, show that synthesized AgNPs had shown an excellent activity against fourth instar larvae of A. stephensi, C. quinquefasciatus, and A. aegypti with low LC50 and LC90 values (Table 2). The LC50 values of 24 and 48 h of AgNPs are 37.70, 31.29, and 20.65 and 25.47, 21.10, and 7.41 ppm. The AgNP LC90 values of 24 and 48 h are 73.67, 59.54, and 45.58 and 53.18, 44.34, and 20.11 ppm respectively. During 48 h observation, 100% mortality was observed in A. aegypti at all concentrations of extracts (Fig. 9). Similar kind of work has been done by several researchers and reported the plant mediated silver nanoparticles have better larvicidal activity against selected mosquitoes viz. Pedilanthus tithymaloides [27], Rhipicephalus microplus, Pediculus humanus capitis [41], Chrysosporium tropicum [42], Tinospora cordifolia [43], and Heliotropium indicum [44] which supports the findings of present investigation. 4. Conclusion
Fig. 9. Mortality of A. aegypti mosquito larvae after 24 h exposure of AgNPs.
This study reports a green, eco-friendly, low cost, nontoxic and single step synthesis of AgNPs using A. muricata leaf extract. The physical properties of synthesized AgNPs were characterized using relevant techniques like HRTEM and XRD reveals AgNPs are in spherical shape with an average size of 22 nm. The outcome of this study confirms that the plant mediated AgNPs are having an excellent larvicidal potency against selected mosquitoes A. stephensi, C. quinquefasciatus
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