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Feb 9, 2018 - Ali, 2011; Ali et al., 2015, 2016b,e,f, 2017a) but AgNPs have proved to be the ... Leaves of the plant are reported to cure scabies (Prajapati et al., 2004). .... of AgNPs in 500 ml capacity of the plastic bowl for 48 h at 28 ◦C RT.

Process Safety and Environmental Protection 116 (2018) 137–148

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Strong larvicidal potential of silver nanoparticles (AgNPs) synthesized using Holarrhena antidysenterica (L.) Wall. bark extract against malarial vector, Anopheles stephensi Liston Dinesh Kumar a , Gaurav Kumar b , Ram Das b , Veena Agrawal a,∗ a b

Department of Botany, University of Delhi, New Delhi 110007, India National Institute of Malaria Research, Dwarka, New Delhi 110077, India

a r t i c l e

i n f o

Article history: Received 18 September 2017 Received in revised form 29 January 2018 Accepted 1 February 2018 Available online 9 February 2018 Keywords: Anopheles stephensi Silver nanoparticles Holarrhena antidysenterica Biosynthesis Larvicidal activity

a b s t r a c t The present study highlights the strong larvicidal potential of silver nanoparticles (AgNPs) synthesized using bark extract of Holarrhena antidysenterica against third instar larvae of Anopheles stephensi over the other bark extract prepared in chloroform, hexane, ethyl acetate, methanol, aqueous and acetone individually. AgNPs were prepared by mixing of 90 ml of silver nitrate (AgNO3 ) with 10 ml of aqueous bark extract of H. antidysenterica. Optimization of various physical parameters such as temperature, pH, time duration and AgNO3 concentrations was done and 1 mM of AgNO3 , 7.5 pH, 50 ± 2 ◦ C temp and time 120 min proved optimum for best synthesis of AgNPs. Characterized of AgNPs was done by ultravioletvisible spectroscopy (UV–vis), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), field emission scanning electron microscope (FE-SEM) and transmission electron microscope (TEM). XRD pattern of such AgNPs revealed characteristics Bragg’s reflection peaks at (38.34) 111, (44.54) 200, (64.36) 220 and (76.9) 311 lattice planes indicating the crystalline nature of biologically synthesized AgNPs. FT-IR analysis of AgNPs exhibited the presence of functional groups of various compounds including phenols, alcohols, amine, amide which were responsible for the reduction and capping of AgNPs. The FE-SEM and TEM images showed that most of the AgNPs were spherical, hexagonal and triangular in shape varying from 40 to 60 nm in size. Larvicidal activity of these AgNPs and bark extracts prepared in different solvents such as hexane, ethyl acetate, methanol, water and chloroform were tested separately against the A. stephensi larvae for 24 h. Maximum larval mortality was seen with bark extract synthesized AgNPs having LC50 and LC90 value of 2.672 ppm and 4.482 ppm, respectively compared to chloroform, hexane, ethyl acetate, methanol, water and acetone bark extracts where the LC50 values were 3.0, 31.56, 41.92, 96.40, 121.53 and 1.91E3 ppm, respectively. Incidentally, AgNPs proved non-toxic against the nontarget organism, Mesocyclops thermocyclopoides. GC–MS analysis of bark extract identified 41 compounds having a range of activities which might have helped in the bio-reduction of AgNPs. These AgNPs have tremendous applications in pharmaceutical and biomedical industries such as cancer therapies, targeted drug delivery, as antiseptic agents and as an imaging agent. © 2018 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction Mosquitoes transmit several diseases such as malaria, dengue, chikungunya, filariasis, Japanese encephalitis and leishmaniasis throughout the world (WHO, 2009; Udayabhanu et al., 2018). Among all the mosquito-borne diseases, malaria causes millions of causalities every year. According to World Health Organization (WHO, 2015), 214 million cases of malaria were reported with an

∗ Corresponding author. E-mail address: drveena [email protected] (V. Agrawal).

estimated 438,000 deaths during the year 2015 including mainly the children from different regions of Africa (Sujitha et al., 2017). Anopheles stephensi is one of the most important species, which serves as a vector for malaria parasite transmission in India and other West Asian countries (Burfield and Reekie, 2005; Ali et al., 2018b). Recently, the occurrence of vector-borne diseases increased due to favorable environmental conditions caused due to global warming, high relative humidity, uneven rainfall and sanitation facility all over the world (Kumar et al., 2017a; Benelli, 2018). Incidentally, development of vaccines against this vector proved to be ineffective due to its high cost and long trial process required. Killing mosquitoes at larval stages is the only effective measure for 0957-5820/© 2018 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.


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controlling this vector-borne disease (Kumar et al., 2017a). Different chemical insecticides and synthetic agents used to control such mosquito-borne diseases have many major drawbacks, as they are expensive, non-selective and harmful to other organisms in the environment. Their effects on human health and toxicity to nontargeted organisms are the main concerns (Kalimuthu and Pandian, 2010; Quinquefasciatus, 2017). Use of such chemicals also leads to the generation of resistance in vector species (Pandey et al., 2007, 2011; Sharma et al., 2014; Rai et al., 2017). Contrary to the above, biologically synthesized nanoparticles have proved to exhibit strong antimalarial activity (Chitra et al., 2015; Das et al., 2017) against various mosquito vectors. The nanoparticles synthesized using plant extracts have major advantages over those of biological organisms including fungi, algae and bacteria as the former are easily available, safe, nontoxic in most cases and less number of steps are required in downstream processing (Mittal et al., 2013; Ali et al., 2016a,c,d; Ali et al., 2017b, 2018a; Pavela et al., 2017; Salari et al., 2017). Plants possess a broad variety of metabolites that can aid in the biosynthesis, reduction and capping of metal ions (Kovendan et al., 2012). Though different types of nanomaterials like copper, zinc, titanium, magnesium, gold and silver have come up with different utilities (Sharma and Ali, 2011; Ali et al., 2015, 2016b,e,f, 2017a) but AgNPs have proved to be the most effective as they possess good antimicrobial efficacy against bacteria, viruses and other harmful eukaryotic microorganisms. Few reports have been published related to synthesis of AgNPs using several medicinal plants like Azadirachta indica, Glycine max, Cinnamon zeylanicum, Pongamia pinnata, Annona squamosa and Murraya koenigii (Sathishkumar et al., 2009; Tripathi et al., 2009; Vivekanandhan et al., 2009; Rajesh et al., 2010; Kumar et al., 2011; Suganya et al., 2013). However, there is no report on the synthesis of silver nanoparticles using H. antidysenterica bark extract and their larvicidal activity against mosquito species. A member of Apocynaceae Holarrhena antidysenterica (Linn.) Wall. contains many important phytochemicals useful for the treatment of various human ailments and can be utilized in the preparation of different drugs. This plant is widely distributed throughout the tropical and subtropical regions of the world. The bark of this plant commonly known as “Kurchi” has many medicinal properties such as stringent, antidysenteric, antibacterial, stomachic, antimalarial (Kumar et al., 2017b) and febrifugal and is used in the treatment of amoebic-dysentery and diarrhoea (Ahmad et al., 1998; Chakraborty and Brantner, 1999; Bakshi et al., 2001). Leaves of the plant are reported to cure scabies (Prajapati et al., 2004). The current investigation has been undertaken to develop an efficient method for AgNPs synthesis using aqueous bark extract of H. antidysenterica and their evaluation against A. stephensi, a malaria vector vis-à-vis other bark extracts prepared in different solvents. 2. Materials and methods 2.1. Plant material Fresh green bark of Holarrhena antidysenterica (L.) Wall. was collected from trees growing near the Department of Physics, University of Delhi (latitude 28.68◦ N; longitude 77.21◦ E), Delhi, India (Fig. 1A). 2.2. Bark extracts preparation The green bark was washed twice with running tap water followed by washing using double distilled water to remove impurities. Further, the bark was cut into small pieces and air-dried at room temperature for 48 h. The dried bark was grounded into a fine powder using mortar and pestle and divided into six parts of 20 g

each for making extract with different solvents. Subsequently, 20 g barks powder was extracted with different solvents such as chloroform, ethyl acetate, hexane, acetone and methanol in 400 ml of each in separate beakers. All the beakers containing mixture were kept on incubator shaker at room temperature for 24 h and was filtered through Whatman filter paper No. 1. For aqueous extract, 20 g bark powder was boiled with 500 ml of double distilled water and filtered through Whatman filter paper No. 1. The filtrates were then concentrated and stored at 4 ◦ C for further experiments. 2.3. Synthesis of AgNPs For nanoparticles synthesis (Kumar et al., 2017a), the powder (10 g) was extracted with 100 ml of double distilled water and was incubated in water bath at 50–60 ◦ C temperature for 30 min. The extract was filtered through Whatman filter paper No.1 and 10 ml of fresh bark extract was added to conical flasks containing 90 ml of different concentrations of aqueous AgNO3 solution. The mixture was heated at 50 ± 2 ◦ C with continuous stirring for 5 min. The silver ions were reduced to AgNPs within few minutes by adding aqueous bark extract which can be seen by the gradual change in colour of the solution. The conversion of solution color showed the formation of AgNPs. 2.3.1. Effect of time on AgNPs synthesis In order to determine the effect of time on the biosynthesis of AgNPs, absorption spectra of the solution was measured at the different time interval of 0, 5, 15, 30, 60 and 120 min. During the biosynthesis of AgNPs, 10 ml of plant extract was added to 90 AgNO3 (1 mM) solution in a conical flask and the reaction was allowed to occur at pH 7.5 and 50 ± 2 ◦ C. 2.3.2. Effect of concentrations on AgNPs synthesis In order to ensure the optimum concentration of AgNO3 for the biosynthesis of AgNPs, different concentrations (0, 0.5, 1, 2, 3, 4 and 5 mM) of AgNO3 were used. The reaction was carried out at pH 7.5 and temperature 50 ± 2 ◦ C using 10 ml plant extract and 90 ml of aqueous AgNO3 over the for 120 min. 2.3.3. Effect of temperatures on AgNPs synthesis To investigate the effect of temperatures, biosynthesis of AgNPs was carried out at different temperature 25, 50, 75 and 100 ◦ C. The optimum concentration of AgNO3 (1 mM) was selected for the synthesis of AgNPs (pH 7.5, 120 min) using the results obtained from the effect of concentrations on AgNPs synthesis section. 2.3.4. Effect of pH on AgNPs synthesis Effect of pH on the biosynthesis of AgNPs under optimum condition was determined by varying the pH (3, 7 and 11) of the solution. Optimum conditions for AgNPs synthesis were 1 mM AgNO3 solution, temperature 50 ± 2 ◦ C and time 120 min. The pH of the solution was adjusted by adding 0.1 N HNO3 or 0.1 N NaOH. 2.4. Characterization of AgNPs The bio-reduction of AgNO3 was determined using UV–vis spectrophotometer (Shimadzu 250 1 PC, version 2.33) at 370–800 nm wavelength. The extract of silver nanoparticles prepared under optimal conditions (1 mM AgNO3 ; pH 7.5; Temp, 50 ± 2 ◦ C and time, 120 min) was centrifuged at 10000 rpm for 20 min and the pellet was collected, freeze-dried to obtain a dried powder which was subjected to X-ray diffraction analysis (XRD) (Bruker D8 Discover) in order to confirm the crystalline nature of AgNPs. The operating voltage of XRD was 40 kV and current of 40 mA with Cu k␣ radiation of 0.1541 nm wavelength in the 2␪ range 10–80◦ with a step size of 0.02/␪. The morphological features of synthesized

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Fig. 1. (A) Holarrhena antidysenterica (Linn.) Wall. (B) Chromatic variation of the H. antidysenterica bark extract after adding 0.5, 1, 2, 3 and 4 mM of AgNO3 solution at pH 7.5 and 50C for 120 min. (C) UV–vis spectra of silver nanoparticles synthesized using H. antidysenterica bark extract with aqueous solution AgNO3 (1 mM, pH 7.5, 50 ± 2 ◦ C) at different time intervals. (D) UV–vis spectra of H. antidysenterica bark extract with different concentrations of AgNO3 solution at pH 7.5 and 50 ± 2 ◦ C for 120 min. (E) UV–vis spectra of silver nanoparticles obtained at different reaction temperature (25, 50, 75 and 100 ◦ C) with aqueous solution of 1 mM AgNO3 at pH 7.5 for 120 min. (F) Absorption spectra of silver nanoparticles recorded at different pH values (pH 3, 7 and 11) with aqueous solution of AgNO3 (1 mM) at 50 ± 2 ◦ C for 120 min.

nanoparticles from the plant extract were studied under the field emission scanning electron microscope (FE-SEM) (TESCAN MIRA3). Few drops of solution containing nanoparticles were loaded on the sputter-coated copper stub and air dried completely. After that, samples were characterized under FE-SEM at an accelerating

voltage of 20 kV. The size and shape of the nanoparticles were determined under transmission electron microscope (TEM) (TECNAI G2 T30, U TWIN). A drop of aqueous silver nanoparticles sample was loaded on carbon-coated copper TEM grid and allowed to evaporate for complete dryness at room temperature. TEM micrograph


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images were recorded on an accelerating voltage of 50–300 kV. FTIR measurements were carried out to identify the functional groups of biomolecules involved in bio-reduction and capping of AgNPs using FT-IR Spectrometry (Perkin Elmer, Spectrum RXI, resolution: 400 cm−1 , detector: LiTaO3). One mg bark extract of H. antidysenterica was dissolved in 1 ml of solvent and a drop of the given extract was placed on one of the KBr plates in order to obtain a translucent sample disc. 2.5. Mosquito culture Cyclic colonies of Anopheles stephensi were maintained in an insectory at 28 ± 2 ◦ C temperature and 70–80% relative humidity and fitted with a simulated dawn and dusk machine with a photoperiod of 14 h light and 10 h dark. Adult mosquitoes were kept in 30 cm × 30 cm × 30 cm organdy cloth cages tied onto the iron frame. Mosquitoes were offered water soaked raisins and 10% glucose solution soaked cotton pads as a source of energy. For ovarian development, the females were fed on rabbit blood after 4–6 days of their emergence. The gravid females oviposited their eggs on the water surface in small plastic containers lined with filter paper. The eggs were kept undisturbed for 48 h for hatching. The hatched larvae were reared in enamel trays consisting of boiled cooled water. The larvae were fed on powdered dog biscuits and fish food (6:4) mixture. The water was changed daily as this species of mosquito prefer fresh water. After the development of larva to pupa, pupae were collected separately in water-filled plastic bowls. The pupal stage lasted for about 48 h. The pupae were transferred into the organdy cloth cages for adult emergence.

2.8. Gas chromatography-mass spectrometry (GC–MS) One mg of bark sample was dissolved in a solvent and subjected to GC–MS analysis. The GC–MS unit (Shimadzu QP2010 Plus) equipped with MS detector was operated under the following parameters: Rtx- 5 MS capillary column (0.25 mm × 0.25 mm × 30 m), injector temperature: 260 ◦ C; oven temperature: 70–280 ◦ C; carrier gas: helium; flow rate: 1.21 ml/min. The identification of compounds was carried out by comparing mass spectra and retention indices with National Institute of Standards and Technology (NIST) library. 2.9. Statistical analysis The larval mortality was recorded after 24 h AgNPs treatment and their corresponding lethal doses (LC50 and LC90 values), upper and lower confidence limits (UCL-LCL), chi-square values, 95% confidence limits and regression equations were calculated according to Probit Analysis (Finney, 1971) using the SPSS software, window 16. Corrected percentage mortality was calculated using Abbott’s formula. P < 0.05 was used to determine the significance of differences among the means. We also conducted the principal component analysis (PCA) to determine differences in bark extracts prepared in different solvents (chloroform, hexane, ethyl acetate, methanol, water and acetone) and AgNPs in terms of their corresponding LC50 and LC90 values. PCA was conducted using Canoco 4.5 software. 3. Results 3.1. Synthesis of silver nanoparticles

2.6. Larval bioassay Larvae of A. stephensi were reared at insectory of National Institute of Malaria Research (Delhi), India for regular availability for bioassays. Stocks of the desired concentrations were prepared in ethanol for each extract (hexane, ethyl acetate, methanol, water and chloroform) from respective concentrated residues. The stock solutions were further diluted to prepare a range of various test concentrations in ppm. One ml of these test concentrations were added to 249 ml of water in 500 ml beakers to obtain the desired concentration of above-mentioned extracts to which the larvae were exposed. Each test contained a set of control (1 mM silver nitrate, de-chlorinated water and corresponding solvent concentration). Twenty-five numbers of 3rd instar larvae of A. stephensi were exposed to the above-mentioned concentrations of each extract along with control according to the standard procedure (WHO, 2009). Experiments were carried out in triplicates along with the control in each series at 27 ± 2 ◦ C temperature and 85 ± 5% relative humidity. 2.7. Bioassay for non-target organisms, Mesocyclops thermocyclopoides Harada Toxic effect of biosynthesized silver nanoparticles was tested against the non-targeted organism, M. thermocyclopoides which were collected from ponds, ditches and pools of Burari village (North Delhi). These species were brought and acclimatized to laboratory conditions of National Institute of Malaria Research (NIMR, New Delhi). Twenty-five number of M. thermocyclopoides organism were exposed to 250 ml of tested concentrations (LC50 and LC90 ) of AgNPs in 500 ml capacity of the plastic bowl for 48 h at 28 ◦ C RT with the protocol adopted by Patil et al. (2012). Experiments were performed in three replicates with a set of control (dechlorinated water). Mortality was recorded after 24 h of AgNPs treatments.

UV–vis absorption spectrophotometer is one of the most efficient, simple, sensitive and widely used technique to confirm the synthesis of AgNPs at the initial stage. The change in colour of aqueous bark extract from yellow to dark brown after addition of AgNO3 confirmed its reduction AgNO3 into AgNPs (Fig. 1B). Further, AgNPs synthesized using H. antidysenterica showed characteristic surface plasmon resonance (SPR) peaks at 420 nm which confirmed their synthesis. Hence, from the above observation, it can be concluded that H antidysenterica bark extract has the ability to reduce silver nitrate to silver nanoparticles. But no colour change was observed when AgNO3 was not amended in the aqueous bark extracts. 3.1.1. Parameters affecting the biosynthesis of silver nanoparticles Different parameters were found to affect the conversion of silver nitrate to silver nanoparticles using aqueous bark extract of H. antidysenterica. In this investigation, factors affecting the biosynthesis of silver nanoparticles such as temperature of the reaction, pH, AgNO3 concentrations and reaction time were studied and optimized. Effect of time and concentrations on the biosynthesis of silver nanoparticles. Synthesis of AgNPs has been observed when 10 ml plant extract was added in 90 ml of different concentrations (0.5, 1, 2, 3, 4 and 5 mM) of silver nitrate solution. In each concentration, the conversion of yellow to characteristic brown colour of the reaction mixture showed visual confirmation of silver nanoparticles synthesis. Intensity of the absorbance peaks of AgNPs were found to increase with increasing concentrations (0.5–5 mM) of AgNO3 due to maximum availability of substrate for the bio-reduction by aqueous bark extract (Fig. 1C). We also observed that UV–vis spectra shifted towards higher wavelength as the concentration of silver nitrate increased. The absorption peaks were found to vary with different concentrations (0.5, 1, 2, 3, 4 and 5 mM) of AgNO3 . In order

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Fig. 2. Characterizations of silver nanoparticles synthesized using H. antidysenterica bark extract with 1 mM aqueous solution AgNO3 at pH 7.5 and 50 ± 2 ◦ C for 120 min. (A) XRD pattern of AgNPs synthesized using aqueous bark extract of H. antidysenterica. (B) Transmission electron microscopy micrograph of AgNPs derived from H. antidysenterica bark extract. (C) Histogram showing Particle size distribution of AgNPs. This data is based on the image analysis of more than 400 particles. (D) Scanning electron microscopy image showing the morphological feature of AgNPs synthesized using the aqueous bark extract H. antidysenterica.

to ensure the complete reduction of AgNO3 , UV–vis spectra were observed at different time intervals. No absorbance peak was seen at the initial stage (0 min), but after 5 min of incubation period, a wide peak was recorded at 420 nm. Further, the absorbance peaks were found to be increasing with rise in the time of reaction. After some time (120 min), the peak became stable and no further change in colour was observed showing complete reduction of AgNO3 by aqueous bark extract. Formation of silver nanoparticles was confirmed by taking absorption spectra at different time intervals and recording peaks at different time intervals at 0.52 a.u. after 15 min, 0.74 a.u. after 30 min, 1.27 a.u. after 60 min, and 2.23 a.u. after 120 min (Fig. 1D). Finally, AgNPs were seen to be synthesized using aqueous bark extract of H. antidysenterica were stable in solution up to six weeks. Effect of pH and temperature on AgNPs synthesis. Temperature is one of the most important factors affecting the size of AgNPs by altering the rate of their synthesis. Another important parameter which affected the synthesis of AgNPs was the temperature of the reaction. Fig. 1F depicts the UV visible spectra of AgNPs formation at different temperatures (25–100 ◦ C). The absorption peaks observed at 25 ◦ C, 50 ◦ C, 75 ◦ C and 100 ◦ C were 434, 427, 420 and 415 nm, respectively. Absorption peaks shifted towards lower wavelength as the temperature of the solution increased due to smaller size of AgNPs. Further, rapid change in colour of reaction was observed at

higher temperature indicating reduction of silver salt within few minutes. pH is another factor which affects the shape and size of AgNPs by altering the charge of biomolecules of aqueous extract. The changing of the pH of reaction mixture influences the reduction of silver nitrate by affecting chemical state (ionization) of plant constituents. At low pH (pH 3), a flat UV spectrum (no absorption peak) was observed which increased with the increasing pH of the solution. The absorption peak at pH 7 and pH 11 were 420 nm and 435 nm, respectively (Fig. 1E). No absorption peak was recorded at pH 3, hence it was not suitable for the synthesis of AgNPs while pH 7 and 11 exhibited absorption peaks confirming that these pH are appropriate for biosynthesis of AgNPs. Further, increasing the pH of the reaction mixture enhances the bio-reduction of AgNPs as observed by very fast (within few minutes) colour change when aqueous bark extract was added in 1 mM silver nitrate solution. Shifting in the position of absorption peak towards higher wavelength with increasing pH of the solution was also observed which was directly correlated to larger size of nanoparticles.

3.2. Characterization of aqueous bark derived AgNPs XRD analysis showed a number of Bragg reflections value 38.34, 44.54, 64.36 and 76.9 at the 2␪ angle, which corresponded to 111, 200, 220 and 311, respectively sets of lattice planes (Fig. 2A). This analysis explained that AgNPs synthesized by H. antidysenterica


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Table 1 FT-IR profile of AgNPs show the functional groups responsible for AgNO3 reduction. S. N.

Frequency (CM−1 )

Wave number (CM−1 )

Functional groups


1 2 3

690–515 900–700 1200–900 1075–1020 1250–1020 1275–1200 1600–1300 1670–1600 1750–1720 2140–1990 2175–2140 2600–2550 3000–2500 2820–2850 3500–3300 3550–3200

610.18 830.20 942.14 1024.10 1076.32 1203.13 1480.08 1640.08 1745.27 2045.09 2167.80 2556.12 2621.13 2826.12 3320.79 3418.42

C Br C H, C C C O C, C O C O, N H C N C O, C F C H C N, C C, C O C O N C S S C N S H O H HC O: CH N H O H

Halo compound 1,2,4-trisubstituted, 1,3-disubstituted,alkene Polysaccharides Vinyl ether, amide Amine Alkyl aryl ether, fluoro compound Alkenes Imine/oxime, alkene, conjugated alkenes, ␦-lactone, aldehyde, esters Isothiocyanate Thiocyanate Thiol Carboxylic acid Aldehydes Amines Alcohols, phenols

4 5 6 7 8 9 10 11 12 13 14 15



1745.27 2167.80


942.14 830.20 1203.17 1024.10 1076.32

2621.13 2045.09

90 80

610.18 1480.08



60 50



4000 3500 3000 2500 2000 1500 1000 500

Fig. 3. FT-IR spectrum of silver nanoparticles synthesized using aqueous bark extract of H. antidysenterica.

were crystalline in nature with face-centered cubic structure. Morphological nature of AgNPs was determined by FE-SEM and TEM studies. Their micrographs showed that most of the AgNPs were spherical, hexagonal and triangular in nature with an average size of 40–60 nm (Fig. 2B–D). FT-IR analysis of synthesized AgNPs was carried out to identify the possible functional groups, responsible for its reduction, capping and stabilization (Table 1). The FT-IR spectrum of the AgNPs showed broad and strong absorbance peaks at 3418.42, 2045.09, 1640.08, 1076.32 and 610.08 cm−1 , which might be corresponding to O H, N C S, C H, C C, C N and C Br moieties, respectively (Fig. 3). 3.3. Mosquitocidal activity of AgNPs against A. stephensi Larvicidal activity of H. antidysenterica bark extracts in different solvents such as acetone, hexane, ethyl acetate, methanol, chloroform and of AgNPs (prepared with aqueous bark extract of H. antidysenterica) were tested against the malaria vector A. stephensi (Table 2). The highest larval mortality was observed in the green synthesized AgNPs with LC50 and LC90 values being 2.672 ppm and 4.482 ppm, respectively. In our experiments, no mortality was observed in (AgNO3 , de-chlorinated water and solvent solution) control. Chloroform extract of H. antidysenterica also showed moderate larvicidal activity (LC50 3.004 ppm: LC90 6.86 ppm) against mosquito vector. Hexane, ethyl acetate and methanol derived bark extracts showed poor efficacy against A. stephensi vector with LC50 31.56, 41.92, 96.40, 121.53 and LC90 55.26, 70.51 639.99 and 688.99,

respectively. Acetone derived bark extract of H antidysenterica proved least effective as its higher doses (LC50 1.99E3 ppm: LC90 4.84E3 ppm) could not control the vector’s lethality. AgNPs proved to be non-toxic against non-target organism M. thermocyclopoides when exposed to tested concentration (LC50 and LC90 values of A. stephensi) of AgNPs. In the GC–MS analysis, 41 compounds were identified in the bark extract of H. antidysenterica which were clearly matched with those found in NBT/NIST spectral database. The major components in the bark extract were piperidine (36.62%), betulin (13.13%), 5-hydroxymethylfurfural (9.26%), cycloartenol (5.86%), methyl hexafuronoside (5.73) lanosterol (4.78%) (Fig. 4) and remaining were in the range of 0.1–2% (Table 3). From the above observation, bioactive compounds identified from bark extract of H. antidysenterica may be considered effective in designing new larvicidal drugs that can be effectively used in controlling the mosquito vector, A. stephensi. PCA results grouped the bark extract prepared in different solutions into two different clusters separated by the first axis explaining 99% variation in terms of LC50 and LC90 . Cluster l consisted of methanol, aqueous extract and cluster 2 had AgNPs, chloroform, hexane, ethyl acetate. On the other hand, acetone extract separated out. LC50 and LC90 were found to be positively correlated with the first axis (Fig. 5). The two axis of PCA explained 100% variance. 4. Discussion Nanoparticles have opened a new insight in targeted delivery of the compounds to influence efficacy because of their reduced size and deep penetration. Present study for the first time highlights the efficacy of AgNPs prepared by aqueous bark extract of H. antidysenterica against the A. stephensi, a most dreaded malaria vector. AgNPs were prepared by addition of AgNO3 into the bark extracts of H. antidysenterica. The change in colour of aqueous bark extract may be due to the reduction of AgNO3 to Ag ion associated with the excitation of surface plasmon resonance (SPR) (Suman et al., 2013; Ahmed et al., 2018; Hashemi et al., 2018). The combined vibration of free electrons of nanoparticles in resonance with the light wave gives rise to the SPR absorption band (Selvi Barnabas et al., 2018). It is interpreted that the plant extract contains different bio-molecules, which act as both reducing and capping agents to form stable and shape-controlled nanoparticles. According to Anand et al. (2017) phenolics, terpenoids, polysaccharides, flavones, alkaloids, proteins, enzymes, amino acids and alcoholic compounds are such main compounds that participate in this type of reaction. Also, quinol, chlorophyll pigments, linalool, methyl chavicol, eugenol, caffeine, theophylline, ascorbic acid and other vitamins have been reported to show similar effects (Kesharwani

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Table 2 Larvicidal activity of H. antidysenterica bark extracts and AgNPs against 3rd instar larvae of A. stephensi after 24 h treatment. Extracts

Concentrations (ppm)

Mortality(%) ± SD

LC50a (LCLb and UCLc ) ppm

LC90d (LCL and UCL) ppm

Regression equation

X2 e (d. f.)f


Control 200 400 600 800 1000

0 ± 00 0 ± 00 0 ± 00 2.76 ± 0.56a 7.37 ± 0.57b 15.0 ± 1.31c

1.9991 E3 (1291.981–134897.144)

4.484E3 (2044.233–1.352E7)

y = 11.988 + 3.634x

0.302 (3)


Control 50 100 200 400 800 1000

0 ± 00 35 ± 0.76a 56 ± 0.65b 60 ± 2.01c 84 ± 0.43d 92 ± 1.2e 100 ± 00

96.402 (22.500–176.605)

639.991 (523.620–849.808)

y = 0.227 + 0.002x

6.475 (4)


Control 10 20 30 40 50 60

0 ± 00 20.0 ± 0.81a 25.0 ± 0.21b 42.32 ± 0.39c 52.12 ± 0.68d 87.5 ± 1.01e 100.0 ± 00

31.562 (22.510–39.932)

55.267 (45.368–80.683)

y = 1.706 + 0.054x

9.767 (4)

Ethyl acetate

Control 10 20 30 40 50 60 70 80

0 ± 00 5.0 ± 0.87a 22.3 ± 1.87b 30.0 ± 0.32c 47.5 ± 0.87d 62.67 ± 0.61e 70.0 ± 0.93f 90.0 ± 0.76g 100.0 ± 00

41.925 (38.179–45.592)

70.517 (64.983–78.106)

y = 1.879 + 0.045x

5.439 (6)


Control 2 4 6 8 10

0 ± 00 10 ± 0.38a 90 ± 1.21b 96 ± 0.73c 100 ± 00 100 ± 00

3.004 (2.189–3.750)

6.863 (3.883–8.450)

y = 2.926 + 6.126x

2.479 (6)

Aqueous extract

Control 50 100 200 400 800 1000

0 ± 00 28.71 ± 0.33a 48.53 ± 0.46b 60.86 ± 0.71c 75 ± 1.43d 96.1 ± 1.6e 100 ± 00

121.53 (79.33–167.24)

688.79 (453.96–998.053)

y = 39.103 + 0.066x

3.350 (4)


Control 2 4 6 8 10

0 ± 00 25 ± 1.84a 95 ± 0.13b 100 ± 00 100 ± 00 100 ± 00

2.672 (1.842–3.270)

4.482 (3.814–6.825)

y = 1.893 + 0.708x

6.190 (6)

a b c d e f g

LC50 , lethal concentration that kills 50% of the exposed larvae. LCL, lower confidence limit. UCL upper confidence limit. LC90 , lethal concentration that kills 90% of the exposed larvae. ␹2 ,chi-square test, Significant value at P < 0.05. (d.f.), degree of freedom. Indicates the significant difference among the variables.

et al., 2009; Mallikarjuna et al., 2011; Bindhu and Umadevi, 2013; Chahardoli et al., 2017; Patil and Kim, 2017; Zhou et al., 2017). Among these compounds, flavonoids and phenols have the unique chemical power to effectively wrap the nanoparticles, preventing their agglomeration (Ahmad et al., 2010). It is therefore suggested that H. antidysenterica extract used in this have comprises different compounds which might have played a crucial role in the formation of AgNPs. In our finding, the absorption peak increased with the increase in concentrations from 0.5 to 5 mM of AgNO3 in the solution in between the range of 370–800 nm for UV–vis spectrum. This result indicated that synthesis of nanoparticles continues to enhance with

the increasing concentrations of AgNO3 due to their reduction by aqueous bark extract. Zaki et al. (2011) reported that higher concentration (10 mM) of AgNO3 increased the synthesis of silver nanoparticles as compared to lower concentrations. Similar to this work, in our case also the peak intensity increased with the increasing concentration of AgNO3. At higher concentrations of silver nitrate, UV–vis spectrum peaks shifted towards higher wavelength (red shift) indicating the larger size of AgNPs. Absorption peak shifted towards higher wavelength (from 408 to 421 nm) with higher concentrations of silver nitrate in Azadirachta indica leaf derived AgNPs (Verma and Mehata, 2016). Similar findings were also observed in Achyranthes aspera synthesized AgNPs (Sharma


D. Kumar et al. / Process Safety and Environmental Protection 116 (2018) 137–148

Fig. 4. Chemical structure of the six major constituents of H. antidysenterica barks extract.

et al., 2017). From above observation, it can be concluded that size and peak intensity of AgNPs were directly correlated to AgNO3 concentration. It has been found that the time duration too play crucial role in the synthesis of AgNPs. The reduction of silver nitrate using H. antidysenterica bark extract was observed at different time intervals. Change in colour of the reaction mixture started after 5 min of amending silver salt into plant extract indicating the AgNPs synthesis. Incidentally, after 120 min, no further change in colour was seen proving that the complete reduction of silver salt by aqueous bark extract. Shankar et al. (2004) reported that complete reduction of silver nitrate was observed using Azadirachta indica leaf extract after half an hour. This is similar to the recent work of Veisi et al. (2018) also reported the complete reduction of AgNO3 with in 60 s using aqueous extract of Thymbra spicata and silver nitrate solution. Amin et al. (2012) reported that optimum time (30 min) required for completion of reaction employing Solanum xanthocarpum Berry extract and AgNO3 solution. From above observations, it can be concluded that the time required for completion of reaction depend upon the reduction potential of plant species used. pH is one of the important parameters that affects size and shape of the nanoparticles by altering the charge of phytoconstituents. No absorption peak was recorded under acidic conditions (pH 3), while neutral and alkaline conditions showed absorption peaks in H. antidysenterica bark extract and AgNO3 solution indicating that these conditions were suitable for AgNPs biosynthesis. Higher pHinduced the synthesis of the large number of AgNPs with smaller size due to bioavailability of functional groups in bark extract and electrostatic repulsion among AgNPs (Sathishkumar et al., 2009). Acidic pH inhibited the synthesis of AgNPs and at low pH large

sized nanoparticles were produced due to agglomeration or nucleation and reduction of reducing power of functional groups. As the pH of solution increased from 7 to 11, the absorption peaks or band shifted from 420 to 435 nm, due to the excitation of SPR. As the size of AgNPs increased, the energy required for excitation of surface plasmon electrons decreased, consequently the absorption peak or band shifted towards the longer wavelength region (Ibrahim, 2016). Khalil et al. (2014) reported that the intensity of absorbance peak of AgNPs synthesized using Olea europaea leaf extract increased with increasing pH of the solution (2–8) and maximum absorption was recorded at pH 8. Vanaja and Annadurai (2013) while working with Coleus aromaticus leaf derived AgNPs observed that higher pH is favourable for the synthesis of AgNPs. Similarly slightly alkaline pH (pH 7) was optimum for the synthesis of AgNPs employing Olea europaea leaf extract (Rashidipour and Heydari, 2014). In our studies, it was concluded that acidic pH was unfavourable for AgNPs synthesis while alkaline pH was favourable for AgNPs synthesis. Silver nanoparticles synthesis was carried out using H. antidysenterica aqueous bark extract at different temperatures (25, 50, 75 and 100 ◦ C). The reduction of silver nitrate increased with the increase in temperature as shown by rapid change in the colour of solution. Rashidipour and Heydari (2014) reported while working with different temperature (20, 30, 45 and 60 ◦ C) of the reaction reported that rapid reduction of silver ions was also observed at higher temperature (45 ◦ C) using Olea europaea leaf extract. In our experiment, the absorption band maxima shifted towards smaller wavelength from 434 to 420 nm, as temperature varied from 25 to 100 ◦ C due to localization of SPR of AgNPs. Shifting of absorption peaks towards lower wavelengths was also observed at different temperature (10, 20, 30, 40 and 50 ◦ C) in Azadirachta indica

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Table 3 GC–MS analysis of bark extract of H. antidysenterica. Peak No.

R. time





1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

6.942 7.02 8.428 10.159 10.415 11.674 11.818 13.409 14.88 15.708 16.396 16.994 17.584 18.723 19.460 19.623 22.582 24.982 28.922 29.095 29.771 31.880 32.022 32.505 32.955 33.514 33.765 35.783 37.301 37.989 38.183 38.745 40.200 40.873 42.396 42.669 43.447 43.807 43.997 44.783 44.977

26,72,922 7,46408 7124337 63734677 2662078 4737614 2047447 981085 2971126 10370683 779671 1016425 8321913 39343075 11082170 4259743 6268469 11852947 2605391 1415389 7237096 792852 5555650 8445589 251425235 661663 2436381 8544682 2401583 989570 2383515 1386502 1917586 10424127 7811478 3590112 7081603 32842091 11031068 40231874 90156491

0.39 0.11 1.04 9.29 0.39 0.69 0.3 0.14 0.43 1.51 0.11 0.15 1.21 5.73 1.61 0.62 0.91 1.73 0.38 0.21 1.05 0.12 0.81 1.23 36.62 0.1 0.35 1.24 0.35 0.14 0.35 0.2 0.28 1.52 1.14 0.52 1.03 4.78 1.61 5.86 13.13

Cyclopentane,1-acetyl-1,2 epoxy Orcinol Pyranone 5-Hydroxymethylfurfural 1-Monoacetin 3-Heptanol Cis-dimethyl morpholine Hydroquinone Methylheptanoate D-allose Embanox 4-Allylsyringol Decanoic acid Methyl hexafuronoside 3-O-Methylhexose Tetradecanoic acid Hexadecanoic acid Cis-vacenic acid Alpha-ethynyl-17-hydroxyester 2-(Octadecylamino)-1-phenyl-1-propanol 1-Monostearin L-Prolinol Coumarin,3-benzoyl-4-phenyl NCNH Conessine Piperidine Squalene Conessimine E-3-Pentadecen-2-ol Solanesol (−)-Cholesterol ␣-Tokopharm Desmosterol Campesterin 22-Stigmasten-3-one Sitosterol Spinasterone ␣-Amyrin Lanosterol Epi-Psi-taraxastanonol Cycloartenol Betulin

Treatment of chronic HCV infection Treat of acne and other greasy skin No activity reported Treatment of sickle cell disease Antifungal agent Antiarrhythmic agents Treatment of autoimmune diseases Larvicidal and anticancer No activity reported Treatment of urinary tract infection Antioxidant activity No activity reported Antimicrobial activity Antimicrobial activity No activity reported Antimicrobial activity Antimicrobial activity Cholesterolytic activity No activity reported Treatments of asthma Skin treatments No activity reported Antibacterial activity Larvicidal activity Larvicidal activity Anticancer activity Antimicrobial activity No activity reported Fungicidal activity Antiviral activity Antioxidant activity anti-inflammatory activity Antidiarrhoeal activity No activity reported Antioxidant activity Antitumor activity Anti-microbial and anti-fungal activity anti-fungal activity Antibacterial activity Antimicrobial activity Anticancer activity

leaves derived AgNPs (Verma and Mehata, 2016). The absorption peak showed that the size of synthesized AgNPs decreased with the increasing temperature of the reaction. At higher temperature, molecules have maximum kinetic energy and high rate of silver ion reduction, thus less possibility of growth in size of AgNPs. Premasudha et al. (2015) observed the smaller size of AgNPs using Eclipta alba leaf extract when the reaction was carried out at higher temperature. It has also been suggested by Khan et al. (2017) that the shape and size of metal nanoparticles using plant extract depends upon concentration of extracts and metal salt, temperature, pH and incubation time. The structure of bark synthesized AgNPs was analyzed by XRD. XRD pattern clearly indicated that the biologically synthesized AgNPs were of crystalline nature. Similar observations were also reported by various authors using different plants (Murugan et al., 2015; Sebastian et al., 2018). The size of the AgNPs was in the range of 18 to 60 nm, full width at half maximum of the (111) peak of silver using the Scherrer formula. The sharp peaks in XRD pattern might be due to the presence of capping agent in plant extract, which stabilized AgNPs (Krishnaraj et al., 2010). Abnormal peak positions in XRD pattern indicated the existence of strain in the AgNPs, which is a characteristic feature of nanocrystallites. Few smaller peaks were also observed in the XRD pattern which might be due to some impurities of various biological macromolecules. Anomalous peak positions, heights and widths in XRD patterns are size-dependent properties of AgNPs. The broadening of peak in XRD pattern illustrates that AgNPs present in the samples were smaller in size. Dubey

Fig. 5. PCA plot showing LC50 and LC90 values of bark extracts in different solvents. The extracts were grouped in two different clusters separated by first axis explaining 99% variation in terms of LC50 and LC90 . Cluster l consisted of methanol, aqueous extract and cluster 2 had AgNPs, chloroform, hexane, ethyl acetate.

et al. (2009) also estimated the size of silver nanocrystallites using the Scherrer formula to be 20–60 nm. The surface morphology of the AgNPs was determined by FESEM. FE-SEM micrograph illustrated that the AgNPs synthesized using aqueous bark extract of H. antidysenterica were spherical, hexagonal and triangular in shape. Visual observation of the FESEM image indicated that size of nanoparticles was in the range of


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40–60 nm. FE-SEM results also indicated that AgNPs were stable in the aqueous solution which could be due to the presence of various capping agents in the aqueous bark extract. FE-SEM showed the irregular distribution of AgNPs on the surface of the pellets. Previous studies also reported that green synthesized nanoparticles were mostly spherical in shape (Roni et al., 2015) with the exception of Azadirachta indica synthesized AgNP, which have flat plate and spherical morphology with an average size 5–35 nm (Shankar et al., 2004). The shape and size of AgNPs were also determined by TEM. TEM images showed that most of the AgNPs were separated from each other by an interparticle distance. Cruz et al. (2010) reported similar separation in nanoparticle using Lippia citriodora plant extract. The FT-IR spectrum of bark synthesized AgNPs exhibited intense vibration stretching at 3418.42 cm−1 (O H stretching alcohol, phenols), 3320.79 cm−1 (N H stretching aliphatic compounds), 2826.12 cm−1 (C H stretching aldehydes), 2621.13 cm−1 (O H stretching carboxylic acid), 2556.12 cm−1 (S H stretching thiol compound), 2167.80 cm−1 (S C N stretching thiocyanate compounds), 2045.09 cm−1 (N C = S stretching isothiocyanate), 1640.08 cm−1 (C N, C C, C O stretching imine/oxime, alkene, conjugated alkenes), 1076.32 cm−1 (C O, N H stretching vinyl ether) and 610.18 (C Br stretching halo compounds). FT-IR spectrum supported the presence of phenols, alcohol, aromatic and aliphatic amide and carbonyl functional groups in the synthesized AgNPs (Ahluwalia et al., 2014; Chouhan et al., 2017). Hence, it may be possible that these functional groups of compounds are responsible for reduction, capping and stabilization of synthesized AgNPs (Salari et al., 2017; Saratale et al., 2017). In the current scenario, biologically synthesized nanoparticles have tremendous larvicidal potential against different mosquito vectors (Santhoshkumar et al., 2011; Subramaniam et al., 2015; Benelli, 2016; Murugan et al., 2016; Rawani, 2017; Ghramh et al., 2018). It has been observed that biologically synthesized nanoparticles have higher vector mortality as compared to crude extracts prepared in different solvents. Nanoparticles extract was tested against third instar larvae of A. stephensi which showed maximum larvicidal activity with LC50 and LC90 values being 2.672 ppm and 4.482 ppm, respectively. However, considerably high values were reported in Chomelia asiatica and Caulerpa scalpelliformis against Culex quinquefasciatus with LC50 and LC90 values of 20.92, 4.64 and 37.41, 10.43 ppm, respectively (Muthukumaran et al., 2015; Murugan et al., 2015). AgNPs synthesized using the aqueous extract of Phyllanthus niruri were highly effective against Aedes aegypti with the LC50 value of 3.90 ppm (Suresh et al., 2015). It is thus interpreted that smaller size of metal nanoparticles allows them to pass through the insect cuticle and into individual cells, where they can bind to sulfur of proteins or to phosphorus of DNA, leading to the rapid denaturation of enzymes, organelles proteins and nucleic acids. Subsequently, the decrease in membrane permeability and disturbance in proton motive force takes place. It may cause loss of cellular functions, damaged molting and physiological processes and at the last results in cell death (Arjunan et al., 2012; Murugan et al., 2015; Crisponi et al., 2017; Kumar et al., 2017a). Contrary to above bioassays conducted with the bark crude extract of H. antidysenterica prepared in different solvents viz. chloroform, methanol, hexane, ethyl acetate and acetone revealed high LC50 and LC90 against A. stephensi. It was observed that the maximum vector mortality was achieved with bark chloroform extract, which showed 3.004 ppm LC50 and LC90 6.863 ppm values for A. stephensi. In yet another report, a very high LC50 and LC90 values were observed in leaf chloroform extracts of Hippophae rhamnoides (LC50 1389.74 and LC90 2718.32 ppm) and Pithecellobium dulce (LC50 160.71 and LC90 282.35 ppm) against A. stephensi (Ahmad and Ali, 2013; Govindarajan et al., 2014). Chloroform is a good solvent for extraction of these bioactive compounds for enhanced larvicidal activity (Bagavan et al., 2008). Hexane bark extract of H.

antidysenterica showed moderate larvicidal activity with LC50 and LC90 values 31.56 and 55.26 ppm respectively. Similarly, Singh et al. (2007), observed lower mortality rates in larvae of A. stephensi exposed to crude hexane extract of Eucalyptus citriodora with LC50 and LC90 of 69.86 and 221.80 ppm, respectively. Kovendan et al. (2012) studied the larvicidal activity of the Orthosiphon thymiflorus extract against A. stephensi, with high LC50 and LC90 values 201.39 and 490.86 ppm respectively. In another study, a high LC50 (79.58 ppm) value was observed using hexane extracts of Azadirachta against A. stephensi (Siddiqui et al., 2003). GC–MS analysis data showed that piperidine, betulin, 5-hydroxymethylfurfural, cycloartenol, methyl hexafuronoside and lanosterol were the main constituents of bark extract of H. antidysenterica and reported to possess larvicidal, anticancer, antimicrobial and antifungal activities (Lee, 2000; Manral et al., 2016). Hydroquinone (Ioset et al., 2000) and conessine (Thappa et al., 1989) have also been previously reported for their larvicidal activity against Culex pipiens pallens and Aedes aegypti, respectively. Thus, the larvicidal activity against A. stephensi of the extract may be due to the synergistic effect of larvicidal agent present in the bark of H. antidysenterica. The PCA analysis revealed that larvicidal behaviour of bark extract prepared in methanol was comparable with aqueous extract as both of them clustered together. Whereas AgNPs was comparable to the chloroform, hexane and ethyl acetate extract in terms of LC50 and LC90 values. The current investigation clearly proves that H. antidysenterica bark mediated AgNPs efficiently controlled the third instar larvae of, A. stephensi at minimum dozes as compared to other solvents. Beside this, AgNPs prepared using aqueous bark extract of H. antidysenterica were non-toxic against the non-target organism, M. thermocyclopoides at calculated LC50 and LC90 values of A. stephensi. Silver nanoparticles synthesized using Berberis tinctoria did not exhibit any toxic effect against Toxorhynchites splendens and Mesocyclops thermocyclopoides after 48 h of exposure (Maheshkumar et al., 2016). Prior to this, similar results were also observed in H. antidysenterica derived AgNPs against M. thermocyclopoides after 72 h exposure (Kumar et al., 2017a). 5. Conclusion Study reveals the successful synthesis of AgNPs using aqueous bark extract of H. antidysenterica. AgNPs synthesized by this technique are crystalline in nature, spherical in shape and showed SPR band around 400 to 450 nm (depending upon various controllable factors). It is also concluded that the shape and size of AgNPs can be altered by changing the reaction conditions such as AgNO3 concentrations, temperature, pH and reaction time. Higher temperature and alkaline condition of reaction enhanced the production of AgNPs. The AgNPs thus synthesized have excellent larvicidal efficacy against malaria vector with LC50 and LC90 values being 2.672 and 4.482 ppm, respectively without harming non-target organism, M. thermocyclopoides. Thus, AgNPs synthesized by this method can be effectively used in different biomedical devices, optoelectronics devices and also play an important role in the production of nano-based pesticides for mosquito control in near future. Conflict of interest The authors declare that they have no competing interests. Acknowledgments Authors are grateful to SERB, DST PURSE Grant and the University of Delhi for providing R&D Grant. Dinesh Kumar is indebted to University Grants Commission, New Delhi for the award of JRF & SRF.

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