Green synthesis of silver nanoparticles using ...

13 downloads 10 Views 1MB Size Report
Dec 17, 2017 - Benelli G, Pavela R, Maggi F, Petrelli R, Nicoletti M (2017) ... Jinu U, Rajakumaran S, Senthil-Nathan S, Geetha N, Venkatachalam P.

Green synthesis of silver nanoparticles using Holarrhena antidysenterica (L.) Wall.bark extract and their larvicidal activity against dengue and filariasis vectors Dinesh Kumar, Gaurav Kumar & Veena Agrawal

Parasitology Research Founded as Zeitschrift für Parasitenkunde ISSN 0932-0113 Volume 117 Number 2 Parasitol Res (2018) 117:377-389 DOI 10.1007/s00436-017-5711-8

1 23

Your article is protected by copyright and all rights are held exclusively by SpringerVerlag GmbH Germany, part of Springer Nature. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at”.

1 23

Author's personal copy Parasitology Research (2018) 117:377–389


Green synthesis of silver nanoparticles using Holarrhena antidysenterica (L.) Wall.bark extract and their larvicidal activity against dengue and filariasis vectors Dinesh Kumar 1 & Gaurav Kumar 2 & Veena Agrawal 1 Received: 13 October 2017 / Accepted: 5 December 2017 / Published online: 17 December 2017 # Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract The present study was carried out to evaluate the larvicidal potential of methanol, hexane, acetone, chloroform, and aqueous bark extracts of Holarrhena antidysenterica (L.) Wall. and silver nanoparticles (AgNPs) synthesized using aqueous bark extract against the third instar larvae of Aedes aegypti L. and Culex quinquefasciatus Say. AgNPs were prepared by adding 10 ml of aqueous bark extract in 90 ml of 1 mM silver nitrate (AgNO3) solution. After 5 min of mixing, a change in color from yellow to dark brown occurred indicating the synthesis of AgNPs. Their further characterization was done through ultraviolet-visible spectroscopy (UV–Vis), X-ray diffraction analysis (XRD), field emission scanning electron microscope (FE-SEM), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FT-IR). UV–Vis spectrum of synthesized AgNPs showed a maximum absorption peak at 420 nm wavelength. Crystalline nature of AgNPs was confirmed by the presence of characteristic Bragg reflection peaks in XRD pattern. TEM images have shown that most of the AgNPs were spherical in shape with an average size of 32 nm. FT-IR spectrum of AgNPs showed prominent absorbance peaks at 1012.2 (C–O) and 3439.44 cm−1 (O–H) which represent the major constituents of phenolics, terpenoids, and flavonoids compounds. LC-MS analysis of the bark extract confirmed the presence of carbonyl and hydroxyl functional groups which were directly correlated with FT-IR results. These AgNPs were assayed against different mosquito vectors, and the maximum mortality was recorded against the larvae of A. aegypti with LC50 and LC90 values being 5.53 and 12.01 ppm, respectively. For C. quinquefasciatus, LC50 and LC90 values were 9.3 and 19.24 ppm, respectively, after 72 h of exposure. Bark extracts prepared in different solvents such as methanol, chloroform, hexane, acetone, and water showed moderate larvicidal activity against A. aegypti their respective LC50 values being 71.74, 94.25, 102.25, 618.82, and 353.65 ppm and LC90 values being 217.36, 222.24, 277.82, 1056.36, and 609.37 ppm. For C. quinquefasciatus, their LC50 values were 69.43, 112.39, 73.73, 597.74, and 334.75 ppm and LC90 values of 170.58, 299.76, 227.48, 1576.98, and 861.45 ppm, respectively, after 72 h of treatment. AgNPs proved to be nontoxic against the non-target aquatic organism, Mesocyclops thermocyclopoides Harada when exposed for 24, 48, and 72 h. The results showed that bark extract-derived AgNPs have extremely high larvicidal potential compared to other organic solvents as well as aqueous bark extract alone. These AgNPs, therefore, can be used safely for the control of dengue and filarial vectors that cause severe human health hazards. Keywords Aedes aegypti L. . Culex quinquefasciatus Say . Bark extract . Holarrhena antidysenterica (L.) Wall. . Silver nanoparticles . Larvicidal activity

* Veena Agrawal [email protected] 1

Department of Botany, University of Delhi, Delhi 110 007, India


National Institute of Malaria Research, Dwarka, New Delhi 110077, India

Introduction Mosquitoes (Diptera: Culicidae) are known to transmit several lethal human diseases such as Japanese encephalitis, malaria, dengue fever, yellow fever, chikungunya, and lymphatic filariasis which affect the contemporary civilization in terms of

Author's personal copy 378

the economic loss, morbidity, and mortality. Among vectorborne diseases, dengue and filariasis are of major health concern in the urban and semi-urban areas. Dengue is a viral infection transmitted through Aedes aegypti. About 390 million of dengue infection cases were reported in 2016, and over 96 million persons were at risk due to favorable environmental conditions for the vector (Benelli and Mehlhorn 2016). Culex quinquefasciatus is a carrier of Western equine encephalitis virus, Wuchereria bancrofti (Cobbold) Seurat, West Nile virus, Zika virus, arboviruses and transmits a number of zoonotic diseases such as lymphatic filariasis, St. Louis encephalitis and avian malaria. Among them, lymphatic filariasis is the most common, and it has been reported that approximately 120 million people from 83 countries are affected (Rawani 2017). In recent decades, the incidence of vector-borne diseases dramatically increased worldwide due to the rapid changes in environmental conditions (humidity, temperature, and precipitation), water supply, and sanitation facilities. The development of vaccines against viral diseases often turns ineffective due to the high mutation rate of the parasites, cost, the long trial process required, and developing quick resistance towards the vaccine. Vector management, therefore, is the only effective measure to prevent and control such diseases (Pandey et al. 2007, 2011; Sharma et al. 2014a; Sujitha et al. 2015; Roni et al. 2015; Benelli et al. 2017a, b; Benelli 2018). Disturbing breeding sites and killing mosquitoes at larval stage are the prominent strategies for controlling such lethal diseases. A few years back, chemical insecticides such as methoprene, pyrethroids, carbamates, temephos, and organophosphates were largely being used in pest management programs. Currently, such chemical insecticides are not listed in integrated vector management programs due to their harm to non-target organisms, emerging avoidance and resistance behavior of mosquitoes towards insecticides, high-risk environmental protection from insecticides and sustainable development (WHO 2004; Tesfazghi et al. 2016; Benelli and Beier 2017). The combined use of plant extract and nanotechnology may be an alternative strategy for controlling mosquito vectors since they are non-toxic, cheap, and biodegradable (Amer and Mehlhorn 2006; Rahuman et al. 2009; Elumalai et al. 2016; Kumar et al. 2017). Nowadays, plant-mediated synthesis of silver nanoparticles has received an extraordinary attention over other biological organisms like bacteria, algae, and fungi as they are easily available, safe to handle, nontoxic, and only a few steps are required in downstream processing and have the faster rate of AgNPs synthesis. Besides, these are inexpensive, eco-friendly, allow a better manipulation or control of crystal growth during synthesis and stabilization (Salam et al. 2012; Benelli 2016a; Murugan et al. 2017; Pavela et al. 2017). Till date, only a few reports have been published related to synthesis of silver nanoparticles using plant extracts and their larvicidal activity against various mosquito species. Silver nanoparticles

Parasitol Res (2018) 117:377–389

synthesized using Eclipta prostrata L. (Rajakumar and Rahuman 2011), Pergularia daemia Forsk. (Patil et al. 2012a), Cadaba indica Lam. (Kalimuthu et al. 2013), Heliotropium indicum L. (Veerakumar et al. 2014b), Feronia elephantum Correa (Veerakumar et al. 2014a), Achyranthes aspera L. (Elumalai et al. 2016), Polianthus tuberosa L. (Rawani 2017), Cleistanthus collinus (Roxb.) Benth. ex Hook.f. (Jinu et al. 2017), and Derris trifoliataLour. (Kumar et al. 2017) have shown very strong larvicidal activity against different mosquito vectors. Besides, AgNPs had no side effects against non-targeted organisms as compared to conventional insecticides and are environmentally sustainable as reported by various authors (Rawani et al. 2013; Duan et al. 2015; Benelli 2016a). Incidentally, so far, there is no published report on the larvicidal activity of AgNPs synthesized using bark extract of Holarrhena antidysenterica. Holarrhena antidysenterica (L.) Wall., commonly known as tellicherry bark or conessi (English) or ‘kurchi’ or ‘inderjab’ (Hindi), belongs to the family Apocynaceae and is considered as one of the most important medicinal plants in India in Hindu mythology. Every part of this plant has medicinal value as mentioned in the Vedas, and its main medicinal property is to relieve amebic dysentery. Several tribes have used this species to cure serious ailments such as diarrhea, cholera, dysentery, skin infections, epilepsy, stomach pain, and skin diseases for hundreds of years and it is still in use today (Thakur et al. 2016). Plants or extracts of plant parts have various therapeutic potentials like anti-amoebiasis activity (Shahabuddin et al. 2006), diuretic (Khan et al. 2012a), anti-urolithic property (Khan et al., 2012b), central nervous system depressant (Nahar et al. 2012), analgesic (Shwetha et al. 2014), anticancerous activity (Sharma et al. 2014b). Considering its immense medicinal uses, it was planned to (i) synthesize silver nanoparticles (AgNPs) using bark extract of H. antidysenterica; (ii) their characterization through various techniques such as UV–Vis spectroscopy, X-ray diffraction, field emission scanning electron microscope, transmission electron microscope, and Fourier transform infrared spectroscopy; and (iii) evaluation of bioefficacy of synthesized AgNPs and solvent extracts against the larvae of Culex quinquefasciatus, Aedes aegypti, and non-target organism (M. thermocyclopoides). In addition, LC-MS analysis of the bark extract was also carried out to identify the possible biomolecules which might be involved in the bio-reduction of AgNPs.

Materials and methods Collection of plant materials and preparation of bark extracts Fresh green bark of Holarrhena antidysenterica (L.) Wall. was obtained from trees growing near the Department of

Author's personal copy Parasitol Res (2018) 117:377–389

Physics, University of Delhi (latitude 28.68°N; longitude 77.21°E), Delhi, India, and washed thoroughly thrice using running tap water to remove the impurities. These barkpieces were then air-dried at room temperature (28 °C) for 72 h and ground into a rough powder using a mortar and pestle. The powder was divided into several parts of 20 g each for making extracts with different solvents. For preparing the aqueous extract, 20 g of the aforesaid powder was boiled with 500 ml of water for 45 min and filtered through Whatman filter paper No. 1. The filtrate was air-dried and stored at 4 °C for further experiments. For solvent bark extracts, each time 20 g of the powder was extracted with 500 ml of different solvents, e.g., chloroform, ethyl acetate, hexane, acetone, and methanol separately. These were filtered individually using Whatman filter paper No. 1. Subsequently, the filtrates were dried and stored separately at 4 °C for future experiments.


Field emission scanning electron microscope and transmission electron microscope measurement The surface features of synthesized AgNPs were monitored using the field emission scanning electron microscope (FESEM) (TESCAN MIRA-3) operated at 20 kV voltages. Transmission electron micrographs of AgNPs were taken by transmission electron microscope (TEM) (TECNAI G2T30, U TWIN) operated at 50 to 300 kV voltages. A drop of AgNPs was used for FE-SEM and TEM analysis. Fourier-transform infrared spectroscopy analysis One drop of the synthesized AgNPs solution was put on the KBr plates and subjected to FT-IR analysis (Perkin Elmer, Spectrum RXI, detector: LiTaO3, resolution: 400 cm−1) to determine the possible functional groups involved in AgNPs reduction. Liquid chromatography-mass spectrometry analysis

Synthesis of AgNPs The synthesis of AgNPs was done by adding 10 ml of aqueous bark (prepared above) extract in 90 ml of 1 mM aqueous silver nitrate solution (pH 7.5) at 50 ± 2 °C temperature for 120 min. The change in the color of the reaction mixture or solution was observed after 5 min of mixing. Subsequently, the reaction mixture was centrifuged for 20 min at 10,956×g. The residue was collected and the supernatant was discarded. The residue was washed with double distilled water and air-dried. The dried residue was used for the characterization of AgNPs and further experiments.

Characterization of AgNPs Ultraviolet-visible spectroscopy (UV–Vis) analysis The bio-reduction of silver nitrate solution was observed primarily with the formation of dark brown color and then determined by UV–Vis spectrophotometer (Shimadzu 250 1 PC, version 2.33) at the range of 360 to 800 nm wavelengths. AgNPs spectra showed unique optical properties, depending upon size, shape, and distribution.

LC-MS of the bark sample was carried out using 2D nano ACQUITY system operated with autosampler, column oven, a binary pump, and in line degasser on Waters Synapt G2. This system was attached to SynaptG2Q-TOF system operated with an electrospray ionization source.The LC-MS data were compared to the available literature to identify the peaks and relative compounds. Mosquito culture and larvicidal bioassay The larvae of Aedes aegypti L. and Culex quinquefasciatus Say were maintained in the insectary of National Institute of Malaria Research, Dwarka at the given conditions (temperature, 28 ± 2 °C; relative humidity, 70–80%; photoperiod, 14:10 h light and dark) and fed with fish food and dog biscuits (4: 6). Bioassays were conducted according to standard protocols recommended by the World Health Organization (WHO 1988). Stock solutions were prepared by dissolving 1 g of the bark extract in 50 ml ethanol. The stocks were further diluted with dechlorinated water to prepare a range of test concentrations. Larvae were exposed to desired concentrations in 500-ml beakers containing 1-ml test concentration and 249 ml of water. Each set of experiments was conducted in triplicate, along with the controls (solvent concentration, de-chlorinated water, and 1 mM silver nitrate).

X-ray diffraction analysis The nature of silver nanoparticle powder was monitored by Xray diffraction analysis (XRD) (Bruker D8 Discover) which was operated under the following conditions: step size, 0.02/ θ; voltage, 40 kV; 2θ range, 10–80°; current, 40 mA with Cu kα radiation of 0.1541 nm wavelength.

Toxicity of silver nanoparticles to non-target organism, Mesocyclops thermocyclopoides Harada To evaluate the toxic effect of silver nanoparticles, a nontargeted organism, M. thermocyclopoides was selected. These organisms were brought to the laboratory of National

Author's personal copy 380

Institute of Malaria Research (NIMR, New Delhi) from ditches, ponds, and pools of Burari village (North Delhi) and acclimatized to the laboratory environment. Toxic effect of silver nanoparticles (LC50 and LC90) was evaluated following the protocol by Patil et al. (2012b). Twenty-five numbers of M. thermocyclopoides were placed in 500-ml capacity plastic bowl and exposed to 250 ml of test solution of AgNPs at 28 °C RT. Each set of experiments contained a control (dechlorinated water) and three replicates. Mortality was recorded after 24, 48, and 72 h of treatment. Statistical analysis The mortality rate was recorded after 24, 48, and 72 h. Their LC50 and LC90, Chi-square values, lower confidence limits (LCL), upper confidence limits (UCL), and other statistics at 95% intervals were calculated using probit analysis (Finney 1971). Data were analyzed using the SPSS software, window 16.

Results and discussion Synthesis and characterization of AgNPs When the H. antidysenterica bark extract was incubated with 1 mM of silver nitrate solution, the color of the solution changed from yellow to dark brown due to the reduction of silver nitrate solution to silver nanoparticles (Fig. 1a). The change in color of the solution is reported to occur due to the surface plasmon resonance excitation (SPR) of AgNPs. SPR band arises due to combined vibration of free e−1 of AgNPs in resonance to light wave (Noginov et al. 2007). Ahmad et al. (2003) also reported that the excitation of SPR of AgNPs was the major reason behind the color change in the nanoparticles synthesized using Fusarium oxysporum Schlecht. extract. The color change was seen to be directly correlated with the synthesis of AgNPs as reported in Berberis tinctoria Lesch. (Mahesh Kumar et al., 2016) and Pseudomonas aeruginosa (Schroeter) Migula (Siddhardha et al. 2014). Similar color change was also observed after adding extracts of different plant parts such as leaves of Eclipta prostrata (Rajakumar and Rahuman 2011), roots of Salvia miltiorrhiza Bunge (Lee et al. 2017), rhizomes of Hedychium coronarium Koenig (Kalimuthu et al. 2017b), and leaves of Derris trifoliata Lour. (Kumar et al. 2017) in 1 mM silver nitrate solution. The AgNPs, thus synthesized, when observed under UV–Vis spectrophotometer, the maximum absorbance peak was recorded at 420 nm (Fig. 1b) which is considered their characteristic feature. Our observation is similar to the work of Shameli et al. (2012), who reported that UV–Vis spectra of AgNPs exhibited an absorption band at 400–480 nm. The shape, dielectric environment,

Parasitol Res (2018) 117:377–389

composition, and size of AgNPs are also determined by theSPR. The width of the peak is said to be directly correlated to the larger or smaller size of AgNPs (Petit et al. 1993). According to Kong and Jang (2006), the absorption peaks in UV–Vis spectra became broader as the size of nanoparticles decreased. In yet another work, Sujitha et al. (2015) reported that the optimum absorption peaks have been recorded at 450 nm in Moringa oleifera Lam synthesized AgNPs.Similarly, AgNPs synthesized using various plant extracts such as Nelumbo nucifera Gaertn., Merremia emarginata (L.) Burm., and Pteridium aquilinum (L.) Kuhn exhibited optimum absorbance peaks at a given range (420–480 nm) of wavelength (Santhoshkumar et al. 2011; Panneerselvam et al. 2016; Azarudeen et al. 2017). Though the underlying mechanism for reduction of AgNO3 using plant extracts is not clearly understood, some authors assumed that plant extracts contain various primary (amino acids, proteins, carbohydrates, and lipids) and secondary metabolites (terpenoids, alkaloids, flavonoids, and phenolics) which might be responsible for the reduction of AgNO3 to AgNPs (Aromal and Philip 2012; Chung et al. 2016). According to Mahendran and Kumari (2016), the phenolic compounds are used to transfer the electron to Ag+ ion and generate Ag0 during reduction of AgNO3 to silver nanoparticles. After the reaction, resonance-stabilized quinine is produced by phenolics during the bio-reduction of silver nitrate solution. Chung et al. (2016) reported that the carbonyl and hydroxyl groups of primary and secondary metabolites were the main functional groups involved in the bio-reduction of AgNO3. A similar finding has also been reported in Satureja intermedia (C.A.Mey) mediated AgNPs by Firoozi et al. (2016). In present case also, such phenolics, flavonoids, terpenoids, and proteins in aqueous bark extract of H. antidysenterica might be responsible for the bio-reduction and stabilization of silver nanoparticles. Furthermore, the XRD pattern of synthesized AgNPs showed four diffraction peaks of 38.34°, 44.28°, 64.22°, and 77.76° at 2 representing the (111), (200), (220), and (311) sets of lattice planes, respectively, and these bands could be assigned to a face-centered cubic nature (Fig. 1c). Lukman et al. (2011), while working on the Medicago sativa L. synthesized AgNPs, reported the Bragg reflection values of 38.32°, 44.48°, 64.68°, and 77.64° which were related to (111), (200), (220), and (311) sets of crystalline planes, respectively. Similar results were also obtained in Bauhinia variegata L. synthesized AgNPs by Govindarajan et al. (2016). The mean size of AgNPs (28 nm) was calculated using Scherrer formula from the full width of halfmaximum of the (111) diffraction peak. Besides, some smaller peaks were also observed in the XRD pattern which could be due to the unidentified impurities in the bark extract. The slight shifts in peak positions are associated with strain in nanocrystallites which is the characteristic of

Author's personal copy Parasitol Res (2018) 117:377–389 Plant extract

5 min

25 min

50 min

120 min


Absorbance (a.u.)

AgNO3 solution

UV-vis spectrum after 120 min




0.0 350









Wavelength (nm)


A 250 200 Intensity (a.u.)

Fig. 1 a Chromatic variation of the H. antidysenterica bark extract after adding 1 mM of silver nitrate solution at different time intervals. b UV–Vis spectrum of silver nanoparticles synthesized using H. antidysenterica bark extract with 1 mM aqueous solution of silver nitrate at 50 ± 2 °C for 120 min. c X-ray diffraction pattern of silver nanoparticles synthesized with aqueous bark extract of H. antidysenterica. d, e Field emission scanning electron microscopy images of silver nanoparticles synthesized using aqueous bark extract H. antidysenterica at 1 μM and 500 nm magnifications. f Transmission electron microscopy micrograph of silver nanoparticles derived from aqueous bark extract H. antidysenterica


150 100 50 0 30


50 2   (Degree)








AgNPs. Similar abnormal peak positions and impurities were also reported in AgNPs synthesized using Eclipta prostrata leaf extract (Rajakumar and Rahuman 2011). Fouriertransform infrared spectroscopy (FT-IR) measurements were carried out to confirm the presence of different functional groups which might be involved in the bio-reduction and stabilization of silver ions to silver nanoparticles. The spectrum obtained with FT-IR spectrum showed prominent absorbance bands at 1012.2, 1254.21, 1365.32, 2835.31, 2932.32, and

3439.44 cm−1, assigning vinyl ether, aromatic amines, secondary amines, aldehydes, aliphatic compounds, and alcohols, respectively. These absorbance peaks were seen to be associated with the following C–O, C–N, C–N, C–H, and O–H functional groups. Yadav and Rai (2011) reported that FT-IR spectrum of H. antidysenterica leaves synthesized AgNPs exhibited absorption peaks at 1047, 1414, 2341, 2359, and 3335 cm−1 which could be attributed to the vinyl ether (C–O), amines (N–H) alkynes (C–H), alcoholic (O–H),

Author's personal copy 382

and phenolic compounds (O–H), respectively. Benelli (2016a, b) also observed that flavonoids and terpenoids were the main compounds in the FT-IR spectrum of the plant extract which could be responsible for the reduction, capping, and stabilization of AgNPs. It is also reported that the AgNPs thus synthesized remained stable up to 6 weeks in the aqueous medium. The LC-MS analysis of bark extract revealed the presence of several molecules such as proteins, terpenoids, phenolics, and flavonoids, which perhaps could be responsible for the bioreduction and stabilization of AgNPs (Table 1). However, Ballottin et al. (2016) reported that protein molecules were actively involved in the stabilization of AgNPs. FE-SEM images showed that most of the AgNPs were polydispersed and spherical in nature as depicted by the SPR band in the UV–Vis spectrum (Fig. 1d, e). The average particle size of AgNPs was about 40–80 nm as calculated by FE-SEM images. AgNPs were also seen to be agglomerated in FE-SEM images. According to previous studies, the SEM micrograph of phyto-synthesized AgNPs showed cubic and spherical forms with a size range of 18 to 50 nm (Chandran et al. 2006; Huang et al. 2007; Dinesh et al. 2015; Suresh et al. 2015). The size and shape of H. antidysenterica mediated AgNPs were observed by TEM and it was reported that they are dispersed, crystalline, and mostly spherical in shape with 32 nm (Fig. 1f). Our results were similar to those of various studies performed earlier using different plant extracts such as Sargassum muticum (Yendo) Fensholt, Leucas aspera (Willd.) Link,Melia azedarach L. Pteridium aquilinum, Hypnea musciformis (Wulfen) JVLamouroux, Annona muricata L., Azadirachta indica A. Juss, and Centroceras clavulatum L. for the synthesis of AgNPs and reported similar morphology with the size of 20 to 80 nm (Suganya et al. 2014; Madhiyazhagan et al. 2015; Santhosh et al. 2015; Chandramohan et al. 2016; Murugan et al. 2016).

Larvicidal activity of AgNPs and bark extracts The larvicidal activity of bark extract of H. antidysenterica prepared in different solvents individually, aqueous bark extract and bark extract synthesized AgNPs were evaluated against the third instar larvae of C. quinquefasciatus and A. aegypti. The maximum larvicidal activity was observed with the bark synthesized AgNPs with LC50 and LC90 values being 9.3 and 19.24 ppm, respectively, for C. quinquefasciatus and LC50 and LC90 values were 5.53 and 12.01 ppm, respectively, for A. aegypti. These were the minimum doses as compared to aqueous bark extract as well as bark extracts prepared with different solvents (Table 2) after 72 h of treatments. Similar to this in another member of the Apocynaceae, Benelli and Govindarajan (2017) reported that AgNPs synthesized employing Aganosma cymosa (Roxb.)G. Don.leaf extract exhibited potential larvicidal activity against A. stephensi Liston, C. quinquefasciatus, and A. aegypti having LC50 and LC90 values of 12.45, 14.79, 13.58, and 24.25, 27.30, 26.11 μg/ml, respectively, after 24 h of treatment. Likewise, LC50 (15.69,

Parasitol Res (2018) 117:377–389

14.33, and 16.95 μg/ml) and LC 90 (31.15, 28.29, and 30.39 μg/ml) values were reported in AgNPs synthesized from other species of the same family, Carissa carandas L.,against the larvae of C. quinquefasciatus, A. stephensi, and A. aegypti by Govindarajan and Benelli (2017). Similarly, AgNPs prepared with Avicennia marina Forsk. have been shown to exhibit larvicidal activity against the larvae of A. aegypti, C. quinquefasciatus, A. stephensi with LC50 and LC90 values of 18.05, 19.52, 16.58, and 34.04, 36.07, 32.11 μg/ml, respectively, after 24 h of treatment (Azarudeen et al. 2016). Contrary to above, different solvent extracts when tested alone showed moderate larvicidal activity against these two vectors, A. aegypti and C. quinquefasciatus. The maximum mortality was observed with the methanol extract followed by chloroform and hexane extracts of H. antidysenterica bark against A. aegypti and their LC50 and LC90 value being 71.74, 94.25, 102.64 and 217.36, 222.82, 277.82 ppm, respectively. For C. quinquefasciatus, the maximum larvicidal mortality was observed with the methanol extract followed by hexane and chloroform with their LC50 values being 69.43, 73.79, 112.39 and LC 90 values being 170.58, 227.48, 299.76 ppm, respectively, after 72 h of treatment. The log-probit analysis showed that the LC50 and LC90 values decreased with the increase in concentrations as well as the exposure time period (Table 2). Acetone extract of H. antidysenterica was least toxic to A. aegypti and C. quinquefasciatus. Similar to our result, hexane extract of Nerium oleander L. exhibited higher larvicidal activity against C. quinquefasciatus with LC50 and LC90 values of 61.11 and 4916.44 ppm, respectively, compared to aqueous extract (LC50; 168.84: LC90; 7882.93 ppm) after 48 h of exposure (Raveen et al. 2014).Suganya et al. (2014) reported that the methanol extract of the Leucas aspera leaf showed high larvicidal activity against the larvae of A. aegypti with LC50 value of 10.036 mg/l as compared to other solvents. Our results showed that methanol is an ideal solvent for extraction of larvicidal compounds from the bark extract of H. antidysenterica as compared to other solvents. Kamaraj et al. (2009) while working with different solvent extracts of Cassia auriculata L., Leucas aspera, and Rhinacanthus nasutus (L.) Kurz reported that the methanolic extract exhibited good larvicidal potential against A. subpictus Grassi and C. tritaeniorhynchus Giles. The bioefficacy of the bark extract against mosquitoes may be due to the presence of different active larvicidal compounds (Table 1). Thus, of the different type of solvents used (hexane, chloroform, acetone, and water), methanolic extract proved more effective in causing morbidity at lower concentration, e.g., LC50; 71.74 ppm for A. aegypti and LC50; 69.43 ppm for C. quinquefasciatus whereas hexane, acetone, chloroform, and aqueous extract require LC 50 ; 102.64, 94.25, 618.82, 353.65 ppm for A. aegypti and L C 5 0 ; 7 3 . 7 3 , 11 2 . 3 9 , 5 9 7 . 7 4 , 3 3 4 . 7 5 p p m f o r

Author's personal copy Parasitol Res (2018) 117:377–389 Table 1 Compounds identified from the bark extract of H. antidysenterica through LCMS analysis



Molecular weight


Molecular formula



Butadiene-styrene rubber

2 3 4 5

298.2508 298.2508 298.2508 298.2508

9-hydroxy-12Z-octadecenoic acid Ricinoleic acid Ricinelaidic acid 12-hydroxy-10E-octadecenoic acid

C12H14 C18H34O3 C18H34O3 C18H34O3 C18H34O3

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508

9-hydroxy-10Z-octadecenoic acid 9-hydroxy-12-octadecenoic acid 17-hydroxy-9Z-octadecenoic acid 18-hydroxy-9Z-octadecenoic acid 12R-hydroxy-9Z-octadecenoic acid 5-hydroxy-2-octadecenoic acid 8-hydroxy-9-octadecenoic acid 8R–hydroxy-9Z-octadecenoic acid 9R-hydroxy-10E-octadecenoic acid 9-hydroxy-10E-octadecenoic acid 9R-hydroxy-12E-octadecenoic acid 9R-hydroxy-12Z-octadecenoic acid 10-hydroxy-8-octadecenoic acid 10R-hydroxy-8E-octadecenoic acid 11-hydroxy-9-octadecenoic acid 12R-hydroxy-9E-octadecenoic acid 12S-hydroxy-9E-octadecenoic acid 12S-hydroxy-9Z-octadecenoic acid

C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508

3-keto stearic acid 4-keto stearic acid 5-keto stearic acid 6-keto stearic acid 7-keto-stearic acid 9-keto stearic acid 10-keto stearic acid cis-9,10-Epoxystearic acid 9R,10S-epoxy-stearic acid 3S,7,11-Trimethyl-6,10-dodecadienoic acid 2R-hydroxy-oleic acid Isoricinoleic Acid 12-hydroxy-10E-octadecenoic acid (10Z)-10-Heptadecenoic acid 9-HOME(12) 17-Hydroxyoleic acid (9Z)-18-Hydroxy-9-octadecenoic acid 5-HOME(2)

C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18HO3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3

42 43 44 45 46 47 48 49

298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508

8-hydroxyoctadec-9-enoic acid 8R-hydroxy-9Z-octadecenoic acid. 9R-HOME(10E) 9-HOME(10E) 9R-HOME(12E) 9R-Hydroxy-10E,12Z-Octadecatrienoic Acid 10-HOME(8) 10R-hydroxy-8E-octadecenoic acid

C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3

Author's personal copy 384 Table 1 (continued)

Parasitol Res (2018) 117:377–389


Molecular weight


Molecular formula

50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508

11-HOME(9) 12S-hydroxy-9E-octadecenoic acid 16-methyl-10-oxo-heptadecanoic acid 2-methyl-4-oxo-heptadecanoic acid 11-oxo-octadecanoic acid 12-oxo-octadecanoic acid 13-oxo-octadecanoic acid 14-oxo-octadecanoic acid 15-oxo-octadecanoic acid 16-oxo-octadecanoic acid 17-oxo-octadecanoic acid 2-oxo-octadecanoic acid 8-oxo-octadecanoic acid 6R,7S-epoxy-octadecanoic acid 5-Hexyltetrahydro-2-furanoctanoic acid 8Z-decen-4,6-diynoic acid

C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84

298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 298.2508 315.2674 328.2977 328.2977 328.2977

10R-hydroxy11Z-octadecenoic acid 10S-hydroxy11Z-octadecenoic acid 12R-HOME(13Z) 12S-HOME(13Z) 11R-hydroxy12E-octadecenoic acid 11S-hydroxy12Z-octadecenoic acid 12R-hydroxy10E-octadecenoic acid 12-Hydroxy-8,10,14-icosatrienoate 12R-HOME(13E) 12S-HOME(13E) 13R-HOME(11E) 13S-hydroxyoctadecadienoic acid 11R-hydroxy12E-octadecenoic acid 11S-hydroxy12E-octadecenoic acid 13-HOME(14E) Panamine 3R-hydroxy-eicosanoic acid 2-hydroxyphytanic acid (2S)-2-hydroxyphytanic acid

C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C18H34O3 C20H33N3 C20H40O3 C20H40O3 C20H40O3

85 86 87 88 89 90 91 92 93 94 95 96 97 98

328.2977 328.2977 328.2977 328.2977 329.293 329.293 329.293 329.293 330.3134 330.3134 342.3134 342.3134 342.3134 342.3134

2-hydroxy-eicosanoic acid 20-hydroxy-eicosanoic acid Eicosanoic acid, 3-hydroxy-, (R) Polyoxyethylene 40 monostearate Penaresidin A Penaresidin B Dihydroceramide Palmitoyl Serinol 1-Hexadecyl-2-O-methyl-glycerol Hexadecyl Methyl Glycerol 21-hydroxy-heneicosanoic acid 1-(1Z-octadecenyl)-sn-glycerol 1-O-Octadec-9-enyl glycerol 15-hydroxy-heneicosanoic acid

C20H40O3 C20H40O3 C20H40O3 C20H40O3 C19H39NO3 C19H39NO3 C19H39NO3 C19H39NO3 C20H42O3 C20H42O3 C21H42O3 C21H42O3 C21H42O3 C21H42O3

Author's personal copy Parasitol Res (2018) 117:377–389 Table 1 (continued)



Molecular weight


Molecular formula

99 100 101 102

342.3134 344.329 782.6061 760.6346

Propylene glycol stearate 1-O-Octadecyl-sn-glycerol 16:2-Glc-Cholesterol 1-eicosyl-2-heneicosanoyl-glycero-3-phosphate

C21H42O3 C21H44O3 C49H82O7 C44H89O7P

S.No. serial number, HOME hydroxy-octadecenoic acid Geometric isomers denoted by E or Z configuration (E means two groups with the higher priorities are on the same side of the double bond: Z means two groups with the higher priorities are on the opposite side of the double bond), stereochemical configuration of compound defined by R and S conformation (R, right-handed: S, lefthanded)

C. quinquefasciatus, respectively, after 72 h. Therefore, it is quite clear that the larvicidal activity of AgNPs in the present investigation is much higher (LC 50 ; 9.93 ppm for C. quinquefasciatus, LC50; 5.53 ppm for A. aegypti) as compared to the solvents tried alone. The probable mechanism of inhibition by AgNPs could be based on the assumption that Ag+ ions are released from the AgNPs by oxidation and these ions then interact with the negatively charged DNA of the insect, disturbing their normal replication, transcription, and protein synthesis thereby inactivating the vital enzymes (Feng et al. 2000; Foldbjerg et al. 2015). Singh et al. (2016), however, hypothesized that silver acts as a soft acid and cells act as a natural base due to the presence of phosphorus and sulfur. The reaction between the soft acid and soft base disrupts the cell function and consequently leads to cell death. A. aegypti larvae when were exposed to Gracilaria firma Chang and Xia derived nanoparticles, its midgut epithelium cells became vacuolated, swollen, losing their normal appearance and passing the epithelium gut contents into lumen and mixing with hemolymph that caused larval death (Kalimuthu et al. 2017a). Larvae of A. aegypti treated with Halodule uninervis (Forsk.) Aschers synthesized AgNPs exhibited similar morphological changes such as swelling of apical cell, shrinkage of the internal cuticle, and accumulation of nanoparticles in the midgut (Mahyoub et al. 2017). Notwithstanding, the smaller size of AgNPs too allows them to pass through individual cells and cuticle, whereas the bioactive constituents wrapping around the AgNPs interfere with physiological and molting processes of insects (Suganya et al. 2014). According to Benelli (2016a, b) due to the smaller size of nanoparticles, they penetrate the insect exoskeleton, intracellular space, and individual cells and interact with phosphorus of DNA and sulfur of protein, leading to alteration of DNA, denaturation of enzymes, organelles, and proteins. Subsequently, the decrease in membrane permeability and disturbance in proton motive force took place. This may cause loss of cellular functions, damaged molting, and physiological processes and eventually results in cell death. The current study thus revealed that larval mortality is increased by ten to twenty times employing biosynthesized

AgNPs, over the bark extract (prepared in different solvents viz. aqueous extract, hexane, chloroform, acetone, and methanol) of H. antidysenterica t ested alone. Subarani et al. (2013) observed that the leaf synthesized silver nanoparticles were more toxic as compared to the leaf extract of Carissa spinarum L. tested alone against A. subpictus, A. albopictus Skuse, and C. tritaeniorhynchus. Recently, it has been reported that the addition of biosynthesized AgNPs in the plant extracts improved the bioefficacy by many folds against various mosquito vectors (Govindarajan and Benelli 2016; Benelli et al. 2017). Furthermore, the larval mortality by plant extract in the presence of AgNPs increased as compared to plant extract alone, indicating that AgNPs increase the maximum bioactivity and interaction of plant product with larval cells due to their smaller size (nanosize). Nevertheless, the green synthesized AgNPs exhibited no significant toxic effects on the non-target organism M. thermocyclopoides, when exposed to tested concentrations of AgNPs (LC 50 and LC 90 value of A. aegypti and C. quinquefasciatus) for 24, 48, and 72 h exposure. Similar findings were also reported earlier with Vinca rosea L. leaf derived silver nanoparticles on Poecilia reticulata Peters, a non-targeted fish (Subarani et al. 2013). Mahesh Kumar et al. (2016) also did not observe toxic effects with Berberis tinctoria-fabricated silver nanoparticles against Mesocyclops thermocyclopoides and Toxorhynchites splendensWiedemann after 42 h of exposure. In another recent publication, Subramaniam et al. (2016) found that AgNPs were non-toxic to other organisms and enhanced the predator efficacy of larvivorous fish species against mosquito’s vectors. The use of AgNPs for controlling the mosquito vectors is inexpensive, simple, and creates a pathway to future innovative research in this field.

Conclusion It is hereby concluded that the aqueous bark extract of H. antidysenterica proved effective both for capping as well as a reducing agent for synthesis of AgNPs. Further, AgNPs produced by this method have a spherical shape with an average size of 32 nm measured by TEM micrographs. After

Author's personal copy 386

Parasitol Res (2018) 117:377–389

Table 2 Log probit and regression analysis of H. antidysenterica bark extracts against the 3rd instar larvae of Aedes aegypti and Culex quinquefasciatus Larvae


Time Regression equations

X2 (d.f.)a LC50b (LCLc and UCLd) ppm LC90e (LCL and UCL) ppm

Aedes aegypti


24 h y = − 9.490 + 3.220 x

2.639 (4)

884.72 (697.77–1242.91)


48 h y = − 7.480 + 3.010 x 72 h y = − 4.940 + 2.662 x

3.175 (4) 1.094 (4)

305.40 (239.11–388.96) 71.74 (45.88–95.99)

813.97 (601.74–1315.62) 217.36 (156.91–395.57)

24 h y = − 10.112+ 3.525 x


739.40 (593.35–958.92)

1708.19 (1236.21–3249.90)

48 h y = − 11.302 + 4.484 x

5.625 (4)

331.50 (269.81–405.77)

640.17 (507.45–929.63)

72 h y = − 5.961 + 2.964 x 2.638 (4) 24 h y = − 17.698 + 6.0224 x 4.261 (4)

102.64 (76.29–132.29) 868.83 (735.78–1043.3)

277.82 (203.08–483.28) 1418.25 (1157.21–2232.48)

7.221 (4)

456.0 (262.5–869.2)

1374.53 (758.95–9161.79)

0.607 (4) 0.468 (4)

94.25 (71.67–119.29) 1956.2 (1532.1–4312.1)

222.24 (166.9–375.4) 4417.1 (3918.2–6271.23)



48 h y = − 7.113+ 2.675 x Acetone

72 h 24 h 48 h 72 h

y = − 6.792 + 3.440 x y = − 11.925 + 3.623 x y = − 9.915 + 3.256 x y = − 10.036 + 3.595 x

0.395 (4) 1108.33 (861.63–1820.73) 1.525 (4) 618.82 (445.8–777.36)

2742.77 (1713.79–10,612.45) 1406.2 (1056.36–2356.6)

0.517 (4) 1325.31 (1031.36–3105.02) 6.237 (4) 587.31(465.84–785.63)

2869.87 (1748.75–29,694.42) 1480.4 (1068.05–2663.83)

72 h y = − 6.484 + 2.544 x 24 h y = − 5.189 + 2.721 x 48 h y = − 1.824+ 1.618 x

8.106 (4) 2.68 (3) 0.238(3)

353.65 (185.59–680.59) 80.79 (61.12–103.7) 13.41 (8.37–183.45)

1128.6 (609.37–8100.14) 238.31 (170–443.12) 83.09 (25.66–258,019.01)

72 h y = − 2.827 + 3.804 x 24 h y = − 8.147 + 2.855 x 48 h y = − 5.222 + 2.416 x

1.831 (4) 0.895 (4) 2.418 (4)

5.53 (4.57–6.61) 714.6 (556.72–975.76) 145.08 (105.85–191.19)

12.01 (9.37–19.51) 2009.72 (1350.78–4455.57) 492.16 (347.15–873.16)

72 h y = − 6.045 + 3.283 x 24 h y = − 7.253 + 2.460 x

0.802 (4) 0.647 (4)

69.43 (47.60–89.37) 888.25 (665.67–1391.6)

170.58 (127.56–303.39) 2948.68 (1744.71–9588.19)

Aqueous extract 24 h y = − 11.925 + 3.819 x 48 h y = − 8.844+ 3.194 x AgNPs

Culex quinquefasciatus Methanol




48 h 72 h 24 h 48 h

y = − 5.981 + 2.531 x y = − 4.892 + 2.619 x y = − 9.915 + 3.256 x y = − 7.138 + 2.674 x

72 h y = − 6.169 + 3.008 x 24 h y = − 11.015 + 3.185 x 48 h y = − 7.826 + 2.443 x

72 h Aqueous extract 24 h 48 h 72 h AgNPs 24 h 48 h 72 h

y = − 8.453 + 3.044 x y = − 11.823 + 3.550 x y = − 10.440 + 3.585 x y = − 7.882 + 3.122 x y = − 6.153 + 2.100 x y = − 1.824+ 1.618 x y = − 1.764 + 1.821 x

4.606 (4) 230.75 (175.09–301.11) 1.622 (4) 73.73 (47.44–98.54) 0.395 (4) 1108.33 (861.63–1820.73) 7.672 (4) 467.63 (267.78–941.84)

740.85 (526.03–1278.79) 227.48 (163.09–426.21) 2742.29 (1713.79–10,612.45) 1410.6 (762.62–11,451.44)

0.205 (4) 112.39 (84.37–144.22) 1.130(4) 2873.12 (2567.21–3411.9) 0.689(4) 1597.45 (1072.13–5655.88)

299.76 (220.91–504.92) 7250.5 (6789.12–8321.87) 5343.27 (2434.22–110,453.11)

0.723(4) 0.196 (4) 3.115 (4) 3.809(4) 0.488 (3) 0.238(3) 1.178 (3)

1576.98 (1120.71–2940.51) 4910.2 (4553.1–6172.32) 1863.88 (1334.30–3743.5) 861.45 (640.07–1382.74) 190 .32 (146.63–361.23) 96.04 (27.3–177.42) 19. 24 (17.93–89.43)

597.74 (470.52–778.50) 2141 (1934.2–2743.12) 817.69 (656.70–1075.78) 334.75 (263.81–425.11) 76.897 (77.41–131.24) 14.97 (8.93–43.81) 9.3 (6.59–25.23)

Control, zero percent mortality (1 mM silver nitrate, respective solvents, and distilled water) a

Degree of freedom


Lethal concentration that kills 50% of the exposed larvae


95% lower confidence limit


95% upper confidence limit

Lethal concentration that kills 90% of the exposed larvae; χ 2 = chi square, (α = 0.05). Bold letter (LC50 and LC90)-maximum larvicidal activity at minimum concentration e

evaluating the toxicity against non-target organisms, it can be said that the AgNPs produced by this method are non-toxic, clean, environmentally acceptable, stable, and have an

excellent larvicidal activity against the larvae of A. aegypti and C. quinquefasciatus with the LC50 values in the range of 5.53 to 19.24 ppm for 72 h exposure.

Author's personal copy Parasitol Res (2018) 117:377–389 Acknowledgements Authors are grateful to the University of Delhi for providing Research and Development and DSTPURSE grants. Dinesh Kumar is indebted to University Grants Commission, New Delhi for the award of JRF. Compliance with ethical standards Conflict of interest The authors declare that they have no competing interests.

References Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan MI, Kumar R, Sastry M (2003) Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids Surf B 28(4):313– 318. Amer A, Mehlhorn H (2006) Larvicidal effects of various essential oils against Aedes, Anopheles and Culex larvae (Diptera: Culicidae). Parasitol Res 99(4):466–472. Aromal AS, Philip D (2012) Green synthesis of gold nanoparticles using Trigonella foenum-graecum and its size-dependent catalytic activity. Spectrochim Acta A Mol Biomol Spectrosc 97:1–5. 10.1016/j.saa.2012.05.083 Azarudeen RMST, Govindarajan M, AlShebly MM, AlQahtani FS, Amsath A, Senthilmurugan S, Vijayan P, Benelli G (2017) Sizecontrolled biofabrication of silver nanoparticles using the Merremia emarginata leaf extract: toxicity on Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) and non-target mosquito predators. J Asia Pac Entomol 20(2):359–366. Azarudeen RMST, Govindarajan M, Amsath A, Kadaikunnan S, Alharbi NS, Vijayan P, Muthukumaran U, Benelli G (2016) Size-controlled fabrication of silver nanoparticles using the Hedyotis puberula leaf extract: toxicity on mosquito vectors and impact on biological control agents. RSC Adv 6(99):96573–96583. C6RA23208F Ballottin D, Fulaz S, Souza ML, Corio P, Rodrigues AG, Souza AO, Gaspari PM, Gomes AF, Gozzo F, Tasic L (2016) Elucidating protein involvement in the stabilization of the biogenic silver nanoparticles. Nanoscale Res Lett 11(1):313–322. s11671-016-1538-y Benelli G (2016a) Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: a review. Parasitol Res 115(1):23–34. s00436-015-4800-9 Benelli G (2016b) Green synthesized nanoparticles in the fight against mosquito-borne diseases and cancer – a brief review. Enzym Microb Technol 95:58–68. Benelli G (2018) Gold nanoparticles–against parasites and insect vectors. Acta Trop 178:73–80. 021 Benelli G, Beier JC (2017) Current vector control challenges in the fight against malaria. Acta Trop 174:91–96. actatropica.2017.06.028 Benelli G, Govindarajan M (2017) Green-synthesized mosquito oviposition attractants and ovicides: towards a nanoparticle-based "lure and kill" approach? J Clust Sci 28(1):287–308. s10876-016-1088-6 Benelli G, Mehlhorn H (2016) Declining malaria, rising of dengue and Zika virus: insights for mosquito vector control. Parasitol Res 115(5):1747–1754.

387 Benelli G, Pavela R, Maggi F, Petrelli R, Nicoletti M (2017) Commentary: making green pesticides greener? The potential of plant products for nanosynthesis and pest control. J Clust Sci 28(1):3–10. Chandramohan B, Murugan K, Panneerselvam C, Madhiyazhagan P, Chandirasekar R, Dinesh D, Kumar PM, Kovendan K, Suresh U, Subramaniam J, Rajaganesh R (2016) Characterization and mosquitocidal potential of neem cake-synthesized silver nanoparticles: genotoxicity and impact on predation efficiency of mosquito natural enemies. Parasitol Res 115:1015–1025 Chandran SP, Chaudhary M, Pasricha R, Ahmad A, Sastry M (2006) Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract. Biotechnol Prog 22(2):577–583. 10.1021/bp0501423 Chung M, Park I, Seung-Hyun K, Thiruvengadam M, Rajakumar G (2016) Plant-mediated synthesis of silver nanoparticles: their characteristic properties and therapeutic applications. Nanoscale Res Lett 11(1):40–54. Dinesh D, Murugan K, Madhiyazhagan P, Panneerselvam C, Nicoletti M, Jiang W, Benelli G, Chandramohan B, Suresh U (2015) Mosquitocidal and antibacterial activity of green-synthesized silver nanoparticles from Aloe vera extracts: towards an effective tool against the malaria vector Anopheles stephensi? Parasitol Res 114(4):1519–1529. Duan H, Wang D, Li Y (2015) Green chemistry for nanoparticle synthesis. Chem Soc Rev 44(16):5778–5792. C4CS00363B Elumalai D, Kaleena PK, Ashok K, Suresh A, Hemavathi M (2016) Green synthesis of silver nanoparticle using Achyranthes aspera and its larvicidal activity against three major mosquito vectors. Engineering in Agriculture, Environment and Food 9:1–8 Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO (2000) A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 52(4):662– 666.;2-3 Finney DJ (1971) Probit Analysis. Cambridge University Press, London, pp 68–72 Firoozi S, Jamzad M, Yari M (2016) Biologically synthesized silver nanoparticles by aqueous extract of Satureja intermedia CA Mey and the evaluation of total phenolic and flavonoid contents and antioxidant activity. Journal of nanostructure inChemistry 6:357–364 Foldbjerg R, Jiang X, Miclăus T, Chunying C, Autrup H, Beer C (2015) Silver nanoparticles-wolves in sheep’s clothing? Toxicol Res 4(3): 563–575. Govindarajan M, Benelli G (2016) One-pot green synthesis of silver nanocrystals using Hymenodictyon orixense: a cheap and effective tool against malaria, chikungunya and Japanese encephalitis mosquito vectors? RSC Adv 6(64):59021–59029. 1039/C6RA10228J Govindarajan M, Benelli G (2017) A facile one-pot synthesis of ecofriendly nanoparticles using Carissa carandas: ovicidal and larvicidal potential on malaria, dengue and filariasis mosquito vectors. J Clust Sci 28(1):15–36. Govindarajan M, Rajeswary M, Veerakumar K, Hoti SL, Mehlhorn H, Barnard DR, Benelli G (2016) Novel synthesis of silver nanoparticles using Bauhinia variegata: a recent eco-friendly approach for mosquito control. Parasitol Res 115(2):723–733. 1007/s00436-015-4794-3 Huang J, Li Q, Sun D, Lu Y, Su Y, Yang X, Wang H, Wang Y, Shao W, He N, Hong J, Chen C (2007) Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf. Nanotechnology 18(10):105104.

Author's personal copy 388 Jinu U, Rajakumaran S, Senthil-Nathan S, Geetha N, Venkatachalam P (2017) Potential larvicidal activity of silver nanohybrids synthesized using leaf extracts of Cleistanthus collinus (Roxb.) Benth. Ex Hook. f. And Strychnos nuxvomica L. nuxvomica against dengue, chikungunya and Zika vectors. Physiol Mol Plant Pathol 0:1–9 Kalimuthu K, Panneerselvam C, Chou C, Lin SM, Tseng LC, Tsai KH, Murugan K, Hwang JS (2017a) Predatory efficiency of the copepod Megacyclops formosanus and toxic effect of the red alga Gracilaria firma-Synthesized Silver nanoparticles against the dengue vector Aedes aegypti. Hydrobiologia 785(1):359–372. 1007/s10750-016-2943-z Kalimuthu K, Panneerselvam C, Chou C, Tseng LC, Murugan K, Tsai KH, Alarfaj AA, Higuchi A, Canale A, Hwang JS, Benelli G (2017b) Control of dengue and Zika virus vector Aedes aegypti using the predatory copepod Megacyclops formosanus: synergy with Hedychium coronarium-Synthesized Silver nanoparticles and related histological changes in targeted mosquitoes. Process Saf Environ Prot 109:82–96. 1016/j.psep.2017.03.027 Kalimuthu K, Panneerselvam C, Murugan K, Hwang JS (2013) Green synthesis of silver nanoparticles using Cadaba indica lam leaf extract and its larvicidal and pupicidal activity against Anopheles stephensi and Culex quinquefasciatus. J Entomol Acarol Res 45(2):11. Kamaraj C, Bagavan A, Rahuman AA, Zahir AA, Elango G, Pandiyan G (2009) Larvicidal potential of medicinal plant extracts against Anopheles subpictus Grassi and Culex tritaeniorhynchus Giles (Diptera: Culicidae). Parasitol Res 104(5):1163–1171. https://doi. org/10.1007/s00436-008-1306-8 Khan A, Bashir S, Gilani AH (2012a) An In vivo study on the diuretic activity ofHolarrhena antidysenterica. Afr J Pharm Pharmacol 6: 454–458 Khan A, Khan SR, Gilani AH (2012b) Studies on the in vitro and in vivo antiurolithic activity of Holarrhena antidysentery. Urol Res 40(6): 671–681. Kong H, Jang J (2006) One-step fabrication of silver nanoparticle embedded polymer nanofibers by radical-mediated dispersion polymerization. Chem Commun 28:3010–3012 Kumar VA, Ammani K, Jobina R, Subhaswaraj P, Siddhardha B (2017) Photo-induced and phytomediated synthesis of silver nanoparticles using Derris trifoliata leaf extract and its larvicidal activity against Aedes aegypti. J Photochem Photobiol B 171:1–8. 10.1016/j.jphotobiol.2017.04.022 LeeJH, VelmuruganP, ParkJH, MuruganK, LovanhN, ParkYJ, OhBT, VenkatachalamP, BenelliG (2017) A novel photo-biological engineering method for Salvia miltiorrhiza-mediated fabrication of silver nanoparticles using LED lights sources and its effectiveness against Aedes aegyptiMosquito larvae and microbial pathogens.Physiol Mol plant Pathol xx:1–9 j.pmpp.2017.03.010 Lukman AI, Gong B, Marjo CE, Roessner U, Harris AT (2011) Facile synthesis, stabilization, and anti-bacterial performance of discrete ag nanoparticles using Medicago sativa seed exudates. J Colloid Interface Sci 353(2):433–444. 09.088 Madhiyazhagan P, Murugan K, Naresh Kumar A, Nataraj T, Dinesh D, Panneerselvam C, Subramaniam J, Mahesh Kumar P, Suresh U, Roni M, Nicoletti M, Alarfaj AA, Higuchi A, Munusamy MA, Benelli G (2015) Sargassum muticum-Synthetized Silver nanoparticles: an effective control tool against mosquito vectors and bacterial pathogens. Parasitol Res 114(11):4305–4317. 1007/s00436-015-4671-0 Mahendran G, Kumari BR (2016) Biological activities of silver nanoparticles from Nothapodytes nimmoniana (Graham) Mabb. Fruit extracts. Food Sci Human Wellness 5(4):207–218. 1016/j.fshw.2016.10.001

Parasitol Res (2018) 117:377–389 Mahesh Kumar P, Murugan K, Madhiyazhagan P, Kovendan K, Amerasan D, Chandramohan B, Dinesh D, Suresh U, Nicoletti M, Saleh Alsalhi M, Devanesan S, Wei H, Kalimuthu K, Hwang JS, Lo Iacono A, Benelli G (2016) Biosynthesis, characterization and acute toxicity of Berberis tinctoria-Fabricated Silver nanoparticles against the Asian tiger mosquito, Aedes albopictus, and the mosquito predators Toxorhynchites splendens and Mesocyclops thermocyclopoides. Parasitol Res 115(2):751–759. Mahyoub JA, Aziz AT, Panneerselvam C, Murugan K, Roni M, Trivedi S, Nicoletti M, Hawas UW, Shaher FM, Bamakhrama MA, Canale A, Benelli G (2017) Sea grasses as sources of mosquito nano-larvicides? Toxicity and uptake of Halodule uninervis-Biofabricated Silver nanoparticles in dengue and Zika virus vector Aedes aegypti. J Clust Sci 28(1):565–580. Murugan K, Benelli G, Ayyappan S, Dinesh D, Panneerselvam C, Nicoletti M, Hwang JS, Kumar PM, Subramaniam J, Suresh U (2016) Toxicity of seaweed-synthesized silver nanoparticles against the filariasis vector Culex quinquefasciatus and its impact on predation efficiency of the cyclopoid crustacean Mesocyclops longisetus. Parasitol Res 114:2243–2253 Murugan K, Wei J, Saleh Alsalhi M, Nicoletti M, Paulpandi M, Samidoss CM, Dinesh D, Chandramohan B, Paneerselvam C, Subramaniam J, Vadivalagan C, Wei H, Amuthavalli P, Jaganathan A, Devanesan S, Higuchi A, Kumar S, Aziz AT, Nataraj D, Vaseeharan B, Canale A, Benelli G (2017) Magnetic nanoparticles are highly toxic to chloroquine-resistant Plasmodium falciparum, dengue virus (DEN- 2), and their mosquito vectors. Parasitol Res 116(2):495– 502. Nahar UJ, Bhuiyan MMR, Samsudduzah ANM, Uddin MR, Maryam Z (2012) Phytochemical screening, cytotoxic and CNS depressant activities of Holarrhena antidysentericaleaves and seeds. Int J Pharm Sci Res 6:620–623 Noginov MA, Zhu G, Bahoura M, Adegoke J, Small C (2007) The effect of gain and absorption on surface plasmos in metal nanoparticles. Appl Phys B-Lasers O 86(3):455–460. s00340-006-2401-0 Pandey V, Agrawal V, Raghavendra K, Dash AP (2007) Strong larvicidal activity of three species of Spilanthes (Akarkara) against malaria (Anopheles stephensi Liston, Anopheles culicifacies, species C) and filaria vector (Culex quinquefasciatussay). Parasitol Res 102(1):171–174. Pandey V, Chopra M, Agrawal V (2011) In vitro isolation and characterization of biolarvicidal compounds from micropropagated plants of Spilanthes acmella. Parasitol Res 108(2):297–304. 10.1007/s00436-010-2056-y Panneerselvam C, Murugan K, Roni M, Suresh U, Rajaganesh R, Madhiyazhagan P, Subramaniam J, Dinesh D, Nicoletti M, Higuchi A, Alarfaj AA (2016) Fern-synthesized nanoparticles in the fight against malaria: LC/MS analysis of Pteridium aquilinum leaf extract and biosynthesis of silver nanoparticles with high mosquitocidal and antiplasmodial activity? Parasitol Res 115(3): 997–1013. Patil CD, Borase HP, Patil SV, Salunkhe RB, Salunke BK (2012a) Larvicidal activity of silver nanoparticles synthesized using Pergularia daemia plant latex against Aedes aegypti and Anopheles stephensi and non-target fish Poecillia reticulate. Parasitol Res 111(2):555–562. Patil CD, Patil SV, Borase HP, Salunke BK, Salunkhe RB (2012b) Larvicidal activity of silver nanoparticles synthesized using Plumeria rubra plant latex against Aedes aegypti and Anopheles stephensi. Parasitol Res 110(5):1815–1822. 1007/s00436-011-2704-x Pavela R, Murugan K, Canale A, Benelli G (2017) Saponaria officinalisSynthesized Silver nanocrystals as effective biopesticides and

Author's personal copy Parasitol Res (2018) 117:377–389 oviposition inhibitors against Tetranychus urticae Koch. Ind Crop Prod 97:338–344. Petit C, Lixon P, Pileni MP (1993) In situ synthesis of silver nanocluster in AOT reverse micelles. J Phys Chem 97(49):12974–12983. Rahuman AA, Bagavan A, Kamaraj C, Vadivelu M, Zahir AA, Elango G, Pandiyan G (2009) Evaluation of indigenous plant extracts against larvae of Culex quinquefasciatussay (Diptera: Culicidae). Parasitol Res 104(3):637–643. Rajakumar G, Rahuman AA (2011) Larvicidal activity of synthesized silver nanoparticles using Eclipta prostrata leaf extract against filariasis and malaria vectors. Acta Trop 118(3):196–203. https://doi. org/10.1016/j.actatropica.2011.03.003 Raveen R, Kamakshi KT, Deepa M, Arivoli S, Tennyson S (2014) Larvicidal activity of Nerium oleanderL. (Apocynaceae) flower extracts against Culex quinquefasciatus Say (Diptera: Culicidae). Int J Mosq Res 1:38–42 Rawani A (2017) Mosquito larvicidal activity of green silver nanoparticle synthesized from extract of bud of Polianthus tuberosa L. Int J Nanotechnol Appl 11:17–28 Rawani A, Ghosh A, Chandra G (2013) Mosquitolarvicidalandantimicrobialactivityofsynthesizednanocrystallinesilverparticlesusingleaves and green berry extract of Solanum nigrum L. (Solanaceae: Solanales). Acta Trop 128(3):613–622. Roni M, Murugan K, Panneerselvam C, Subramaniam J, Nicoletti M, Madhiyazhagan P, Dinesh D, Suresh U, Khater HF, Wei H, Canale A (2015) Characterization and biotoxicity of Hypnea musciformisSynthesized Silver nanoparticles as potential eco-friendly control tool against Aedes aegypti and Plutella xylostella. Ecotoxicol Environ Saf 121:31–38. 005 Salam H, Rajiv A, Kamaraj P, Jagadeeswaran M, Sangeetha P, Gunalan Sivaraj R (2012) Plants: green route for nanoparticle synthesis. Int Res J Biol Sci 1:85–90 Santhosh SB, Yuvarajan R, Natarajan D (2015) Annona muricata leaf extract-mediated silver nanoparticles synthesis and its larvicidal potential against dengue, malaria and filariasis vector. Parasitol Res 114(8):3087–3096. Santhoshkumar T, Rahuman AA, Rajakumar G, Marimuthu S, Bagavan A, Jayaseelan C, Zahir AA, Elango G, Kamaraj C (2011) Synthesis of silver nanoparticles using Nelumbo nucifera leaf extract and its larvicidal activity against malaria and filariasis vectors. Parasitol Res 108(3):693–702. Shahabuddin KU, Sarwar MS, Mohiuddin EJAZ (2006) Clinical evaluation of some herbal medicine for amoebiasis. Pak. J Pharmacol Sci 23:9–12 Shameli K, Ahmad MB, Jazayeri SD, Shabanzadeh P, Sangpour P, Jahangirian H, Yn G (2012) Investigation of antibacterial properties silver nanoparticles prepared via green method. Chem Cent J 6:73 Sharma G, Kapoor H, Chopra M, Kumar K, Agrawal V (2014a) Strong larvicidal potential of Artemisia annua leaf extract against malaria (Anopheles stephensi Liston) and dengue (Aedes aegypti L.) vectors and bioassay-driven isolation of the marker compounds. Parasitol Res 113(1):197–209. Sharma V, Hussain S, Bakshi M, Bhat N, Saxena AK (2014b) In vitro cytotoxic activity of leaves extracts of Holarrhena antidysenterica against some human cancer cell lines. Indian J Biochem Biophys 15: 46–51 Shwetha C, Latha KP, Asha KA (2014) Study on analgesic activity of Holarrhena antidysenterica leaves. J Herb Med 2:14–16 Siddhardha B, Jobina R, Bibhuti R, Sukumar K (2014) Green rapid biogenic synthesis of bioactive silver nanoparticles (AgNPs) using Pseudomonas aeruginosa. IET Nanobiotechnol 8:267–274

389 Singh J, Kaur G, Kaur P, Bajaj R, Rawat M (2016) A review on green synthesis and characterization of silver nanoparticles and their applications: a green nanoworld. World J Pharm Pharm Sci 7:730–762 Subarani S, Sabhanayakam S, Kamaraj C (2013) Studies on the impact of biosynthesized silver nanoparticles (AgNPs) in relation to malaria and filariasis vector control against Anopheles stephensi Liston and Culex quinquefasciatussay (Diptera: Culicidae). Parasitol Res 112(2):487–499. Subramaniam J, Murugan K, Panneerselvam C, Kovendan K, Madhiyazhagan P, Dinesh D, Kumar PM, Chandramohan B, Suresh U, Rajaganesh R, Alsalhi MS (2016) Multipurpose effectiveness of Couroupita guianensis-synthesized gold nanoparticles: high antiplasmodial potential, field efficacy against malaria vectors and synergy with Aplocheilus lineatus predators. Environ Sci Pollut Res 23(8):7543–7558. 10.1007/s11356-015-6007-0 Suganya G, Karthi S, Shivakumar MS (2014) Larvicidal potential of silver nanoparticles synthesized from Leucas aspera leaf extracts against dengue vector Aedes aegypti. Parasitol Res 113(3):875– 880. Sujitha V, Murugan K, Paulpandi M, Panneerselvam C, Suresh U, Roni M, Nicoletti M, Higuchi A, Madhiyazhagan P, Subramaniam J, Dinesh D (2015) Green synthesized silver nanoparticles as a novel control tool against dengue virus (DEN-2) and its primary vector Aedes aegypti. Parasitol Res 114(9):3315–3325. 1007/s00436-015-4556-2 Suresh U, Murugan K, Benelli G, Nicoletti M, Barnard DR, Panneerselvam C, Mahesh Kumar P, Subramaniam J, Dinesh D, Chandramohan B (2015) Tackling the growing threat of dengue: Phyllanthus niruri-mediated synthesis of silver nanoparticles and their mosquitocidal properties against the dengue vector Aedes aegypti (Diptera: Culicidae). Parasitol Res 114(4):1551–1562. Tesfazghi K, Hill J, Jones C, Ranson H, Worrall E (2016) National malaria vector control policy: an analysis of the decision to scale-up larviciding in Nigeria. Health Policy Plan 31:9–101 Thakur P, Chawla R, Narula A, Goel R, Arora R, Sharma RK (2016) Anti-hemolytic, hemagglutination inhibition and bacterial membrane disruptive properties of selected herbal extracts attenuate virulence of Carbapenem resistant Escherichia coli. Microb Pathog 95:133–141. 2016.04.005 Veerakumar K, Govindarajan M, Rajeswary M, Muthukumaran U (2014a) Low-cost and eco-friendly green synthesis of silver nanoparticles using Feronia elephantum (Rutaceae) against Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti (Diptera: Culicidae). Parasitol Res 113(5):1775–1785. https://doi. org/10.1007/s00436-014-3823-y Veerakumar K, Govindarajan M, Rajeswary M, Muthukumaran U (2014b) Mosquito larvicidal properties of silver nanoparticles synthesized using Heliotropium indicum (Boraginaceae) against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus (Diptera: Culicidae). Parasitol Res 113(6):2363–2373. 1007/s00436-014-3895-8 WHO (1988) Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides. WHO/VBC/81.807, Geneva WHO (2004) Global Strategic Framework for Integrated Vector Management. World Health Organization (In WHO/CDS/CPE/ PVC/2004), pp 12 Yadav A, Rai M (2011) Bioreduction and mechanistic aspects involved in the synthesis of silver nanoparticles using Holarrhena antidysenterica. J Bionanoscience 5(1):70–73. 1166/jbns.2011.1051