Larvicidal and antibacterial activity of aqueous leaf extract of Peepal (Ficus religiosa) synthesized nanoparticles

In this study, the zinc oxide nanoparticles (ZnO NPs) and titanium dioxide nanoparticles (TiO2 NPs) were synthesized using the aqueous leaf extract of Ficus religiosa (Peepal tree). The synthesized nanoparticles were tested as larvicides against the larvae of Anopheles stephensi. Further, the synthesized nanoparticles were tested as antibacterial agents against the Escherichia coli (gram negative) and Staphylococcus aureus (gram positive) bacteria. The synthesized nanoparticles were characterized with UV-visible spectroscopy, X-rays powder diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). The larvicidal mortality was observed after 24 h and 48 h by probit analysis. The antibacterial activity was evaluated using the well diffusion method. The synthesized nanoparticles were irregular shape and varied size. The larvae of An. stephensi were found highly susceptible against the ZnO NPs than the TiO2 NPs and aqueous leaves extract. The highest mortality was observed in synthesized ZnO NPs against first to third instars of (LC50 50, 75, and 5 ppm) and 100% mortality in fourth instars of An. stephensi. The higher zone of inhibition was occurred against the E. coli. This report of present investigation revealed that the rapid biological synthesis of ZnO NPs and TiO2 NPs using aqueous leaf extract of F. religiosa would be effective potential larvicides for mosquito control as well as antimicrobial agents with eco-friendly approach


Introduction
F. religiosa is commonly known as Peepal tree belonging to the family moraceae. It is also known as bodhi tree, pippala tree, peepul tree, pepal tree and ashwattha tree. It is a medicinal plant which is emerged as a good source of traditional medicine for the treatment of asthma, diabetes, diarrhea, epilepsy, gastric problems, inflammatory disorders, infectious and sexual disorders (Singh et al., 2011a). The leaves of F. religiosa contain tannic acid, leucine, isoleucine, methionine, tryptophan, threonine, glycine asparatic acid, serine and arginine, bark comprises of bergaptol and bergapten, the fruits consists of tyrosine and asparagines and seeds contain threonine, alanine and valine (Gupta and Singh, 2012), which are the good source of secondary metabolites used as larvicides and antimicrobial agents.
The larvicidal activity of crude hexane, ethyl acetate, petroleum ether, acetone, and methanol extracts of the leaf and bark of Ficus racemosa (Moraceae) has been assayed for their toxicity against the early fourth-instar larvae of Culex quinquefasciatus (Abdul Rahuman et al., 2008). The larvicidal efficacy of different extracts of F. benghalensis against Cx. quinquefasciatus, Ae. aegypti and An. stephensi has been investigated (Govindarajan, 2010). Further, larvicidal efficacy of different solvent leaf extracts of F. benghalensis against Cx. tritaeniorhynchus and An. subpictus has been determined (Govindarajan et al., 2011). Thereafter, the Larvicidal activity of Indian medicinal plants, Commiphora berryi, Commiphora pentandra, Pelargonium graveolens, Thevetia peruviana, Sesamum indicum, Ficus microcarpa, Melia dubia, C. bonplandianus, F. religiosa and Croton lacryma has been tested against Ae. aegypti mosquito (Deepa et al., 2015). The antimicrobial properties of Ficus extract have been reported (Mandal et al., 2000;Ao et al., 2008;Oyeleke et al., 2008;Annan and Houghton, 2008;Kuete et al., 2008;Kuete et al., 2009;Usman et al., 2009;Alimuddin et al., 2010;Lazreg Aref et al., 2010). Unfortunately, the secondary metabolites of plants have the slow reaction against the mosquitoes. Therefore, it is needed to develop the eco-friendly and rapid technology for the mosquito control as well as antimicrobial agent, so that people can be protected from the bacterial and vector borne diseases.
The larvicidal activity of plant synthesized TiO 2 NPs has been assessed (Rajakumar et al., 2015;Suman et al., 2015;Gandhi et al., 2016). The antimicrobial activity of synthesized TiO 2 NPs using plants has been studied (Maurya et al., 2012;Malarkodi et al., 2013;Santhoshkumar et al., 2014;Murphin Kumar et al., 2014;Hariharan et al., 2017). Till now no review is available on the larvicidal and antibacterial activities of ZnO NPs and TiO 2 NPs synthesized using aqueous leaf extract of Peepal (F. religiosa). In the present investigation, the ZnO NPs and TiO 2 NPs were synthesized using the aqueous leaf extract of F. religiosa and access their larvicidal and antibacterial properties. This ZnO NPs and TiO 2 NPs technology could be a rapid, green and, eco-friendly approach for mosquito control and used as antimicrobial also.

Collection and leaf extract preparation
The fresh and green leaves of F. religiosa were collected from the nearest area of ICMR-National Institute of Malaria Research, India. The leaves were rinsed with tap water and then with distilled water to remove dust and other particles. The rinsed leaves were then air dried for 1-2 h. After then, approximately 20 g of leaves were cut into fine pieces and put into a 250 ml conical flask which containing 100 ml of distilled water. Boil the flask for 1 h at 50°C on a magnetic stirrer. After 1 h, cooled the extract and filtered through the whatman-1 filter paper and store the leaves filtrate for the experiment.

Synthesis of ZnO and TiO 2 NPs
The ZnO NPs were biosynthesized by co-precipitation method described by Singh et al. (2011a) with some modification. 20 ml of leaf extract was heated at 60°C for 10 min on a magnetic stirrer. After then, 50 ml of 0.1 M of Zinc nitrate solution and 50 ml of 0.2 M sodium hydroxide solution were added drop wise under stirring. The mixture was continued stirred for 1 h on magnetic stirrer which resulting cream colored precipitate of zinc hydroxide formed. Then, the precipitate was collected by centrifugation at 4000 rpm for 15 min and washed with deionized water and ethanol. The ZnO NPs were collected after dried in hot air oven for 48 h at 45°C.
The TiO 2 NPs were biosynthesized by the following method described by Suman et al. (2015) with some modification. The aqueous solution of TiO(OH) 2 (5 mmol/L) was prepared and used for synthesis of TiO 2 NPs. 20 ml leaf extract of F. religiosa was boiled at 50°C. Then 80 ml of aqueous solution of 5 mmol/L TiO(OH) 2 were added in the leaf extract and boil for 4 h with continuous stirring. After then reduction mixture was centrifuged at 4000 rpm for 15 min and resulting pellet was collected. The TiO 2 NPs were dried in hot air oven for 48 h at 45°C.

Characterization of ZnO and TiO 2 NPs
Optical properties of synthesized ZnO and TiO 2 NPs were confirmed by UV-visible double beam spectroscopy (HALO DB-20) in 300-500 nm wavelength range. The XRD pattern of synthesized ZnO and TiO 2 NPs were carried out using X-ray diffractometer (Bruker X-ray diffractometer D-8 Advance) Cu-Kα radiations (λ = 0.15406 nm) in 2θ range from 20°to 80°. The average size and shape of synthesized ZnO and TiO 2 NPs were obtained by transmission electron microscopy (Tecnai G 2 ). The morphology of synthesized ZnO and TiO 2 NPs were examined by scanning electron microscope (model no. Zeiss EVO MA 10). The synthesized ZnO and TiO 2 NPs were analyzed for elemental analysis by energy dispersive X-ray spectroscopy (Oxford Inca Energy 250).

Rearing of larvae
The larvae of An. stephensi were reared in deionized water containing glucose and yeast powder. The colony of An. stephensi was maintained in the laboratory at 27°C with relative humidity of (75 ± 5%) and 14 h of photoperiod using the standard method with some modifications (Geberg et al., 1994).

Bioassay, data management and statistical analysis
ZnO NPs and TiO 2 NPs, synthesized using aqueous leaves extract of F. religiosa were tested for their killing activities against the An. stephensi larvae (I-IV instar). The bioassay was assessed using the standard method (World Health Organization, 2005). An. stephensi larvae were placed in a container in micro-free deionized water. After that, ZnO and TiO 2 NPs with different test concentrations in 100 mL deionized water were prepared in 250 mL beakers. Bioassays were conducted separately at five different concentrations using serial dilution method, of synthesized ZnO and TiO 2 NPs (25, 50, 100, 150 and 250 ppm). To test the larvicidal activity of ZnO and TiO 2 NPs, 20 larvae were separately exposed to 100 mL of test concentration. Similarly, the control (without ZnO and TiO 2 NPs) was run to test the natural mortality. The experiments were replicated thrice to validate the results. Thereafter, we examined their mortality after 24 h and 48 h. The data on the efficacy was subjected to probit analysis (Finney, 1971). The control mortality was corrected by Abbott's formula (Abbott, 1925).

Antibacterial activity of ZnO and TiO 2 NPs
The antibacterial activity of synthesized ZnO and TiO 2 NPs was evaluated against E. coli and S. aureus. The antibacterial activity was determined using the well diffusion method. The wells were prepared on plates with Muller-Hinton agar (MHA) medium. Then, the plates were seeded with different bacterial strains using sterile swab. Four wells were prepared using gel puncture in each plate. Each well was loaded with 50 μL of different concentration of ZnO and TiO 2 NPs (50, 150, 250 and 500 ppm). Then, the plates were incubated at 35°C for 24 h and zone of inhibition was observed.

Results and discussion
3.1. Proposed mechanism of synthesis of ZnO and TiO 2 NPs through F. religiosa Based on the experimental work that has been done, there are series of chemical reaction that takes place. The complete hydrolysis of zinc nitrate and dihydroxy(oxo)titanium with the aid of F. religiosa aqueous leaves extract solution should result in the formation of ZnO and TiO 2 nanoparticles. The richly available carbohydrates, tannin, alkaloids, steroids, terpenoids, saponin, reducing sugar and favonoids in the plant extract acted as stabilizing and capping agents, respectively. Hence, the proposed principle of formation of ZnO and TiO 2 NPs involves the ionization of zinc nitrate and hydroxylation of dihydroxy(oxo)titanium in an aqueous medium to give Zn 2+ which was reduced by phytochemical principle present in the aqueous extract of F. religiosa, to generate ZnO, which further aggregates to ZnO and TiO 2 NPs as shown in Eq. 1 and Eq. 2.
TiO(OH) 2 + Ficus religiosa leaves extract Δ Stirring TiO 2 + H 2 O↑.3.2UV-visible analysisThe formation of ZnO and TiO 2 NPs during the synthesis can be observed visually. Fig. 1a is the UV-vis absorption spectrum of ZnO NPs dispersed in deionized water and the figure shows the absorption peak at 358 nm. Shah et al. (2015) stated that the UV absorption spectrum for synthesized ZnO NPs was recorded at 330 nm. Similar results were observed by Yedurkar et al. (2016). Fig. 1b shows the UV-vis absorption spectrum of TiO 2 NPs with an absorption peak at 450 nm. Valli and Geetha (2015) observed the UV absorption spectrum for synthesized TiO 2 NPs at 447.3 nm.3.3XRD analysisThe structure of ZnO and TiO 2 NPs were determined in this study using a powder diffraction system with Cu-Ka x-ray tube (λ = 1.541836 A) was used. Fig. 2 (110), (002), (101), (102), (110), (103), (112), (201) and (202) plane. The strong and narrow peak denotes that the product has well crystalline nature of particles. Narendhran and Sivakumar, (2016) Vanathi et al. (2014) in which particles were synthesized using E. crassipes leaf extract. Fig. 3 shows the XRD pattern of synthesized TiO 2 NPs scanned at 2θ range from 0 to 80 degree. Diffraction peaks at 25.28°, 36.91°, 53.85°, 55.03°, 62.6°, 68.70°and 75.1°can be assigned to (110), (101), (211), (220), (204), (112) and (215) plane. Similar results were reported by Khadar et al. (2015).3.4TEM analysisThe shape and size of synthesized ZnO and TiO 2 NPs were obtained using the TEM. Fig. 4a shows the TEM images of synthesized ZnO NPs, which depict the irregular shape and varied size nanoparticles. Fig. 4b depicts the TEM images of TiO 2 NPs, which were spherical in shape and size from 70.29 to 84.93 nm with calculated size of 77.61 nm and polydisperse. Zahir et al. (2014) recorded the TEM images of the synthesized Ag NPs and TiO 2 NPs spherical, quite polydisperse and individual particles showed an average size of 12.82 ± 2.50 and 83.22 ± 1.50 nm, respectively. Similar results were obtained by (Rajakumar et al., 2012) and previous work (Table 1).3.5SEM-EDX analysisThe size and distribution of synthesized ZnO and TiO 2 NPs were also confirmed by SEM shown in Fig. 5a-b. From the result it is evident that the morphology of ZnO NPs was irregular and TiO 2 NPs was spherical in shape and polydisperse in nature, which is very similar to previous studies (Zahir et al., 2014;Rajakumar et al., 2012). Fig. 6 shows the EDX analysis of ZnO NPs 72.57% zinc and 27.42% of oxygen which confirm the elemental composition of ZnO NPs. Narendhran and Sivaraj (2016) showed the EDX analysis of ZnO nanoparticles   37.22% of zinc and 62.78% of oxygen which confirms the elemental composition of ZnO nanoparticles. The strong signals from the zinc atoms in the nanoparticles recorded and other signals from C and O atoms were observed using EDX analysis in Partheniummediated ZnO nanoparticles (Rajiv et al., 2013). The EDX analysis display the optical absorption peaks of ZnO nanoparticles and these absorption peaks were due to the surface plasmon resonance of ZnO nanoparticles (Ankanna and Savithramma, 2011). Fig. 7 depicts the EDX analysis of TiO 2 NPs 71.99% titanium and 28.01% of oxygen which confirm the elemental composition of TiO 2 NPs. Santhoshkumar et al. (2014) showed the energy dispersive X-ray analysis study (EDX) which proves that the particles are crystalline in nature and indeed metallic TiO 2 NPs. The similar results were reported in the previous studies by (Suman et al., 2015).3.6Larvicidal activity of ZnO and TiO 2 NPsLarvicidal activity of F. religiosa leaf extract, synthesized ZnO and TiO 2 NPs were evaluated against the larvae (I-IV) of An. stephensi at different concentrations (25, 50, 100, 150 and 250 ppm).The larvae of An. stephensi were found highly susceptible to the ZnO NPs. The fourth instar larvae have shown the 100% mortality after 24 h of exposure. Whereas, the first instar (LC 50 50 ppm), second instar (LC 50 75 ppm) and third instar (LC 50 5 ppm) larvae were observed with their probit equations and 95% confidential limit, R 2 , chi-square and p value after 24 h (Table 2). No mortality was observed in control group. The anti-parasitic activities have been assessed to determine the efficacies of synthesized zinc oxide nanoparticles (ZnO NPs) prepared by wet chemical method using zinc nitrate and sodium hydroxide as precursors and soluble starch as stabilizing agent against the larvae of cattle tick Rhipicephalus (Boophilus) microplus, Canestrini (Acari: Ixodidae); head louse Pediculus humanus capitis, De Geer (Phthiraptera: Pediculidae); larvae of malaria vector, An. subpictus, Grassi; and filariasis vector, Cx. quinquefasciatus, Say (Diptera: Culicidae) (Kirthi et al., 2011). The maximum efficacy was observed in zinc oxide against the R. microplus, P. humanus capitis, and the larvae of An. subpictus, Cx. quinquefasciatus with LC(50) values of 29.14, 11.80, 11.14, and 12.39 mg/L; r (2) = 0.805, 0.876, 0.894, and 0.904, respectively. The synthesized ZnO NPs showed the LC (50) and r (2) values against the R. microplus (13.41 mg/L; 0.982), P. humanus capitis (11.80 mg/L; 0.966), and the larvae of An. subpictus (3.19; 0.945 mg/L), against Cx. quinquefasciatus (4.87 mg/L; 0.970), respectively.The TiO 2 NPs were found effective against the larvae of An. stephensi. The mortality was recorded after 48 h of exposure. The first instar (LC 50 15 ppm), second instar (LC 50 50 ppm), third instar (LC 50 25 ppm) and fourth instar (LC 50 25 ppm) larvae were observed with their probit equations and 95% confidential limit, R 2 , chi-square and p value after 24 h ( Table 2). The larvicidal activity of titanium dioxide nanoparticles (TiO 2 NPs) synthesized from the root aqueous extract of M. citrifolia against the larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus has been assessed (Suman et al., 2015). The biosynthesized TiO 2 NPS showed maximum activity against the larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus when compared to the aqueous extract of M. citrifolia. Similarly, the antiparasitic activity of TiO 2 NPs against the larvae of R. microplus, H. anatolicum anatolicum and H. bispinosa, fourth instar larvae of An. subpictus, and Cx. quinquefasciatus has been assessed by (Rajakumar et al., 2015). The maximum efficacy was observed in synthesized TiO 2 NPs against the larvae of R. microplus, H. anatolicum anatolicum, H. bispinosa, An. subpictus, and Cx. quinquefasciatus with LC value of 28. 56, 33.17, 23.81, 5.84, and 4.34 mg/L, respectively. Recently, the larvicidal and the pediculicidal activity of synthesized titanium dioxide nanoparticles (TiO 2 NPs) using the leaf aqueous extract of V. negundo against the fourth instar larvae of the malaria vector, An. subpictus Grassi and filariasis vector, Cx. quinquefasciatus Say and the head louse, P. humanus capitis De Geer has been carried out by (Gandhi et al., 2016). The maximum activity has been observed in the synthesized TiO 2 NPs against An. subpictus, Cx. quinquefasciatus and lice, (LC 50 = 7.52, 7.23 and 24.32 mg/L; χ 2 = 0.161, 2.678 and 4.495; r 2 = 0.663, 0.742 and 0.924), respectively. The larvicidal activity of synthesized ZnO and TiO 2 has been reported by other researchers (Table 3) The larvae of An. stephensi have also shown the mortality against the aqueous leaves extract of F. religiosa and mortality was recorded after 48 h. The first instar (LC 50 250 ppm), second instar (LC 50 250 ppm), third instar (LC 50 200 ppm) and fourth instar (LC 50 200 ppm) larvae were observed with their probit equations and 95% confidential limit, R 2 , chi-square and p value after 24 h ( Table 1). The larvicidal efficacy of different extracts of F. benghalensis against Cx. quinquefasciatus, Ae. aegypti and An. stephensi has been investigated (Govindarajan, 2010 Fig. 8. Well diffusion method was used to provide the evidence for and validate the antibacterial activity of ZnO and TiO 2 NPs against E. coli (Gram negative) and S. aureus, (Gram positive) bacteria. The antibacterial activity of the ZnO and TiO 2 NPs was indicated by the formation of the zone. The diameter of the inhibition zone was measured in millimetre. The maximum zone of inhibition was observed against ZnO NPs in E. coli (8, 10, 12 and 14 mm) and S. aureus (6, 8, 10 and 12) ( Table 4 and Fig. 8). Several research confirming antimicrobial activity of ZnO NPs against the food related bacteria B. subtilis, E. coli, P. fluorescens, S. typhimurium and S. aureus has been reported (Russell and Hugo, 1994;Ip et al., 2006). ZnO NPs are also known to exhibit antimicrobial activities against L. monocytogenes, S. enteritidis and E. coli (Russell and Hugo, 1994). The formation of hydrogen peroxide from the surface of ZnO is considered to be mainly responsible for its antimicrobial property (Rai et al., 2009).While, TiO 2 NPs has shown the zone of inhibition in E. coli (7, 9, 10, and 13) and S. aureus (5, 6, 8 and 10) (Table 4 and Fig. 8). The higher zone of inhibition occurred at 500 ppm concentration of synthesized ZnO and TiO 2 NPs. The antibacterial   using the biosynthesized and chemically synthesized titania nanoparticles and other researchers also (Table 5).4ConclusionThe present study, synthesis of ZnO NPs and TiO 2 NPs from the F. religiosa is a green,