Materials Science and Engineering C 58 (2016) 44–52

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Bark extract mediated green synthesis of silver nanoparticles: Evaluation of antimicrobial activity and antiproliferative response against osteosarcoma Debasis Nayak, Sarbani Ashe, Pradipta Ranjan Rauta, Manisha Kumari, Bismita Nayak ⁎ Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology, Rourkela, Odisha 769008, India

a r t i c l e

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Article history: Received 10 December 2014 Received in revised form 6 July 2015 Accepted 12 August 2015 Available online 15 August 2015 Keywords: Silver Nanoparticles Green synthesis Antiproliferative Antimicrobial

a b s t r a c t In the current investigation we report the biosynthesis potentials of bark extracts of Ficus benghalensis and Azadirachta indica for production of silver nanoparticle without use of any external reducing or capping agent. The appearance of dark brown color indicated the complete nanoparticle synthesis which was further validated by absorbance peak by UV–vis spectroscopy. The morphology of the synthesized particles was characterized by Field emission- scanning electron microscopy (Fe-SEM) and atomic force microscopy (AFM). The X-ray diffraction (XRD) patterns clearly illustrated the crystalline phase of the synthesized nanoparticles. ATR-Fourier Transform Infrared (ATR-FTIR) spectroscopy was performed to identify the role of various functional groups in the nanoparticle synthesis. The synthesized nanoparticles showed promising antimicrobial activity against Gram negative (Escherichia coli, Pseudomonas aeruginosa and Vibrio cholerae) and Gram positive (Bacillus subtilis) bacteria. The synthesized nano Ag also showed antiproliferative activity against MG-63 osteosarcoma cell line in a dose dependent manner. Thus, these synthesized Ag nanoparticles can be used as a broad spectrum therapeutic agent against osteosarcoma and microorganisms. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Osteosarcoma is one of the most commonly diagnosed primary malignant bone tumors in humans. It is the 3rd most frequently occurring cancer having a bimodal age distribution having first peak in adolescent and second peak in early adults [1,2]. It primarily arises from mesenchymal cells and the majorly affected body parts are distal femur, proximal tibia and distal humerus [3]. At present, the most commonly used therapy for osteosarcoma is a treatment cycle that consists of preoperative chemotherapy, tumor amputation and postoperative chemotherapy. The most effective chemotherapeutic agents used for treatment includes a combination of high dose-methotrexate, cisplatin and doxorubicin. But these chemical compounds demonstrate significant side effects such as anemia, neutropenia, thrombocytopenia and heart damage along with relatively decrease in patient survival rate [4–7]. Silver being a noble metal maintains its exceptionally well defined optical and electronic properties in quantum size for which it has paved its course and curiosity towards application in nano regime [8,9]. Silver has been employed extensively for various biomedical purposes since time immemorial. The interest in silver nanoparticles has gained prominence owing to its excellent plasmonic activity,

⁎ Corresponding author. E-mail address: [email protected] (B. Nayak).

http://dx.doi.org/10.1016/j.msec.2015.08.022 0928-4931/© 2015 Elsevier B.V. All rights reserved.

electromagnetic, optical and catalytic properties, and bacteriostatic and bactericidal effects along with antiproliferative effects compared with other metal nanoparticles. Its versatile application in dentistry, clothing, catalysis, mirrors, optics, photography, electronics and in the food packaging industry has tremendously increased its market value [10]. Conventional physical and chemical methods of stable nanomaterial synthesis face challenges of nanoparticle aggregation, harsh reaction conditions and the toxicity of the reagents used. Therefore, new synthetic methods based on green chemistry principles have been explored for the synthesis of stable monodispersed nanoparticles with reduced toxicity concerns [11,12]. Green chemistry principles maximize safety and efficiency and minimize the environmental and societal impact of toxic raw materials. Green synthesis of nanoparticles focuses on three important aspects i.e., (i) use of green solvents, (ii) use of an eco-friendly benign reducing agent, and (iii) use of a nontoxic material as a stabilizer [13] Green synthesis of silver nanoparticles using various plant extracts has been reported [11–14]. The extracts contain different enzymes/proteins, amino acids, polysaccharides, vitamins, poly- phenols, etc., which act as both reducing and capping agents during the nanoparticle synthesis [15]. Ficus benghalensis (family Moraceae) commonly known as ‘banyan’ is an evergreen tree found all over India. Its various parts are used in Ayurveda for the treatment of diarrhea, dysentery, piles, rheumatism and as an astringent, haemostatic and antiseptic agent. The bark has been reported to contain leucopelargonidin-3-0-α-L rhamnoside, leucocynidin-3-0-α-D galactosyl

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cellobioside, glucoside, beta glucoside, pentatriacontan-5-one and beta sitosterol-α-D-glucose [16–20]. Azadirachta indica (family Meliaceae) is commonly called as ‘village dispensary’ in traditional medicine as the tree has its efficacy in every disease. Different compounds have been isolated from the bark extract such as Nimbin, Nimbinin, Deacetyl nimbin, Nimbinene, 6-Deacetyl nimbinene, Nimbandiol, polysaccharides G1A, G1B, G2A, and G3A, and NB-2 peptidoglucan [21–24]. Neem bark has antibacterial, antiviral, antifungal, antimalarial, antioxidant and anticancer activity [25]. Various plant parts have been used for the synthesis of silver nanoparticles but rarely the barks have been explored to its full potential. In the present study the barks of F. benghalensis and A. indica have been employed for the synthesis of silver nanoparticles. The leaves of A. indica were used as reference material for available reports on the ability of A. indica leaves to be used for biosynthesis of AgNPs [26]. 2. Materials and methods Silver nitrate, Mueller Hinton agar and Mueller Hinton broth, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Hi-media (Mumbai, India), Minimum Essential Medium (MEM) fetal bovine serum (FBS), antibiotic solution (penicillin– streptomycin), Bisbenzimide H 33342 from Sigma-Aldrich (Mumbai, India) and deionized water was used throughout the experiment.

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were performed at 15 kV. The surface morphology of the sample was also examined using an Atomic Force Microscope (AFM, Dimension D3100, Veeco) with a conducting P(n) doped silicon tip under normal atmospheric condition in contact mode. The X-ray powder diffraction (XRD) patterns of silver nanoparticles were obtained using X-ray diffractometer (PANalytical X'Pert, Almelo, Netherlands) equipped with Ni filter and Cu Kα (l = 1.54056 Å) radiation source. The diffraction angle was varied in the range of 20–80° while the scanning rate was 0.05°/s. The Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy analysis was conducted to corroborate the possible role of the various phytochemicals present in the bark extract on the surface modification of the synthesized nanoparticles. The ATRFTIR was performed on a Bruker ALPHA spectrophotometer (Ettlinger, Germany) with a resolution of 4 cm−1. The samples were scanned in the spectral region between 4000 and 500 cm−1 by taking an average of 25 scans per sample. 1 drop of sample was kept on the sample holder and the samples were scanned and the result obtained was analyzed through OPUS software. 2.4. Antimicrobial activity

The nanoparticles were synthesized by following the protocol described by Zargar et al., with little modifications [28]. 90 ml of silver nitrate solution (1 M) was mixed with 10 ml of bark extract and the reaction mixture was kept in a water bath at different temperature and incubation time till the appearance of dark reddish brown color in the reaction mixture was observed. The resultant colored reaction mixture was then centrifuged at 10,000 rpm for 45 min (C24-BL centrifuge, REMI, India). The pellet obtained was washed thrice with deionized water to remove any traces of un-utilized bark phyto-constituents. The resultant pellet was lyophilized and stored for further characterizations. All the conditions were optimized for its reproducibility.

The antimicrobial activity of the green synthesized AgNPs against the nosocomial Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis collected from SCB Medical College, Cuttack, Odisha, India and Vibrio cholerae (classical 0139) Strain no. 3906 obtained from MTCC, Chandigarh, India were investigated by agar well diffusion method. Briefly, the bacterial strains were grown on Mueller Hinton Broth (MHB) at 37 °C for 24 h (30 °C for B. subtilis). The colony forming unit (CFU) was made to 2.5 × 10 − 5 CFU by adjusting it with 0.5 McFarland constant and observing the OD at 600 nm in a UV–vis Spectrophotometer [29]. Then, the stains were swabbed onto Mueller Hinton Agar (MHA) plate (in triplicates) and wells were formed by using a cork borer. 100 μl of the synthesized AgNPs was added to each well having a concentration of 100 μg/ml and the plates were incubated at 37 °C for 24 h (30 °C for B. subtilis). The mean diameter of the inhibition zone was measured in mm. Minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) was examined using the standard broth dilution method (CLSI-M07-A8). Briefly, 100 μl of overnight growth bacterial cultures (2.5 × 10− 5 CFU) were placed into 96 well plates followed by 100 μl of the synthesized silver nanoparticles by serial dilutions from the initial concentration of 100 μg/ml to 0.782 μg/ml and incubated at optimal temperature for 24 h. The MIC was calculated based on the lowest concentration of synthesized nanoparticles that inhibit the bacterial growth. To confirm bacterial growth inhibition the wells showing no visible growth were swabbed on MHA plates and incubated for 24 h at optimal temperature [30]. The MBC was calculated to be the concentration at which 100% of the bacterial growth was inhibited [31].

2.3. Characterization of the silver nanoparticles

2.5. Antiproliferative activity against cancer cell lines

To investigate the ideal temperature and time required for the synthesis of silver nanoparticles the reaction mixture was monitored periodically in a UV–visible spectrophotometer (Lambda 35® (PerkinElmer, Waltham, MS, USA)) operated at a resolution of 1 nm at room temperature scanned in the wavelength range of 400–600 nm. The hydrodynamic (Z-Average) size, polydispersity index (PDI) and surface zeta potential (charge) of the synthesized nanoparticles were analyzed by Zeta sizer (ZS 90, Malvern Instruments Ltd., Malvern, UK) and the results were obtained by the Malvern ZS nano software. The surface morphology of the synthesized nanoparticles was investigated by field emission scanning electron microscopy (Nova NanoSEM 450/ FEI, USA). The nanoparticles were fixed on adequate support and coated with gold using gold sputter module in a vacuum evaporator. Observations under different magnifications

2.5.1. Determination of cell viability by MTT assay To determine the cytotoxic effect of green synthesized silver nanoparticles, cell viability study was done with the conventional MTT reduction assay with slight modifications [32]. Briefly, MG-63 cells were purchased from NCCS, Pune, India and were seeded in 96 well plates at the density of 3000 cells/well based on the doubling time in presence of 200 μl MEM supplemented with 10% FBS and 1% penicillin–streptomycin solution and incubated for 24 h in an incubator containing 5% CO2 at 37 °C. After 24 h of seeding, the existing media was removed and replaced by fresh media along with various concentrations of silver nanoparticles viz., 10, 20, 40, 60, 80, 100, 150, and 200 μg/ml and incubated for 48 h at 37 °C, 5% CO2. To detect the cell viability, MTT working solution was prepared from a stock solution of 5 mg/ml in growth medium without FBS to the final concentration of

2.1. Preparation of bark extract The barks of F. benghalensis and A. indica were collected from the campus of NIT, Rourkela. They were washed properly with deionized water to remove any traces of dust and impurities. The extracts were prepared by slightly modifying the protocol described by Prasad et al. [27]. Briefly, bark powders of F. benghalensis and A. indica (5 g) were dissolved in 50 ml of deionized water and were boiled in a water bath at 50 °C for 1 h. The extracts were filtered using Whatman filter paper and kept at 4 °C until used. 2.2. Synthesis of silver nanoparticles

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Fig. 1. UV–vis spectra of the synthesized silver nanoparticles with respect to time at 80 °C from the bark extracts of (a) Azadirachta indica and (b) Ficus benghalensis.

0.8 mg/ml. 100 μl of MTT solution was added and incubated for 4 h. After 4 h of incubation the MTT solution was discarded and 100 μl of DMSO solvent was added in each well under dark followed by an incubation of 15 min and the optical density of the formazan product was read at 595 nm in a microplate reader (PerkinElmer, Waltham, MS, USA). 2.5.2. Chromatin condensation assay by Hoechst 33342 staining An average of 1 × 104 cells were plated in 60 mm petri plates. After 24 h of seeding when cells get properly adhered to the plate surface, they were treated with silver nanoparticles at their respective IC50 values. After 24 h of drug treatment the cells were stained with Hoechst 33342 stain (1 mg/ml) and incubated for 10 min at 37 °C and images were taken under UV filter using Epi-fluorescent Microscope (Olympus IX71). 3. Results and discussion The optimal temperature and time required for the biosynthesis of silver nanoparticles from the bark extracts of F. benghalensis and A. indica was monitored by UV–vis spectroscopy. The UV–vis spectra results are an indirect but most efficient method for detecting the formation of nanoparticles. The progress of the reaction leading to the conversion of Ag+ from AgNO3 to reduced nanosilver was monitored by observing the color change and absorbance maxima peak in the range of 420–460 nm. For the rapid synthesis of nanoparticles various parameters has to be taken into consideration along with their optimization such as the temperature, pH and incubation time. Fig. 1 shows the

gradual progress of nanoparticle synthesis when the reaction mixture was incubated at different temperature conditions viz., 20 °C, 40 °C, 60 °C and 80 °C. After confirming the temperature required for nanoparticles synthesis, the incubation period for nanoparticle synthesis was monitored. The reaction mixture was incubated at 80 °C and the absorbance was measured at different time intervals till the occurrence of a broad absorbance peak for the synthesized nanoparticles (Fig. 2). In biological systems, pH plays a crucial role in balancing the reaction conditions and directing the reaction to move forward for product formation, so it was vital to study the role of pH in the process of nanoparticle synthesis. The reaction mixture was incubated at 80 °C for 30 min at different pH conditions and the rate of nanoparticle synthesis was monitored. Rapid synthesis of nanoparticles at alkaline pH (pH 10) was monitored by UV–vis spectroscopy (Fig. 3). Similar kind of results regarding the effect of alkaline pH on nanoparticle synthesis was earlier reported by Andreescu et al. [33]. The absorption peaks were observed at 426 nm and 420 nm for the silver nanoparticles synthesized from the bark extracts of F. benghalensis and A. indica respectively within 30 min of incubation suggested the process to be fairly rapid and spontaneous. The occurrence of absorption peak is due to the surface plasmon resonance (SPR) property of the metallic nanoparticles which occurs due to the oscillations of free electrons on the surface of the metallic nanoparticles when they align in resonance with the wavelength of irradiated light [34]. Dynamic light scattering (DLS) studies were conducted to investigate the hydrodynamic size, polydispersity index and surface zeta

Fig. 2. UV–vis spectra of the synthesized silver nanoparticles during different incubation times at 80 °C from the bark extracts of (a) Azadirachta indica and (b) Ficus benghalensis.

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Fig. 3. UV–vis spectra of the synthesized silver nanoparticles from different pH conditions at 80 °C from the bark extracts of (a) Azadirachta indica and (b) Ficus benghalensis.

potential of the synthesized silver nanoparticles in a colloidal aqueous environment. When particles were dispersed in a medium, it exhibited Brownian motion measured by fluctuations in the intensity of scattered light in the system out of which translational diffusion co-efficient is calculated by applying the Stokes–Einstein equation that gives the hydrodynamic size of the particle [35]. Fig. 4a and b shows the size of the silver nanoparticles synthesized by bark extracts of F. benghalensis (85.95 nm) and A. indica (90.13 nm) respectively. To correlate the role of temperature with nanoparticle synthesis obtained from UV–vis spectroscopy, the reaction mixture incubated at different temperature conditions was scanned for its Z-average size (hydrodynamic size) and surface

zeta potential (Fig. 5a, b). From the graph it can be clearly deduced that with gradual increase in temperature, the size of the nanoparticle decreases and the surface zeta potential increases. This indicates that temperature has a role to play in nanoparticle synthesis. The polydispersity index (PDI) is the measure of the width of the particle size distribution calculated from a cumulant analysis of the DLS measured intensity autocorrelation function where a single particle size is assumed and a single exponential fit is applied to the autocorrelation function [36]. The PDI value ‘0’ represents monodisperse distribution whereas value ‘1’ represents polydisperse distribution. Fig. 4c and d shows the surface zeta potential of the synthesized silver nanoparticles from the

Fig. 4. (a, b) Dynamic light scattering studies; (c, d) surface zeta potential studies of the silver nanoparticles synthesized from the bark extracts of Ficus benghalensis and Azadirachta indica respectively.

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Fig. 5. (a) Mean particle size of the synthesized nanoparticles at different temperature conditions. (b) Surface zeta potential of the synthesized nanoparticles at different temperature conditions from the bark extracts of Azadirachta indica and Ficus benghalensis respectively.

respective bark extracts of F. benghalensis and A. indica. Zeta potential is the measurement of the magnitude of electrostatic charge repulsion or attraction between particles in a liquid suspension. It is one of the essential parameters for characterizing the stability of nanoparticles in an aqueous environment. Particles with zeta potentials greater than +30 mV and less than −30 mV are considered stable for colloidal dispersion in the absence of steric stabilization [37]. Table 1 shows the hydrodynamic size, PDI and zeta potential of the silver nanoparticles synthesized by bark extracts of F. benghalensis and A. indica. Fig. 6 shows the typical image of the surface morphology of the synthesized nanoparticles by field emission scanning electron microscopy (FE-SEM). The average sizes of the particles were around 60 nm for both the bark mediated synthesized silver nanoparticles. The surface roughness of the spherical silver nanoparticles was clearly illustrated by the FE-SEM images. Fig. 7 shows the pictographs of the 3D surface morphology and size analysis graphs obtained from atomic force microscopy (AFM). The size obtained from the AFM pictographs in the contact mode from the line analysis measurement by using the SPMLab programmed Veeco diInnova software were 68 nm and 73.8 nm for silver nanoparticles synthesized from bark extracts of F. benghalensis and A. indica respectively. However results obtained from FE-SEM and DLS may tend to vary because both the methods are based on different techniques of characterization as well as the sample preparation methods are also completely different [38]. X-ray powder diffraction (XRD) technique was used to identify the crystalline phase, orientation and grain size of the synthesized nanoparticles. Fig. 8 shows a typical XRD diffractogram showing Bragg peaks (angle 2θ) at 32.19°, 38.15°, 44.28°, 64.46°, and 77.37° and 32.11°, 37.96°, 44.18°, 64.37°, and 77.23° for the silver nanoparticles synthesized from the bark extracts of F. benghalensis and A. indica respectively which corresponds to (111), (200), (220), (311) and (222) miller indices thus, confirming the formation of face centered cubic (FCC) crystalline elemental silver indexed with the JCPDS data 04-0783. Many unassigned peaks were seen which might be due to the crystallization of the bioorganic phases that occur on the surface of the synthesized nanoparticles [39,40]. The average grain size of the synthesized silver nanoparticles was determined by using Scherer's equation [d = Kλ / β cos θ] where, ‘d’ is the mean diameter of the particle; ‘K’ is the

shape factor (0.9); ‘λ’ is the X-ray radiation source (0.154 nm); ‘β’ is (π / 180) ∗ FWHM and ‘θ’ is the Bragg angle [34] which was approx. 29 nm and 39 nm for the silver nanoparticles synthesized by bark extracts of F. benghalensis and A. indica. The ATR-FTIR measurements were carried out to identify the chemical transformation that occurred during the interaction between the functional groups present in bark extract and formation of the nanoparticles. Fig. 9 shows a typical ATR-FTIR spectrum of the silver nanoparticles synthesized from the bark extracts of F. benghalensis and A. indica. Almost similar peaks were observed in both types of synthesized silver nanoparticles. FTIR peaks were observed at 3590 cm−1, 3340 cm−1, 2310 cm−1, 1693 cm−1, 1519 cm−1 and 615 cm−1 for silver nanoparticles synthesized from the bark extract of F. benghalensis and at peaks 3617 cm− 1, 3332 cm− 1, 2319 cm−1, 1663 cm− 1, 1523 cm−1, 1523 cm−1 and 635 cm−1 for silver nanoparticles synthesized by bark extracts of A. indica. The absorption peaks were assigned to the presence of the following functional groups: O–H stretching (presence of alcohols and phenols), N–H stretching (presence of primary and secondary amines), C≡N stretching (presence of nitriles), C_C stretching (presence of aromatic rings) and C–H stretching (presence of alkynes). From Fig. 9 it can be clearly seen that the O–H and N–H functional group has a definite role in the fabrication of silver nanoparticles which are the main constitutional components present in the flavonoids, terpenoids and phenols. Although the exact mechanism for the reduction of silver nanoparticles is not known, Ajitha et al. proposed that the flavonoids present in Tephrosia purpurea leaf extract may act as powerful reducing agent and the carboxylate group present in the proteins may act as surfactant to attach on the surface of the nanoparticles resulting in their stabilization during the synthesis reaction [41]. The results obtained from the mangrove leaf bud extract of Rhizophora mucronata were quite similar to our ATR-FTIR results thus furnishing a coherent role of the bark extract as reducing and capping agents to prevent agglomeration of the synthesized silver nanoparticles [42]. However, various preparation methods and functional biomolecules like enzymes, proteins, amino acids and polysaccharides present in the reaction mixture might help in better stabilization of the nano silver. Various reductases like NADH-dependant reductases, nitrate-dependant reductases present in the working system channelize the electron transfer

Table 1 Dynamic light scattering studies of the synthesized silver nanoparticles. Sl. no

Plant used for synthesis

Hydrodynamic diameter (nm)

Polydispersity index (PDI)

Zeta potential (mV)

1 2

Ficus benghalensis Azadirachta indica

85.95 90.13

0.247 0.314

−29.4 −27.9

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Fig. 6. Surface morphology of the synthesized silver nanoparticle by scanning electron microscopy (a) Ficus benghalensis and (b) Azadirachta indica.

shuttle in order to produce reduced nanosilver. Single crystalline silver of various sizes can also be obtained by controlling the reaction time only. It is also demonstrated by studies that other factors like the presence of air (O2) can lead to dissolution of silver particles in solution and reduction of silver would compete with oxidation dissolution process resulting in slow ion nucleation and growth of nanoparticles synthesis which might affect the size, morphology and crystallinity of the synthesized nanosilver. Thus, these factors influence the yield and reactivity of the silver nanoparticle produced. The antibacterial potential of the synthesized nanoparticles was investigated by the agar well diffusion assay. Fig. 10a and b shows well defined zones of inhibition (diameter in mm) against Gram positive bacterial strain of B. subtilis and Gram negative bacterial strains of E. coli, P. aeruginosa and V. cholerae when 100 μl of 100 μg/ml of the synthesized nanoparticles were supplied to the agar wells (9 mm). In this experiment the silver nanoparticles synthesized from the leaves extract of A. indica was used as a standard as its antimicrobial potential has already been demonstrated by Nazeruddin et al. [26]. The MIC was calculated to be in between 12.5 and 25 μg/ml against Gram negative strains of E. coli, P. aeruginosa and V. cholerae and 25 μg/ml against B. subtilis for both the synthesized nanoparticles from bark extracts of F. benghalensis and A. indica. The MBC value was calculated based on the concentration of synthesized silver nanoparticles that completely inhibited any visible bacterial colony growth which was 100 μg/ml for almost all the bacterial samples. Agnihotri et al. reported similar results of MIC and MBC which were within 20 μg/ml to 200 μg/ml against MTCC

strains of E. coli and B. subtilis. The MBC/MIC ratio is a parameter that reflects the bacteriostatic and bacteriocidal capacity of the compound that can be used as an antibacterial agent [43]. However it is not feasible to compare all MBC/MIC values reported in other investigation due to various factor variations such as, initial CFU of the bacterial samples used, different origins of bacterial strains, precursor material used for the synthesis of nanoparticles, use of different reducing and capping agents and variation in the size of the silver nanoparticles used in the study [44]. In the well diffusion assay our results show slightly higher zone of inhibition (15 ± 2 mm) against gram negative strains as compared to gram positive isolates. This may be attributed to differences in structure and composition of cell wall between Gram positive and Gram negative bacteria. The thin peptidoglycan layer enveloped by the lipopolysaccharide layer lacks strength and rigidity, facilitating easy penetration of silver nanoparticles into the cells. While a Gram positive bacterium possesses a thick and rigid peptidoglycan layer in the cell wall which makes the entry of silver nanoparticles into the cell difficult [45]. Though the antimicrobial activity is very prominent by the silver nanoparticles, its mode of action is still debatable. It has been proposed that silver nanoparticles has the ability to attach with the bacterial cell membrane causing structural changes in its membrane leading to the formation of ‘pits’ where they accumulate [46]. Feng et al. and Matsumura et al. proposed that silver nanoparticles release silver ions which interact with the thiol groups of many enzymes thus inactivating most of the respiratory chain enzymes leading to the formation of reactive oxygen species (ROS) which causes the self destruction of the bacterial cell [47,48]. According

Fig. 7. Atomic force microscopy pictographs of the synthesized silver nanoparticles from the bark extract of (a) Ficus benghalensis and (b) Azadirachta indica.

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Fig. 8. XRD diffractogram of the synthesized silver nanoparticles from the bark extracts of Ficus benghalensis and Azadirachta indica. Fig. 11. Cytotoxic effect of synthesized silver nanoparticles from the bark extracts of A. indica and F. benghalensis against MG-63 osteosarcoma cell lines.

Fig .9. FTIR spectrum of the synthesized silver nanoparticles from the bark extracts of Ficus benghalensis and Azadirachta indica.

to Morones et al., silver acts as soft acid which acts upon the sulfur and phosphorus bases of DNA and inactivates its replication thus disabling the nuclear machinery of the cell [49]. It can be assumed that the surface area to volume ratio of nanoparticles is playing a crucial role in

furnishing antimicrobial activity against pathogenic bacteria. The effect of capping due to presence of phytochemicals around nanoparticles gives particular type of surface functionality to behave in a specific way to different cell types. The smaller the particles are higher is the efficiency of antimicrobial activity. The zone of inhibition also depends upon the concentration of nanoparticle and the initial bacterial number. The overall effect comes out as a result of interaction between the silver ions with that of ribosome and suppression or expression of different enzymes and proteins taking part essential roles in cell maintenance and metabolism. The effects are loss of membrane function leading to cell permeability, uncontrolled cellular transport, loss of ATP synthesis and DNA replication ability. Silver nanoparticles have promising application in therapeutic efficacy for wound healing, skin cancer and breast cancer and are being used in bone cementing material and prosthetic materials helpful in fast recovery. Therefore, in order to develop better anti-neoplastic drugs and to check if silver nanoparticle can modulate too osteosarcoma, study on antiproliferative effect of silver nanoparticles against the MG-63 osteosarcoma cell was performed. The cell viability of MG-63 cell line with green synthesized nanoparticles was determined by analyzing the spectroscopic reading of MTT assay. The inhibition of the cells (%) treated with various concentrations of silver

Fig. 10. (a) Bactericidal activities of silver nanoparticles synthesized from F. benghalensis bark (F), A. indica bark (NB) and A. indica leaf (NL) against Gram-positive and Gram-negative bacterial isolates and (b) mean zone of inhibition (diameter in mm) (n = 3).

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Fig. 12. Fluorescence microscopic image of condensed chromatin of MG-63 cells (a) control, (b) cells treated with AgNPs synthesized from bark extracts of A. indica, and (c) cells treated with AgNPs synthesized from bark extracts of F. benghalensis after 24 h of incubation.

nanoparticles is shown in Fig. 11. The IC50 values were calculated to be 81.8 ± 2.6 μg/ml and 75.5 ± 2.4 μg/ml for the silver nanoparticles synthesized from the bark extracts of A. indica and F. benghalensis respectively. To further conform the results obtained from MTT assay the chromatin condensation assay by Hoechst 33342 stain was done. After treatment of MG-63 cells with silver nanoparticles synthesized from the bark extracts of A. indica and F. benghalensis for 24 h, the plates were analyzed for observable change in the morphology of nuclei. The formation of granulations in the nuclei is a measure of chromatin condensation which is well documented in the nanoparticle treated samples. The result indicated that there were formations of more condensed chromatin after drug treatment (Fig. 12). The silver nanoparticles were found to exhibit anticancer activity against osteosarcoma cell line having nearly similar IC50 values with other previously reported results [50–53]. From the above studies it was clear that the synthesized nanoparticles have significant cytotoxic effect against the osteosarcoma cell lines which could be due to the combined effect of the size along with the bioactive molecules attached on the surface of the synthesized nanoparticles. Further studies are required to unravel the molecular mechanism behind the anticancer activity of the synthesized silver nanoparticles. 4. Conclusion Biosynthesis of silver nanoparticles from bark extracts of plant parts is relatively new and different approach. Silver nanoparticles were synthesized using bark extracts of F. benghalensis and A. indica through green synthesis method. The synthesized nanoparticles were characterized by UV-spectrophotometer, DLS, Fe-SEM, AFM and ATR-FTIR. The average particle size was around ~ 40 and ~ 50 nm respectively for both types of nanoparticles synthesized from F. benghalensis and A. indica. The AgNPs demonstrated antibacterial potential against human pathogenic Gram positive bacteria B. subtilis and Gram negative bacteria E. coli, P. aeruginosa and V. cholerae. It was observed that when compared AgNPs synthesized from bark extracts of A. indica showed better antimicrobial activity than leave parts of the same plant in terms of yield and activity. The study further demonstrated the

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