Bark extract mediated green synthesis of silver nanoparticles and their antimicrobial efficacy: A low cost effective synthesis route
Debasis Nayak, Sarbani Ashe, Pradipta Ranjan Rauta, Manisha Singh, Bismita Nayak
In this current investigation we report the biosynthesis potential of the bark extracts of Ficus benghalensis and Azadirachta indica for the synthesis of silver nanoparticles without using any external reducing or capping agent. The occurrence of dark brown color indicated the complete synthesis of the silver nanoparticles which was validated by the absorbance peak in UV-Vis spectroscopy. The morphology of the synthesized particles was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The X-ray diffraction (XRD) patterns clearly illustrated the crystalline phase of the synthesized nanoparticles. Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) was performed to identify the role of various functional groups in the nanoparticle synthesis. The synthesized sliver nanoparticles showed promising results against gram negative and gram positive pathogens which could have a broad therapeutic role against multiple drug resistant bacteria.
Keywords: Green synthesis, silver nanoparticles, scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD), antimicrobial
Silver, a noble metal maintains its exceptional optical and electronic properties in quantum size [1-2] which paved its curiosity towards the nano regime. The interest in silver nanoparticles gained prominence owing to its excellent plasmonic activity, bacteriostatic and bactericidal effects compared with the other metal nanoparticles and its versatile use in dentistry, clothing, catalysis, mirrors, optics, photography, electronics, and in the food industry .Conventional physical and chemical methods for stable nanomaterial synthesis present the problem of nanoparticle aggregation, harsh reaction conditions and the toxicity of the reagents used. So for synthesis of monodispersed and stable nanoparticles with reduced toxicity concerns new synthetic routes based on green chemistry principles have been explored [4, 5]. Synthesis of silver nanoparticles using green chemistry principles maximizes safety and efficiency, and minimises 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 . Green synthesis of silver nanoparticles using various plant extract has been reported [4, 7]. The extracts contains different enzymes/proteins, amino acids, polysaccharides, vitamins, poly phenols, etc., which act as both reducing and capping agents during the nanoparticle synthesis . Ficus benghalensis commonly known as ‘banyan’ is an evergreen tree found all over India and belongs to the family Moraceae. Its various parts are used in ayurveda for the treatment of diarrhoea, dysentery, piles, rheumatism and as an astringent, haemostatic and antiseptic agent. The bark has been reported to contain leucopelargonidin-3-O-?-L rhamnoside, leucocynidin-3-O-?-D galactosyl cellobioside, glucoside, beta glucoside, pentatriacontan-5-one, beta sitostero-?-D-glucose [9-13]. 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, G3A, NB-2 peptidoglucan [14-17]. The neem bark has antibacterial, antiviral, antifungal, anti malarial, antioxidant and anticancer activity .
Various plants parts have been used for the synthesis of silver nanoparticles but rarely the barks have been used. In the present study the barks of Ficus bengalensis and Azadirachta indica have been employed for the synthesis of silver nanoparticles. The leaves of Azadirachta were used as a reference sample as much work has been already been done on the ability of A. indica leaves for synthesis of Ag-NPs .
Silver nitrate, Mueller Hinton agar and Mueller Hinton broth of analytical grade were purchased from Hi-Media laboratories and deionised water was used throughout the experiment.
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 deionised water to remove any traces of dust and impurities. The bark extract of F. benghalensis and A. indica was prepared by dissolving 5g of the bark powder with 50 ml of distilled water and boiled in a water bath at 50?C for 1 hour. The extracts were filtered using whatman filter paper and kept at 4?C until used.
Synthesis of silver nanoparticles
90 ml of silver nitrate solution (1M) was mixed with 10 ml of bark extract and the reaction mixture was kept in a water bath at different temperature conditions (20, 40, 60 and 80?C) till the occurrence of the dark reddish color of the reaction mixture. After the color change inference the nanoparticle solutions were centrifuged at 10,000 rpm for 45 min (C24-BL centrifuge, REMI, India) thrice with successive washing with distilled water to remove any traces of un-utilized bark phyto-constituents. The resultant pellet was lyophilized and stored for further characterizations.
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 morphology of the synthesised silver nanoparticles was investigated by scanning electron microscopy (Jeol 6480LV jsm microscope). The nanoparticles were fixed on adequate support and coated with platinum using platinum sputter module in a higher vacuum evaporator. Observations under different magnifications were performed at 20kv. Further morphological studies were done by atomic force microscopy (AFM, Dimension D3100, Veeco) in contact mode under normal atmospheric conditions. The X-ray powder diffraction (XRD) patterns of silver nanoparticles was obtained using X-ray diffractometer (PANalytical X’Pert, Almelo, The Netherlands) equipped with Ni filter and Cu K? (l = 1.54056 A) radiation source. The diffraction angle was varied in the range of 20-80 degrees while the scanning rate was 0.05degree/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 ATR- FTIR 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 of the sample holder and the samples were scanned and the result obtained was analyzed through OPUS software.
The antimicrobial activity of the green synthesized AgNPs against the nosocomial Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Vibrio cholerae were investigated by agar well diffusion method. Briefly, the pathogenic strains were grown on Mueller Hinton Broth (MHB) (HI-MEDIA, Mumbai) at 37?C for 24 hours. The colony forming unit (CFU) was adjusted to 2.5 X 10-5 CFU by adjusting it with 0.5 McFarland constant and observing the OD at 600 nm in a UV-Vis Spectrophotometer . Then, the stains were swabbed onto Mueller Hinton Agar (MHA) plate (in triplicates) and wells were formed by using a cork borer. 100µl of the synthesized AgNPs were added to each well having a concentration of 1000µg/ml and the plates were incubated at 37?C for 24 hours. The mean surface area of the diameter of the inhibition zone was measured in mm.
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 and most efficient method for detecting the formation of the nanoparticle. The reaction process was followed by observing the color change as well as the absorbance maxima peak in the range of 420-460 nm. Fig. 1 shows the time taken for the total synthesis of the nanoparticles when the reaction mixture was incubated at 80 ?C. 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 minutes of incubation suggesting a very rapid synthesis route. The occurrence of the absorption peak is due to the surface plasmon resonance (SPR) property of the metallic nanoparticles which occurs due to the oscillation of free electrons on the surface of the metallic nanoparticles when they align in resonance to the wavelength of irradiated light .
Dynamic light scattering (DLS) studies were conducted to investigate the hydrodynamic size, poly dispersity index and surface zeta potential of the synthesised silver nanoparticles in a colloidal aqueous environment. When dispersed in a medium the particles move due to the Brownian motion which is measured by the fluctuations in the intensity of scattered light from which the translational diffusion co-efficient is calculated by applying the Stokes-Einstein equation which gives the hydrodynamic size of the particle . Fig. 2 (a,b) shows the size of the silver nanoparticles synthesised by bark extracts of F. benghalensis and A. indica which were 85.95 nm and 90.13 nm respectively. The poly dispersity index (PDI) is the measure of the width of the particle size distribution calculated from a cumulants 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 . The PDI value ‘0’ represents monodisperse distribution where as value ‘1’ represents polydisperse distribution. Fig. 2 (c, d) shows the surface zeta potential of the synthesized silver nanoparticles from the respective bark extracts of F. benghalensis and A. indica. Zeta potential is a measure of the magnitude of the electrostatic or charge repulsion or attraction between particles in a liquid suspension. It is one of the essential parameters for characterization of stability of the nanoparticles in an aqueous environment. Particles with zeta potentials more positive than +30 mV and more negative than ?30 mV are normally considered stable for colloidal dispersion in the absence of steric stabilization. . Table.1 shows the hydrodynamic size, PDI and zeta potential of the silver nanoparticles synthesised by bark extracts of F. benghalensis and A. indica.
Fig. 3 shows the typical image of the surface morphology of the synthesized nanoparticles by scanning electron microscopy (SEM). The roughly spherical surface morphology of the synthesized silver nanoparticles was clearly illustrated by the SEM micrographs. Fig. 4 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 7.38 nm for silver nanoparticles synthesized from bark extracts of F. benghalensis and A. indica respectively.
X-ray powder diffraction (XRD) is a non-destructive technique to identify the crystalline phase, orientation and grain size of the synthesized nanoparticles. Fig. 5 shows a typical XRD diffractogram showing Bragg peaks (angle 2?) at 32.19?, 38.15?, 44.28?, 64.46?, 77.37? and 32.11?, 37.96?, 44.18?, 64.37?, 77.23? for the silver nanoparticles synthesised 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 centred 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 synthesised nanoparticles [25-26]. The average grain size of the synthesized silver nanoparticles was determined by using Scherer’s eqn [d= K?/? cos ?] where, ‘d’ is the mean diameter of the particle; ‘K’ is the shape factor (0.9); ‘?’ is the X-ray radiation source (0.154 nm) ; ‘?’ is (?/180)* FWHM and ‘?’ is the Bragg angle  which was approx. 29 nm and 39 nm for the silver nanoparticles synthesised 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. 6 shows a typical ATR-FTIR spectrum of the silver nanoparticles synthesized from the bark extracts of F. benghalensis and A. indica. Nearly similar peaks were observed in both the synthesized silver nanoparticles some of which occurred at 3590 cm-1, 3340 cm-1, 2310 cm-1, 1693cm-1, 1519cm-1 and 615 cm-1 for silver nanoparticles synthesized from the bark extract of F. benghalensis and 3617cm-1, 3332cm-1, 2319cm-1, 1663 cm-1, 1523 cm-1, 1523 cm-1 and 635cm-1 absorption peaks occurred for silver nanoparticles synthesised 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 4 it can be clearly seen that the O-H and N-H functional group has a clear role in the fabrication of silver nanoparticles which are the main constitutional groups present in the flavonoids, terpenoids and phenols. Although the exact mechanism for the reduction of silver nanoparticles is not know Ajitha et al proposed that the flavonoids present in T. 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 . The results obtained from the mangrove leaf bud extract of R. 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.
The antibacterial potential of the synthesized nanoparticles were investigated by the agar well diffusion assay. Fig. 7 shows well defined zones of inhibition (diameter in mm) against gram positive strains of Bacillus subtilis and gram negative strains of Escherichia coli, Pseudomonas aeruginosa and Vibrio cholera when 100µl of 1000µg/ml of the synthesized nanoparticles were supplied to the agar wells (9mm). 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 . Our results show slightly higher zone of inhibition 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 . 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 . 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 [32-33]. According to Morones et al., silver acts as soft acid which acts upon the sulphur and phosphorus bases of DNA and inactivates its replication and thus inactivating the nuclear machinery of the cell .
The present study on the green synthesis of silver nanoparticles through the bark extracts of F. benghalensis and A. indica is a novel, cost-effective, environmental friendly route of synthesis having large scale production ability where no additional reducing agents or capping agents were employed for the reduction and stabilization of the nanoparticles. The synthesized nanoparticles were highly crystalline, roughly spherical in shape having mean grain size of 29 and 39 nm each. Thus with further modifications these synthesized nanoparticles can be used as suitable candidates for biomedical applications and as therapeutics for targeted drug delivery with minimal side effects. The synthesized silver nanoparticles showed enhanced antimicrobial activity against the gram negative and the gram positive bacterial strains which could boost them as antimicrobial agents with the day to day emerging cases of multiple drug resistant pathogens.
The authors would like to acknowledge Dr. Archana Mallick, Dept of Metallurgical & Materials Engineering for helping in AFM images and NIT, Rourkela for supporting and funding the current research work.