Journal of Photochemistry and Photobiology B: Biology 132 (2014) 45–55

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Photosensitized synthesis of silver nanoparticles using Withania somnifera leaf powder and silver nitrate Rajesh Warluji Raut a,⇑, Vijay Damodhar Mendhulkar a, Sahebrao Balaso Kashid b a b

Department of Botany, The Institute of Science, 15 Madame Cama Road, Mumbai 400032, Maharashtra, India Department of Chemistry, The Institute of Science, 15 Madame Cama Road, Mumbai 400032, Maharashtra, India

a r t i c l e

i n f o

Article history: Received 9 July 2013 Received in revised form 9 January 2014 Accepted 3 February 2014 Available online 12 February 2014 Keywords: Sunlight Withania somnifera Antibacterial Antifungal Blue light Silver nanoparticles

a b s t r a c t The metal nanoparticle synthesis is highly explored field of nanotechnology. The biological methods seem to be more effective; however, due to slow reduction rate and polydispersity of the resulting products, they are less preferred. In the present study, we report rapid and facile synthesis of silver nanoparticles at room temperature. The exposure of reaction mixtures containing silver nitrate and dried leaf powder of Withania somnifera Linn to direct sunlight resulted in reduction of metal ions within five minutes whereas, the dark exposure took almost 12 h. Further studies using different light filters reveal the role of blue light in reduction of silver ions. The synthesized silver nanoparticles were characterized by UV–Vis, Infrared spectroscopy (IR), Transmission Electron Microscopy (TEM), X-ray Diffraction studies (XRD), Nanoparticle Tracking Analysis (NTA), Energy Dispersive Spectroscopy (EDS), and Cyclic Voltammetry (CV). The Antibacterial and antifungal studies showed significant activity as compared to their respective standards. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Biosynthesis of metal nanoparticles using plant materials such as tissues, plant extracts and living plant has received considerable attention due to its environmentally benign nature. The method is, free from use of harsh, toxic and expensive chemicals and very cost effective, therefore can be considered as an economic and valuable alternative for the large-scale synthesis. The phytochemical constituents from plant material may act both as reducing and capping agents in nanoparticle synthesis. The bioreduction involves biomolecules found in plant extracts (e.g. enzymes, proteins, amino acids, vitamins, polysaccharides, and organic acids such as citrates secondary metabolites) hence chemically complex in nature to understand. Many researchers have reported biosynthesis of silver nanoparticles using plant materials. Irvani [1] and others [2] have reviewed the biological synthesis of silver nanoparticles using plant materials. The reduction mechanism, effect of various factors leading the different morphologies of the resulting nanoparticles has scarcely discussed. The bioreduction of the silver ions was reported by many researchers. by leaf extracts of Pelargonium graveolens [3] Azadirachta indica [4,5] Cymbopogon flexuosus Tamarindus indica [6] Aloe Vera [7] ⇑ Corresponding author. Tel.: +91 9869145425. E-mail address: [email protected] (R.W. Raut). http://dx.doi.org/10.1016/j.jphotobiol.2014.02.001 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

Coriandrum sativum Cinnamomum camphora [8] Capsicum annuum [9] Gliricidia sepium[10] pongamia pinnata [11] tea polyphenols [12] coffee and tea extract [13] Datura metel [14] latex of Jatropha curcas [15] Zingiber officinale [16] Geraniol [17] Cinnamon zeylanicum bark extract [18] Eclipta leaf [19] Cycas Leaf [20] Hibiscus rosa sinensis [21] Terminalia chebula [22] Camellia Sinensis [23] Orange peel extract [24]. It is presumed that the morphology of silver nanoparticles depend upon the major constituents of the plants extract and synthesis conditions. Therefore the possibility of controlling the nanoparticle properties and dimensions by changing the composition of the reaction mixture was invoked and led to use of different amount of biomass or plant extract and substrate concentration in order to achieve the formation of nanoparticles with desired shape and size. The other factors responsible for the control of shape and size of metallic nanoparticles are the presence of protective and reductive biomolecules. The synergistic effect of complementary factors such as radiation sensitivity was also studied [14,25–30]. The application of ‘‘green chemistry’’ principles for the nanoparticles synthesis has received considerable attention of nanoscience research community. This aspect embodies salient features such as use of benign reducing agents, eco friendliness, and low cost downstream processing. For the synthesis of silver nanoparticles different approaches are available, for example, chemical, electrochemical, radiation, photochemical methods and biological

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synthesis [31]. Apart from biological methods, all these approaches involve the use of harsh, toxic and expensive chemicals. Moreover, these methods are more energy intensive; therefore the possibilities of development of more efficient and ecofriendly method for fabrication of nanoparticles are constantly being invoked in order to achieve better control of the process and products. Among the various approaches mentioned above, biological approaches are more in compliance to the green chemistry principles [32]. The biological methods for nanoparticles synthesis reported till date include microorganisms such as bacteria and fungi as well as higher organisms like plants. Some well-known examples of bacteria synthesizing inorganic nanoparticles include magnetotactic bacteria and S-layer bacteria. There are several reports by various workers using different types of bacteria [33]. Many fungi produce silver nanoparticles intracellular or extracellular [34]. Plants as resource offer certain advantages for the nanoparticles synthesis as they are free from harmful chemicals as well as provide natural capping agents. Moreover, use of plant extracts also eliminates the grave risk of microorganisms handling. Thus, biological synthesis of silver nanoparticles using plants has been considered as a suitable alternative to chemical procedures and physical methods [1,10]. The above cited work shows considerable rise in reports pertaining to the biological synthesis of metal nanoparticles owning to its convenience. However, for the industrial scale production process optimization is necessary which requires the understanding of the reduction mechanism and the interactions of the biomolecules with metal nanoparticles surfaces. The crucial factors deciding the applicability of the nanoparticles in the commercial products are morphologies, monodispersity and size. Biosynthesis of metal nanoparticles by plants though environmentally benign, simple in operative procedure, yet chemically complex, provide an opportunity to production of nanoparticles with desired morphological characteristics and sizes. The understanding of the phenomena at the molecular level might facilitate researcher’s abilities to overcome many limitations of this field. In this article, we report the ‘rapid and Green’ method for the synthesis of silver metal nanoparticles (SNPs) using important medicinal plant Withania somnifera and possible mechanism on the basis of the role played by the phytochemical constituents present in the plant extract. Withania somnifera contains several groups of chemical constituents such as steroidal lactones, alkaloids, flavonoids and tannin. The plant system, therefore, was selected for fabrication of silver nanoparticles. Here, we also report novel strategy for the achievement of faster reaction that leads to monodispersity. When we carried out experiment under sunlight using different optical filters, the optimum reduction rate was observed under light blue radiation of the visible spectrum which is very exciting fact. The silver nanoparticles synthesized are characterized by techniques such as UV–Vis spectroscopic analysis, Transmission Electron Micrograph (TEM), X-ray Diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy, Energy Dispersive Spectroscopy (EDS), Nanoparticle Tracking Analysis (NTA), Zeta Potential Studies, and Cyclic Voltammetric Studies. The present study was also focused on the evaluation of antimicrobial potential of silver nanoparticles.

2. Materials and methods 2.1. Preparation of leaf extract The mature, undamaged and disease free leaves were selected and washed thoroughly with deionized water. The leaves were then kept for sun drying. After suns drying the leaves were finely powdered, sieved through mesh of size 15 (0.19 mm pore size). This fine powder was used for the preparation of leaf extract. For

preparation of leaf extract 0.05 g of leaf powder was added to the 100 ml deionized water in Dippy’s jar of 250 ml. The biomass was then mixed well by simple agitation and allowed to stand for 5 min. The suspension was subsequently filtered through filter paper, to remove the insoluble plant biomass. The filtrate was used as leaf extract for further experiment. 2.2. Synthesis of silver nanoparticles For the synthesis of SNPs, duplicate reaction mixtures containing 100 ml plant extract and 1 ml, 100 mM, aqueous silver nitrate (AgNO3) solutions were prepared. Two controls, one containing silver nitrate and other the leaf extract were also prepared. One set of jars containing reaction mixture along with controls were exposed to direct sunlight, while another set was kept on the laboratory table for the comparison. Same procedure was repeated to study the effect of different regions of the electromagnetic spectrum in the visible range by exposing the jars to the sunlight in the presence of dark blue, light blue, green, red and yellow optical filters. 2.3. Purification of sample The completely bio reduced sample on treatment with acetone (1:4 proportion) undergoes aggregation which can then be separated by centrifugation and redispersion. The pellet obtained was washed and re-dispersed in sterile distilled to produce nanoparticles free from biochemical constituents. 2.4. UV–Vis spectra analysis Aliquots of the reaction mixtures were quickly taken in quartz cuvette for recording the UV–Vis spectrum (190–1100 nm) on the spectrophotometer (Model-Shimadzu UV 1800). Base line correction was carried out using deionized water. Silver nitrate solution (1 mM) and leaf extract (0.05% w/v) were used as control. 2.5. Transmission Electron Microscopy (TEM) Sample of SNPs for TEM analysis was prepared by loading a drop of the nanoparticles colloids on carbon coated copper grids, blot to remove excess of solution and then was allowed to dry under Infrared light for 30 min. TEM measurements were then performed on instrument operated at an accelerating voltage at 200 kV (PHILIPS model CM 200). 2.6. X-ray Diffraction (XRD) analysis Colloidal aggregates of phytosynthesized SNPs in acetone was placed in the cavity of the glass holder and was allowed to dry. The diffraction patterns were recorded from diffraction angle range of 20–80°. The XRD studies were conducted using X-ray diffractometer (Rigaku mini flexII bench top) equipped with Cu Ka radiation source at The Institute of Science, Mumbai. 2.7. Fourier Transform infra-red (FTIR) spectroscopy Samples of silver nanoparticles, plant extract and refined silver NPs were prepared by mixing the purified sample solution with potassium bromide power (Hi media) in a mortar and pestle. The thoroughly mixed fine powder was allowed to dry and subsequently subjected to FTIR analysis on Shimadzu Japan (Model 8400S). 2.8. Electron Diffraction Spectral analysis (EDS) Sample for EDS analysis was prepared by loading a drop of the nanoparticles solution on brass stub. The drop was eventually

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allowed to dry under Infrared light (Philips) for 30 min. The EDS measurements were performed on Field Emission Scanning Electron Microscope (JEOL JSM-7600F) operating at an accelerating voltage of 15 kV with a probe current of 200 nA. 2.9. Nanoparticle Tracking Analysis (NTA) Samples of silver nanoparticles for NTA were prepared by diluting (5 ll/ml v/v) with deionized water. The diluted samples were then subjected to tracking analysis using LM 20 unit (Nanosight LM 20 UK). The nanoparticle tracking analysis was performed in order to obtained mean size of the particles. In NTA, the sample is illuminated by monochromatic LASER; the Brownian motion of nanoparticles in the solvent is monitored by the high performance CCD camera. The images obtained are the combination of the number of frames taken as a function of time. The displacement of particles from the bin center is measured, and the particles size is calculated by Stoke–Einstein equation. The measurement is mainly governed by the diffusion of the particles and hence by the temperature and viscosity of the dispersion medium and size of the particles. The NanoSight LM20 nanoparticle tracking analysis instrument (NanoSight Ltd., Amesbury, UK), as described by [35] is used to produce videos of a population of nanoparticles moving under Brownian motion in a liquid when illuminated by laser light. All sample preparation and measurements were carried out and analyzed using a beta version of NTA 2.1 software. Multiple videos, each 166 s long were recorded, analyzed in batch mode, to ensure statistical invariance. 2.10. Zeta Potential Studies (ZP) The Zeta potential analysis of purified silver nanoparticles dispersed in deionized water was performed on Malvern UK Zetasizer (Nano ZS) using clear disposable zeta cell. 2.11. Cyclic voltammetry studies Single scan cyclic voltammetry was performed using conventional three electrode system. Three electrodes used were platinum wire as counter electrode, platinum disc (1 mm2) as working electrode and silver/silver chloride as a reference electrode. 5 ml colloidal solution of metal nanoparticle was mixed with 1 ml 0.1 M KNO3 supporting electrolyte followed by purging nitrogen gas for 2 min to remove dissolved oxygen. The measurement was carried out using Versa STAT 3 potentiostat, Princeton Applied Research, UK. 2.12. Antimicrobial studies 2.12.1. Antibacterial The antibacterial activity of silver nanoparticles was studied against Staphylococcus aureus (ATCC 6538P) and Escherichia coli (ATCC 8739) by the agar disc diffusion method [36]. The 18 h overnight grown cultures were prepared in Müller-Hinton broth [composition (w/v) 30.0% beef infusion, 1.75% casein hydrolysate, 0.15% starch, pH adjusted to neutral at 25 °C]. The three replica of the respective microorganism cultures were prepared by spreading 100 ll (inoculum concentration 1.2  108 CFU/ml) of log phase culture on the Müller-Hinton agar plate [composition (w/v) 30.0% beef infusion, 1.75% casein hydrolysate, 0.15% starch and 2.0% agar pH adjusted to neutral at 25 °C] with the help of spreader. Disc of Whatman filter paper No. 40 was made with a diameter of 6 mm. The pure aliquot of plant material (25 ll) was placed on the first disc and silver nitrate (25 ll) on the second disc as control. 25 ll sample of synthesized silver nanoparticles was placed on the third disc. The fourth disc

47

was used for 25 ll Amikacin (Ampilin 250 Hetero healthcare Ltd.) with the concentration, of 125 lg/ml as standard. The Petri plates were incubated in dark at 37 °C. 2.12.2. Determination of minimum inhibition concentration (MIC) by Broth Dilution method Dilution susceptibility testing method was used to determine the minimal concentration of antimicrobial to inhibit or kill the microorganism. The Broth Dilution method is a simple procedure for determination of MIC, for its antibacterial activity. Nutrient broth (10 ml) was dispensed to each test tube. These tubes were sterilized at 121 °C, for 15 min. The nanoparticle containing stock solution (i.e. Silver colloid) was prepared using 1 mM silver nitrate solution. Stock solution of 1 mM silver nitrate and the broth containing dried leaves powder 0.5 g were taken as control. The tubes containing 10 ml of sterilized broth with 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 ml of silver nanoparticles were incubated with 0.1 ml of overnight grown culture of E. coli (ATCC39403) and Staphylococcus aureus (ATCC-25923) with 106 CFU/ml independently. The control broth and 1 mM silver nitrate was maintained in the tubes along with the experimental samples. 2.12.3. Antifungal The SNPs were tested for antifungal activity by disc diffusion method [36], against the test organisms Candida albicans (NCIM 3110) and Aspergillus niger (NCIM 1207) and Aspergillus flavus. The sterilized Sabouraud dextrose agar (SDA) medium plates were prepared and overnight grown C. albicans culture was spread with the use of sterile cotton swab. A. niger and A. flavus cultures were spread on potato dextrose agar (PDA). The antifungal activity was also evaluated against the silver nitrate (1 mM) and leaf extract as control and antibiotic fluconazole (25 lg) as a standard. Sterile discs (Hi-media, Pvt. Ltd. Mumbai) of 6 mm diameter were soaked in SNPs, dried plant extract and silver nitrate solution (1 mM). After spreading, the test organisms on plates, discs were dispensed onto the surface of the inoculated agar plate. Each disc was pressed down to ensure full contact with the agar surface. The plates were incubated at 27 °C after the placing of discs. After the overnight incubation, each plate was examined for the proper growth. The diameter of the zones of complete inhibition (as judged by the unaided eye) was measured, including the diameter of the disc. Zones were measured to the nearest whole millimeter, using sliding calipers, which is held on the back of the inverted petri plates for the measurement. 2.12.4. MIC determination by Broth Dilution method MIC (also known as minimum fungicidal concentration, MFC) of Phytosynthesized SNPs against Candida albicans (NCIM-3110), Aspergillus niger (NCIM-1207) and A. flavus was determined using the microdilution method in accordance with the guidelines (M27-A3 and M38-A respectively for both fungal types) of the Clinical Laboratory Standards Institute (CLSI). All assays were repeated in duplicate on three separate occasions. 3. Results and discussion 3.1. UV–Vis studies The UV–Vis spectra of the reaction mixture consisting 1 mM aqueous Ag+ and W. somnifera broth reveal the production of Ag nanoparticles within 5 min reaction time after Ag ions came in contact with the biomass. The bottles containing plant broth, 1 mM AgNO3 solution and the mixture of leaf broth with 1 mM AgNO3 were exposed to the sunlight. In case of the former two bottles, no color change was observed even after five minutes of exposure

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to sunlight. On the contrary, the bottles containing a mixture of the leaf broth of studied plant system with 1 mM AgNO3 solution turned brown in color after 5 min of exposure to sunlight. The appearance of a brown color to the solution containing the mixture of leaf broth with 1 mM AgNO3 clearly indicates the formation of SNPs in the reaction mixture. The color of the solution is due to the excitation of surface Plasmon vibrations in the SNPs. The exposure of the reaction mixture to the sunlight resulted in the rapid reduction of silver ions, which was confirmed by the UV–Vis studies whereas; it requires almost 24 h for complete reduction in the dark. The absorption band at 440 nm (Fig. 1) is a characteristic feature of spherical silver nanoparticle, attributed to the Surface Plasmon band of silver nanoparticles [11]. Silver nanoparticles show intense yellowish brown color in water, and this intense color arises from the surface Plasmon, which are oscillation modes arising when an electromagnetic field in the visible range is coupled to the collective oscillations of conduction electrons [10]. The environmental cues have regulatory influence on the development and behavior of plants. Sunlight as one of the environmental factor is not only source of energy for photosynthesis but also as a means of signal transduction in the various developmental activities [37]. Even though the reaction conditions such as biomass concentration, temperature, pH being optimized [1], the progress achieved in the field of fabrication is not appealing. The role of the external environmental factors such as radiation on the plant metabolism is well known. In this context, we carried out the synthesis of silver nanoparticles in the presence of sunlight. In the present study the reaction mixture containing leaf broth and silver nitrate when exposed to direct sunlight, led to rapid reduction of Ag+ ions. Further, having insights about the role of different regions of sun light in the photosensitised synthetic processes, the reaction was repeated under different optical filters. It has revealed that the reduction of silver ions is rapid in the presence of blue light filter, indicating crucial role of blue light in the reduction process. The visible region absorption of these reaction mixtures as a function of the time furnishes information regarding the rate of the reduction process (Fig. 1).

nanoparticles is composed of a large quantity of nanoparticles. At low magnification very large density of silver nanoparticles can be seen (Fig. 2a). The TEM images also show that the silver nanoparticles are in the size range from 5 to 30 nm. At higher resolution prominent capping layer of about 2–3 nm is seen around the nanoparticles (Fig. 2b).

3.3. X-ray Diffraction (XRD) analysis The XRD pattern of powder sample of phytofabricated SNPs in the studied plant system exhibited peaks at 38°, 44°, 64° and 77°, these 2h values that indexes the 1 1 1, 2 0 0, 2 2 0 and 3 1 1 facets of silver, respectively confirmed the ‘‘face centered cubic’’ (fcc) crystalline structure of metallic silver (Fig. 3). The values are in agreement with the JCPDS (Joint Committee on Powder Diffraction Standard) file No. 04-078. The diffraction patterns of fcc crystals are characterized by the presence of either an odd or even number of Miller indices [38]. Besides the peaks due to silver, there are two additional peaks with marginal intensity. These peaks might be attributable to the proteins which are capped with the SNPs [3].

3.4. Fourier Transform infra-red (FTIR) spectroscopy studies FTIR measurements were carried out to determine the potential biomolecules responsible for the reduction and capping of the silver nanoparticles synthesized. The IR spectra of leaf broth, bio-reduced silver nanoparticle and refined silver nanoparticles samples

(a)

3.2. Transmission Electron Microscopy (TEM) TEM analysis reveals that the Ag nanoparticles are predominantly spherical Fig. 2a and b. The overall morphology of the silver

1.8 Silver Nitrate Plant Extract Direct Sunlight Light Blue Filter Dark Blue Filter Green Filter Yellow Filter Red Filter

1.6 1.4

Absorbance

1.2

(b)

1.0 0.8 0.6 0.4 0.2 0.0

200 300 400 500 600 700 800 900 1000 1100 Wavelength (nm) Fig. 1. UV–Vis spectra recorded after 5 min of exposure to sunlight and in presence of various optical filters for the reaction mixture.

Fig. 2. (a and b) TEM images of SNP’s synthesized using dried leaves extract Withania somnifera.

R.W. Raut et al. / Journal of Photochemistry and Photobiology B: Biology 132 (2014) 45–55

160 140

*

100 80 *

Counts

120

60 40 20 30

40

50

60

70

80

2θ Fig. 3. XRD spectrum of SNP’s synthesized using dried leaves extract Withania somnifera.

were recorded in the range of 400–4000 cm 1. The major absorption peaks are manifested in the fig 4. IR spectra of leaf powder of Withania somnifera (Table 1, Fig. 4) shows absorption bands characteristics of functional groups such as alcohol, phenols, amines, amides, carbonyl group, CAO linkage, esters, and acids as well as characteristics absorptions of the hydrocarbon part. Though, the IR spectrum of purified SNPs shows fewer bands. The fall in number of bands for specific functional groups is attributed to the involvement of these entities in the capping, and therefore, in the stabilization. The major shifts were observed in stretching frequencies on CAO (1052.2, 1205.07– 1031.47, 1119.71 cm 1), CAN (1259.08–1267.76 cm 1), NAH (3382.29, 1530.08–3391.45, 1556.61 cm 1) and C@O of amides (1676.2–1684.39 cm 1) due to involvement of these functional sites of proteins in weak interactions with SNPs signifying protein molecules are playing a vital role in capping and stabilization of refined SNPs. Nevertheless, the presence of IR band corresponding to geminal dimethyl group does not rule out the possibility of the sitoindoside as a capping and stabilizing agents. This observation does not provide any evidence about the role of biomolecules in the reduction of silver ions, but observed bands for separated SNPs confirm their presence in capping around the particles. 3.5. Energy Dispersive X-ray Spectroscopy studies To have better analysis of the elemental composition of the phytosynthesized SNPs, the samples, were loaded on brass studs

49

instead of carbon. EDX analysis of the sample showed the presence of the elemental silver, carbon, oxygen, phosphorus, chlorine, iron and sodium (Fig. 5). The elements like iron and sodium could have come from the brass, on which the sample was prepared. The presence of the silver element is attributed to the phytosynthesized SNPs whereas, the carbon and oxygen, may be assigned to the bioorganic compounds present in the shell. The details of the elemental composition and their atomic percentage are tabulated in the inset. EDX analysis showed a peak in the silver region in the studied plant system, confirming the formation of silver nanoparticles (Fig. 5). The optical absorption peak is observed approximately at 3 keV, which is typical for the absorption of metallic silver nanocrystallites due to surface Plasmon resonance [39]. [40] reported variable minor amounts of Mg, Si, P, S Cl and Ca in the EDX analysis of gold–silver–copper alloy accumulated in Brassica juncea, which were assigned, to the capping around the metal nanoparticles. 3.6. Nanoparticle Tracking Analysis (NTA) NTA analysis of bio-reduced nanoparticles reveals the minimum mean size 28 nm with standard deviation (SD) of ±8 nm and maximum mean size 22 nm. The concentration of silver nanoparticle was found to be maximum (2.18  1008 particles/ml). The results are consistent with the TEM findings. [35] have summarized some of the applications and sample types to which NTA analysis was applied. Recently, [41] have documented specific applications of NTA for determination of size; size distributions and concentrations of particles in nanotoxicology, ecotoxicology and environmental studies. 3.7. Zeta potential analysis Zeta potential measured for the silver nanoparticles in the present study is -31.5 ± 8.19 (mV) (Table 2). The general dividing line between stable and unstable suspensions is usually taken at either +30 or 30 mV. Particles with zeta potentials more positive than +30 mV or more negative than 30 mV are usually considered stable and zeta potential more positive than 15 mV indicates suspension at the threshold of agglomeration. Coagulation or flocculation is most rapid when the zeta potential is 30 ± 3 mV [42]. The observed zeta potential indicates that the particles are extremely stable. The zeta potential of silver colloids that contain no silver ions will have a higher negative potential of about 50 mV. Higher is the ionic content, less is the negative zeta potential since positive charge of the silver ions counteract negative charge on the particles. The surface charge present on the particle gives rise to potential distribution. Zeta potential is expressed in millivolts (mV) and

Element CK OK Na K PK Cl K KK Ag L Total

Weight% 19.43 26.71 6.54 1.15 1.61 1.08 43.47 100.00

Atomic% 39.60 40.86 6.97 0.91 1.11 0.68 9.87

Fig. 4. Energy dispersive spectra of synthesized using dried leaves extract Withania somnifera.

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Table 1 The FTIR characteristic absorption frequency range for various functional groups and major absorption bands value of leaf extracts, phyto reduced SNP’s as well as refined SNP’s sample synthesized using Withania somnifera. 1

Range group

Frequency (cm

Bond

Leaf powder

SNP in broth

Separated SNPs

Functional groups of the biomolecules

I

3595–3425 (v)

)

OAH stretch

3561.19 3524.55 3478.74 3450.28

3518.76 3564.57 3448.36

3501.88 3442.57

Alcohols and phenols

II

3400–3250 (m)

NAH stretch

3276.68 3320.57 3351.91 3382.29

3275.24 3389.52

3391.45

Primary, secondary amines and amides of proteins

III

3000–2850 (m)

CAH stretch

2857.64 2888.5 2929.97

2864.39 2930.93

2857.15 2924.66

Hydrocarbon part of biomolecules

IV

2325–2500 (v)

CANH+ Stretch

2361.43

2357.57

2357.57

Charged amines

V

1750–1735 (s)

C@O stretch

1737.92

1737.44

Aldehydes, ketones and fatty acids

VI

1710–1665 (s)

C@O stretch

1676.2

1684.39

Amide I band of proteins

VII

1680–1620 (m)

AC@CA stretch

1635.69

1625.56

1638.58

Hydrocarbon part of molecule

VIII

1650–1550 (s)

NAH bending

1530.08

1538.28 1573.48

1556.61

Amide II band of proteins

IX

1500–1400 (m)

CAC stretch (in–ring)

1424.96 1463.06

1425.92 1462.57 1385.42

1418.21 1459.68 1384.45

Aromatics Aliphatic

X

1335–1250 (s)

CAN stretch

1259.08

1254.74

1267.76

Amide III band of protein

XI

1320–1000 (s)

CAO stretch

1052.2 1205.07

1030.02 1114.89

1031.47 1119.71

Alcoholic, carboxylic acids, esters and ethers functional sites of biomolecules

CAC stretch

4000

3500

3000

2500

2000

1750

1500

1250

1000

750

500

1/cm Fig. 5. Overlaid IR spectra of leaf broth, SNP’s and separated SNP’s synthesized using Withania somnifera Linn. dried leaves extract.

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R.W. Raut et al. / Journal of Photochemistry and Photobiology B: Biology 132 (2014) 45–55 Table 2 Zeta potential studies of SNP’s synthesized by Withania somnifera Linn. Mean (mV) 31.5 8.98

Peak 1: Peak 2: Good

32.3 8.84

Area (%)

Width (mV)

95.3 4.7

7.89 3.57

Current Amp

Zeta potential (mV): Zeta deviation (mV): Result quality

Current (A)

0.00001

usually falls in the range of 70 mV to +70 mV. Potentials more positive than +30 mV or more negative than 30 mV are normally considered stable and zeta potential more positive than 15 mV indicates a suspension at the threshold of agglomeration. Coagulation or flocculation is most rapid when the zeta potential is 0 mV ± 3 mV [43,44].

0.00000

-0.00001

-0.00002 -0.1 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Potential V 3.8. Cyclic voltammetry studies

Fig. 6b. Multiple scan CV of silver nanoparticles.

The cyclic voltammetry measurements were carried out at a scan rate of 50 mV/s in aqueous solution. The CV curves showed prominent anodic and poor cathodic peak (Fig. 6a). The anodic peak is ascribable to the oxidation of metal nanoparticles in the size of quantum dots at platinum disc electrode. The sharp anodic peak indicates the oxidation is rapid while the possibility of the reversible reduction of the silver metal ion is very little which might be due to the incompatible medium or the metal ions may be highly solvated. Further, fall in the anodic current for successive cycles in multiple scan CV indicates the decrease in concentration of diffusible particles (Fig. 6b). The particles of bigger size cannot diffuse towards the electrode surface. Thus the silver nanoparticles show irreversible or quasi-reversible redox behavior as shown by the wide separation of anodic and cathodic peaks with very poor cathodic peak. The contribution to the Faradic current due to the side reactions and/or other functional groups of bioactive compounds is remarkably little as the concentration is extremely low compared to the metal concentration. The above observation indicates that the silver nanoparticle shows quasi-reversible or irreversible redox behavior [45]. 3.9. Probable mechanism of bioreduction

3.10. Anti-microbial studies

Though last decade has witnessed the rapid rise in the biological synthesis of metal nanoparticles due to its convenience, the glimpse involved in the mechanism and stabilization of metal nanoparticles is not yet fully explored. The biological synthesis of

Current (A)

0.00003 0.00002

Current Amp

0.00001 0.00000 -0.00001 -0.00002 -0.00003 -0.00004 -0.00005 -0.1

0.0

0.1

0.2

0.3

metal nanoparticles using plant materials has undergone extensive exploration, but the mechanism involved is still not clear. Many researchers have proposed possible mechanisms; however, the conclusive evidences are far from reality [29]. When reaction mixture, containing silver salt and leaf extract were exposed to the sunlight, the metal ions reduced immediately. Among the various components of the visible region of EMR spectrum, the blue light is found to be more effective. Therefore, it construed that the blue light sensitive phytochemical constituents are responsible for the reduction of metal ions. In the decade of 1990, various types of blue light sensitive proteins have been reported. These proteins have serine/threonine protein kinase domains having two different structural units’ N-terminal and C-terminal domains. The N-terminal plays photo-sensory action while C terminal is responsible for ancillary action. The exact mechanism of photo-cycle and thus signal transduction is not yet fully understood [46]. Here, we emphasize that this photo receptors are playing pre dominant role in the reduction of metal ions. The reaction is schematically represented in graphical abstract.

0.4

0.5

0.6

Potential V Fig. 6a. Single scan CV of silver nanoparticles.

0.7

0.8

The synthesized nanoparticles were tested for their efficacy against the pathogens-Staphylococcus aureus (ATCC 6538P) and Escherchia coli (ATCC 8739), Candida albicans (NCIM 3110), Aspergillus niger (NCIM 1207) and Aspergillus flavus. The study shows that the biosynthesized nanoparticles have potent activity against S. aureus and A. niger, while, against the other pathogens it showed moderate activity Fig. 7a and Table 3A. The MIC for SNPs (experimental) and 1 mM silver nitrate (control) against S. aureus was found to be 1.0 ml and 2.5 ml respectively and MIC of silver colloid and silver nitrate (1 mM) against E. coli were found to be 1.5 ml and 2.5 ml respectively Table 3B. MIC determined clearly reveals that the phytosynthesized silver colloid shows superior activity against gram positive bacteria (S. aureus) as compared to gram negative bacteria (E. coli). The study clearly demonstrates that phytosynthesized SNPs shows significant activity as compared to silver ions (silver nitrate) of similar concentration. In the present study, the concentration of SNPs was determined on the basis of either the salt (silver nitrate) concentration used for the synthesis or on the basis of the number of SNPs present in the colloid. In literature, the MIC of SNPs is reported in terms of weight by volume. Synthesis of SNPs by biological route leads to the formation of silver colloids, but the conversion of colloid to powder

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(a)

(b)

Fig. 7a. Antibacterial studies against (a) E. coli and (b) S. aureus.

Table 3A MIC of phytosynthesized SNP’s by Withania somnifera Linn. Sr. No.

Name of microorganism

1 2

E. coli (ATCC-39403) S. aureus (ATCC-25923)

Minimum inhibitory concentration (lg/ml) SNP’s

Silver nitrate

1.8 ± 0.20 1.2 ± 0.14

5.0 ± 0.24 3.4 ± 0.18

Note ± = standard deviation.

form is not reported in any of the literature available. The phytosynthesized silver colloid was converted into powder form by treating the silver colloid with acetone in 1:5 ratios, followed by centrifugation at 10,000 g for 5 min. The SNPs in colloid form settle at the bottom due to treatment with acetone. After centrifugation pellet was collected and resuspended in acetone and again centrifuged. 1–2 ml acetone was added to the pellet and acetone was allowed to evaporate, and powder form was weighed. The MIC values calculated in terms of weight by volume against E. coli and S. aureus is given in Table 3B. These MIC values demonstrate the effectiveness of phytosynthesized silver nanoparticle against E. coli and S. aureus compared with silver ions (silver nitrate). Phytosynthesized SNPs exhibited better antimicrobial activity against gram-positive microorganism than gram-negative ones. The increase in fold area was assessed by calculating the average surface area of the inhibition zone of silver nitrate and phytosynthesized SNPs. The increase Fold area (c) was calculated by the equation c = a2–b2/b2, where a and b are zone of inhibitions by phytosynthesized SNPs and silver nitrate, respectively. Disk diffusion analysis of phytosynthesized SNPs against E. coli strain ATCC-39403 reveals the fold area increase of 0.30. Whereas it was 0.49 against the S. aureus strain ATCC-25923 (Tables 4A and 4B). The SNPs showed better activity against gram positive bacteria i.e. S. aureus as compared to that of gram negative bacteria E. coli strain. The findings corroborate with the reports given by [47].

On the contrary, there are reports of better activity of SNPs against gram negative bacteria (E. coli) by [48,49]. The antibacterial activities shown by SNPs synthesized using different Plant systems may differ from species to species due to variations in shape and size of SNPs, bacterial load, exposure time and nutrient media. SNPs have been widely used as antimicrobial agents; however in many products their antimicrobial mechanism is still unclear. Recently, Marambio-Jones and Hoek [50] have documented the three most common mechanisms of toxicity: (1) uptake of free silver ions followed by disruption of ATP production and DNA replication, (2) SNPs and silver ion generation of reactive oxygen species (ROS) and (3) silver nanoparticle direct damage to cell membranes. Since there is no confirmatory evidence, it is difficult to assign any one of them as operating mechanism. The nanoparticles release silver ions in the bacterial cells, which enhance their bactericidal activity. Silver ions interact with enzymes of the respiratory chain reaction such as NADH dehydrogenase resulting in the uncoupling of respiration from ATP synthesis. Silver ions also bind with transport proteins leading to proton leakage, inducing collapse of the proton motive force [51]. It also inhibits the uptake of phosphate and causes the efflux of intracellular phosphate [52]. The interaction is likely due to the high affinity of silver ions with thiol groups present in the cysteine residues of those proteins [53] and also with phosphorus containing compounds like DNA. Kim et al. [48] have provided evidence of toxicity related to ROS generated from SNPs and silver ions, released from or absorbed on its surface. They observed that the activity of SNPs against S. aureus and E. coli were abolished in the presence of an antioxidant, suggesting the formation of free radicals from the surface of SNPs and subsequent free radical induced membrane damage. SNPs can even directly damage the cell membranes. SNPs interact with the bacterial membrane and are able to penetrate inside the cell. Sondi and Salopek-Sondi [54] by TEM analysis has shown that SNPs adhere to and penetrate into E. coli cells and also are able to induce the formation of pits in the cell membrane. SNPs were

Table 3B Antibacterial activity Phytosynthesized SNP’s by Disk diffusion test. Increase in fold area (c = a2–b2/b2)

Sr. No.

Microorganism

Zone of inhibition in mm SNP’s (a)

Plant extract

Silver nitrate (b)

Amikacin (25 lg/ml)

1 2

E. coli S. aureus

08 11

–a –

07 9

14 9

All experiments were done in triplicate, and standard deviations were negligible. a In the absence of the growth inhibition zones, the disc diameters (6 mm) were used to calculate the fold area increase.

0.30 0.49

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R.W. Raut et al. / Journal of Photochemistry and Photobiology B: Biology 132 (2014) 45–55 Table 4A Antifungal activity Phytosynthesized SNP’s by Disk diffusion test. Sr. No.

Microorganism

1 2 3

C. albicans Aspergillus niger Aspergillus flavus

Increase in fold area (c = a2–b2/b2)

Zone of inhibition in mm SNP’s (a)

Plant extract

Silver nitrate (b)

Standard fluconazole disk

–a – –

– – –

7 12 7

– – –

0.36 3 0.36

All experiments were done in triplicate, and standard deviations were negligible. a In the absence of the growth inhibition zones, the disc diameters (6 mm) were used to calculate the fold area increase.

Fig. 7b. Antifungal activity against (a) A. niger, (b) A. flavus and (c) C albicans.

observed within E. coli cells, moreover, SNPs with oxidized surfaces induce the formation of ‘‘huge holes’’ in E. coli surfaces after the interaction and large portions of the cellular content seemed to be ‘‘eaten away’’ [55]. The exact, detailed mechanism by which SNPs interact with cytoplasmic membranes and are able to penetrate inside cells is not fully understood. Thiel et al. [56] have hypothesized that the interaction between nanoparticles and bacterial cells are due to electrostatic attraction between negatively charged cell membranes and positively charged silver ions. Nevertheless, this mechanism does not explain the adhesion and uptake of negatively charged SNPs. Even though, significant progress has been made to elucidate the mechanisms of silver nanomaterial toxicity, further research is required to understand the processes fully involved and to exploit the tremendous antimicrobial properties of silver safely without affecting human health, critical infrastructure, and the environment. 3.11. Antifungal Disk diffusion analysis of phytofabricated SNPs in the present plant system against C. albicans and A. flavus fungal pathogens

showed the fold area increase of 0.36, whereas it was 3.0 for A. niger (Table 4A). The inhibition zones were shown only by phytofabricated SNPs, whereas fungal filtrate, silver nitrate and antibiotics (fluconazole disc 25 lg) did not reveal any activity (Fig. 7b). SNPs are known to possess antifungal activity, which was demonstrated against the fungi such as Aspergillus niger, Candida albicans, C. glabarata, Saccharomyces cerevisia, Trichophyton mentagrophytes, Penicillium citrinum, Trichoderma sp. and Phoma glomerata. The results are conclusive with antifungal activity performed against A. niger. The MFC of the phytofabricated SNPs is reduced when tested against A. niger than C. albicans or in other words phytofabricated SNPs were more effective against A. niger compared with C. albicans and A. flavus (4.8, 4.1 and 4.9 lg/ml for C. albicans, A. niger and A. flavus respectively) Table 4B. MFC values of phytofabricated SNPs against C. albicans were similar to research carried out by Kim et al. [48]. In this study, they reported IC80 (lowest concentration that causes 80% inhibition) in the range of 1–7 lg/ml against against C. albicans (ATCC 90028), C. glabrata (ATCC 90030), C. tropicalis and T. mentagrophytes. Lower MFC values of 2 lg/ml were reported by the same group Kim et al. [57] against fungal strains S. cerevisiae (KCTC 7296), T. beigelii (KCTC 7707), C. albicans (ATCC

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Table 4B MFC of phytofabricated SNP’s by Withania somnifera against C. albicans, A. niger and A. flavus. Sr. No.

Name of Microorganism

1 2 3

C. albicans (NCIM-3110) A. niger (NCIM-1207) A. flavus

Minimum inhibitory concentration (lg/ml) SNP’s

Silver nitrate

4.8 ± 0.20 4.1 ± 0.18 4.9 ± 0.17

8.4 ± 0.24 6.8 ± 0.18 7.2 ± 0.19

Note ± = standard deviation

90028) and Raffaelea sp. Panácek et al. [58] also reported the lower MFC of SNPs against Candida sp. to be in the range of 0.21–1.69 lg/ ml. The variation in the MFC values may be due to the nature of the strain, type of isolate and physiological variation in the microbial species. Egger et al. [59] have reported the MIC of 2 mg/ml for silver nanocomposites, 125 lg/ml for silver zeolite and 15.6 lg/ml for silver nitrate against the A. niger. Finding by Tomšicˇ et al. [60] have clearly demonstrated the significant antifungal activity of SNPs against A. niger. Recently, Monteiro et al. [61] have reported that fungicidal activity is species dependent in case of yeast. These workers reported lowest MFC’s at a concentration of 0.4–0.8 lg/ ml, against C. albicans (324LA/94) and C. glabrata (ATCC 90030). On the other hand, C. albicans (ATCC 10231) and C. glabrata (D1) were found to be less sensitive, with MFC values corresponding to 0.8–1.6 lg/ml and 1.6–3.3 lg/ml, respectively. Studies on comparing the effectiveness of SNPs on bacteria and fungi are reported and most of these studies suggest a comparatively higher MIC for fungi compared to bacterial strains when studied over the same time period. 4. Conclusion In conclusion, we report simple, rapid and green method for the synthesis of silver nanoparticles. The process neither requires higher temperature boiling nor addition of stabilizing and accelerating agents. The size and shape and composition and stability have been elucidated by the TEM, EDS, NTA, IR, Zeta and CV analysis. The rapid reduction and very fine particles are key outcomes which is an advantage gained over other methods. Further, the study reveals the possible involvement of blue light sensitive phytochemical constituents responsible for the reduction of metal ions. The rapid rate of reduction is attributed to the sunlight driven electron shuttling from biomolecules to the metal ion. The silver nanoparticles show significant activity against studied bacterial and fungal species. References [1] S. Iravani, Green synthesis of metal nanoparticles using plants, Green Chem. 13 (2011) 2638. [2] R. Vaidyanathan, K. Kalishwaralal, S. Gopalram, S. Gurunathan, Nanosilver— The burgeoning therapeutic molecule and its green synthesis, Biotechnol. Adv. 27 (2009) 924–937. [3] S.S. Shankar, A. Ahmad, M. Sastry, Geranium leaf assisted biosynthesis of silver nanoparticles, Biotechnol. Progr. 19 (2003) 1627–1631. [4] S.S. Shankar, A. Rai, A. Ahmad, M. Sastry, Rapid synthesis of Au, Ag, and bimetallic Au core-Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth, J. Colloid Interface Sci. 275 (2004) 496–502. [5] T. Santhoshkumar, A.A. Rahuman, G. Rajakumar, S. Marimuthu, A. Bagavan, C. Jayaseelan, et al., Synthesis of silver nanoparticles using Nelumbo nucifera leaf extract and its larvicidal activity against malaria and filariasis vectors, Parasitol. Res. 108 (2011) 693–702. [6] B. Ankamwar, M. Chaudhary, M. Sastry, Gold Nanotriangles Biologically Synthesized using Tamarind Leaf Extract and Potential Application in Vapor Sensing, Synth. React. Inorganic, Met. Nano-Metal Chem. 35 (2005) 19–26. [7] S.P. Chandran, M. Chaudhary, R. Pasricha, A. Ahmad, M. Sastry, Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract, Biotechnol. Progr. 22 (2006) 577–583.

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