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Non-cytotoxic effect of green synthesized silver nanoparticles and its antibacterial activity
Journal of Photochemistry & Photobiology, B: Biology 177 (2017) 1–7
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Non-cytotoxic effect of green synthesized silver nanoparticles and its antibacterial activity
MARK
B. Senthila, T. Devasenaa,⁎, B. Prakashb, A. Rajasekara a b
Centre for Nanoscience and Technology, Anna University, Chennai 600025, India Kings Institute of Preventive Medicine and Research, Chennai 600032, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Silver nanoparticles AgNPs Staphylococcus aureus Escherichia coli HaCaT
Silver nanoparticles (AgNPs) were green synthesized using ethanolic extract of fenugreek leaves and characterized using UV–Vis spectroscopy, Fourier transform infra-red spectroscopy (FTIR), X-ray diffraction (XRD), high resolution transmission electron microscopy HRTEM and energy dispersive X-ray analysis (EDX) techniques. The HRTEM results revealed the formation of highly stable, mono dispersed, spherical shaped AgNPs with the size ranging from 20 to 30 nm. The presence of flavonoids and their interaction with the AgNPs were confirmed using FTIR. Antibacterial activities of the AgNPs were studied against pathogenic gram-positive Staphylococcus aureus (S. aureus) and gram negative Escherichia coli (E. coli) bacteria. The synthesized AgNPs displayed the enhancement of antibacterial activity against E. coli. The morphological changes in the bacterial cell membrane was observed using SEM analysis. Leakage of protein from the bacterial cells increased at every time intervals (2 and 4 h). MTT assay was carried out for the AgNPs against human skin cell line (HaCaT). Interestingly, cytotoxicity of the synthesized AgNPs was less toxic to HaCaT cells as compared to bacteria cells, which suggests that the synthesized AgNPs by this method is eco-friendly in nature.
1. Introduction The antimicrobial properties of silver have been well-known to the civilization all around the world for many centuries [1]. In 1893, researchers have proved that the silver has the most effective antimicrobial activity and less toxic to the living cells. Silver became most common material in medical industry, for example silver was effectively used during the World War I to prevent the microbial growth of the wounded soldiers [2–4]. The emergence of nanotechnology has made the silver compound to flourish its growth in the medicine due to its efficacy against the microbes. Nanomaterials demonstrate unique and significant physical and chemical properties as compared to bulk materials, which makes them interesting medicinal materials [5,6]. In recent years, AgNPs have been researched with keen interest because of its superior antibacterial activity. The potential applications of AgNPs includes house disinfectant, medical devices for water purification, wound treatment, sterilization, food sanitation and more recently in drug delivery [7,8]. However, exposures to environment of AgNPs cause health and ecological issues [9]. The level of impact of chemically synthesized nanoparticles in the bio solids increases their concentration in the ecosystem [10]. Specifically, nanoparticles increase the changes in
⁎
Corresponding author. E-mail address: [email protected] (T. Devasena).
http://dx.doi.org/10.1016/j.jphotobiol.2017.10.010 Received 3 August 2017; Received in revised form 4 October 2017; Accepted 6 October 2017 Available online 07 October 2017 1011-1344/ © 2017 Published by Elsevier B.V.
microbial structures, biomass and cell based enzyme activities, as well as species-specific effects on to above ground vegetation. To reduce the effect of AgNPs on ecosystem, a new innovative technique called “green synthesis” has been considered and found to be ecofriendly [11,12]. This could be used not only for the treatment procedure but also reduces the effects on the ecosystem, by increasing the biomass of the earth. Fenugreek, a spice used in Indian food has many phytochemicals possessing several pharmacological activities like anti dermatitis (AD), anticancer (AC), anti-cholesterol, anti-hepatitis, antioxidant. These phytochemicals may be used as a reducing agent to blemishes [13–15]. In this present work, a novel method was employed using the ethanolic extract of fenugreek leaves for the synthesis of AgNPs. This ethanolic extract may be an effective reducing agent as well as a better capping agent because it contains polyphenols, alkaloids, flavonols, tannins, terpenoids etc., which are nontoxic to the health and environment. The structure, morphology and chemical composition of the AgNPs were studied using XRD, HRTEM, FTIR and EDX techniques. The toxicity level of the AgNPs were observed and compared between pathogenic bacteria and HaCaT cell lines.
Journal of Photochemistry & Photobiology, B: Biology 177 (2017) 1–7
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XRD pattern was recorded using a powder X-ray diffractometer – Rigaku Mini flux II using the CuKα radiation with the wavelength (λ) of 1.5406 Å. The peak positions were matched with the existing standard data of silver (JCPDS no: 87–0720). The surface morphology and particle size were confirmed using HRTEM, FEI Tecnai TM G2 F20, USA, with the operating voltage of 120 kV. For this, a drop of AgNPs solution was dripped on a copper grid and allowed it to dry. The EDX analysis was carried out to analyze the chemical composition.
2. Materials and Methods 2.1. Chemicals Silver nitrate (AgNO3) and ethanol were procured from Alfa Aesar. The pathogenic microorganisms (S. aureus and E. coli) were acquired from the Microbial Type Culture Collection (MTCC) Centre, Chandigarh, India. The human skin cell lines (HaCaT) were purchased from NCCS, Pune, India.
2.5. Antimicrobial Activity 2.2. Preparation of Ethanolic Fenugreek Leaf Extract
The antimicrobial activities of green synthesized AgNPs were studied by well diffusion method against pathogenic organisms like gram positive S. aureus (ATCC 29736) and gram negative E. coli (ATCC 8739) bacteria. The pure cultures of organism were sub cultured on a Muller–Hinton broth at 35 °C on a rotary shaker at 200 rpm. Each strain was swabbed uniformly on the individual petri plates using sterile cotton swab. 5 wells with the diameter of 6 mm on each Muller–Hinton agar plates were made using gel puncture. 1 μL of green extract (I), AgNO3 (II), AgNPs (III) and Streptomycin control (IV) (Fig. 5) were inoculated into their respective wells. These plates were incubated at 35 °C for 24 h [17,18]. After incubation, the obtained zones of inhibition (ZOI) were measured.
Fresh fenugreek leaves were used for the preparation of ethanolic extract [16]. In order to remove the dust particles, the leaves were washed using double distilled water and dried in a room atmosphere. These leaves were further ground mechanically to make a fine powder. 5 g of fine powder was added into a beaker which contains 100 ml of 90% ethanol. The solution was kept in a shaker at 30 °C for 15 h, and then it was filtered using Whatman filter paper. The final solution was kept in a freezer at 4 °C for further studies. 2.3. Synthesis of Silver Nanoparticles Initially, 5 ml of ethanolic fenugreek extract was added to 100 ml of 1 mM AgNO3 in an Erlenmeyer flask at room temperature (30 °C). This solution was incubated at 37 °C under continuous stirring (200 rpm) and the bioreduction of AgNPs was monitored, at a regular interval was done up to 96 h (1, 3, 6, 12, 24, 48 and 96 h). The color change was observed from yellow to dark brown, which indicated the bio reduction of AgNPs (Fig. 1). After the completion of the bio reduction, the synthesized nanoparticles were centrifuged at 10,000 rpm for 30 min to remove residues. This bioreduction of AgNPs were attained without utilizing any polymers as a stabilizing and capping agents. Synthesis of AgNPs was continuously monitored at regular intervals as mentioned above using UV–Vis spectrophotometer.
2.6. Minimal Inhibition Concentration of Ag-NPs The minimum inhibitory concentration (MIC) of Ag-NPs was evaluated using the plate count method. AgNPs sample was weighed and sterilized, by using UV radiation for 1 h. Further, Mueller-Hinton broth containing 105 CFU/ml of S. aureus and E. coli bacterial cells were used as a culture medium. Subsequently the AgNPs were diluted as follows 100, 50, 25, 12.5, 6.25, 3.12, 1.5, 0.78, 0.39 and 0.19 μg/ml. After incubation, the number of colonies on the agar was counted [19]. The experiments were performed in triplicates. 2.7. Protein Leakage Assay of Ag-NPs
2.4. Characterization of Silver Nanoparticles
Protein leakage from bacterial cells was detected using Bradford's protein assay. The known concentration of Ag-NPs was treated with S. aureus and E. coli bacterial cells for about 2 and 4 h. The samples were centrifuged at 4 °C for 30 min at 300 ×g, and the supernatant was frozen at − 20 °C. The supernatant was treated with Bradford's reagent, and then incubated for 15 min. The optical density (OD) was measured at 595 nm using T90 UV–Vis spectrometer. For this experiment, Bovine serum albumin (BSA) was used as the standard protein [20]. Experiments were done in triplicate.
The green synthesized AgNPs was confirmed using UV–Visible spectrophotometer (T90 UV–Vis spectrophotometer, PG instruments Ltd). IR spectrum was obtained using Perkin Elmer FTIR spectrometer with the wavelength from 400 to 4000 cm− 1 at a resolution of 4 cm− 1.
2.8. Imaging of Bacteria by Scanning Electron Microscope (SEM) SEM analysis was used to observe the morphological changes of bacterial cells treated with AgNPs. Bacterial cells (106 CFU/ml) were treated with 10 μg/ml of Ag-NPs for 2 h and 4 h. This solution was centrifuged at 3000 ×g for 30 min. Then the pellets were washed with phosphate buffered saline (PBS) for three times and pre-fixed with 2.5% glutaraldehyde for 30 min. The pre-fixed cells were washed with PBS for two times and post-fixed with 1% Osmium tetroxide for 1 h. Again this solution was three times washed with PBS, then dehydration process was conducted with 100% of ethanol [7,21]. The fixed cell was dried and gold-coated using ion sputter for conducting purpose. The pre-treated samples were subjected to SEM (VEGA 3 Tescan) analysis. 2.9. MTT Assay Fig. 1. UV–Vis spectra of AgNPs synthesized using fenugreek leaves ethanolic extract at different time periods (1 to 96 h) and dot arrow mark shows the increase in the formation of AgNPs. Inlet, the photographic image indicates (a) ethanolic fenugreek leaves extract and (b) AgNPs.
Synthesized AgNPs were used for the assessment of cytotoxicity against HaCaT cell lines measured by MTT (3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay [22]. The MTT assay is a 2
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colorimetric and nonradioactive assay for measuring the cell viability through increased metabolism of tetrazolium salt. The HaCaT cells were plated separately in 96 well plates at a concentration of 105 cells/well. After 24 h, cells were washed twice with 100 μL of serum-free medium and starved for an hour at 37 °C. After starvation, cells were treated with different concentrations of AgNPs (10–500 μg/ml) for 24 h. At the end of the treatment, period the medium was aspirated and serum free medium containing MTT (0.5 mg/ml) was added and incubated for 4 h at 37 °C in a CO2 incubator. The MTT containing medium was then discarded and the cells were washed with PBS (200 μL). Then the crystals were dissolved in 100 μL of DMSO and this was mixed properly by pipetting up and down. Spectrophotometrical absorbance of the purple blue formazan dye was measured in a microplate reader at 570 nm (Biorad 680). The Cytotoxicity results were plotted using Graph pad prism 6 software. 2.10. Statistical Analysis All the experiments were done in triplicate. Data were analyzed using one-way analysis of variance (ANOVA) to determine statistical significance. p values of < 0.05 were considered statistically significant. Graph Pad Prism 6 software was used for the statistical analyses.
Fig. 2. FTIR spectrum of silver nanoparticle synthesis using ethanolic fenugreek extract.
3. Results and Discussion Formation of a stable AgNPs was confirmed using UV–Visible spectrum (Fig. 1). The reduction of AgNO3 to AgNPs using ethanolic extracts of fenugreek leaf was monitored at regular intervals of 1, 3, 6, 12, 24, 48 and 96 h. Fig. 1 shows a gradual increase in the absorbance values, which indicates the formation of AgNPs. An optical absorption band at 410 nm for AgNPs was observed which is due to the surface plasma resonance (SPR) of silver. This SPR absorption band is due to the stimulation of free electron in the outermost orbitals of AgNPs [23]. Evaluation of reaction time for the formation of the AgNPs was noted, as the time of the reaction increases the peak becomes intense and sharper as shown in Fig. 1. Thus, the increase in the absorbance indicates the formation of AgNPs. The inset image in Fig. 1 shows the visual observation of the AgNO3, extract and the green synthesized AgNPs, which clearly shows the dark brown color. Initially, the AgNO3 was colorless solution and the extract was green in color, and finally the synthesized AgNPs was dark brown in color which clearly shows the formation of AgNPs. The FTIR spectra of pure fenugreek leaf extract and green synthesized AgNPs were recorded and the resulting graphs are illustrated in Fig. 2. The pure extract (Fig. 2a) shows five major peaks at 3326, 2974, 1374, 1050 and 868 cm− 1. The band at 3326 cm− 1 was due to the stretching vibrations of the OH functional group. The peak at 2974 cm− 1 corresponds to the strong stretching vibration of CeH functional groups. The band at 1050 cm− 1 corresponds to various functional groups such as alcohol, ether, ester, carboxylic acid, anhydrides. The peak at 868 cm− 1 is due to the CH3 deformations of isopropyl group. FTIR spectrum of AgNPs synthesized using fenugreek extract (Fig. 2b) shows two major peaks at 3307 and 1637 cm− 1. It is noteworthy that the peak shift was observed at 3307 cm− 1 and simultaneously a new peak was seen at 1637 cm− 1. Intensive review on fenugreek reveals that polyphenols are their predominate constituents [24,25]. FTIR spectra shown in the Fig. 2 also reveals the predominance of OH and C]C moieties on the surface of the AgNPs. This is the signature of polyphenols which forms the major constituent phytochemical of the fenugreek leaves. In addition, our previous characterization report on green synthesized AgNPs using fenugreek also revealed the same type of signature [26,27]. The XRD pattern was obtained to confirm the crystalline nature of the synthesized AgNPs. Fig. 3 shows the diffraction peaks of the AgNPs which indicates the polycrystalline nature. The major peaks were
Fig. 3. X-ray diffraction pattern of AgNPs synthesized using fenugreek leaves ethanolic extract.
observed at 2θ = 38.36°, 44.36°, 64.51° and 77.41° which corresponds to the (111), (200), (220), and (311) planes, respectively [28]. These peaks are well attributed to the standard JCPDS data of the silver with FCC crystal lattice structure (JCPDS NO: 87-0720). In addition, the peak observed at 29° might be due to unreduced AgNO3 peaks. Using Debye Scherrer formula, the average crystallite size was estimated around 18 nm from the major diffraction peaks. Surface morphology and crystallography of the AgNPs were studied using HRTEM. Fig. 4 shows the HRTEM images with different magnifications. The results clearly show that the AgNPs were exhibit spherical shape, uniform size and well crystalline nature. The size of the AgNPs was ranging from 20 to 30 nm. Crystallography and crystalline size are in good agreement with the XRD results (Fig. 3) [29]. It can be seen that, agglomeration of the NPs was not observed because of the encapsulation of the AgNPs with fenugreek extract. This encapsulation may be due to the presences of alkaloids, flavonoids, polyphenols, etc., presented naturally in the fenugreek plants. The selected area electron diffraction (SAED) pattern is illustrated in the Fig. 4d. The SAED pattern of AgNPs, exhibits the concentric rings indexed corresponding to the (111), (200), (220), and (311) planes of FCC silver phase. The SAED pattern is in good agreement with the ascribed result of XRD, which also suggest the similar reflections of the AgNPs [18]. The antibacterial activity of the ethanolic fenugreek extract, silver
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Fig. 4. HRTEM images of spherical shaped AgNPs synthesized using fenugreek leaves ethanolic extract at different scale bars (a) 100 nm, (b) 30 nm, (c) 5 nm shows the lattice arrangement (d) SAED pattern of AgNPs and (e) energy dispersive X-ray spectrum shows presence of elemental silver. Fig. 5. Antibacterial activity on S. aureus (a) and E. coli (b) bacteria, I. extract, II. AgNO3, III. AgNPs and IV. Streptomycin antibiotic. The results are expressed as the mean ± SD of triplicates.
16.27 mm and 12.47 mm respectively [18,30]. Instantaneously, there was zero ZOI for ethanolic fenugreek leaves extract at in well I. While, the ZOI for AgNO3 in the well II was about 5.97 mm for S. aureus and 6.67 mm for E. coli at a concentration of 1 μL, which was trivial activity than the synthesized AgNPs. Thus, the results reveal that gram negative E. coli shows the maximum antibacterial activity than gram positive S. aureus (Table 1) [31]. The MIC was used to determine the lowest concentrations that completely inhibit the growth of bacterial cells. The MIC level of AgNPs to inhibit the S. aureus growth was 12.5 μg/ml, whereas, this value was lower for inhibition of E. coli growth about 6.25 μg/ml [30]. The antibacterial activity and MIC of the AgNPs against the gram-positive S. aureus and gram negative E. coli were found to be almost identical. As per statistical analysis we could observe that the AgNPs induces time dependent release of protein from the target bacteria. The leakage was maximum at 4th hour confirming cell death. It was found that AgNPs could enhance the protein leakage by increasing the membrane permeability of S. aureus and E. coli cell wall,
Table 1 Antibacterial activity of synthesized AgNPs against S. aureus and E. coli. ZOI was measured as millimeter ± standard deviation for three independent experiments; (–) shows no activity. Test organism
Crude extract (1 μL)
Silver nitrate (1 μL)
Silver NPs (1 μL)
Streptomycin (1 μL)
Staphylococcus aureus Escherichia coli
–
5.97 ± 0.15
12.47 ± 0.31
9.47 ± 0.21
–
6.67 ± 0.25
16.27 ± 0.51
10.12 ± 0.10
nitrate, AgNPs was studied against S. aureus and E. coli bacteria. AgNPs synthesized from ethanolic fenugreek leaves extract shows considerable antibacterial activity against the test pathogens (Fig. 5). The AgNPs in well III of both the bacteria had comparatively good growth of inhibition when compared with the control antibiotic streptomycin. The ZOI for streptomycin was about 9.41 mm (S. aureus) and 10.12 mm (E. coli), whereas, the ZOI of AgNPs in E. coli and S. aureus was found to be 4
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Fig. 6. Leakage of protein from the bacterial cells S. aureus (a) and E. coli (b) treated with different concentrations of AgNPs at 0, 2 and 4 h. Values represents mean ± SD of three replication. Statistically significant at p < 0.05.
Fig. 7. Scanning electron microscope images of control (untreated) cells (a) S. aureus and (d) E. coli, and treated with AgNPs at 2 and 4 h. (b and c) S. aureus and (e and f) E. coli.
was lower than that of the gram-negative E. coli. This difference was possibly attributable to the thickness of the peptidoglycan layer of S. aureus. The essential function of the peptidoglycan layer is to protect against antibacterial agents such as antibiotics, toxins, chemicals, and degradative enzymes, and hence, lower protein leakage of S. aureus than E. coli [20,31]. The SEM analysis was used to observe the morphological changes on the surface of the cells treated with AgNPs (Fig. 7). The changes on the bacterial cells by AgNPs were observed at two different time intervals (2 and 4 h) of similar concentration. To the best of our knowledge, no reports are available on time dependent experiments. The SEM image of the control group S. aureus cells were characteristically grape shaped (Fig. 7a) where, the surface of the bacteria cells are intact and no damage was observed. After the cells were treated with AgNPs, the morphology of the cell changes showing the damage of cell membrane due to the attachment of nanoparticles (Fig. 7b and c) [32]. The control
which are shown in the Figs. 5 and 6 respectively. Initially, protein leakage from the S. aureus cell membranes treated with AgNPs was almost similar as that from cells in the control group (Fig. 6a). After 2 and 4 h of incubation, protein leakage from cells treated with AgNPs were considerably increased, however, there is no change in the amount of protein leakage from the cells of control group (Fig. 6b). Leakage from cells treated with AgNPs was significantly higher than that of cells in the control group. Furthermore, the initial protein leakage from the membranes of E. coli cells treated with AgNPs was almost similar as that of cells in the control group as shown in Fig. 6. After 2 and 4 hours incubation, protein leakage from E. coli cells treated with AgNPs was significantly increased as compared to the cells from control group, which may be due to the increase in the membrane permeability caused by AgNPs (Fig. 6). Notably, higher quantity of proteins leaked through the E. coli membranes than those of S. aureus membranes, suggest that the sensitivity of the gram-positive S. aureus 5
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Fig. 8. In vitro cytotoxicity effect of green synthesized AgNPs in HaCaT cell lines. (a) Control, (b) 5 μg/ml (c) 250 μg/ml of AgNPs treatment and (d) percentage of cell viability at various concentration of AgNPs. Values represent mean ± SD of three replications. Statistically significant at p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of the AgNPs against HaCaT cells were detected by MTT assay. The cell viability percentage pertaining to serial concentrations of the particles is shown in the Fig. 8. The morphology of the cells treated with the lowest dose and the highest dose were compared with untreated control cells using the optical microscopic images (Fig. 8). In HaCat cells, we identified less cytotoxic effect at the highest concentration (250 μg/ml) of AgNPs. Nonetheless, the same concentration of AgNPs showed a dramatic cytotoxic effect in bacterial cells. Earlier reports stated that the chemically synthesized AgNPs shows a 30% viability towards the epithelium cell lines [36]. This remains noteworthy that the less cytotoxic effect was observed at 250 μg/ml of AgNPs concentration. In this study, AgNPs synthesized using ethanolic extract showed 96% of HaCaT cell viability. Optical microscopic image supports our findings as it showed no morphological alterations in HaCat cells, suggesting that the AgNPs kill bacterial cells alone [37,38].
group of E. coli cells are typically rod shaped (Fig. 7d) with uniform size with no cell surface damage. After AgNPs treatment, irregular fragments were formed (Fig. 7e and f). Report by Kim et al. [20] also suggest that AgNPs have the ability to form free radicals (Reactive oxygen species-ROS), which inturn are capable of inducing membrane damage. Thus, the fragments seen in Fig. 7e and f may be due to the increased oxidative stress in the cell membrane which was caused by AgNPs induced ROS [33]. This study showed not only morphological changes of cell surface but also the time-dependent damage of cell membranes. The damage of the cell membrane could further be confirmed from leakage of cytoplasmic cell contents. The antimicrobial effect of the AgNPs on gram-negative bacteria was dependent on the concentration of Ag in the nanoparticles and it is closely related to the formation of “pits” in the cell walls [34,35]. Kim et al. have reported that Ag-NPs have the ability to form free radicals [20]. Induction of membrane damage by lipid peroxidation is the wellknown feature of free radicals (ROS). Hence we interpret that the permeability of the bacterial cell membrane damage as shown in SEM image may be due to the free radicals generated by the AgNPs. The free radicals induce oxidative stress and cause damage to the cell membrane and the vital biomolecules like lipid, protein and DNA of the intracellular system. The protein leakage from the AgNPs-treated bacterial cells observed in our study (Fig. 6) also supports the membrane damage. This study showed an evidence for concentration and time dependent increase in the protein leakage (Fig. 6) and membrane permeability (Fig. 7). Interestingly, there were some differences between gram positive S. aureus and gram negative E. coli. The S. aureus showed less bactericidal activity than E. coli. This difference might be due to the variation in the thickness of the peptidoglycan layer present on the cell membrane as mentioned above. The gram positive cells, consist of lipoteichoic acid containing a thick and multiple peptidoglycan layer (30 to 100 nm) and cell membrane. But, the gram negative cells consist of a thin layer of peptidoglycan (few nm) [31,32]. The thickness of this peptidoglycan layer plays an important role to protect the cells from the formation of pits or ROS caused by AgNPs. From the current study, it was found that the AgNPs synthesized by ethanolic extract exhibits antimicrobial activity by damaging the cell membranes. This encouraged us to study the AgNPs activity on human cell lines to find out its safety profile. The cytotoxic effect towards HaCaT cells, well known as immortalized human keratinocyte cell lines, was performed using AgNPs at various concentrations. The cytotoxicity
4. Conclusion AgNPs synthesized from ethanolic extract of fenugreek leaves is a novel greener method, which is simple and potential way to develop a sustainable nanoparticle with less toxicity than any other methods, thus being environmental friendly. The UV–Visible spectroscopy, FTIR, XRD, TEM and EDX confirmed the formation and well dispersed nature of the AgNPs. The ethanolic extract of AgNPs has potent antibacterial activities against S. aureus and E. coli bacteria. The maximum ZOIs were found to be 12 and 16 mm for S. aureus and E. coli, respectively. The growth and reproduction of AgNPs treated bacterial cells were rapidly inhibited. AgNPs showed higher antibacterial activity against gram negative E. coli than gram positive S. aureus. The morphological changes on bacterial cells treated with AgNPs at 2 and 4 h were observed by SEM. The cytotoxicity towards the HaCaT cell lines were lesser compared with the cytic effect towards the S. aureus and E. coli bacteria. This study specifies that AgNPs can be used as effective antibacterial materials without affecting epithelial cells thus proving its non-toxicity. References [1] N. Silvestry Rodriguez, E. Sicairos-Ruelas, C. Gerba, K. Bright, Silver as a desinfectant, Rev. Environ. Contam. Toxicol. 191 (2007) 23–45, http://dx.doi.org/10. 1007/978-0-387-69163-3. [2] B.E. Wildt, A. Celedon, E.I. Maurer, B.J. Casey, A.M. Nagy, S.M. Hussain, P.L. Goering, Intracellular accumulation and dissolution of silver nanoparticles in L929 fibroblast cells using live cell time-lapse microscopy, Nanotoxicology 5390
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Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Non-cytotoxic effect of green synthesized silver nanoparticles and its antibacterial activity
MARK
B. Senthila, T. Devasenaa,⁎, B. Prakashb, A. Rajasekara a b
Centre for Nanoscience and Technology, Anna University, Chennai 600025, India Kings Institute of Preventive Medicine and Research, Chennai 600032, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Silver nanoparticles AgNPs Staphylococcus aureus Escherichia coli HaCaT
Silver nanoparticles (AgNPs) were green synthesized using ethanolic extract of fenugreek leaves and characterized using UV–Vis spectroscopy, Fourier transform infra-red spectroscopy (FTIR), X-ray diffraction (XRD), high resolution transmission electron microscopy HRTEM and energy dispersive X-ray analysis (EDX) techniques. The HRTEM results revealed the formation of highly stable, mono dispersed, spherical shaped AgNPs with the size ranging from 20 to 30 nm. The presence of flavonoids and their interaction with the AgNPs were confirmed using FTIR. Antibacterial activities of the AgNPs were studied against pathogenic gram-positive Staphylococcus aureus (S. aureus) and gram negative Escherichia coli (E. coli) bacteria. The synthesized AgNPs displayed the enhancement of antibacterial activity against E. coli. The morphological changes in the bacterial cell membrane was observed using SEM analysis. Leakage of protein from the bacterial cells increased at every time intervals (2 and 4 h). MTT assay was carried out for the AgNPs against human skin cell line (HaCaT). Interestingly, cytotoxicity of the synthesized AgNPs was less toxic to HaCaT cells as compared to bacteria cells, which suggests that the synthesized AgNPs by this method is eco-friendly in nature.
1. Introduction The antimicrobial properties of silver have been well-known to the civilization all around the world for many centuries [1]. In 1893, researchers have proved that the silver has the most effective antimicrobial activity and less toxic to the living cells. Silver became most common material in medical industry, for example silver was effectively used during the World War I to prevent the microbial growth of the wounded soldiers [2–4]. The emergence of nanotechnology has made the silver compound to flourish its growth in the medicine due to its efficacy against the microbes. Nanomaterials demonstrate unique and significant physical and chemical properties as compared to bulk materials, which makes them interesting medicinal materials [5,6]. In recent years, AgNPs have been researched with keen interest because of its superior antibacterial activity. The potential applications of AgNPs includes house disinfectant, medical devices for water purification, wound treatment, sterilization, food sanitation and more recently in drug delivery [7,8]. However, exposures to environment of AgNPs cause health and ecological issues [9]. The level of impact of chemically synthesized nanoparticles in the bio solids increases their concentration in the ecosystem [10]. Specifically, nanoparticles increase the changes in
⁎
Corresponding author. E-mail address: [email protected] (T. Devasena).
http://dx.doi.org/10.1016/j.jphotobiol.2017.10.010 Received 3 August 2017; Received in revised form 4 October 2017; Accepted 6 October 2017 Available online 07 October 2017 1011-1344/ © 2017 Published by Elsevier B.V.
microbial structures, biomass and cell based enzyme activities, as well as species-specific effects on to above ground vegetation. To reduce the effect of AgNPs on ecosystem, a new innovative technique called “green synthesis” has been considered and found to be ecofriendly [11,12]. This could be used not only for the treatment procedure but also reduces the effects on the ecosystem, by increasing the biomass of the earth. Fenugreek, a spice used in Indian food has many phytochemicals possessing several pharmacological activities like anti dermatitis (AD), anticancer (AC), anti-cholesterol, anti-hepatitis, antioxidant. These phytochemicals may be used as a reducing agent to blemishes [13–15]. In this present work, a novel method was employed using the ethanolic extract of fenugreek leaves for the synthesis of AgNPs. This ethanolic extract may be an effective reducing agent as well as a better capping agent because it contains polyphenols, alkaloids, flavonols, tannins, terpenoids etc., which are nontoxic to the health and environment. The structure, morphology and chemical composition of the AgNPs were studied using XRD, HRTEM, FTIR and EDX techniques. The toxicity level of the AgNPs were observed and compared between pathogenic bacteria and HaCaT cell lines.
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XRD pattern was recorded using a powder X-ray diffractometer – Rigaku Mini flux II using the CuKα radiation with the wavelength (λ) of 1.5406 Å. The peak positions were matched with the existing standard data of silver (JCPDS no: 87–0720). The surface morphology and particle size were confirmed using HRTEM, FEI Tecnai TM G2 F20, USA, with the operating voltage of 120 kV. For this, a drop of AgNPs solution was dripped on a copper grid and allowed it to dry. The EDX analysis was carried out to analyze the chemical composition.
2. Materials and Methods 2.1. Chemicals Silver nitrate (AgNO3) and ethanol were procured from Alfa Aesar. The pathogenic microorganisms (S. aureus and E. coli) were acquired from the Microbial Type Culture Collection (MTCC) Centre, Chandigarh, India. The human skin cell lines (HaCaT) were purchased from NCCS, Pune, India.
2.5. Antimicrobial Activity 2.2. Preparation of Ethanolic Fenugreek Leaf Extract
The antimicrobial activities of green synthesized AgNPs were studied by well diffusion method against pathogenic organisms like gram positive S. aureus (ATCC 29736) and gram negative E. coli (ATCC 8739) bacteria. The pure cultures of organism were sub cultured on a Muller–Hinton broth at 35 °C on a rotary shaker at 200 rpm. Each strain was swabbed uniformly on the individual petri plates using sterile cotton swab. 5 wells with the diameter of 6 mm on each Muller–Hinton agar plates were made using gel puncture. 1 μL of green extract (I), AgNO3 (II), AgNPs (III) and Streptomycin control (IV) (Fig. 5) were inoculated into their respective wells. These plates were incubated at 35 °C for 24 h [17,18]. After incubation, the obtained zones of inhibition (ZOI) were measured.
Fresh fenugreek leaves were used for the preparation of ethanolic extract [16]. In order to remove the dust particles, the leaves were washed using double distilled water and dried in a room atmosphere. These leaves were further ground mechanically to make a fine powder. 5 g of fine powder was added into a beaker which contains 100 ml of 90% ethanol. The solution was kept in a shaker at 30 °C for 15 h, and then it was filtered using Whatman filter paper. The final solution was kept in a freezer at 4 °C for further studies. 2.3. Synthesis of Silver Nanoparticles Initially, 5 ml of ethanolic fenugreek extract was added to 100 ml of 1 mM AgNO3 in an Erlenmeyer flask at room temperature (30 °C). This solution was incubated at 37 °C under continuous stirring (200 rpm) and the bioreduction of AgNPs was monitored, at a regular interval was done up to 96 h (1, 3, 6, 12, 24, 48 and 96 h). The color change was observed from yellow to dark brown, which indicated the bio reduction of AgNPs (Fig. 1). After the completion of the bio reduction, the synthesized nanoparticles were centrifuged at 10,000 rpm for 30 min to remove residues. This bioreduction of AgNPs were attained without utilizing any polymers as a stabilizing and capping agents. Synthesis of AgNPs was continuously monitored at regular intervals as mentioned above using UV–Vis spectrophotometer.
2.6. Minimal Inhibition Concentration of Ag-NPs The minimum inhibitory concentration (MIC) of Ag-NPs was evaluated using the plate count method. AgNPs sample was weighed and sterilized, by using UV radiation for 1 h. Further, Mueller-Hinton broth containing 105 CFU/ml of S. aureus and E. coli bacterial cells were used as a culture medium. Subsequently the AgNPs were diluted as follows 100, 50, 25, 12.5, 6.25, 3.12, 1.5, 0.78, 0.39 and 0.19 μg/ml. After incubation, the number of colonies on the agar was counted [19]. The experiments were performed in triplicates. 2.7. Protein Leakage Assay of Ag-NPs
2.4. Characterization of Silver Nanoparticles
Protein leakage from bacterial cells was detected using Bradford's protein assay. The known concentration of Ag-NPs was treated with S. aureus and E. coli bacterial cells for about 2 and 4 h. The samples were centrifuged at 4 °C for 30 min at 300 ×g, and the supernatant was frozen at − 20 °C. The supernatant was treated with Bradford's reagent, and then incubated for 15 min. The optical density (OD) was measured at 595 nm using T90 UV–Vis spectrometer. For this experiment, Bovine serum albumin (BSA) was used as the standard protein [20]. Experiments were done in triplicate.
The green synthesized AgNPs was confirmed using UV–Visible spectrophotometer (T90 UV–Vis spectrophotometer, PG instruments Ltd). IR spectrum was obtained using Perkin Elmer FTIR spectrometer with the wavelength from 400 to 4000 cm− 1 at a resolution of 4 cm− 1.
2.8. Imaging of Bacteria by Scanning Electron Microscope (SEM) SEM analysis was used to observe the morphological changes of bacterial cells treated with AgNPs. Bacterial cells (106 CFU/ml) were treated with 10 μg/ml of Ag-NPs for 2 h and 4 h. This solution was centrifuged at 3000 ×g for 30 min. Then the pellets were washed with phosphate buffered saline (PBS) for three times and pre-fixed with 2.5% glutaraldehyde for 30 min. The pre-fixed cells were washed with PBS for two times and post-fixed with 1% Osmium tetroxide for 1 h. Again this solution was three times washed with PBS, then dehydration process was conducted with 100% of ethanol [7,21]. The fixed cell was dried and gold-coated using ion sputter for conducting purpose. The pre-treated samples were subjected to SEM (VEGA 3 Tescan) analysis. 2.9. MTT Assay Fig. 1. UV–Vis spectra of AgNPs synthesized using fenugreek leaves ethanolic extract at different time periods (1 to 96 h) and dot arrow mark shows the increase in the formation of AgNPs. Inlet, the photographic image indicates (a) ethanolic fenugreek leaves extract and (b) AgNPs.
Synthesized AgNPs were used for the assessment of cytotoxicity against HaCaT cell lines measured by MTT (3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay [22]. The MTT assay is a 2
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colorimetric and nonradioactive assay for measuring the cell viability through increased metabolism of tetrazolium salt. The HaCaT cells were plated separately in 96 well plates at a concentration of 105 cells/well. After 24 h, cells were washed twice with 100 μL of serum-free medium and starved for an hour at 37 °C. After starvation, cells were treated with different concentrations of AgNPs (10–500 μg/ml) for 24 h. At the end of the treatment, period the medium was aspirated and serum free medium containing MTT (0.5 mg/ml) was added and incubated for 4 h at 37 °C in a CO2 incubator. The MTT containing medium was then discarded and the cells were washed with PBS (200 μL). Then the crystals were dissolved in 100 μL of DMSO and this was mixed properly by pipetting up and down. Spectrophotometrical absorbance of the purple blue formazan dye was measured in a microplate reader at 570 nm (Biorad 680). The Cytotoxicity results were plotted using Graph pad prism 6 software. 2.10. Statistical Analysis All the experiments were done in triplicate. Data were analyzed using one-way analysis of variance (ANOVA) to determine statistical significance. p values of < 0.05 were considered statistically significant. Graph Pad Prism 6 software was used for the statistical analyses.
Fig. 2. FTIR spectrum of silver nanoparticle synthesis using ethanolic fenugreek extract.
3. Results and Discussion Formation of a stable AgNPs was confirmed using UV–Visible spectrum (Fig. 1). The reduction of AgNO3 to AgNPs using ethanolic extracts of fenugreek leaf was monitored at regular intervals of 1, 3, 6, 12, 24, 48 and 96 h. Fig. 1 shows a gradual increase in the absorbance values, which indicates the formation of AgNPs. An optical absorption band at 410 nm for AgNPs was observed which is due to the surface plasma resonance (SPR) of silver. This SPR absorption band is due to the stimulation of free electron in the outermost orbitals of AgNPs [23]. Evaluation of reaction time for the formation of the AgNPs was noted, as the time of the reaction increases the peak becomes intense and sharper as shown in Fig. 1. Thus, the increase in the absorbance indicates the formation of AgNPs. The inset image in Fig. 1 shows the visual observation of the AgNO3, extract and the green synthesized AgNPs, which clearly shows the dark brown color. Initially, the AgNO3 was colorless solution and the extract was green in color, and finally the synthesized AgNPs was dark brown in color which clearly shows the formation of AgNPs. The FTIR spectra of pure fenugreek leaf extract and green synthesized AgNPs were recorded and the resulting graphs are illustrated in Fig. 2. The pure extract (Fig. 2a) shows five major peaks at 3326, 2974, 1374, 1050 and 868 cm− 1. The band at 3326 cm− 1 was due to the stretching vibrations of the OH functional group. The peak at 2974 cm− 1 corresponds to the strong stretching vibration of CeH functional groups. The band at 1050 cm− 1 corresponds to various functional groups such as alcohol, ether, ester, carboxylic acid, anhydrides. The peak at 868 cm− 1 is due to the CH3 deformations of isopropyl group. FTIR spectrum of AgNPs synthesized using fenugreek extract (Fig. 2b) shows two major peaks at 3307 and 1637 cm− 1. It is noteworthy that the peak shift was observed at 3307 cm− 1 and simultaneously a new peak was seen at 1637 cm− 1. Intensive review on fenugreek reveals that polyphenols are their predominate constituents [24,25]. FTIR spectra shown in the Fig. 2 also reveals the predominance of OH and C]C moieties on the surface of the AgNPs. This is the signature of polyphenols which forms the major constituent phytochemical of the fenugreek leaves. In addition, our previous characterization report on green synthesized AgNPs using fenugreek also revealed the same type of signature [26,27]. The XRD pattern was obtained to confirm the crystalline nature of the synthesized AgNPs. Fig. 3 shows the diffraction peaks of the AgNPs which indicates the polycrystalline nature. The major peaks were
Fig. 3. X-ray diffraction pattern of AgNPs synthesized using fenugreek leaves ethanolic extract.
observed at 2θ = 38.36°, 44.36°, 64.51° and 77.41° which corresponds to the (111), (200), (220), and (311) planes, respectively [28]. These peaks are well attributed to the standard JCPDS data of the silver with FCC crystal lattice structure (JCPDS NO: 87-0720). In addition, the peak observed at 29° might be due to unreduced AgNO3 peaks. Using Debye Scherrer formula, the average crystallite size was estimated around 18 nm from the major diffraction peaks. Surface morphology and crystallography of the AgNPs were studied using HRTEM. Fig. 4 shows the HRTEM images with different magnifications. The results clearly show that the AgNPs were exhibit spherical shape, uniform size and well crystalline nature. The size of the AgNPs was ranging from 20 to 30 nm. Crystallography and crystalline size are in good agreement with the XRD results (Fig. 3) [29]. It can be seen that, agglomeration of the NPs was not observed because of the encapsulation of the AgNPs with fenugreek extract. This encapsulation may be due to the presences of alkaloids, flavonoids, polyphenols, etc., presented naturally in the fenugreek plants. The selected area electron diffraction (SAED) pattern is illustrated in the Fig. 4d. The SAED pattern of AgNPs, exhibits the concentric rings indexed corresponding to the (111), (200), (220), and (311) planes of FCC silver phase. The SAED pattern is in good agreement with the ascribed result of XRD, which also suggest the similar reflections of the AgNPs [18]. The antibacterial activity of the ethanolic fenugreek extract, silver
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Fig. 4. HRTEM images of spherical shaped AgNPs synthesized using fenugreek leaves ethanolic extract at different scale bars (a) 100 nm, (b) 30 nm, (c) 5 nm shows the lattice arrangement (d) SAED pattern of AgNPs and (e) energy dispersive X-ray spectrum shows presence of elemental silver. Fig. 5. Antibacterial activity on S. aureus (a) and E. coli (b) bacteria, I. extract, II. AgNO3, III. AgNPs and IV. Streptomycin antibiotic. The results are expressed as the mean ± SD of triplicates.
16.27 mm and 12.47 mm respectively [18,30]. Instantaneously, there was zero ZOI for ethanolic fenugreek leaves extract at in well I. While, the ZOI for AgNO3 in the well II was about 5.97 mm for S. aureus and 6.67 mm for E. coli at a concentration of 1 μL, which was trivial activity than the synthesized AgNPs. Thus, the results reveal that gram negative E. coli shows the maximum antibacterial activity than gram positive S. aureus (Table 1) [31]. The MIC was used to determine the lowest concentrations that completely inhibit the growth of bacterial cells. The MIC level of AgNPs to inhibit the S. aureus growth was 12.5 μg/ml, whereas, this value was lower for inhibition of E. coli growth about 6.25 μg/ml [30]. The antibacterial activity and MIC of the AgNPs against the gram-positive S. aureus and gram negative E. coli were found to be almost identical. As per statistical analysis we could observe that the AgNPs induces time dependent release of protein from the target bacteria. The leakage was maximum at 4th hour confirming cell death. It was found that AgNPs could enhance the protein leakage by increasing the membrane permeability of S. aureus and E. coli cell wall,
Table 1 Antibacterial activity of synthesized AgNPs against S. aureus and E. coli. ZOI was measured as millimeter ± standard deviation for three independent experiments; (–) shows no activity. Test organism
Crude extract (1 μL)
Silver nitrate (1 μL)
Silver NPs (1 μL)
Streptomycin (1 μL)
Staphylococcus aureus Escherichia coli
–
5.97 ± 0.15
12.47 ± 0.31
9.47 ± 0.21
–
6.67 ± 0.25
16.27 ± 0.51
10.12 ± 0.10
nitrate, AgNPs was studied against S. aureus and E. coli bacteria. AgNPs synthesized from ethanolic fenugreek leaves extract shows considerable antibacterial activity against the test pathogens (Fig. 5). The AgNPs in well III of both the bacteria had comparatively good growth of inhibition when compared with the control antibiotic streptomycin. The ZOI for streptomycin was about 9.41 mm (S. aureus) and 10.12 mm (E. coli), whereas, the ZOI of AgNPs in E. coli and S. aureus was found to be 4
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Fig. 6. Leakage of protein from the bacterial cells S. aureus (a) and E. coli (b) treated with different concentrations of AgNPs at 0, 2 and 4 h. Values represents mean ± SD of three replication. Statistically significant at p < 0.05.
Fig. 7. Scanning electron microscope images of control (untreated) cells (a) S. aureus and (d) E. coli, and treated with AgNPs at 2 and 4 h. (b and c) S. aureus and (e and f) E. coli.
was lower than that of the gram-negative E. coli. This difference was possibly attributable to the thickness of the peptidoglycan layer of S. aureus. The essential function of the peptidoglycan layer is to protect against antibacterial agents such as antibiotics, toxins, chemicals, and degradative enzymes, and hence, lower protein leakage of S. aureus than E. coli [20,31]. The SEM analysis was used to observe the morphological changes on the surface of the cells treated with AgNPs (Fig. 7). The changes on the bacterial cells by AgNPs were observed at two different time intervals (2 and 4 h) of similar concentration. To the best of our knowledge, no reports are available on time dependent experiments. The SEM image of the control group S. aureus cells were characteristically grape shaped (Fig. 7a) where, the surface of the bacteria cells are intact and no damage was observed. After the cells were treated with AgNPs, the morphology of the cell changes showing the damage of cell membrane due to the attachment of nanoparticles (Fig. 7b and c) [32]. The control
which are shown in the Figs. 5 and 6 respectively. Initially, protein leakage from the S. aureus cell membranes treated with AgNPs was almost similar as that from cells in the control group (Fig. 6a). After 2 and 4 h of incubation, protein leakage from cells treated with AgNPs were considerably increased, however, there is no change in the amount of protein leakage from the cells of control group (Fig. 6b). Leakage from cells treated with AgNPs was significantly higher than that of cells in the control group. Furthermore, the initial protein leakage from the membranes of E. coli cells treated with AgNPs was almost similar as that of cells in the control group as shown in Fig. 6. After 2 and 4 hours incubation, protein leakage from E. coli cells treated with AgNPs was significantly increased as compared to the cells from control group, which may be due to the increase in the membrane permeability caused by AgNPs (Fig. 6). Notably, higher quantity of proteins leaked through the E. coli membranes than those of S. aureus membranes, suggest that the sensitivity of the gram-positive S. aureus 5
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Fig. 8. In vitro cytotoxicity effect of green synthesized AgNPs in HaCaT cell lines. (a) Control, (b) 5 μg/ml (c) 250 μg/ml of AgNPs treatment and (d) percentage of cell viability at various concentration of AgNPs. Values represent mean ± SD of three replications. Statistically significant at p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of the AgNPs against HaCaT cells were detected by MTT assay. The cell viability percentage pertaining to serial concentrations of the particles is shown in the Fig. 8. The morphology of the cells treated with the lowest dose and the highest dose were compared with untreated control cells using the optical microscopic images (Fig. 8). In HaCat cells, we identified less cytotoxic effect at the highest concentration (250 μg/ml) of AgNPs. Nonetheless, the same concentration of AgNPs showed a dramatic cytotoxic effect in bacterial cells. Earlier reports stated that the chemically synthesized AgNPs shows a 30% viability towards the epithelium cell lines [36]. This remains noteworthy that the less cytotoxic effect was observed at 250 μg/ml of AgNPs concentration. In this study, AgNPs synthesized using ethanolic extract showed 96% of HaCaT cell viability. Optical microscopic image supports our findings as it showed no morphological alterations in HaCat cells, suggesting that the AgNPs kill bacterial cells alone [37,38].
group of E. coli cells are typically rod shaped (Fig. 7d) with uniform size with no cell surface damage. After AgNPs treatment, irregular fragments were formed (Fig. 7e and f). Report by Kim et al. [20] also suggest that AgNPs have the ability to form free radicals (Reactive oxygen species-ROS), which inturn are capable of inducing membrane damage. Thus, the fragments seen in Fig. 7e and f may be due to the increased oxidative stress in the cell membrane which was caused by AgNPs induced ROS [33]. This study showed not only morphological changes of cell surface but also the time-dependent damage of cell membranes. The damage of the cell membrane could further be confirmed from leakage of cytoplasmic cell contents. The antimicrobial effect of the AgNPs on gram-negative bacteria was dependent on the concentration of Ag in the nanoparticles and it is closely related to the formation of “pits” in the cell walls [34,35]. Kim et al. have reported that Ag-NPs have the ability to form free radicals [20]. Induction of membrane damage by lipid peroxidation is the wellknown feature of free radicals (ROS). Hence we interpret that the permeability of the bacterial cell membrane damage as shown in SEM image may be due to the free radicals generated by the AgNPs. The free radicals induce oxidative stress and cause damage to the cell membrane and the vital biomolecules like lipid, protein and DNA of the intracellular system. The protein leakage from the AgNPs-treated bacterial cells observed in our study (Fig. 6) also supports the membrane damage. This study showed an evidence for concentration and time dependent increase in the protein leakage (Fig. 6) and membrane permeability (Fig. 7). Interestingly, there were some differences between gram positive S. aureus and gram negative E. coli. The S. aureus showed less bactericidal activity than E. coli. This difference might be due to the variation in the thickness of the peptidoglycan layer present on the cell membrane as mentioned above. The gram positive cells, consist of lipoteichoic acid containing a thick and multiple peptidoglycan layer (30 to 100 nm) and cell membrane. But, the gram negative cells consist of a thin layer of peptidoglycan (few nm) [31,32]. The thickness of this peptidoglycan layer plays an important role to protect the cells from the formation of pits or ROS caused by AgNPs. From the current study, it was found that the AgNPs synthesized by ethanolic extract exhibits antimicrobial activity by damaging the cell membranes. This encouraged us to study the AgNPs activity on human cell lines to find out its safety profile. The cytotoxic effect towards HaCaT cells, well known as immortalized human keratinocyte cell lines, was performed using AgNPs at various concentrations. The cytotoxicity
4. Conclusion AgNPs synthesized from ethanolic extract of fenugreek leaves is a novel greener method, which is simple and potential way to develop a sustainable nanoparticle with less toxicity than any other methods, thus being environmental friendly. The UV–Visible spectroscopy, FTIR, XRD, TEM and EDX confirmed the formation and well dispersed nature of the AgNPs. The ethanolic extract of AgNPs has potent antibacterial activities against S. aureus and E. coli bacteria. The maximum ZOIs were found to be 12 and 16 mm for S. aureus and E. coli, respectively. The growth and reproduction of AgNPs treated bacterial cells were rapidly inhibited. AgNPs showed higher antibacterial activity against gram negative E. coli than gram positive S. aureus. The morphological changes on bacterial cells treated with AgNPs at 2 and 4 h were observed by SEM. The cytotoxicity towards the HaCaT cell lines were lesser compared with the cytic effect towards the S. aureus and E. coli bacteria. This study specifies that AgNPs can be used as effective antibacterial materials without affecting epithelial cells thus proving its non-toxicity. References [1] N. Silvestry Rodriguez, E. Sicairos-Ruelas, C. Gerba, K. Bright, Silver as a desinfectant, Rev. Environ. Contam. Toxicol. 191 (2007) 23–45, http://dx.doi.org/10. 1007/978-0-387-69163-3. [2] B.E. Wildt, A. Celedon, E.I. Maurer, B.J. Casey, A.M. Nagy, S.M. Hussain, P.L. Goering, Intracellular accumulation and dissolution of silver nanoparticles in L929 fibroblast cells using live cell time-lapse microscopy, Nanotoxicology 5390
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