Department of Food processing Technology, Central Institute of Technology, BTAD, Kokrajhar 783370, Assam, IndiaDepartment of Biotechnology, Haldia Institute of Technology, Purba Medinipur Haldia 721657, WB, IndiaDepartment of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata 700032, IndiaDepartment of Chemistry, Sree Chaitanya College, 24 Parganas (North), Habra 743268, WB, India
i g h l i g h t s
Very stable aqueous silver nanocol-loids have been synthesized usingsingle step green biosyntheticmethod.TEM micrograph reveals that the sizeof the synthesized spherical silvernanoparticle is 8.9 ± 3.6 nm.The as-synthesize silver nanocolloidsshow significant antibacterial activityagainst several bacterial strains.In-vitro and in-vivo DNA damageassay show silver nanocolloids haveno genotoxic effect.
g r a p h i c a l a b s t r a c t
r t i c l e i n f o
rticle history:eceived 12 August 2013eceived in revised form 5 February 2014ccepted 10 February 2014vailable online 18 February 2014
eywords:ilver nanoparticles
a b s t r a c t
Aqueous silver nanocolloids were synthesized using single step and completely green biosyntheticmethod employing aqueous leaf extracts of Paederia foetida as both the reducing and capping agent.Crystalline silver nanoparticles (AgNPs) having average diameter (8.9 ± 3.6) nm have been obtained. Thenanocolloids are very stable and no precipitation was observed in 6 months. The absorption spectra ofcolloidal silver nanoparticles showed characteristic surface plasmon resonance (SPR) peak centered at awavelength of 424 nm. The activity of the AgNPs colloidal suspension as an antibacterial agent againstboth gram negative (Shigella dysenteriae, Salmonella infantis, Vibrio parahaemolyticus and Escherichia coli)
iosynthesiseaf extractICenomic DNAalf thymus DNA
and gram positive (Staphylococcus aureus) bacteria was investigated following MIC as well as disc diffu-sion technique. Good antibacterial activity was found against all test bacteria. In-vitro and in-vivo DNAdamage assay was performed by silver nanocolloid. Interestingly silver nanocolloid showed no geno-toxic effect and this results lead to conclude that inhibition of bacterial growth occurred without anyDNA damage by silver nanoparticle.
The antimicrobial properties of metals were known since
ancient times [1,2]. Especially silver is traditionally well-knownantimicrobial materials and it is less toxic to human cells andenvironmentally benign in low concentration [3,4]. For this rea-son silver was used as antiseptic materials specifically for the
reatment of open wounds and burns before the introduction ofntibiotic therapy and silver containers have been used for cen-uries to purify potable water [5–7]. Along other unique propertiesuch as electronic, optical, catalytic etc metal nanoparticle alsoxhibit significant antimicrobial activity. The enhanced antimicro-ial effect of metal nanoparticle is owing to their high surface toolume ratio, which allows the nanoparticle to interact closely withhe microbial membrane [8]. Several studies have been reportedn the antimicrobial activity of silver nanoparticle of various sizend shape against different antimicrobial strain [9–11]. In mostf these studies silver nanoparticles have been synthesized byeduction of silver ions in aqueous phase using environmentallyenign reagents such as biological reducing agents and stabi-
izer [12,13,4]. The advantage of such ‘green synthesis’ is thathe as-synthesized silver nanocolloids can be directly introducedn the application fields as the colloids do not contain any toxiceducing agent and stabilizer. Moreover, the method is also lowost. The use of silver nanoparticles as an antimicrobial agentas been already practiced in several application fields includingloths, fabrics, washing machines, water purification, toothpaste,eodorants, filters, kitchen utensils, toys, wound dressings etc andiomedical areas [14–16]. Very recently some of us successfully fab-icated antibacterial and antifungal silver-gelatin nanocomposites a prospective applicant for bio-packaging [17].
However, very few studies have been reported on the mechanis-ic pathway of antimicrobial action of silver nanoparticle [18–21].ut such studies are important to know the toxic effects of silveranoparticle at that particular concentration on human cells.
In the present study, initially we have synthesized very stableilver nanocolloids in aqueous phase by employing plant extract as
reducing and stabilizing agent. The leaf extract of Paederia foetidaas been used in this purpose. The antibacterial activity of sucholloidal silver nanoparticle was tested against several bacterialtrains by ‘spectroscopic analyses and ‘zone of inhibition’ tech-iques. Minimum inhibition concentration (MIC) is also reported
or each bacterial strain. To know the killing-mechanism, we havenvestigated the effect of silver nanocolloid on calf thymas (CT) DNAin-vitro) and on genomic DNA of Escherichia coli (in-vivo).
. Materials and methods
.1. Preparation of leaf extract
P. foetida was collected from Sonarpur, Kolkata-700150, Westengal. The fresh leaf extract was prepared by taking 50 g of thor-ughly washed and finely cut leaves in a 500 ml flask with 100 ml ofterile distilled water and then boiling the mixture for 5 min beforenally filtering it through Whatman No. 1 filter paper. The extractas stored at 4 ◦C and used within a week.
.2. Synthesis of silver nanocolloids
Silver nitrate (AgNO3, 99.99%) was purchased from Sigma. For typical synthesis, 0.40 ml of leaf extract was added to a 5 ml ofreshly prepared 1 mM aqueous AgNO3 solution. The mixture wasaken in a sealed Teflon container and it was kept in an incubatorith vigorous shaking at 50 ◦C for maximum 7 days. The volume
f leaf extract was varied from 0.16 to 0.48 ml where the concen-ration and amount of AgNO3 solution was kept constant. The finalolume of the solution mixture was maintained 6 ml in each set.
.3. Characterization of AgNPs
The UV–vis absorption spectra were measured as a function ofeaction time at room temperature on a Perkin Elmer UV spec-rophotometer (model �-25) using a quartz cell (1 cm path). 24 h
cochem. Eng. Aspects 449 (2014) 82–86 83
aged sample was taken for TEM and antimicrobial study. The TEMstudy was carried out in a JEOLJEM 2100 microscope, working at anacceleration voltage of 200 kV. Sample for TEM analysis was pre-pared by placing a small drop of colloidal solution on carbon coatedcopper grid. After 2 min of deposition of the film on TEM grid, theexcess solution was removed using a blotting paper and the gridwas allowed to dry in room temperature prior to measurement.
2.4. Microbial strains used and growth conditions
The microorganisms used in this study were clinical isolates ofS. dysenteriae, V. parahaemolyticus, S. aureus, S. infantis and E. coli.
Bacterial strains stock cultures were maintained at 4 ◦C on nutri-ent agar medium. Active cultures were prepared by inoculatingfresh nutrient broth medium with a loopful of cells from the stockcultures at 37 ◦C for overnight. To get desirable cell counts for bioas-says, overnight grown bacterial cells were sub-cultured in freshnutrient broth at 37 ◦C. In vitro antimicrobial activity of the silvernanocolloid was screened against a total of five above mentionedbacterial strains.
2.4.1. Determination of MIC by spectroscopic analysisThe silver nanocolloids were later tested to determine the
Minimal Inhibitory Concentration (MIC) for each bacterial strain.Freshly, grown bacterial strains 100 �l (1 × 106 cells/ml) in nutrientbroth was inoculated in tubes with nutrient broth supplementedwith different concentrations (0–100 �l/ml) from the stock sample(0.899 �g/ml) and incubated for 24 h at 37 ◦C. Presence of turbiditydenoted presence of micro organism in the test tube after the periodof incubation whereas the complete absence of any turbidity indi-cates complete inhibition of microbial growth. The test tube withthe lowest dilution with no detectable growth by visual inspectionwas considered the MIC. The MIC was calculated for the individualbacterial species.
2.4.2. Disc diffusion methodThe antimicrobial activity of silver nanocolloids was screened
using disc diffusion technique [17]. The agar plates were preparedby pouring 15 ml of molten nutrient agar media into sterile petri-plates. The plates were allowed to solidify and 0.1% inoculumssuspension was swabbed uniformly with sterile cotton and wasallowed to stand for 15 min. The different dilutions of the test sam-ple (0%, 25%, 50% and 75%) from stock concentration (89.9 �g/ml)were loaded on 6 mm autoclaved filter paper discs. The loadeddisc was placed on the surface of medium and the compound wasallowed to diffuse for 5 min and the plates were incubated at 37 ◦Cfor 24 h. At the end of incubation, inhibition zones formed aroundthe disc were measured with ruler in millimeter. These studies wereperformed in triplicate.
2.4.3. Determination of genotoxic effect of silver nanoparticles onthe in-vitro damage of genomic DNA
50 �g of Calf Thymus (CT) DNA and varying concentration(0–100 �l/ml) of silver nanocolloids was mixed, and incubated at37 ◦C for 1 h. After treatment, the DNA was immediately subjectedto electrophoresis in 0.9% agarose gel, in 1× TAE buffer (40 mM-Trise-acatate, 1 mM EDTA), followed by ethidium bromide staining(50 �g/ml) and visualized by gel documentation system. The result-ing fragmentation and inhibition of fragmentation were analyzed.
2.4.4. Determination of in-vivo genotoxic effect of silver
nanocolloids on E. coli cells
1 × 106 cells/ml of E. coli were treated with and without sil-ver nanocolloids (10–100 �l/ml) and incubated at 37 ◦C for 24 h.After the incubation is over, cells were treated with 200 �l cell
8 Physicochem. Eng. Aspects 449 (2014) 82–86
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4 S. Kumar et al. / Colloids and Surfaces A:
ysis buffer (50 mM Tris HCl, pH 8.0, 10 Mm ethylenediaminetet-aacetic acid, 0.1 M NaCl, and 0.5% sodium dodecyl sulfate) for 1 ht 37 ◦C. The lysate was incubated with 0.5 mg/ml RNase A at 37 ◦Cor 1 h, and then with 0.2 mg/ml proteinase K at 40 ◦C for 2 h. Afterompletion of incubation, the sample was centrifuged for 10 min at0,000g. The aqueous portion, containing the DNA was transferredo new eppendrof tube. DNA was subjected to electrophoresis in.9% agarose gel, in 1× TAE buffer (40 mM-Trise-acatate, 1 mMDTA), followed by ethidium bromide staining (50 �g/ml) and visu-lized by gel documentation system. The resulting fragmentationnd inhibition of fragmentation were analyzed.
. Results and discussions
.1. Characterization of AgNPs
The formation of silver nanoparticle by reduction of the aque-us silver ions during the exposure to the broth of boiled P. foetidaeaves was followed by UV–vis spectroscopy. The colorless aque-us silver nitrate solution became yellow within few minutes whenreated with the broth of boiled P. foetida leaves at 60 ◦C. It is wellnown that this yellow color of the silver nanoparticle arises owingo excitation of surface plasmon vibration in the silver nanoparti-le [22]. Fig. 1a and b shows the UV–vis spectra recorded from thequeous silver nitrate–P. foetida leaf broth reaction medium as aunction of time of reaction and concentration of leaf broth respec-ively. Fig. 1a shows that the surface plasmon resonance (SPR) bandf silver nanoparticles centered at 424 nm and increases in inten-ity as a function of time of reaction. The inset of Fig. 1a representshe plot of absorbance at � max (i.e. at 424 nm) against time ofeaction and it shows that the reduction was almost completedt 8 h. To know the optimum amount of reducing agent requiredor the reduction of all the silver ions present in the solution, wetudied the intensity of the plasmon band as a function of increas-ng amount of leaf broth against a fixed amount (5 ml, 1 mM) ofilver nitrate solution. Fig. 1b exhibits that 0.40 ml of leaf-broths sufficient to reduce all the silver ions present in our experi-
ental solution. No increase in the intensity of plasmon band wasbserved for further increase of the amount of reducing agent (leaf-roth). Interestingly the silver nanocolloids solutions are highlytable and no precipitation was observed over 6 month storage atoom temperature. The results suggest that the aqueous extract of P.oetida leaf-broth which contains phenolic compounds, flavinoids,
onoterpenes, carbohydrates etc simultaneously acts as a very effi-ient reducing agent for silver ions as well as good capping agent
or silver nanoparticle. Recently, Iravani et al. [23] has been exten-ively reviewed the synthesis of metal nanoparticles using plantxtracts. In this review they found that the phenolic compounds,avinoids, terpinoids, polysaccharides, enzymes and other proteins
Fig. 2. (a) TEM image of sample containing 0.40 ml of leaf extract (inset). The
Fig. 1. (a) UV–vis spectra of the mixture of aqueous silver nitrate–P. foetida leafbroth (a) as a function of time and (b) as function of concentration of leaf broth.
etc are responsible for reduction of metal ions and stabilization ofnanoparticles.
Representative TEM image of sample containing 0.40 ml of leafextract and corresponding size distribution are shown in Fig. 2aand b, respectively. TEM micrograph reveals that the particles arealmost spherical having diameter within 2–20 nm and the averagediameter of particle is around (8.9 ± 3.6) nm. The selected area elec-tron diffraction (SAED) pattern (inset of Fig. 2a) with bright spotsindicates that the silver nanoparticles are crystalline in nature.
3.2. Antimicrobial activity of AgNPs
The silver nanocolloid containing 0.40 ml of leaf extract wastested as antimicrobial agent. The antibacterial activity of sample
selected area electron diffraction (SAED) pattern; (b) size distribution.
S. Kumar et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 449 (2014) 82–86 85
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lane 1) showed control sample DNA (with no treatment) whichwas compact with a high molecular weight exhibiting its integrity.Nanoparticles treated CT DNA also showed similar gel profile as
ig. 3. Agar plat exhibited zones of inhibition by different dilution of AgNPs (0%,arahaemolyticus, S. aureus and S. infantis).
ith different dilution for four clinical bacterial isolates (S. dysen-eriae, V. parahaemolyticus, S. aureus and Salmonella nfantis) was
easured by disc diffusion technique (Fig. 3). No growth inhi-ition ring was observed for control i.e. without any silveranoparticle and growth inhibition ring increases with increase ofilver nanoparticle concentration against all experimental bacte-ia (Fig. 3). Growth inhibition curve of tested clinical isolates ofram-positive and Gram-negative bacteria against the amount ofilver nanocolloid are presented in Fig. 4. Concentration of silveranoparticles in the tested nanocolloids solution was 89.9 �g/ml.he similar MIC value (4.454 �g/ml) was observed for S. dysente-iae, S. infantis, S. aureus. The MIC values for V. parahaemolyticusnd E. coli were 5.394 and 6.293 �g/ml respectively. Different MICalues for silver nanoparticle against different bacterial strains haveeen reported in literature [24–28]. MIC values obtained in ourtudies for E. coli and S. aureus are lower than most of the reportedalues in literature [29–31].
Since our experimental conditions reduced all the silver ionsnto silver nanoparticle and leaf extract alone did not show anyntimicrobial activity against tested bacteria, so it can be concludedhat only silver nanoparticles act as an antibacterial agent here.
.3. Determination of genotoxic effect of AgNPs in-vitro andn-vivo
To explore the mechanism of antimicrobial activities of sil-er nano particles we studied in-vitro and in-vivo DNA damagessay by silver nanocolloids. Fig. 5A demonstrate Calf thymus DNA
50% and 75%) against the clinical bacterial isolates under study (S. dysenteriae, V.
profile treated with/without synthesized silver nano particles tocheck its genotoxic property. Agarose gel electrophoresis (Fig. 5A,
Fig. 4. Growth inhibition curve of the tested clinical isolates of bacteria (S. dysente-riae, V. parahaemolyticus, S. aureus, S. infantis and E. coli). Isolates were grown in LBmedium supplemented with different concentrations from the stock AgNPs sample(89.9 �g/ml). The values are mean ± SD of three replicas.
86 S. Kumar et al. / Colloids and Surfaces A: Physi
Fig. 5. In-vitro and in-vivo DNA damage assay performed by AgNPs. (A) In-vitroeffects of AgNPs on calf thymus (CT) DNA as monitored by electrophoretic mobility.50 �g/ml of CT DNA was treated by different concentration (0–50 �l/ml) from thestock sample (89.9 �g/ml) for 1 h at 37 ◦C. (B) In-vivo effects of AgNPs on genomicDNA of E. coli. 1 × 106 cells/ml of E. coli was treated by different concentration(0–50 �l/ml) for 24 hours at 37 ◦C. After completion of incubation, the genomic DNAof E. coli was isolated and monitored by electrophoretic mobility. DNA profiles ofba(
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[29] J.P. Ruparelia, A.K. Chatterjee, S.P. Duttagupta, S. Mukherji, Acta Biomater. 4
oth in-vivo and in-vitro were visualized by electrophoresis on 0.9% agarose gelnd stained with ethidium bromide (50 �g/ml). Lane 1, control (0 �l/ml), 2–9, DNA10–100 �l/ml).
een from nature of the native DNA band (Fig. 5A, lanes 2–9).he result indicates no genotoxic effect of this nanoparticle withinhis concentration range. Similarly Agarose gel electrophoresis ofacterial genomic DNA isolated from E. coli treated with severaloncentration of AgNP solution shows no damage of DNA (Fig. 5B)hough cell death occurs within that concentration range of AgNPolution (Fig. 4). For biomedical application, it is important to checkor genotoxic property of any sample. DNA laddering assay is sim-le and in short period of time the effect on genome can be tested
n-vitro.There are different opinions and finding regarding the mecha-
ism of antimicrobial activities of silver nanoparticles as mentionedbove. Our findings suggest that as DNA was not damaged by AgNPs,
o rapture of cell membrane by AgNPs may one of the causes ofltimate cell death of microbes. However, further studies, like cyto-lasmic material release will be needed to explore the detailedechanism of antimicrobial activity of silver nanoparticle.
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cochem. Eng. Aspects 449 (2014) 82–86
4. Conclusion
In summary we described a cost efficient and green syntheticmethod leading to a very stable colloid of silver nanoparticles usingaqueous extract of P. foetida leaf. These nanocolloids exhibit goodantibacterial activity against several bacterial strains. Employingsimple DNA laddering assay we have shown that inhibition of bac-terial growth occurred by silver nanoparticle without any damageof DNA. Such highly stable aqueous silver nanocolloids may findpotential applications in various fields of science and technology.
Acknowledgment
One of us (A. Mitra) acknowledges UGC for financial supportthrough minor research project (Ref No. PSW-127/10-11)
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