Research Article Biosynthesis, Antimicrobial and Cytotoxic ...Cytotoxic Activity. Cell viability was evaluated by the MTT colorimetric technique. Human HeLa cancer cell lines (cells/well)

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 287638 9 pageshttpdxdoiorg1011552013287638

Research ArticleBiosynthesis Antimicrobial and Cytotoxic Effect of SilverNanoparticles Using a Novel Nocardiopsis sp MBRC-1

Panchanathan Manivasagan1 Jayachandran Venkatesan2 Kalimuthu Senthilkumar2

Kannan Sivakumar3 and Se-Kwon Kim12

1 Marine Biotechnology Laboratory Department of Chemistry and Marine Bioprocess Research Center Pukyong National UniversityBusan 608-737 Republic of Korea

2Department of Chemistry and Marine Bioprocess Research Center Pukyong National University Busan 608-737 Republic of Korea3 Centre of Advanced Study in Marine Biology Faculty of Marine Sciences Annamalai University ParangipettaiTamil Nadu 608 502 India

Correspondence should be addressed to Se-Kwon Kim sknkimpknuackr

Received 4 May 2013 Revised 18 June 2013 Accepted 20 June 2013

Academic Editor Maria Alice Zarur Coelho

Copyright copy 2013 Panchanathan Manivasagan et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The biosynthesis of nanoparticles has been proposed as a cost effective environmental friendly alternative to chemical and physicalmethods Microbial synthesis of nanoparticles is under exploration due to wide biomedical applications research interest innanotechnology and microbial biotechnology In the present study an ecofriendly process for the synthesis of nanoparticles usinga novel Nocardiopsis sp MBRC-1 has been attempted We used culture supernatant of Nocardiopsis sp MBRC-1 for the simple andcost effective green synthesis of silver nanoparticles The reduction of silver ions occurred when silver nitrate solution was treatedwith the Nocardiopsis sp MBRC-1 culture supernatant at room temperature The nanoparticles were characterized by UV-visibleTEM FE-SEM EDX FTIR andXRD spectroscopyThenanoparticles exhibited an absorption peak around 420 nm a characteristicsurface plasmon resonance band of silver nanoparticles They were spherical in shape with an average particle size of 45 plusmn 015 nmThe EDX analysis showed the presence of elemental silver signal in the synthesized nanoparticles The FTIR analysis revealed thatthe protein component in the formof enzymenitrate reductase produced by the isolate in the culture supernatantmay be responsiblefor reduction and as capping agents The XRD spectrum showed the characteristic Bragg peaks of 1 2 3 2 0 4 0 4 3 1 4 4 and 3 1 1facets of the face centered cubic silver nanoparticles and confirms that these nanoparticles are crystalline in nature The preparedsilver nanoparticles exhibited strong antimicrobial activity against bacteria and fungi Cytotoxicity of biosynthesized AgNPs againstin vitro human cervical cancer cell line (HeLa) showed a dose-response activity IC

50value was found to be 200 120583gmL of AgNPs

against HeLa cancer cells Further studies are needed to elucidate the toxicity and the mechanism involved with antimicrobial andanticancer activity of the synthesized AgNPs as nanomedicine

1 Introduction

Nanotechnology is emerging as a rapidly growing field withits application in science and technology [1] Noble metalnanoparticles such as gold silver and platinum are widelyapplied in medicinal applications Marine actinobacteriaare high Guanine+Cytosine content Gram-positive bacteriawith an unparalleled ability to produce diverse secondarymetabolites such as antibiotics immunosuppressors and

many other biologically active compounds [2] Exploitationof marine actinobacteria in nanotechnology has recentlyreceived considerable attention [3 4] Nanotechnology holdspromising application in biosensing drug delivery andcancer therapy [5ndash7]The expensive and extensive use of toxicsolvents and hazardous reducing agents in chemical proce-dures to synthesize nanoparticles has augmented the neces-sity in view of ecofriendly and green chemistry approachHence a well established nontoxic and ecofriendly potent

2 BioMed Research International

methodology for the synthesis of nanoparticles has mountedto a level of supreme importance [8ndash11] An alternativeapproach for the synthesis of metal nanoparticles is to applybiomaterials such as plants microorganisms encompassinggroups such as bacteria fungi and actinobacteria as nanofac-tories [12ndash14] Emerging multidrug resistant (MDR) bacteriahas raised a demand for the urgent need to identify novelantimicrobial agents It was reported that silver had beenused as antimicrobial agents since ancient times [3] Withthe advancements in nanotechnology AgNPs have found itssignificant applications as antimicrobial agents in fields ofmicroelectronics catalysis and biomolecular detection [15ndash17] Although the antibacterial activity of AgNPs has beenproved in the recent years the actual mechanism of actionis not yet clear They may inactivate microorganisms byinteracting with their enzymes proteins or DNA to inhibitcell proliferation [18] It is also evident that the increasedantimicrobial activity of AgNPs may be attributed to itsspecial characteristics of small size and high surface area tovolume ratio [19] The advantage of adapting biosynthesisof AgNPs is the simplicity of extracellular synthesis anddownstream processing [20 21]

Nanoparticles have a wide range of applications asin combating microbes [22] biolabelling [23] and in thetreatment of cancer [24] The antibacterial activity of silverspecies is known since ancient times [25] and it has beendemonstrated that at low concentrations silver is nontoxicto human cells [26] It has also been reported that Ag+ions uncouple the respiratory chain from oxidative phos-phorylation or collapse the proton-motive force across thecytoplasmic membrane [27] The interaction of Ag+ withbacteria is directly related to the size and shape of thenanoparticles [26 28]

Sastry et al [29] reported on the biosynthesis of metalnanoparticles using the mycelial extract of fungi and acti-nobacteria [29] In addition the time required for completionof the reaction using both bacteria and fungi ranges betweenapproximately 24 hrs and 120 hrs whereas maximum syn-thesis of AgNPs can be achieved after 24 hrs of incubationMoreover metal accumulation is dependent on the growthphase of the cells [30] Sadhasivam et al [3] reported onthe extracellular biosynthesis of NPs by Streptomyces hygro-scopicus and antimicrobial activity against medically impor-tant pathogenic micro-organisms [3] Sivalingam et al [31]reported on the biosynthesis of bactericidal silver nanoparti-cles (AgNPs) using a novel Streptomyces sp BDUKAS10 anisolated mangrove sediment [31] Though the mechanism ofsilver resistance offered by bacteria using the silver bindingprotein is well documented their extraction and purificationneed to be elucidated further for large-scale productionHowever only a few studies have examined the componentsof marine actinobacteria that mediated the reduction ofsilver ions into AgNPs In this study we examined andcharacterized the extracellular biosynthesis of AgNPs usinga novel Nocardiopsis sp MBRC-1 which is a very importantmicro-organism to the production of several antibiotics andenzymes of commercial value To the best of our knowledgethis marine actinobacterium (Nocardiopsis sp MBRC-1) hasnever been used for nanoparticles biosynthesis

2 Materials and Methods

21 Chemicals All analytical reagents and media compo-nents were purchased from Sigma-Aldrich (St Louis USA)

22 Microbial Synthesis of AgNPs The Nocardiopsis spMBRC-1 strain was isolated from the marine sedimentsamples from the Busan coast (Lat 35∘091015840 N Long 129∘071015840E) South Korea Their partial 16S rRNA gene sequenceswere deposited in GenBank under the accession numberKC179785 For the synthesis of silver nanoparticles the activeNocardiopsis sp MBRC-1 culture was freshly inoculated onsterile starch casein medium and the flasks were incubated at25ndash28∘C and 180 rpm for 96 hrs (pH 70) After the incubationperiodwas complete the culture was centrifuged at 5000 rpmfor 30min and the supernatant was used for the biosynthesisof AgNPs Deionized water was used as a solvent in thesynthesis of AgNPs The collected supernatant (pH 70) wasadded separately to the reaction vessel containing silvernitrate at a concentration of 10minus3M (1 (vv)) and incubatedon an orbital shaker (dark condition) for 96 hrs at 30∘C Thereaction was carried out in the dark after the addition of theAgNO

3 and color change appeared transparent It confirmed

the synthesis of AgNPs The formation of the AgNPs wasmonitored by UV-vis spectroscopy using Shimadzu (ModelNo-UV 1800) double beam UV-vis spectrophotometer [3]All the experiments were carried out in triplicate and averagevalues have been reported

23 Characterization of AgNPs The synthesized AgNPswere freeze dried powdered and used for XRD analysisThe spectra were evaluated using an X-ray diffractometer(PHILIPS XrsquoPert-MPD diffractometerThe Netherlands) andCu-K120572 radiation 15405 A over an angular range of 5 to80∘ a step size of 002 a scan speed of 4∘mminus1 at a 40 kVvoltage and a 30mA current The dried powder was dilutedwith potassium bromide in the ratio of 1 100 and recordedthe Fourier transform infrared spectroscopy (FTIR) (PerkinElmer Inc USA) and spectrum GX spectrometry within therange of 400 to 4000 cmminus1 SynthesizedAgNPsweremountedon specimen stubs with double-sided adhesive tape coatedwith platinum in a sputter coater and examined under fieldemission scanning electron microscopy (FE-SEM) (JSM-6700 JEOL Japan) For transmission electron microscopy(TEM) imaging a drop of aqueous solution containing theAgNPs was placed on carbon coated copper grids and driedunder an infrared lamp (JEM 1010 JEOL Japan) (AC voltageminus800 kV) In addition the presence of silver metals in thesample was analyzed by energy dispersive X-ray analysis(EDX) combined with FE-SEM Finally the size distributionof the nanoparticles was evaluated using dynamic lightscatteringmeasurements conductedwith aMalvern ZetasizerZS compact scattering spectrometer (Malvern InstrumentsLtd Malvern UK)

24 Particle-Size Distribution of AgNPs Particle-size distri-bution analysiswas carried out after treatment of a 1mMsolu-tion of AgNO

3with the culture supernatant of Nocardiopsis

sp MBRC-1 at room temperature for 98 hrs The organism

BioMed Research International 3

was grown in starch casein broth under incubation at 30∘Cfor 98 hrs After the incubation period the culture wascentrifuged at 10000 rpm and the supernatant was used toreduce the AgNO

3solution For the DLS measurements the

supernatant thus obtained was a clear brown homogenoussuspension of AgNPs diluted 10-fold for all experimentsinvolving measurement of DLS The solutions were thenfiltered through syringemembrane filters with pores less than04 120583m then centrifuged at 5000 rpm for 30min

25 Antimicrobial Activity of the AgNPs The antimicrobialactivity of the microbiologically synthesized AgNPs againstpathogenic organisms such as bacteria (Escherichia coliBacillus subtilis Enterococcus hirae Pseudomonas aerugi-nosa Shigella flexneri and Staphylococcus aureus) and fungi(Aspergillus niger A brasiliensis A fumigates and Candidaalbicans) wasmeasured using the well-diffusionmethod [26]Pure cultures of bacteria and fungi were grown in Mueller-Hinton broth (Sigma USA) for bacteria and Sabouraud-broth for fungi at 35∘C and 30∘C respectively on a rotaryshaker at 180 rpm Wells that were 6mm in diameter weremade on the Mueller-Hinton agar and Sabouraud agar platesusing a gel puncture and each well was inoculated withindividual culturesTheAgNPs in various concentrations (1020 30 40 and 50120583gmL) were loaded in each well Thepositive and negative controls were also maintained and theplates (triplicates) were incubated at 35∘C and 30∘C for 24and 48 hrs Simultaneously the synergistic effects of differentcommercial antibiotics (Amoxicillin and Nystatin SigmaUSA)withAgNPs againstmultidrug resistant pathogenswerealso checked in well diffusion method After incubation thesusceptibility pattern of the test organisms was determinedby measuring the diameter of the zone of inhibition for welldiffusion method

26 Determination of Minimum Inhibitory ConcentrationThe synthesized silver nanoparticles were tested (triplicates)for minimum inhibitory concentration by microtiter brothdilution method [32] Muller-Hinton broth was used asdiluents for bacterial strains and Sabouraud broth for fungalspecies About 106 CFUmL cells were inoculated and thefinal volume in each microtiter plate well was 01mL Afterincubation for 24 h at 35∘C for bacterial strains and 30∘Cfor fungal strains the microtiter plates were read at 450 nmusing TRIADmultimode reader prior to and after incubationto determine the minimum inhibitory concentration (MIC)values The MIC is defined as the lowest concentrationof compound which inhibited 90 of the growth whencompared with that of the growth control

27 Cell Culture Human cervical cancer cell line (HeLa) wascultured in Dulbeccorsquos Modified Eagle Medium (DMEM)Culture media were supplemented with 10 fetal bovineserum (FBS) and 1 antibiotic and antimycotic (Penicillin-Streptomycin cocktail) solution The cells were grown ina humidified atmosphere containing 5 CO

2at 37∘C and

subcultured by detaching with trypsin-EDTA solution atabout 70ndash80 confluent

28 Cytotoxic Activity Cell viability was evaluated by theMTT colorimetric technique Human HeLa cancer cell lines(5000 cellswell) were seeded in 96 well tissue culture platesStock solutions of nanoparticles (5mgmL) were preparedin sterile distilled water and diluted to the required con-centrations (50 100 150 200 and 250 120583gmL) using thecell culture medium Appropriate concentrations of AgNPsstock solution were added to the cultures to obtain respec-tive concentration of AgNPs and incubated for 24 hrs at37∘C Nontreated cells were used as control After 24 hrscells were washed with PBS and then 100 120583L of the yel-low tetrazolium MTT solution (3-(45-dimethylthiazolyl-2)-25-diphenyltetrazolium bromide) without phenol red(05mgmL in phosphate buffer solution) was added to eachwell The plates were incubated for 3-4 hrs at 37∘C forreduction of MTT by metabolically active cells in part bythe action of dehydrogenase enzymes to generate reducingequivalents such as NADH and NADPH For solubilizationof the MTT crystals 100120583L of DMSOwas added to the wellsThe plates were placed on a shaker for 15min to completesolubilization of crystals and then the optical density of eachwell was determined The quantity of formazan product asmeasured by the amount of 545 nm absorbance is directlyproportional to the number of living cells in culture Eachexperiment was done in triplicate The relative cell viability() related to control wells containing cell culture mediumwithout nanoparticles as a vehicle was calculated as followsPercentage of cell viability () = Sample absorbancecontrolabsorbance times 100

29 Cytomorphological Changes in HeLa Cells by AgNPsHeLa cells (1 times 105 cellswell) were seeded in a 6 well plate for24 hrs After 24 hrs theywere treatedwith 100 and 200120583gmLof synthesized AgNPs and incubated for 24 hrs at 37∘C in5 CO

2atmosphere After the incubation the cells were

washed twice with PBS and morphological changes in thecells were visualized and photographed under phase contrastmicroscope (CTR 6000 Leica Wetzlar Germany)

210 Statistical Analysis The grouped data were statisticallyevaluated using ANOVA with SPSS14 software Values arepresented as the mean plusmn SD of the three replicates of eachexperiment

3 Results and Discussion

31 Isolation and Identification of Marine ActinobacteriaA marine actinobacterium MBRC-1 strain was isolatedfrom the marine sediment samples from the Busan coastSouth Korea and was used for the synthesis of silvernanoparticles The marine actinobacterium MBRC-1 showsthat the presence of meso-diaminopimelic acid as theamino acid in the cell wall and arabinose and galactoseas whole cell sugars and the absence of characteristicglycine in their cell credibly categorized the cell wall of thisstrain belonged to the cell wall type-IV [33] This isolatewas identified as Nocardiopsis sp MBRC-1 based on themorphological physiological and biochemical characteris-tics and it was confirmed by the 16S rDNA sequencing


MBRC-l

002

896367

517537

68

Nocardiopsis dassonvillei subsp dassonvillei (NR 029314)Nocardiopsis synnemataformans (NR 029343)Nocardiopsis sp (KC160824)Nocardiopsis dassonvillei (KC119570)Nocardiopsis lucentensis (NR 026342)Nocardiopsis aegyptia (NR 025589)Nocardiopsis alba (NR 026340)Nocardiopsis halotolerans (NR 025422)Bacillus subtilis (KC257097)

Figure 1 Phylogenetic tree of the 16S rDNA sequence of strain Nocardiopsis sp MBRC-1 and related strains

(Figure 1) The sequence was submitted to GenBank in NCBI(httpwwwncbinlmnihgovnuccore443501390) with theaccession number KC179785

32 UV-Vis Analysis of AgNPs In this study AgNPswere suc-cessfully synthesized in the culture supernatant ofNocardiop-sis sp MBRC-1 Interestingly the culture supernatant incu-bated with the silver nitrate mediated the biosynthesizing ofAgNPs within 24 hrs of incubation During the experimentthe pH of the sample was adjusted to 70 The appearanceof a yellowish brown color in the silver nitrate treated flaskindicated the formation of silver nanoparticles whereas nocolor change was observed in either the culture supernatantwithout silver nitrate or the silver nitrate control experi-ments Notably the intensity of the brown color increaseddramatically up to 24 hrs and was maintained throughoutthe experiment This may have been due to the excitationof surface plasmon resonance (SPR) and the reduction ofAgNO

3 In the UV-visible spectrum a strong and broad

peak was observed between 420 nm indicating the presenceof AgNPs This may have occurred due to the reductionof metal ions by secondary metabolites present in the cellsThe 24 48 72 and 96 hrs peaks indicate the absorptionspectra of biosynthesizedAgNPs at different incubation times(Figure 2) Numerous reports have discussed the biosynthesisof silver nanoparticles [3 31 34] but to the best of knowledgethis was the first report on biosynthesis of silver nanoparticlesusing a novel Nocardiopsis sp MBRC-1

33 FTIR Analysis of AgNPs FTIR spectrum analysis ofAgNPs showed intense absorption bands at 3440 2923 28531655 1460 and 685 cmminus1 The intense broad absorbanceat 3440 cmminus1 (OndashH stretch) is the characteristic of the H-bonded functional group in alcohols and phenolic com-pounds The band at 2923 and 2853 cmminus1 (CndashH stretch)can be assigned to the alkanes group The intense mediumabsorbance at 1655 cmminus1 (ndashC=Cndash stretch) is the characteristicof the alkenes group The intense medium absorbance at1460 cmminus1 (CndashH bend) is the characteristic of the alkanesgroup The intense broad absorbance at 685 cmminus1 (ndashC=CndashH CndashH bend) is the characteristic of the alkynes group Aprevious report reveals that the alcohols phenolic alkynes

(a) (b) (c) (d) (e) (f)25

2

15

1

05

0

400 500 600 700 800

Abso

rban

ce (a

u)

420 nm

Wavelength (nm)

Culture supernatantAgNO3 control24 hrs

48 hrs72 hrs96 hrs

Figure 2 UV-Vis spectra of AgNPs synthesized using cell freesupernatant of Nocardiopsis sp MBRC-1 (a) Culture supernatant(b) AgNO

3control ((c)ndash(f)) correspond to the AgNO

3treated with

culture supernatant incubated for 24 48 72 and 96 hrs respectively

and alkanes groups have a strong ability to interact withnanoparticles [31 35 36]

34 XRD Analysis of AgNPs The XRD pattern of the silvernitrate-treated sample (Figure 3) corresponds to that of silvernanoparticles The XRD pattern shows five intense peaksin the whole spectrum of 2120579 values ranging from 30 to80 It is important to know the exact nature of the silverparticles formed and this can be deduced from the XRD


3844

4438

5677

6438 775

(1 2 3)

(2 0 4)

(0 4 3)

(1 4 4) (3 1 1)

Cou

nts

30 40 50 60 70 802120579 (degrees)

Figure 3 X-ray diffraction pattern of the AgNPs obtained fromNocardiopsis sp MBRC-1

spectrum of the sample XRD spectra of pure nanoparticlessilver structures and pure silver nitrate have been publishedby the Joint Committee on Powder Diffraction Standards (fileno 04-0783) A comparison of our XRD spectrum with thestandard confirmed that the silver particles formed in ourexperiments were in the form of nanoparticles as evidencedby the peaks at 2120579 values of 3844∘ 4438∘ 5677∘ 6438∘and 7750∘ corresponding to 1 2 3 2 0 4 0 4 3 1 4 4 and3 1 1 planes for silver respectively The full width at halfmaximum (FWHM) values measured for 1 2 3 2 0 4 0 4 31 4 4 and 3 1 1 planes of reflection was used with the Debye-Scherrer equation to calculate the size of the nanoparticlesThe particle sizes obtained fromXRD line broadening agreedwell with those obtained from SEM From these the averageparticle size was found to be around 45 plusmn 005 nm

35 FE-SEM Analysis of AgNPs FE-SEM determinationsof the above-mentioned sample showed the formation ofnanoparticles which were confirmed to be of silver by EDXAs shown in Figures 4(a) and 4(b) well-dispersed nanopar-ticles could be seen in the samples treated with silver nitrateEDX analysis also showed a peak in the silver region con-firming the formation of silver nanoparticles (Figure 4(c))The optical absorption peak is observed approximately at3 keV which is typical for the absorption of metallic silvernanoparticles due to surface Plasmon resonance [37] Inaddition other peaks for Cl and O were observed which arepossibly due to emissions from proteins or enzymes presentin the culture supernatant [30]

36 TEM Analysis of AgNPs The TEM image analysis (Fig-ures 5(a) and 5(b)) revealed that silver nanoparticles werespherical in shapeThemicrograph showedNPswith variableshape most of them present in spherical in nature TheTEM micrograph also confirmed the size of NPs which

(a)

(b)

Cl

ClAg

Spectrum 1

0 2 4 6 8 10 12 14 16 18 20(keV)

Full scale 3874 cts cursor 0

(c)

Figure 4 ((a) and (b)) FE-SEM images of AgNPs synthesized byNocardiopsis sp MBRC-1 (a) 100 nm scale (b) 1 120583m scale and (c)EDX analysis of AgNPs synthesized by Nocardiopsis sp MBRC-1

were in the range of 30ndash90 nm with an average particle sizeof 45 plusmn 015 nm Majority of the AgNPs were aggregateswith only a few of them showing scattering of varyingsizes as observed under TEM The particle size distributionhistogram plot constructed from the TEM micrograph isshown in Figure 5(c) Synthesis of AgNPs by treating AgNO

3

solution with the culture supernatant of K pneumonia(belonging to the family Enterobacteriaceae) has also beenreported in which the particles range in size from 282 to122 nm and possess an average size of 525 nm [14] A studyon synthesis of AgNPs usingMorganella sp (belonging to thefamily Enterobacteriaceae) reported spherical nanoparticlesof sim20 nm size [38]

37 Antimicrobial Activity of the AgNPs In this study theantimicrobial activity of AgNPs using a novel biosyntheticmethod was evaluated In this analysis the AgNPs displayed


Table 1 Antimicrobial activity of the AgNPs against various pathogenicmicro-organismsThe data is presented as themeanplusmn value standarddeviation of three replicates

Micro-organisms Zone of inhibition (mm in diameter)10120583gmL 20120583gmL 30 120583gmL 40120583gmL 50120583gmL Antibiotics 30 120583gmL

Bacteria AmoxicillinEscherichia coli ATCC 10536 75 plusmn 035 152 plusmn 031 188 plusmn 030 233 plusmn 020 273 plusmn 015 193 plusmn 010Bacillus subtilis ATCC 6633 112 plusmn 035 194 plusmn 025 225 plusmn 010 281 plusmn 020 332 plusmn 020 238 plusmn 025Enterococcus hirae ATCC 10541 63 plusmn 020 133 plusmn 014 172 plusmn 015 218 plusmn 030 254 plusmn 025 195 plusmn 010Pseudomonas aeruginosa ATCC 27853 91 plusmn 015 177 plusmn 030 194 plusmn 020 236 plusmn 035 283 plusmn 020 213 plusmn 030Shigella flexneri ATCC 12022 52 plusmn 020 112 plusmn 021 154 plusmn 015 193 plusmn 025 225 plusmn 010 175 plusmn 030Staphylococcus aureus ATCC 6538 78 plusmn 025 151 plusmn 032 191 plusmn 020 242 plusmn 020 271 plusmn 015 213 plusmn 010

Fungi NystatinAspergillus niger ATCC 1015 67 plusmn 032 136 plusmn 022 173 plusmn 025 214 plusmn 020 253 plusmn 015 181 plusmn 010A brasiliensis ATCC 16404 48 plusmn 025 102 plusmn 015 146 plusmn 020 194 plusmn 010 234 plusmn 015 158 plusmn 030A fumigates ATCC 1022 72 plusmn 035 154 plusmn 022 193 plusmn 020 243 plusmn 010 263 plusmn 030 214 plusmn 015Candida albicans ATCC 10231 95 plusmn 020 181 plusmn 021 224 plusmn 025 252 plusmn 025 284 plusmn 025 245 plusmn 020

Table 2 Minimum inhibitory concentration of the AgNPs againstvarious bacterial and fungal strains The data is presented as themean plusmn value standard deviation of three replicates

Micro-organismsMinimum inhibitory

concentrationAgNPs(120583gmL)

Antibiotics(120583gmL)

Bacteria AmoxicillinEscherichia coli ATCC 10536 13 11Bacillus subtilis ATCC 6633 7 6Enterococcus hirae ATCC 10541 16 14Pseudomonas aeruginosa ATCC 27853 10 9Shigella flexneri ATCC 12022 18 15Staphylococcus aureus ATCC 6538 14 12

Fungi NystatinAspergillus niger ATCC 1015 16 14A brasiliensis ATCC 16404 18 16A fumigates ATCC 1022 13 12Candida albicans ATCC 10231 10 7

antimicrobial activity against a range of different pathogenicmicroorganisms (Table 1) The mean of three replicates ofthe diameter of the zone of inhibition (30120583gmL) for eachmicroorganism was determined to be about 188 plusmn 030225 plusmn 010 172 plusmn 015 194 plusmn 020 154 plusmn 015 191 plusmn 020173 plusmn 025 146 plusmn 020 193 plusmn 020 and 224 plusmn 025mmrespectively forEscherichia coli Bacillus subtilisEnterococcushirae Pseudomonas aeruginosa Shigella flexneri Staphylo-coccus aureus Aspergillus niger A brasiliensis A fumigatesand Candida albicans The highest antimicrobial activity wasobserved against Bacillus subtilis Pseudomonas aeruginosaand Candida albicans These findings are in agreement withprevious studies that examined the antimicrobial activity ofAgNPs against Bacillus subtilis and Candida albicans [3] Theantimicrobial activity of silver nanoparticles was reported

to be due to the penetration into the bacteria damage ofcell membrane and release of cell contents [39] Anotherpossibility suggested that [40 41] was the release of silverions from the nanoparticles which may contribute to thebactericidal properties of silver nanoparticles

38 Determination of Minimum Inhibitory ConcentrationMinimum inhibitory concentration of AgNPs (Table 2)was evaluated against various pathogenic bacteria andfungi The silver nanoparticles exhibited lowest minimuminhibitory concentration (MIC) against Bacillus subtilis at7 120583gmL Bacillus subtilis 10120583gmL and Candida albicansat 10 120583gmL suggesting the broad spectrum nature of theirminimum inhibitory concentration Kumar and Mamidyala[35] reported the minimum inhibitory concentration ofAgNPs against Gram-positive Gram-negative and differentCandida species at concentrations ranging between 4 and32 120583gmL

39 Cytotoxic Activity The in vitro potential cytotoxic activ-ity of AgNPs against cervical cancer cell lines HeLa The useof synthetic AgNPs there are only a few studies to determinethat the cytotoxic effects of biologically synthesized AgNPsMTT assay was used to assess the effect of AgNPs onthe cytotoxicity of cancer cells This study to evaluate themarine sediment samples isolated species Nocardiopsis spMBRC-1 derived AgNPs cytotoxicity against HeLa cancercell lines AgNPs inhibit the viability of the HeLa can-cer cell lines in dose dependent manner The IC

50value

of biosynthesized AgNPs against HeLa cells at 200120583gmLconcentrations (Figure 6(a)) Previously synthesized AgNPsinducing cytotoxicity were discussed by Sriram et al [42] andSafaepour et al [43]

310 Cytomorphological Changes of HeLa Cells Induced byAgNPs Themorphological examinations of the HeLa cancercells were observed and photographed using phase contrastmicroscope The morphological alteration was observed in


(a)

(b)

05

101520253035404550

30ndash40 40ndash50 50ndash60 60ndash70 70ndash80 80ndash90

Inte

nsity

()

Average particle size (nm)

(c)

Figure 5 HR-TEM images of AgNPs formed by Nocardiopsis spMBRC-1 (a) 10 nm scale (b) 2 nm scale and selected area diffractionpattern (c) Particle-size distribution under unoptimized conditionsThe particle-size distribution revealed that the particles rangingfrom 30 to 90 nm had the maximum intensity and thereafter theintensity was reducedThe average particle size was found to be 45 plusmn015 nm

control and AgNPs treated HeLa cancer cells The HeLacells were treated with AgNPs at 100 and 200120583gmL con-centrations for 24 hrs showing that significant morphologicalchanges which are characteristic features of apoptotic cellssuch as loss of membrane integrity cell shrinkage andreduced cell density (Figures 6(b) and 6(c))

0

20

40

60

80

100

120

Control 50 100 150 200 250

Cel

l via

bilit

y (

)

Concentration (120583gmL)

(a)

(b)

(c)

Figure 6 (a) MTT assay results confirming the in vitro cytotoxicityof AgNPs against HeLa cell lines ((b) and (c)) Morphology ofcontrol and AgNPs treated HeLa cell lines (10x magnification) (b)Control (c) IC

50concentration (200120583gmL)

4 Conclusions

In conclusion silver nanoparticles are synthesized by thebiomass of the marine actinobacterium Nocardiopsis spMBRC-1 Marine actinobacteria are easy to handle and canbe manipulated genetically without much difficulty Consid-ering these advantages a bacterial system could prove to bean excellent alternative for synthesis of AgNPs Nocardiopsissp MBRC-1 can be a good candidate for the synthesisof the AgNPs using silver nitrate of average size 45 plusmn015 nm Nocardiopsis sp MBRC-1 genetics and enzymaticactivities sophisticated molecular breeding can producestrains and biotechnological processes which could eliminate


many types of contaminants in an economical efficientand simple process and environmentally friendly mannerThe biosynthesized silver nanoparticles showed excellentantimicrobial activity and possessed considerable cytotoxiceffect against in vitro HeLa cancer cell lines IC

50value

was found to be 200 120583gmL of AgNPs against HeLa celllines The data represented in our study contribute to anovel and unexplored area of nanomaterials as alternativemedicine Furthermore the biosynthesized AgNPs displayeda pronounced antimicrobial and cytotoxicity activity againstclinical pathogenic microorganisms and HeLa cancer celllines Taken together the data collected in this study suggeststhat it would be important to understand the mode of actionof the biosynthesized nanoparticles prior to their use innanomedicine applications

Acknowledgment

This research was supported by a grant fromMarine Biopro-cess Research Center of the Marine Biotechnology Programfunded by the Ministry of Oceans and Fisheries Republicof Korea One of the authors Kannan Sivakumar expresseshis thanks to the Director Centre of Advanced Study inMarine Biology Faculty of Marine Sciences and AnnamalaiUniversity authorities for facilities and encouragement

References

[1] MAAlbrecht CW Evans andC L Raston ldquoGreen chemistryand the health implications of nanoparticlesrdquo Green Chemistryvol 8 no 5 pp 417ndash432 2006

[2] K F Chater ldquoGenetics of differentiation in StreptomycesrdquoAnnual Review of Microbiology vol 47 pp 685ndash713 1993

[3] S Sadhasivam P Shanmugam and K Yun ldquoBiosynthesis of sil-ver nanoparticles by Streptomyces hygroscopicus and antimicro-bial activity against medically important pathogenic microor-ganismsrdquo Colloids and Surfaces B vol 81 no 1 pp 358ndash3622010

[4] S Sadhasivam P Shanmugam M Veerapandian R Subbiahand K Yun ldquoBiogenic synthesis of multidimensional goldnanoparticles assisted by Streptomyces hygroscopicus and itselectrochemical and antibacterial propertiesrdquoBioMetals vol 25no 2 pp 351ndash360 2011

[5] K A Willets and R P Van Duyne ldquoLocalized surface plasmonresonance spectroscopy and sensingrdquoAnnual Review of PhysicalChemistry vol 58 pp 267ndash297 2007

[6] D I Gittins D Bethell D J Schiffrin and R J Nichols ldquoAnanometre-scale electronic switch consisting of a metal clusterand redox-addressable groupsrdquo Nature vol 408 no 6808 pp67ndash69 2000

[7] P K Jain I H ElSayed and M A El-Sayed ldquoAu nanoparticlestarget cancerrdquo Nano Today vol 2 no 1 pp 18ndash29 2007

[8] D Yu ldquoFormation of colloidal silver nanoparticles stabilized byNa+-poly(120574-glutamic acid)-silver nitrate complex via chemicalreduction processrdquo Colloids and Surfaces B vol 59 no 2 pp171ndash178 2007

[9] K Mallick M J Witcomb and M S Scurrell ldquoSelf-assemblyof silver nanoparticles in a polymer solvent Formation of ananochain through nanoscale solderingrdquo Materials Chemistryand Physics vol 90 no 2-3 pp 221ndash224 2005

[10] Y-C Liu and L-H Lin ldquoNewpathway for the synthesis of ultra-fine silver nanoparticles from bulk silver substrates in aqueoussolutions by sonoelectrochemical methodsrdquo ElectrochemistryCommunications vol 6 no 11 pp 1163ndash1168 2004

[11] A B Smetana K J Klabunde and C M Sorensen ldquoSynthesisof spherical silver nanoparticles by digestive ripening stabiliza-tion with various agents and their 3-D and 2-D superlatticeformationrdquo Journal of Colloid and Interface Science vol 284 no2 pp 521ndash526 2005

[12] M Kowshik S Ashtaputre S Kharrazi et al ldquoExtracellularsynthesis of silver nanoparticles by a silver-tolerant yeast strainMKY3rdquo Nanotechnology vol 14 no 1 pp 95ndash100 2003

[13] S Senapati A Ahmad M I Khan M Sastry and R KumarldquoExtracellular biosynthesis of bimetallic AundashAg alloy nanopar-ticlesrdquo Small vol 1 no 5 pp 517ndash520 2005

[14] A R Shahverdi S Minaeian H R Shahverdi H Jamalifar andA Nohi ldquoRapid synthesis of silver nanoparticles using culturesupernatants of Enterobacteria a novel biological approachrdquoProcess Biochemistry vol 42 no 5 pp 919ndash923 2007

[15] M Liong B France K A Bradley and J I Zink ldquoAntimicrobialactivity of silver nanocrystals encapsulated inmesoporous silicananoparticlesrdquo Advanced Materials vol 21 no 17 pp 1684ndash1689 2009

[16] K-H Cho J-E Park T Osaka and S-G Park ldquoThe studyof antimicrobial activity and preservative effects of nanosilveringredientrdquo Electrochimica Acta vol 51 no 5 pp 956ndash9602005

[17] H Wei C Chen B Han and E Wang ldquoEnzyme colorimetricassay using unmodified silver nanoparticlesrdquo Analytical Chem-istry vol 80 no 18 pp 7051ndash7055 2008

[18] A K SinghM Talat D P Singh andO N Srivastava ldquoBiosyn-thesis of gold and silver nanoparticles by natural precursorclove and their functionalization with amine grouprdquo Journal ofNanoparticle Research vol 12 no 5 pp 1667ndash1675 2010

[19] A R Shahverdi A Fakhimi H R Shahverdi and S MinaianldquoSynthesis and effect of silver nanoparticles on the antibacterialactivity of different antibiotics against Staphylococcus aureusand Escherichia colirdquo Nanomedicine vol 3 no 2 pp 168ndash1712007

[20] A Ingle M Rai A Gade and M Bawaskar ldquoFusarium solania novel biological agent for the extracellular synthesis of silvernanoparticlesrdquo Journal of Nanoparticle Research vol 11 no 8pp 2079ndash2085 2009

[21] H Bai B Yang C Chai G Yang W Jia and Z Yi ldquoGreensynthesis of silver nanoparticles usingRhodobacter sphaeroidesrdquoWorld Journal of Microbiology and Biotechnology vol 27 no 11pp 2723ndash2728 2011

[22] N Duran P D Marcato O L Alves G I H De Souzaand E Esposito ldquoMechanistic aspects of biosynthesis of silvernanoparticles by several Fusarium oxysporum strainsrdquo Journalof Nanobiotechnology vol 3 article 8 2005

[23] T Klaus R Joerger E Olsson and C Granqvist ldquoSilver-basedcrystalline nanoparticles microbially fabricatedrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 96 no 24 pp 13611ndash13614 1999

[24] S Arora J Jain J M Rajwade and K M Paknikar ldquoCellularresponses induced by silver nanoparticles in vitro studiesrdquoToxicology Letters vol 179 no 2 pp 93ndash100 2008

[25] S Gurunathan K Kalishwaralal R Vaidyanathan et alldquoBiosynthesis purification and characterization of silvernanoparticles using Escherichia colirdquo Colloids and Surfaces Bvol 74 no 1 pp 328ndash335 2009


[26] S Pal Y K Tak and J M Song ldquoDoes the antibacterial activityof silver nanoparticles depend on the shape of the nanoparticleA study of the gram-negative bacterium Escherichia colirdquoApplied and EnvironmentalMicrobiology vol 73 no 6 pp 1712ndash1720 2007

[27] K B Holt and A J Bard ldquoInteraction of silver(I) ions withthe respiratory chain of Escherichia coli an electrochemical andscanning electrochemical microscopy study of the antimicro-bial mechanism of micromolar Ag+ rdquo Biochemistry vol 44 no39 pp 13214ndash13223 2005

[28] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[29] M Sastry A Ahmad M Islam Khan and R Kumar ldquoBiosyn-thesis of metal nanoparticles using fungi and actinomyceterdquoCurrent Science vol 85 no 2 pp 162ndash170 2003

[30] P Mukherjee A Ahmad D Mandal et al ldquoFungus-mediatedsynthesis of silver nanoparticles and their immobilization inthemycelial matrix a novel biological approach to nanoparticlesynthesisrdquo Nano Letters vol 1 no 10 pp 515ndash519 2001

[31] P Sivalingam J J Antony D Siva S Achiraman and KAnbarasu ldquoMangrove Streptomyces sp BDUKAS10 as nanofac-tory for fabrication of bactericidal silver nanoparticlesrdquoColloidsand Surfaces A vol 98 pp 12ndash17 2012

[32] S D Sarker L Nahar and Y Kumarasamy ldquoMicrotitre plate-based antibacterial assay incorporating resazurin as an indica-tor of cell growth and its application in the in vitro antibacterialscreening of phytochemicalsrdquo Methods vol 42 no 4 pp 321ndash324 2007

[33] M P Lechevalier and H Lechevalier ldquoChemical compositionas a criterion in the classification of aerobic actinomycetesrdquoInternational Journal of Systematic Bacteriology vol 20 no 4pp 435ndash443 1970

[34] A V Kirthi A A Rahuman C Jayaseelan et al ldquoNovel ap-proach to synthesis silver nanoparticles using plant pathogenicfungi Puccinia graminisrdquo Materials Letters vol 81 pp 61ndash722013

[35] C G Kumar and S K Mamidyala ldquoExtracellular synthesis ofsilver nanoparticles using culture supernatant of Pseudomonasaeruginosardquo Colloids and Surfaces B vol 84 no 2 pp 462ndash4662011

[36] C Krishnaraj E G Jagan S Rajasekar P Selvakumar P TKalaichelvan and N Mohan ldquoSynthesis of silver nanoparticlesusing Acalypha indica leaf extracts and its antibacterial activityagainst water borne pathogensrdquo Colloids and Surfaces B vol 76no 1 pp 50ndash56 2010

[37] P Magudapathy P Gangopadhyay B K Panigrahi K GM Nair and S Dhara ldquoElectrical transport studies of Agnanoclusters embedded in glass matrixrdquo Physica B CondensedMatter vol 299 no 1-2 pp 142ndash146 2001

[38] K Kalishwaralal V Deepak S Ramkumarpandian H Nella-iah and G Sangiliyandi ldquoExtracellular biosynthesis of silvernanoparticles by the culture supernatant of Bacillus licheni-formisrdquoMaterials Letters vol 62 no 29 pp 4411ndash4413 2008

[39] A Panacek L Kvıtek R Prucek et al ldquoSilver colloid nanoparti-cles synthesis characterization and their antibacterial activityrdquoJournal of Physical Chemistry B vol 110 no 33 pp 16248ndash162532006

[40] KJ Kim W S Sung B K Suh et al ldquoAntifungal activity andmode of action of silver nano-particles on Candida albicansrdquoBioMetals vol 22 no 2 pp 235ndash242 2009

[41] W-R Li X-B Xie Q-S Shi H-Y Zeng Y Ou-Yang andY-B Chen ldquoAntibacterial activity and mechanism of silvernanoparticles on Escherichia colirdquo Applied Microbiology andBiotechnology vol 85 no 4 pp 1115ndash1122 2010

[42] M I Sriram S B M Kanth K Kalishwaralal and SGurunathan ldquoAntitumor activity of silver nanoparticles inDaltonrsquos lymphoma ascites tumor modelrdquo International Journalof Nanomedicine vol 5 no 1 pp 753ndash762 2010

[43] M Safaepour A R Shahverdi H R Shahverdi M R Khor-ramizadeh and A R Gohari ldquoGreen synthesis of small sil-ver nanoparticles using geraniol and its cytotoxicity againstFibrosarcoma-Wehi 164rdquo Avicenna Journal of Medical Biotech-nology vol 1 no 2 pp 111ndash115 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


methodology for the synthesis of nanoparticles has mountedto a level of supreme importance [8ndash11] An alternativeapproach for the synthesis of metal nanoparticles is to applybiomaterials such as plants microorganisms encompassinggroups such as bacteria fungi and actinobacteria as nanofac-tories [12ndash14] Emerging multidrug resistant (MDR) bacteriahas raised a demand for the urgent need to identify novelantimicrobial agents It was reported that silver had beenused as antimicrobial agents since ancient times [3] Withthe advancements in nanotechnology AgNPs have found itssignificant applications as antimicrobial agents in fields ofmicroelectronics catalysis and biomolecular detection [15ndash17] Although the antibacterial activity of AgNPs has beenproved in the recent years the actual mechanism of actionis not yet clear They may inactivate microorganisms byinteracting with their enzymes proteins or DNA to inhibitcell proliferation [18] It is also evident that the increasedantimicrobial activity of AgNPs may be attributed to itsspecial characteristics of small size and high surface area tovolume ratio [19] The advantage of adapting biosynthesisof AgNPs is the simplicity of extracellular synthesis anddownstream processing [20 21]

Nanoparticles have a wide range of applications asin combating microbes [22] biolabelling [23] and in thetreatment of cancer [24] The antibacterial activity of silverspecies is known since ancient times [25] and it has beendemonstrated that at low concentrations silver is nontoxicto human cells [26] It has also been reported that Ag+ions uncouple the respiratory chain from oxidative phos-phorylation or collapse the proton-motive force across thecytoplasmic membrane [27] The interaction of Ag+ withbacteria is directly related to the size and shape of thenanoparticles [26 28]

Sastry et al [29] reported on the biosynthesis of metalnanoparticles using the mycelial extract of fungi and acti-nobacteria [29] In addition the time required for completionof the reaction using both bacteria and fungi ranges betweenapproximately 24 hrs and 120 hrs whereas maximum syn-thesis of AgNPs can be achieved after 24 hrs of incubationMoreover metal accumulation is dependent on the growthphase of the cells [30] Sadhasivam et al [3] reported onthe extracellular biosynthesis of NPs by Streptomyces hygro-scopicus and antimicrobial activity against medically impor-tant pathogenic micro-organisms [3] Sivalingam et al [31]reported on the biosynthesis of bactericidal silver nanoparti-cles (AgNPs) using a novel Streptomyces sp BDUKAS10 anisolated mangrove sediment [31] Though the mechanism ofsilver resistance offered by bacteria using the silver bindingprotein is well documented their extraction and purificationneed to be elucidated further for large-scale productionHowever only a few studies have examined the componentsof marine actinobacteria that mediated the reduction ofsilver ions into AgNPs In this study we examined andcharacterized the extracellular biosynthesis of AgNPs usinga novel Nocardiopsis sp MBRC-1 which is a very importantmicro-organism to the production of several antibiotics andenzymes of commercial value To the best of our knowledgethis marine actinobacterium (Nocardiopsis sp MBRC-1) hasnever been used for nanoparticles biosynthesis

2 Materials and Methods

21 Chemicals All analytical reagents and media compo-nents were purchased from Sigma-Aldrich (St Louis USA)

22 Microbial Synthesis of AgNPs The Nocardiopsis spMBRC-1 strain was isolated from the marine sedimentsamples from the Busan coast (Lat 35∘091015840 N Long 129∘071015840E) South Korea Their partial 16S rRNA gene sequenceswere deposited in GenBank under the accession numberKC179785 For the synthesis of silver nanoparticles the activeNocardiopsis sp MBRC-1 culture was freshly inoculated onsterile starch casein medium and the flasks were incubated at25ndash28∘C and 180 rpm for 96 hrs (pH 70) After the incubationperiodwas complete the culture was centrifuged at 5000 rpmfor 30min and the supernatant was used for the biosynthesisof AgNPs Deionized water was used as a solvent in thesynthesis of AgNPs The collected supernatant (pH 70) wasadded separately to the reaction vessel containing silvernitrate at a concentration of 10minus3M (1 (vv)) and incubatedon an orbital shaker (dark condition) for 96 hrs at 30∘C Thereaction was carried out in the dark after the addition of theAgNO

3 and color change appeared transparent It confirmed

the synthesis of AgNPs The formation of the AgNPs wasmonitored by UV-vis spectroscopy using Shimadzu (ModelNo-UV 1800) double beam UV-vis spectrophotometer [3]All the experiments were carried out in triplicate and averagevalues have been reported

23 Characterization of AgNPs The synthesized AgNPswere freeze dried powdered and used for XRD analysisThe spectra were evaluated using an X-ray diffractometer(PHILIPS XrsquoPert-MPD diffractometerThe Netherlands) andCu-K120572 radiation 15405 A over an angular range of 5 to80∘ a step size of 002 a scan speed of 4∘mminus1 at a 40 kVvoltage and a 30mA current The dried powder was dilutedwith potassium bromide in the ratio of 1 100 and recordedthe Fourier transform infrared spectroscopy (FTIR) (PerkinElmer Inc USA) and spectrum GX spectrometry within therange of 400 to 4000 cmminus1 SynthesizedAgNPsweremountedon specimen stubs with double-sided adhesive tape coatedwith platinum in a sputter coater and examined under fieldemission scanning electron microscopy (FE-SEM) (JSM-6700 JEOL Japan) For transmission electron microscopy(TEM) imaging a drop of aqueous solution containing theAgNPs was placed on carbon coated copper grids and driedunder an infrared lamp (JEM 1010 JEOL Japan) (AC voltageminus800 kV) In addition the presence of silver metals in thesample was analyzed by energy dispersive X-ray analysis(EDX) combined with FE-SEM Finally the size distributionof the nanoparticles was evaluated using dynamic lightscatteringmeasurements conductedwith aMalvern ZetasizerZS compact scattering spectrometer (Malvern InstrumentsLtd Malvern UK)

24 Particle-Size Distribution of AgNPs Particle-size distri-bution analysiswas carried out after treatment of a 1mMsolu-tion of AgNO

3with the culture supernatant of Nocardiopsis

sp MBRC-1 at room temperature for 98 hrs The organism








2at 37∘C and










MBRC-l

002

896367

517537

68








(a) (b) (c) (d) (e) (f)25

2

15

1

05

0

400 500 600 700 800

Abso

rban

ce (a

u)

420 nm

Wavelength (nm)


48 hrs72 hrs96 hrs



3treated with





3844

4438

5677

6438 775

(1 2 3)

(2 0 4)

(0 4 3)

(1 4 4) (3 1 1)

Cou

nts

30 40 50 60 70 802120579 (degrees)





(a)

(b)

Cl

ClAg

Spectrum 1

0 2 4 6 8 10 12 14 16 18 20(keV)


(c)



3


















50value




(a)

(b)

05

101520253035404550


Inte

nsity

()


(c)



0

20

40

60

80

100

120

Control 50 100 150 200 250

Cel

l via

bilit

y (

)


(a)

(b)

(c)



4 Conclusions




50value


Acknowledgment


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








2at 37∘C and










MBRC-l

002

896367

517537

68








(a) (b) (c) (d) (e) (f)25

2

15

1

05

0

400 500 600 700 800

Abso

rban

ce (a

u)

420 nm

Wavelength (nm)


48 hrs72 hrs96 hrs



3treated with





3844

4438

5677

6438 775

(1 2 3)

(2 0 4)

(0 4 3)

(1 4 4) (3 1 1)

Cou

nts

30 40 50 60 70 802120579 (degrees)





(a)

(b)

Cl

ClAg

Spectrum 1

0 2 4 6 8 10 12 14 16 18 20(keV)


(c)



3


















50value




(a)

(b)

05

101520253035404550


Inte

nsity

()


(c)



0

20

40

60

80

100

120

Control 50 100 150 200 250

Cel

l via

bilit

y (

)


(a)

(b)

(c)



4 Conclusions




50value


Acknowledgment


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


MBRC-l

002

896367

517537

68








(a) (b) (c) (d) (e) (f)25

2

15

1

05

0

400 500 600 700 800

Abso

rban

ce (a

u)

420 nm

Wavelength (nm)


48 hrs72 hrs96 hrs



3treated with





3844

4438

5677

6438 775

(1 2 3)

(2 0 4)

(0 4 3)

(1 4 4) (3 1 1)

Cou

nts

30 40 50 60 70 802120579 (degrees)





(a)

(b)

Cl

ClAg

Spectrum 1

0 2 4 6 8 10 12 14 16 18 20(keV)


(c)



3


















50value




(a)

(b)

05

101520253035404550


Inte

nsity

()


(c)



0

20

40

60

80

100

120

Control 50 100 150 200 250

Cel

l via

bilit

y (

)


(a)

(b)

(c)



4 Conclusions




50value


Acknowledgment


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


3844

4438

5677

6438 775

(1 2 3)

(2 0 4)

(0 4 3)

(1 4 4) (3 1 1)

Cou

nts

30 40 50 60 70 802120579 (degrees)





(a)

(b)

Cl

ClAg

Spectrum 1

0 2 4 6 8 10 12 14 16 18 20(keV)


(c)



3


















50value




(a)

(b)

05

101520253035404550


Inte

nsity

()


(c)



0

20

40

60

80

100

120

Control 50 100 150 200 250

Cel

l via

bilit

y (

)


(a)

(b)

(c)



4 Conclusions




50value


Acknowledgment


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















50value




(a)

(b)

05

101520253035404550


Inte

nsity

()


(c)



0

20

40

60

80

100

120

Control 50 100 150 200 250

Cel

l via

bilit

y (

)


(a)

(b)

(c)



4 Conclusions




50value


Acknowledgment


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

(b)

05

101520253035404550


Inte

nsity

()


(c)



0

20

40

60

80

100

120

Control 50 100 150 200 250

Cel

l via

bilit

y (

)


(a)

(b)

(c)



4 Conclusions




50value


Acknowledgment


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



50value


Acknowledgment


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Biosynthesis, Antimicrobial and Cytotoxic ...Cytotoxic Activity. Cell viability was evaluated by the MTT colorimetric technique. Human HeLa cancer cell lines (cells/well)