Materials Science and Engineering C 34 (2014) 115–122

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Evaluation of antioxidant, antibacterial and cytotoxic effects of green synthesized silver nanoparticles by Piper longum fruit N. Jayachandra Reddy, D. Nagoor Vali, M. Rani, S. Sudha Rani ⁎ Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Pondicherry, India

a r t i c l e

i n f o

Article history: Received 1 April 2013 Received in revised form 13 August 2013 Accepted 29 August 2013 Available online 6 September 2013 Keywords: Piper longum Nanoparticles Condensed tannins Antioxidant Antimicrobial MCF-7 cell line

a b s t r a c t Silver nanoparticles synthesized through bio-green method has been reported to have biomedical applications to control pathogenic microbes as it is cost effective compared to commonly used physical and chemical methods. In present study, silver nanoparticles were synthesized using aqueous Piper longum fruit extract (PLFE) and confirmed by UV–visible spectroscopy. The nanoparticles were spherical in shape with an average particle size of 46 nm as determined by scanning electronic microscopy (SEM) and dynamic light scattering (DLS) particle size analyzer respectively. FT-IR spectrum revealed the capping of the phytoconstituents, probably polyphenols from P. longum fruit extract and stabilizing the nanoparticles. Further the ferric ion reducing test, confirmed that the capping agents were condensed tannins. The aqueous P. longum fruit extract (PLFE) and the green synthesized silver nanoparticles (PLAgNPs) showed powerful antioxidant properties in in vitro antioxidant assays. The results from the antimicrobial assays suggested that green synthesized silver nanoparticles (PLAgNPs) were more potent against pathogenic bacteria than the P. longum fruit extract (PLFE) alone. The nanoparticles also showed potent cytotoxic effect against MCF-7 breast cancer cell lines with an IC 50 value of 67 μg/ml/24 h by the MTT assay. These results support the advantages of using bio-green method for synthesizing silver nanoparticles with antioxidant, antimicrobial and cytotoxic activities those are simple and cost effective as well. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Nanotechnology is emerged as an interdisciplinary approach in biochemical applications with a focus on the synthesis of nanoparticles with the improved antioxidant and antimicrobial activities against degenerative diseases like Alzheimer's and Cancer [1,2]. Nanoparticles are commonly synthesized using physical and chemical methods in a short period of time and demonstrated for new properties based on size, shape and distribution [3]. Lithography, ultrasonic fields, UV irradiation and photochemical reduction [4–9] are commonly used methods, but the usage of non biodegradable toxic chemicals as reducing agents in some of these methods are potentially dangerous to the environment and biological system. At this juncture, the bio-green method of synthesizing nanoparticles using either microorganisms or extracts of medicinal plants holds unique attention among researchers. Synthesis of nanoparticles using microorganisms [10–12] consume more time for the maintenance of microorganisms whereas the plant extract mediated methods require less processing time. Mervat et al. [13] has reported the bioreductive activity of leaf extracts from selected five plants Malva parviflora, Beta vulgaris sub sp. Vulgaris, Anethum graveolens, Allium kurrat and Capsicum frutescens. From the above five plants, the synthesis of nanoparticles from M. parviflora (Malvaceae) was ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, Pondicherry University, Pondicherry 605014, India. Tel.: +91 9443768726. E-mail addresses: [email protected], [email protected] (S.S. Rani). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.08.039

found to be effective bioactive, stable and monodispersed. The effects of natural and Ayurvedic plants like green tea (Camellia sinensis) [14], neem (Azadirachta indica) leaf broth [15], natural rubber [16], starch [17], aloe Vera [18], Mongrass leaf extract [19,20] leguminous shrub (Sesbania drummondii) [21], and latex of Jatropha curcas [22] etc., were also reported for the synthesis of nanoparticles. In the quest for effective natural plant constituents against various diseases caused by the different kinds of pathogenic microbes [23], the primary active constituents namely piperine, piplartine, and piperlongumine are found to be present in Piper longum. Piperine consists of four to five percent essential oil derived from the catkins in addition to the active chemicals including several piperidine alkaloids, tannins, dihydrostigmasterol, sesamim, terpenines and isobutyl deca-trans-2-trans-4-dienamide [24]. Silver plays a vital role in antimicrobial, catalytic and biological systems [25] among the other metals and the synthesis of silver nanoparticles as an antimicrobial agent has gained more importance against the increasing threat posed by antibiotic resistant microbes [26,27]. Though there are reports on the synthesis of silver nanoparticles with desirable size and shape exhibiting antimicrobial activity using physical and chemical methods [28,29], but their potential use in biomedical field is uncertain owing to their toxic nature. Hence, the present study was designed to prepare silver nanoparticles using bio-green method with aqueous fruit extract of P. longum as bio reducing agent of Ag + to Ag to synthesize the silver nanoparticles. The formed silver nanoparticles were characterized using UV–visible

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spectrophotometer, scanning electron microscope, Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) particle size analyzer. At the same time in vitro antioxidant, antimicrobial and cytotoxic effects of green synthesized silver nanoparticals were evaluated. 2. Materials and methods 2.1. Materials AgNO3 (99%) was purchased from Merck bioscience, India and P. longum fruit powder purchased from local Ayurvedic market in Puducherry. AgNO3 was used as a silver precursor and the natural fruit powder from P. longum was used as a bio reducing agent. The reagents used in all the experiments were purely analytical-grade and used without further purification. 2.2. Preparation of the extract One gram of dried P. longum fruit fine powder was added to 100 ml double distilled water in 250 mL Erlenmeyer flask. The preparation of aqueous P. longum fruit extract was done by using magnetic heatingstirrer at 80 °C for 10 min. The extract was then filtered through Whatman filter paper no.1 and stored at 4 °C for further work. 2.3. Bio-green synthesis of silver nanoparticles The synthesis of nanoparticles by bio-green method was carried out by adding 10 ml of P. longum fruit aqueous extract (PLFE) to 50 ml of 1 mM silver nitrate solution and kept for incubation at room temperature for 2 h. The overall reaction process was carried out in dark to avoid unnecessary photochemical reactions. The color change of the silver nitrate solution from colorless to brownish yellow was observed by naked eye and the bio reduced sample component was confirmed by UV–Visible spectroscopy. The obtained P. longum silver nanoparticles (PLAgNPs) were purified through repeated centrifugation at 11,500 rpm for 20 min and washed with distilled water. The PLAgNPs were collected and redispersed in deionized water for characterization. 2.4. Characterization of Silver nanoparticles

dropping very little amount of the sample. The extra solution was removed by using blotting paper and the film on the SEM grid was allowed to dry for 5 min under mercury lamp for the observation of shape of the particles. FT-IR analysis done by using Thermo Nicolet Nexus 670 spectrometer to identify the bioactive compounds of PLFE associated with the PLAgNPs, and the FT-IR spectrum was recorded in the range of 4000–400 cm−1. The various vibration stretches were observed in spectrum analysis to determine different functional groups associated with the PLAgNPs. The size of the PLAgNPs and distribution was determined using DLS (Dynamic Light Scattering) particle size analyzer [ZETA Seizers Nanoseries (Malvern Instruments Nano ZS)]. 2.5. Test for phenol compounds Ferric ion reducing test was performed to identify the presence of phenol compounds in PLFE and PLAgNPs. In this test FeCl3 solution (30 mM) was added to PLFE and PLAgNPs then observed for color change. Hydrolysable tannins gave blue black color and condensed tannins brownish green color while forming ferrous compound [30]. 2.5.1. Test for bound phenols The synthesized silver nanoparticles were washed twice with distilled water and residue was treated with methanol. The supernatant was collected after centrifugation and the absorbance was measured using UV–visible spectrophotometer [31]. 2.6. In vitro antioxidant assays 2.6.1. Quantitative determination of total phenol content in PLFE and PLAgNPs The total phenol content was estimated by standard Folin–Ciocalteau method [32]. Aqueous PLFE (1 mg/ml) and PLAgNPs (1 mg/ml) were mixed with Folin–Ciocalteau reagent (5 ml diluted to 1: 10 with double distilled water) for 5 min and 4 ml of 1 M sodium carbonate to maintain alkaline environment followed by the incubation for 15 min at room temperature. The phenol content was determined by spectrophotometric method at 765 nm. Gallic acid was used as standard at different concentrations (10 to 250 μg/ml in50% methanol). The phenol content was expressed as Gallic acid (GA) equivalents (mg GA/g dry weight).

The synthesized PLAgNPs were analyzed by using UV-1700 Shimadzu UV–Visible spectrophotometer and their spectral analysis was done in the range of 300 to 700 nm at different time intervals. The shape of the PLAgNPs was determined by Scanning Electron Microscope (SEM) using Hitachi S-4500 SEM machine. In this experiment, on the carbon coated copper grid thin film of the sample was prepared by

2.6.2. DPPH free radical scavenging assay DPPH radical scavenging assay for both PLFE and PLAgNPs was performed as described by Chang et.al [33]. Different concentrations (100 to 600 μg/ml) of PLFE and PLAgNPs were separately mixed with

Fig. 1. UV–visible spectra of PLAgNPs at different time intervals.

Fig. 2. SEM image of PLAgNPs.

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Table 1 Total phenolic content of PLFE and PLAgNPs.

Fig. 3. Size distribution of PLAgNPs with maximum intensity at 46 nm.

3 ml of 0.1 mM DPPH and incubated in dark for 15 min. After incubation, the absorbance of the samples was measured by using UV–vis spectrophotometer at 517 nm against methanol as blank. Rutin was used as standard and DPPH methanol reagent without sample was used as control and percentage of inhibition was calculated by the following formula

% of inhibition ¼

Absorbancecontrol ‐Absorbancetest Absorbancecontrol

2.6.3. Reducing power assay The total reducing power of the PLAgNPs and PLFE was determined based on the previously reported Yildrim et al. [34]. Different concentrations (100–500 μg/ml) of PLFE and PLAgNPs of 0.5 ml was separately mixed with 2.5 ml of 0.2 M (pH 6.6) phosphate buffer and 2.5 ml of 1% potassium ferricyanide then incubated for 20 min at 500 C. To this 2.5 ml of 10% trichloroacetic acid (TCA) was added and then centrifuged at 3000 rpm for 10 min. To 2.5 ml of supernatant, 2.5 ml of de ionized water followed by 0.5 ml of FeCl3 (0.01%) solution was added and the absorbance was measured at 700 nm against phosphate buffer blank. Rutin was used positive control in this test. An increase in the absorbance with increasing concentration is directly proportional to the reducing power.

Fig. 4. The FT-IR spectra of PLFE and PLAgNPs.

Sample

Phenol content (mg GA/g sample)

PLFE PLAgNPs

216 ± 0.7 104 ± 0.8

2.6.4. Superoxide radical scavenging activity Superoxide anion radical scavenging activity was estimated according to the previously reported method by Nishimiki et al. [35]. The purple formazan formed by nitrobluetetrazolium (NBT) by reacting with the superoxide radicals generated from phenazine methosulfate–nicotinamide adeninedinucleotide (PMS/NADH) non-enzymatic system was measured spectrophotometrically. In this assay, the 1 ml reaction mixture contained phosphate buffer (100 mM, pH 7.4), NADH (468 μM), NBT (156 μM), PMS (60 μM) and various concentrations (10–200 μg/ml) of PLAgNPs and PLFE. After incubation for 5 min at room temperature the absorbance at 560 nm was measured against appropriate blank to determine the quantity of formazan generated. Rutin was used as positive control in this test. 2.6.5. Nitric oxide radical scavenging The NO Scavenging activity of PLAgNPs and PLFE was assessed according to the method described by Griess Illosvoy reaction [36]. In this method, the nitric oxide generated from sodium nitroprusside interacts with oxygen and the resulting nitrite ions are quantified. In this test 3 ml of the reaction mixture contained sodium nitroprusside (10 mM), phosphate buffered saline (pH 7.4) and various concentrations of (50–500 μg/ml) of PLAgNPs and PLFE and incubated at room temperature for 90 min. To 0.5 ml of this solution was added 1 ml of sulfanilamide (0.33% in 22% glacial acetic acid) followed by 1 ml of napthyl ethylenediamine dihydrochloride (NED) (0.1% w/v) and the resulting pink chromophore was read at 540 nm spectrophotometrically. Rutin was used as positive control. 2.6.6. Hydrogen peroxide scavenging assay Hydrogen peroxide scavenging activity PLAgNPs and PLFE slightly modified method of Avani patel et al. [37]. In this test, H2O2 (100 mM) was prepared freshly in phosphate buffer saline (pH 7.4). 300 μl of test samples containing various concentrations of PLAgNPs and PLFE (10 to 60 μg/ml) was added to 600 μl of H2O2 (100 mM) and the final volume was made up to 1 ml with PBS. The absorbance was measured

Fig. 5. The reducing power of PLAgNPs, PLFE and rutin.

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Fig. 6. The DPPH assay of PLAgNPs, PLFE and Rutin.

Fig. 8. The NO radical scavenging activity of PLAgNPs, PLFE and rutin.

at 230 nm against the separate sample blanks. The percentage of inhibition was calculated and quercetin used as positive control.

2.7.2. Growth curve studies To study the effect PLAgNPs on the growth pattern of bacteria, E. coli was cultured in nutrient broth with and without PLAgNPs (4 μg/ml) and the growth was measured at 600 nm at 1 h interval for 24 h. Under normal growth conditions, the four phases of growth curve including lag phase, log phase, stationary phase and death phase for E. coli culture was plotted in the graph.

2.7. Tests for antibacterial activity 2.7.1. Disc diffusion method The antibacterial activity of PLAgNPs and PLFE was evaluated by using the Kirby–Bauer method [38]. Nutrient broth/Agar (1 g beef extract, 1 g peptone, 0.5 g NaCl dissolved in 100 ml of double distilled water) was used for culturing the bacteria. The Nutrient agar plates were inoculated with cultures of Bacillus subtilis, Bacillus cereus, Staphylococcus aureus and Pseudomonas aeruginosa allowed for overnight growth at 30 °C. Sterile whatman filter paper discs of 5 mm diameter were impregnated with 10 μg and 20 μg of PLAgNPs/PLFE and were placed on to the bacterial lawn in agar plates. Standard streptomycin antibiotic discs were used as a reference drug. The agar plates were incubated at 37 °C for 24 h. After 24 h of incubation, the zone of inhibition was measured.

The cancer cell line MCF-7 was cultured in the DMEM (Dulbecco's modified of Eagle medium with L-glutamine & 1000 mg/L glucose) supplemented with 10% fetal bovine serum, penicillin G (100 units/ml) and streptomycin sulfate (0.1 mg/ml) in a humidified atmosphere consisting of 5% CO2 at 37 °C.

Fig. 7. The superoxide scavenging activity of PLAgNPs, PLFE and rutin.

Fig. 9. The hydrogen peroxide scavenging activity of PLAgNPs, PLFE and quercetin.

2.8. Cell culture

2.8.1. MTT (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium Bromide) assay for Cytotoxicity The cytotoxic effect of PLAgNPs on MCF-7 cells was assessed by MTT method. The monolayer culture was trypsinized and the cell count was adjusted to 50,000 cells/ml with DMEM medium containing 10% fetal

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Table 2 Inhibition zones of PLFE and PLAgNPs. Test sample

Concentration Staphylococcus Bacillus Pseudomonas Bacillus (μg/ml) aureus cereus Diameter of subtilis Diameter of Diameter zone (cm) Diameter zone (cm) of zone of zone (cm) (cm)

Streptomycin

10 20 Plant extract 10 20 Nanoparticles 10 20

2.0 2.6 0.7 0.8 1.0 1.4

3.2 3.4 0.4 0.6 0.8 1.3

2.8 3.4

2.6 3.1

1.0

1.1

1.8

2.0

bovine serum. Subsequently the cells were seeded as triplicates at 104 cells/well (200 μl/well) in 96 well plate, and kept in CO2 incubator for 12 h for attachment and growth. The cells were then exposed to PLAgNPs at different concentrations (10, 20, 30, 40, 50, 60, 70 and 80 μg/ml) for 24 and 48 h separately. After treatment, the medium was removed and cells were incubated with 20 μl of MTT (5 mg/ml in PBS) in fresh medium for 4 h at 37 °C and the resulting formazan crystals from mitochondrial reduction of MTT were solubilized in DMSO (150 μl/well) and the absorbance was read at 570 nm. Percentage of viability was calculated following the formula and expressed as IC50. % of viability ¼

Absorptiontest  100 Absorptioncontrol

3. Results and discussion In the bio-green method, the commencement formation of PLAgNPs was indicated by a change in the color of 1 mM silver nitrate solution from colorless solution to brownish yellow color on addition of PLFE in 5:1 ratio. The color change in the reaction mixture was due to collective oscillation of free electrons present in the reduced PLAgNPs [39]. In UV–visible spectrophotometer, the synthesized PLAgNPs displayed a

Fig. 11. Effect of PLAgNPs on different phases of E. coli growth.

clear and single surface plasmon resonance band with the λ max at 430 ± 6 nm. The formation of PLAgNPs was found to increase with reaction time and broadening of the peak was observed between 400 to 500 nm and this is indicative of the poly dispersed nature of the PLAgNPs formed (Fig. 1).The yield of the PLAgNPs obtained was found to be 140 mg/g of plant extract. Similar spectrum was observed during the time dependent synthesis of silver nanoparticles using Dillenia fruit extract [40]. The synthesized PLAgNPs were spherical in shape as observed in the scanning electron microscope image (Fig. 2) and this is confirmed by the single peak obtained in the SPR spectrum of the UV–visible study(Fig. 1). The size distribution of synthesized PLAgNPs was determined using DLS particle size analyzer and the average size of the PLAgNPs was found to be 46 nm. Though the size distribution of synthesized PLAgNPs ranged between 15 to 200 nm, maximum proportion of the particles

Fig. 10. Images of anti bacterial activities PLAgNPs and PLFE were used at 10 μg/ml concentration against Bacillus cereus (A,G) and Staphylococcus aureus (B,E) and at 20 μg/ml against Bacillus cereus (A,G), Staphylococcus aureus (B,E), Pseudomonas aeruginosa (C,F) and Bacillus subtilis (D, H). (P = PLFE, N = PLAgNPs, EFGH = streptomycin).

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Fig. 12. Cytotoxic effect of PLAgNPs against MCF-7 cell lines at 24 and 48 h.

was observed to be in the size range of 40 to 70 nm (Fig. 3). The broadening of SPR peak obtained in the UV–visible spectra (Fig. 1) confirms the broad size range of PLAgNPs between 15 to 200 nm as determined in DLS analyzer. The functional groups present in the PLFE and PLAgNPs were analyzed using FT-IR spectroscopy. In the FT-IR analysis, PLFE vibration stretches were observed at 3297 cm−1, 2928 cm−1, 1597 cm−1, 1400 cm−1, 1220 cm−1, 1085 cm−1, 1040 cm−1 and PLAgNPs peaks were found at 3315 cm−1, 1624 cm−1, 1390 cm−1, 1220 cm−1, 1031 cm−1(Fig. 4). The band pattern of the functional group as well as finger print region was similar to those of polyphenole specified by Chen and Mu [41]. The bands at 3297 cm−1 and 2928 cm−1 corresponding to O\H stretching vibrations of phenol group and C\H stretching of aromatic compound were observed respectively. The vibration stretch at 1597 cm−1 is because of C_C stretch in aromatic ring is confirmation for the presence of aromatic group. The peak at 1400 cm−1 corresponds to O–H bend of poly phenol. The weaker band at 1220 cm−1 corresponding to C\O\C stretch was observed. The C\O stretching vibrations of IR spectrum observed at 1085 cm−1 and 1040 cm−1. In the PLAgNPs peaks were changed from 3297 cm−1 to 3315 cm−1, 1597 cm−1 to 1624 cm−1, 1400 cm−1 to 1390 cm−1 and weaker bands 1121 cm−1, 1085 cm−1 were disappeared. Based on the FT-IR analysis, it can be assumed that phenol compounds present in the PLFE may be involved in capping and stabilizing the nanoparticles. To confirm the role of phenol compounds in the reduction process, the ferric ion reducing test was performed. Addition of ferric chloride solution to the PLFE caused color change to brownish green and further addition of ferric chloride caused some precipitation. The fast color change and the formation of precipitation indicated the presence of condensed tannins in the fruit extract. Similar color change was not observed with synthesized PLAgNPs on addition of ferric chloride solution (Supplementary data 1). This test confirms that the phenol compounds in the PLFE were responsible for the reduction of silver nitrate to silver and they were not free in the PLAgNPs solution to give the color reaction. To reconfirm the presence of polyphenols bound to PLAgNPs, the PLAgNPs were treated with methanol and the supernatant was analyzed by UV–visible spectrophotometer for the absorbance pattern. The peak was observed at 259 nm (Supplementary data 2) which indicated the presence of polyphenol groups as reported earlier by Theerasin [42]. The free radical scavenging activity of synthesized PLAgNPs was determined by using different in vitro assays. The total phenol content of synthesized PLAgNPs was found to be 104 ± 0.8 mg of GAE/g as compared to PLFE (216 ± 0.7 mg of GAE/g) phenol content (Table 1)

Fig. 5 shows the ferric ion reducing activity of PLAgNPs and the PLFE. PLAgNPs showed more reducing activity than the PLFE and the reducing activity of PLAgNPs was found to increase with increasing concentrations. Similar observations were made by Depankar and Murugan [43] with Iresine herbstii silver nanoparticles. Since the reducing power of compounds is directly proportional to antioxidant activity, antioxidant activity of PLAgNPs was assessed by DPPH scavenging assay by using Rutin as positive control. DPPH was a stable compound and accepts hydrogen or electrons from silver nanoparticles. The results obtained in the DPPH assay showed effective free radical inhibition by both PLAgNPs and PLFE (Fig. 6). The average percentage inhibition of synthesized PLAgNPs was 67% as compared to that of PLFE at different concentrations used in this study and the activity increased with increasing concentrations of PLAgNPs. Similar observations with enhanced DPPH scavenging activity by selenium, platinum, silver nanoparticles [44–47] and by torolex and chitosan coated gold nanoparticles [48,49] have been reported. Fig. 7 shows the superoxide scavenging activity of both the PLAgNPs and PLFE as determined by the PMS-NBT reduction system. Superoxide (O− 2 ) radicals easily react with DNA and protein which necessitate their immediate clearance in living systems. The superoxide radical quenching activity of PLAgNPs was found to be increased with increasing concentrations and the average inhibition was about 60% as compared to the activity of PLFE. Rutin was used as standard in this assay. The potential superoxide scavenging activity of gold and silver nanoparticles reported earlier [50] support our findings in the present study. The role of nitric oxide radicals in carcinomas and inflammatory process is well established. The toxic effects of nitric oxide will increase when reacts with superoxide radicals that lead to vascular system damage and results in conditions including inflammation, juvenile diabetes and multiple sclerosis [51,52]. Because of the less stability of nitric oxide ions, they accept electrons from silver nanoparticles and form formazan when treated with Griess reagent that can be detected spectrophotometrically. The nitric acid quenching activity of PLAgNPs was assessed as compared to that of PLFE. The NO radical quenching activity of PLAgNPs was found to increase with increasing concentrations and the average inhibition was found to be 70% as compared to the activity of PLFE (Fig. 8). Similar effects are reported with Cerium nanoparticles which showed good protective effects against NO. Radicals [53]. The H2O2 scavenging activity of PLAgNPs and PLFE is shown in Fig. 9. PLAgNPs were as effective as PLFE in quenching H2O2 radicals and the average inhibition was found to be 96% as compared to PLFE. In this study, it could be noted that the superoxide radical quenching activity and NO quenching activity of PLAgNPs was 60% and 70% respectively as compared to PLFE which can be explained on the fact that the concentration of phytocompounds responsible for the scavenging activities was higher in the extract than adhered to the nanoparticles. On the other hand, the observed increase in H2O2 scavenging activity of PLAgNPs (96%) may be because of the plant condensed tannins present in the extract that are involved in the formation of nanoparticles. Similar observations were made with silver nanoparticles prepared with stem bark of Shorea roxburghii previously [54]. In the present age, the threat due to spreading of antibiotic resistant microbes has become a global problem and hence there is an urgent need to contemplate and develop new powerful antimicrobial agents to take care of multi drug resistant strains. [55]. Colloidal silver has been used as an antibacterial agent since ancient Greece [56]. Unlike antibiotic drugs, bacteria cannot easily develop resistance because silver targets multiple components in the bacterial cell. As a result, silver is used in medical equipment coatings [57] and dental resin components [58]. It is also reported that the mechanism behind its antibacterial activity is by weakening DNA replication and inactivating proteins [59]. The antibacterial activity of P. longum fruit is also well established [60]. To evaluate the antibacterial effect of PLAgNPs and PLFE, they were tested against four different bacterial species at different concentrations in this study. PLAgNPs and PLFE were used at 10 μg/ml concentration

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against Bacillus cereus and S. aureus and at 20 μg/ml against B. cereus, S. aureus, Pseudomonas aeruginosa and Bacillus subtilis. The antibiotic streptomycin was used as a standard in this assay. The zone of inhibition produced by PLAgNPs in plates containing bacterial lawns was compared with plates treated with PLFE alone. As could be observed in Fig. 10 and Table 2, the antibacterial effect of PLAgNPs was almost twofold higher than that of PLFE alone. This effect could be due to the conformational changes induced in the membrane by positively charged silver nanoparticles that accumulated on the negatively charged membrane of microbes leading to the cell death [61]. To confirm the potential antibacterial effect of PLAgNPs, E. coli culture was treated with PLAgNP at a concentration of 4 μg/mL and its effect on the growth pattern of bacteria was analyzed. As shown in Fig. 11, the PLAgNPs caused disturbance in the growth behavior of bacteria by increasing the lag phase time and by decreasing the number of viable cells in the log phase suggesting the toxic effect of PLAgNPs against E. coli. The biomedical applications of silver nanoparticles are promising with their tremendous effects in the fields of medicine, drug delivery and anti angiogenic property of cancer [62,63]. In the present study the cytotoxic effect of PLAgNPs was tested against MCF-7 cell lines by using MTT assay. The viability of the MCF-7 cell lines was observed after 24 and 48 h of treatment with PLAgNPs. Fig. 12 shows reduced cell viability of MCF-7 cell lines with increasing concentrations (10–80 μg/ml) of PLAgNPs at 24 and 48 h. The IC 50 value for PLAgNPs was calculated and was found to be 67 μg/ml and 51 μg/ml at 24 and 48 h respectively. The reduced cell viability of MCF-7 cells observed in this study is suggestive of anticancer effects of PLAgNPs and further studies are required to be done to understand the process of cell death by apoptosis or necrosis pathway. 4. Conclusion In this study, stable bioactive silver nanoparticles of average size of 46 nm were synthesized using P. longum fruit extract by bio-green method as a cost effective manner. The nanoparticles possessed the added advantage of active phytoconstituents incorporated in them. The biomedical applications of the synthesized silver nanoparticles is substantiated by their potent free radical quenching effect, antibacterial activity and cytotoxic effect against MCF-7 cell lines in this study. The outcomes of this study illustrate a broad range of applications of bioactive silver nanoparticles synthesized by bio-green method. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2013.08.039. References [1] A. Nazem, G.A. Mansoori, Nanotechnology solutions for Alzheimer's disease: advanced in research tools, diagnostic methods and therapeutic agents, J. Alzheimers Dis. 13 (2008) 199–223. [2] Pallab Sanpui, Arun Chattopadhyay, Siddhartha Sankar Ghosh, induction of apoptosis in cancer cells at low silver nanoparticle concentrations using chitosan nanocarrier, Appl. Mater. Interfaces 3 (2011) 218–228. [3] Manish Dubey, Seema Bhadauria, B.S. Kushwah, Green synthesis of nanosilver particles from extract of eucalyptus hybrid leaf, Dig. J. Nanomater. Biostruct. 4 (2009) 537–543. [4] F. Mafune, J. Kohoo, Y. Takeda, T. Kondow, Full physical preparation of size-selected gold nanoparticles in solution: laser ablation and laser-induced size control, J. Phys. Chem. B 106 (2002) 7575–7577. [5] R.R. Naik, S.J. Stringer, G. Agarwal, S.E. Jones, M.O. Stone, Biomimetic synthesis and patterning of silver nanoparticles, Nat. Mater. 1 (2002) 169–172. [6] K. Okitsu, A. Yue, S. Tanabe, H. Matsumoto, Y. Yobiko, Formation of colloidal gold nanoparticles in an ultrasonic field: control of rate of gold(III) reduction and size of formed gold particles, Langmuir 17 (2001) 7717–7720. [7] T.K. Sau, A. Pal, N.R. Jana, Z.L. Wang, T. Pal, Size controlled synthesis of gold nanoparticles using phytochemically prepared seed particles, J. Nanopart. Res. 3 (2001) 257–261. [8] W.M. Tolles, Nanoscience and nanotechnology in Europe, Nanotechnology 7 (1996) 59–105. [9] M.H. Magonnuss, K. Deepert, J. Malm, J. Bovin, L. Samuelson, Size selected gold nanoparticles by aerosol technology, Nanostruct. Mater. 12 (1999) 45–48.

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N. Jayachandra Reddy, Research scholar, Department of Biochemistry and Molecular Biology under supervisor of Dr. S. Sudha rani. He completed his M.Sc degree in Banaras Hindu University, India. Area of interest: Nanoparticles, Cell Signaling, Cancer.

D. Nagoor Vali, M.Sc student at the same department.

M. Rani, Research scholar at Department of Biochemistry and Molecular Biology working under supervision of Dr. S. Sudha rani. Area of interest nanotechnology, cancer biology and immunology.

Dr. S. Sudha Rani, Ph.D, Assistant professor, Department of Biochemistry and Molecular Biology, Pondicherry University, India. Area of interest: Nanotechnology, Immunology, Cell signaling in Degenerative diseases, Geo-microbiology.