Catalytic and various biological activities of green silver nanoparticles synthesized from Plumeria alba (Frangipani) flower extract

Materials Science and Engineering C 51 (2015) 216–225

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Catalytic and biological activities of green silver nanoparticles synthesized from Plumeria alba (frangipani) flower extract Rani Mata, Jayachandra Reddy Nakkala, Sudha Rani Sadras ⁎ Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Pondicherry, India

a r t i c l e

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Article history: Received 22 April 2014 Received in revised form 26 November 2014 Accepted 27 February 2015 Available online 28 February 2015 Keywords: Frangipani flower extract Catalytic Biological activities Cytotoxicity Apoptosis

a b s t r a c t Herein, we report the green synthesis of silver nanoparticles using Plumeria alba (frangipani) flower extract (FFE) and their biological applications. The formation of frangipani silver nanoparticles (FSNPs) was confirmed by UV– visible spectroscopy and characterized by DLS particle size analyzer, SEM/EDAX, FTIR, TGA/DSC and XRD. The synthesized spherical FSNPs were found to be 36.19 nm in size as determined by DLS particle size analyzer. EDAX data and XRD pattern of FSNPs confirmed the presence and face-centered cubic (fcc) phase structure of silver. The bioactive groups C-C and C-N present in FFE were involved in the formation of FSNPs as identified by FTIR analysis. FSNPs exhibited powerful catalytic activity by reducing 4-nitrophenol to 4-aminophenol within 8 min and the other organic dyes namely methylene blue and ethidium bromide were moderately degraded. Biological activities of FSNPs are evaluated by means of antioxidant, antibacterial and cytotoxic effect. Antioxidant potential of FSNPs was assessed by various in vitro assays in which they exhibited moderate antioxidant activity. The antibacterial effect of FSNPs was tested in two different pathogenic bacterial strains and their bacteriostatic effect was confirmed by growth kinetic study in Escherichia coli. The cytotoxic effect of FSNPs in COLO 205 was analyzed by MTT assay and the IC50 concentration was found at 5.5 and 4 μg/ml respectively after 24 and 48 h of incubation. Cytotoxic effect of FSNPs in COLO 205 cells was associated with the loss of membrane integrity and chromatin condensation which might have played a crucial role in the induction of apoptosis as evidenced in AO/EB staining. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Nanotechnology has gained intense attention in the recent past due to its wide application in diverse areas like medicine, catalysis, energy and materials [1–4]. Particularly nanoparticles with small size to large surface area have potential medical as well as industrial applications [5]. Researchers make significant efforts towards the synthesis of nanoparticles by various means, including physical, chemical and biological methods [6,7]. As chemical synthesis involves the use of chemical reducing agents that may be toxic, such nanoparticles become unsuitable for biomedical applications. In the last decade, biological methods by using microbes or plant biomasses for the synthesis of nanometals was explored. Biological method has received increasing attention due to the growing need to develop environmentally benign technologies in material synthesis [8]. Green method of synthesizing nanoparticles with plant extracts is advantageous over physical, chemical and microbial methods as it is simple, convenient, environment friendly and requires less reaction time [9]. Among the noble metals used in green synthesis, silver is widely preferred owing to its antibacterial [10], antifungal, larvicidal, antiparasitic [11] and anticancer [12] properties. In ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, Pondicherry University, Pondicherry 605 014, India. E-mail address: [email protected] (S. Rani Sadras).

http://dx.doi.org/10.1016/j.msec.2015.02.053 0928-4931/© 2015 Elsevier B.V. All rights reserved.

green synthesis capping and stabilization of silver nanoparticles are brought about by bioactive compounds like alkaloids, flavonoids and polyphenols present in plant extracts [13]. Reports on the synthesis of green silver nanoparticles using plant extracts such as Camellia sinensis [14], Aloe vera plant extract [15], latex of Jatropha curcas [16] and lemongrass (Cymbopogon) leaf extract [17] are available. Further, the in vitro antioxidant properties of green silver nanoparticles synthesized from Cassia auriculata flower extract have been reported [18]. Anticancer potential silver nanoparticles from Euphorbia chapmaniana on HL-60 has also been reported recently [19]. Silver nanoparticles owing to their catalytic property can degrade dyes in industrial effluents and convert nitrate and nitrite ions in chemical fertilizers to harmless N2 and thereby find their application in treating environmental pollutants. These nitrate and nitrite ions affect human health by causing methemoglobinemia [20]. The catalytic action of silver nanoparticles can be evaluated by applying many of the chemically synthesized cationic and anionic dyes. Recently, photo catalytic degradation of methyl orange dye by silver nanoparticles synthesized from Ulva lactuca has been reported [21]. In this study, we report the green synthesis of silver nanoparticles using Plumeria alba (frangipani) flower extract (FFE) and their biological applications. The plant P. alba (frangipani) belonging to the family Apocynaceae and the plant parts like leaves, bark, flowers and latex have been shown to possess various biological activities including

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antioxidant, antibacterial, antiarthritic activity, antitumor and anti diabetic activities. The flowers contain several bioactive compounds, including β-sitosterol, scopoletin, iridoids isoplumericin, plumieride, plumieride coumarate and plumieride coumarate glucoside [22]. Recently, the antimicrobial activity of gold nanoparticles synthesized from frangipani flower extract combined with standard antibiotic drugs has been reported [23]. In this study, we focused on the synthesis of frangipani silver nanoparticles (FSNPs) and the evaluation of their potential catalytic, antibacterial, antioxidant and anticancer activities in vitro. The synthesized FSNPs were characterized by UV–visible spectroscopy, dynamic light scattering (DLS) particle size analyzer, scanning electron microscope/energy dispersive analysis of X-rays (SEM/EDAX), fourier transform infrared spectroscopy (FTIR), thermo gravimetric analyzer/differential scanning calorimeter (TGA/DSC) and X-ray diffraction (XRD). 2. Materials and methods 2.1. Chemicals The frangipani (P. alba) flowers were collected from Pondicherry University, Puducherry, India. All the chemicals used were from HiMedia and Sigma Aldrich Bangalore, India and were purely analytical grade used without further purification. Deionized water was used for all experiments and all assays were performed in triplicates. 2.2. Preparation of flower extract Fresh frangipani flowers were collected, rinsed with distilled water, shade dried and fine powder was prepared. 1 g of frangipani flower powder was mixed with 100 ml of double distilled water in an Erlenmeyer flask and boiled for 10 min. Then the mixture was subjected to repeated filtration with Whatman No. 1 filter paper and the extract was stored at 4 °C. 2.3. Syntheses of silver nanoparticles To the 70 ml of silver nitrate (AgNO3)(1 mM) solution 10 ml of FFE was added and incubated at room temperature. The color change of the reaction mixture from pale yellow to yellowish brown was observed after 30 min of incubation indicating the formation of FSNPs which was further confirmed by UV-visible spectrophotometer.

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Nicolet Nexus 6700 spectrometer) analysis. FSNPs and FFE powders were separately mixed with potassium bromide (KBr) to make KBr pellets and then subjected to FTIR analysis. 2.4.5. TGA-DSC Thermal stability and surface weight loss of FSNPs were analyzed by SDT Q600 TG and the thermal behavior was analyzed by Q20 DSC. 2.4.6. XRD Crystalline nature of the synthesized FSNPs was analyzed by XRD analysis. Thin film of FSNPs powder was prepared on a glass slide and run under monochromatic Cu Kα radiation at 40 kV and 30 mA. 2.5. Catalytic activity The catalytic activity of FSNPs was analyzed by using three different dyes namely, methylene blue (MB), 4-nitrophenol (4-NP) and ethidium bromide (EB) in the presence of NaBH4. In this assay, 2.8 ml of MB (1 mM) was mixed with 0.2 ml of colloidal FSNPs (50 μg/ml) solution and the total volume was made up to 3.5 ml with distilled water. The absorbance of the reaction mixture was monitored at regular time intervals of 30 min [24]. The EB dye degradation assay was also performed in a similar way following the above procedure of MB. On the other hand reduction of 4-NP was carried out following the previously reported method [25]. In this analysis, 0.2 ml of FSNPs (50 μg/ml) was added to 0.2 ml of 4-NP (10− 5 M) and 2.5 ml of freshly prepared NaBH4 (0.150 M) and the absorbance of reaction mixture was measured by using a UV–visible spectrophotometer at different time intervals. 2.6. Antioxidant activities 2.6.1. DPPH (di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium) assay The free radical quenching activity of FSNPs and FFE was determined by DPPH assay [26]. 1 ml of FSNPs and FFE (10-100 µg/ml) were separately mixed with 3 ml of methanolic DPPH (0.1 mM). The mixture was incubated at 37 °C for 30 min in the dark and the absorbance was measured by a spectrophotometer at 517 nm. DPPH alone served as control and rutin was used as a standard. The percentage inhibition of DPPH reduction by FFE and FSNPs was calculated by using the formula: % of inhibition ¼

Absorbancecontrol −Absorbancetest  100 Absorbancecontrol

2.4. Characterization 2.4.1. UV–visible spectral analysis The bio-reduction and formation of FSNPs from FFE was monitored at regular time intervals using a UV–visible spectrophotometer (Shimadzu 1700). Further, the synthesized FSNPs were purified and collected by repeated centrifugation at 18,000 rpm. 2.4.2. DLS particle size analysis The average size of the synthesized FSNPs was determined by ZETA Seizers Nanoseries (Malvern Instruments Nano ZS) dynamic light scattering instrument. 2.4.3. SEM/EDAX A thin film of the sample was prepared by adding a pinch of FSNPs powder on a copper coated carbon grid and the surface morphology of FSNPs was analyzed by SEM (Hitachi S-4500). EDAX analysis was performed to determine the elemental composition of FSNPs equipped with SEM machine. 2.4.4. FTIR The functional groups of phytocompounds present in FFE which are involved in the formation of FSNPs was identified by FTIR (Thermo

2.6.2. Total reduction The reducing potential of FSNPs and FFE was analyzed accordingly by the method devised by Yen et al. [27] with simple modifications using rutin as a standard. 1 ml of the samples (25-100 µg/ml) was separately added to the reaction mixture containing 2.5 ml of PBS (0.2 M, pH 6.6) and 2.5 ml of potassium ferricyanide (1%). After incubation at 50 °C for 20 min, 2.5 ml of trichloroacetic acid (TCA, 10%) was added to the reaction mixture and then centrifuged at 3000 rpm. To the 2.5 ml of supernatant, 0.5 ml of ferric chloride (1%) was added and the absorbance was measured at 700 nm by using a UV–visible spectrophotometer. 2.6.3. Superoxide radical scavenging activity The non-enzymatic phenazine methosulphate (PMS)/nicotinamide adenine dinucleotide (NADH) system generated super oxide radicals, which reduce nitro blue tetrazolium (NBT) to purple formazan. The superoxide radical scavenging power of FSNPs and FFE was measured by the standard method of Nishikimi et al. [28] with minor changes. Different concentrations (10–60 μg/ml) of the test samples were added to the reaction mixture containing 1 ml of NBT (312 μm in PBS pH (7.4)) and 1 ml of NADH (936 μm in PBS pH (7.4)). Finally 0.1 ml of PMS (126 μm in PBS pH (7.4)) was added to the reaction mixture

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and incubated in the dark for 5 min. The absorbance of chromophore was measured at 560 nm by UV–visible spectrophotometer using rutin as a reference standard. 2.6.4. Hydrogen peroxide radical scavenging activity The hydrogen peroxide radical scavenging activity of FSNPs and FFE was determined following the previously reported method [29]. In this assay, different concentrations (10–60 μg/ml) of FSNPs and FFE were separately mixed with H2O2 (hydrogen peroxide) (40 mM) and the absorbance was measured at 240 nm by a spectrophotometer using sample blank. Rutin was used as a standard compound. 2.6.5. Nitric oxide radical scavenging activity The nitric oxide radical scavenging potential of FSNPs and FFE was estimated by the method described by Ebrahimzadeh et al. [30]. In this assay, different concentrations (25–150 μg/ml) of FSNPs and FFE were mixed with sodium nitroprusside (10 mM in PBS) and incubated for 150 min at room temperature. After incubation 0.5 ml of Griess reagent was added and the absorbance of the chromophore formed was measured at 546 nm by a UV–visible spectrophotometer using quercetin as a standard. 2.7. Antibacterial activity Agar disc diffusion method is a conventional method to check antibacterial activity. The microorganisms used in this study were obtained from the Microbial Type Culture Collection (MTCC), Chandigarh, India. The antibacterial susceptibility of FSNPs was tested against Escherichia coli and Bacillus subtilis by modified disc diffusion method [31]. The inoculums of bacterial culture was uniformly mixed with nutrient agar and poured in sterile petri plates. 20 μl of FGNPs (200–400 μg/ml) was loaded on to sterile Whatman No. 1 filter paper discs and placed on pre-inoculated agar plates. Streptomycin (400 μg/ml) was used as a standard in this assay. The diameter of the zone of inhibition was measured by a zone scale (Hi-Media) after 12 h of incubation at 37 °C. 2.7.1. Growth kinetics The inhibitory effect of FSNPs on the growth kinetics of bacteria was studied using E. coli culture by constructing the growth curve. Normally bacterial growth curve includes lag phase, log phase, stationary phase and death phase. In this study E. coli was cultured in liquid media (nutrient broth) in the presence of FSNPs at three different concentrations (200, 300 and 400 μg/ml). E. coli cultured in media without FSNPs was served as a control. Bacterial growth was measured at 1 h interval at 600 nm by a UV–visible spectrophotometer and the growth curve was plotted.

reader (Bio-RAD 680, USA). The IC50 values for FSNPs and FFE were obtained by calculating the percentage of inhibition. % of inhibition Mean OD of untreated cells – Mean OD of treated cells ¼  100 Mean OD of untreated cells

2.8.2. Acridine orange/ethidium bromide (AO/EB) staining The AO/EB staining technique has been widely applied to detect apoptotic cell death. COLO 205 (1×105cells/well) cells seeded in a six well plate were treated with an IC50 concentration of FSNPs and incubated for 24 h. Following the treatment cells were stained with AO/EB (1 mg/ml) and incubated for 2 min. Followed by the staining cells were observed under Nikon Eclipse Ti (Japan) fluorescence microscope (20X) for apoptotic changes. 3. Results and discussion The formation of FSNPs was indicated by the change in color of the solution to yellowish brown following the addition of FFE to AgNO3 solution after 30 min of incubation due to surface plasmon resonance (SPR). The formation of FSNPs was further confirmed by UV–visible spectroscopy and the results are shown in Fig. 1. The UV-visible spectral analysis of FSNPs exhibited a SPR peak with λmax at 445 nm after 30 min of incubation at room temperature. The position and shape of the SPR peak depend on the size, shape and dielectric constant of solutions. As the SPR peak position at different time intervals did not show considerable change, this might be indicative that the nanoparticles formed at different incubation time are similar in shape [32]. Our findings were in accordance with the earlier results of Raman Sukirtha et al. [33] which emphasizing the SPR peak of silver nanoparticles. The broadening of SPR peak with increasing time may be attributed to the increased formation of nanoparticles. The particle size distribution was analyzed by the DLS particle size analyzer and the average size of the FSNPs formed was found to be 36.19 nm as shown in Fig. 2. Data also indicate that the synthesized FSNPs were between the range of 5-100 nm and it represent the hydrodynamic diameter of FSNPs in liquid. The surface morphology of FSNPs was analyzed by SEM and they were found to be spherical in shape as shown in the SEM image (Fig. 3(A)). This observation is in good agreement with the data from UV–visible analysis indicating that the FSNPs were in similar shrerical

2.8. Cell culture The COLO 205 cell line was obtained from NCCS, Pune, India. The cells were maintained in DMEM (Dulbecco's Modified Eagle Medium) with L-glutamine and 4.5 g/l glucose, 10% fetal bovine serum, penicillin G (100 units/ml) and streptomycin sulfate (0.1 mg/ml) at 5% CO2 and 37 °C. 2.8.1. Cytotoxic effects The cytotoxic activity of FSNPs and FFE was checked by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The COLO 205 (1 × 104 cells/well) cells seeded in 96 well plates were treated with FSNPs (1-10 μg/ml) as well as FFE (25-250 μg/ml) and the plates were incubated for 24 and 48 h in a CO2 incubator. Following incubation 20 μl of MTT (5 mg/ml) was added and further incubated for 4 h. The purple colored formazan crystals formed were dissolved in 150 μl of dimethyl sulfoxide (DMSO) and the resulted chromophore was measured at 570 nm with reference at 630 nm using a microplate

Fig. 1. UV–visible spectral analysis of FSNPs at different time intervals.

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Fig. 4. FTIR spectrum of FSNPs and FFE.

Fig. 2. Size distribution of FSNPs by DLS particle size analyzer.

shape. EDAX data is helpful as it provides the elemental composition of the sample. The qualitative and quantitative elemental analysis can be estimated by measuring the energy and intensity of X-rays. As shown in Fig. 3(B) synthesized FSNPs showed an intense peak for silver and the other unlabelled peak might be carbon possibly originated from FFE. In the bio-green synthesis of metallic nanoparticles, the active phytochemical groups like amino, carboxylic, sulfhydryls etc. present in plant extracts play a key role in reducing, capping and subsequent formation of metallic nanoparticles. FTIR spectroscopy measurements of FFE and FSNPs indicated that the intense vibrational stretches correspond to the presence of bioactive or functional groups as shown in (Fig. 4). The FTIR spectrum of FFE exhibited vibrational stretches at 3285 cm−1 (O\H stretch of alcohol or phenol), 1690 cm−1 (C_O of aldehydes), 1392 cm−1 (C\H of alkanes) and 1052 cm−1 (C\N stretch of aliphatic amines). The spectrum of FSNPs showed intensive peaks at 1627 cm−1 (C\C stretch aromatic), 1384 cm−1 (C\C bond of aromatic ring or amide group-II), 1067 cm−1 (C\N stretch polyphenols) and 673 cm− 1 (C\H of alkynes). The obtained results indicate that the C\C and C\N vibrational stretches in FSNPs are from polyphenols of FFE which might have involved in the formation of FSNPs by acting as

capping and stabilizing agents. Our findings are substantiated by the observations made with green synthesized silver and gold nanoparticles from tansy fruit which indicated the involvement of phytoconstituents in the formation of silver nanoparticles [34]. Thermal properties of FSNPs by TGA analysis indicated that the FSNPs began to degrade initially at around 46 °C due to moisture loss with an initial 2% weight loss. Further degradation FSNPs occurred in two steps with one occurring between 50 and 150 °C and the second phase of degradation occurred between 200 and 350 °C with a weight loss of 36% and this weight loss may be attributed to the desorption of plant derived phytocompounds that were involved in the formation of FSNPs. Further, no degradation was observed between 350 and 800 °C which indicating the stability of metallic silver. The DSC curve in Fig. 5(A) shows an endothermic peak at 68 °C and this correlates with the denaturation enthalpy of FSNPs at 70 °C observed in TGA curve. Similar findings are reported with green synthesized silver nanoparticles using Ricinus communis leaf extract showed the desorption of less stable phytocompounds that are adhered to the silver nanoparticles [35]. The crystalline metallic nature of synthesized FSNPs was established using XRD analysis. As shown in Fig. 5(B) the FSNPs' XRD pattern indicated that a number of Bragg's reflections with 2θ values — 38.2, 44.3 and 64.6° sets of lattice planes were observed which may be indexed to (111), (200) and (220) facets of Ag0. These observations are

Fig. 3. (A) SEM image of FSNPs (arrows indicate the spherical silver nanoparticles) and (B) EDAX data of FSNPs.

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Fig. 5. (A) TGA and DSC curves of FSNPs and (B) XRD pattern of FSNPs.

Fig. 6. Catalytic activity of FSNPs, (A) (i) 4-nitrophenol + NaBH4 (ii) methylene blue (iii) ethidium bromide without FSNPs; (B) (i) 4-nitrophenol+ NaBH4 + FSNPs (ii) methylene blue + FSNPs (iii) ethidium bromide + FSNPs, shows degradation.

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supported by the EDAX data of our study which confirm the presence of elemental silver. These results of XRD were similar to the observations of Huang et al. which confirm the crystalline nature of silver [36].

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The catalytic activity of FSNPs was analyzed by dye degradation method using UV–visible spectroscopy. Different dyes were used in this study to analyze the catalytic activity of FSNPs including MB, 4-NP

Fig. 7. (A) DPPH radical scavenging activity of FSNPs, FFE and rutin. (B) Total reducing power of FSNPs, FFE and rutin. (C) Superoxide radical scavenging activity of FSNPs, FFE and rutin. (D) Hydrogen peroxide radical scavenging activity of FSNPs, FFE and rutin. (E) Nitric oxide radical scavenging activity of FSNPs, FFE and quercetin, (the data represent mean ± SD of three replicates (n=3).

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and EB (Fig. 6(A(i-iii))). Interestingly, among the three dyes used in this study maximum degradation was observed with 4-NP and the reaction was completed within 8 min as reduction reaction progressed with the yellow colored dye degraded to colorless 4-amino phenol (4-AP) (Fig. 6(B(i)). The other two dyes methylene blue and ethidium bromide were moderately degraded by FSNPs and the reaction was completed in 120 min and 3 h respectively (Fig. 6(B(ii-iii)). These observations indicate that FSNPs possess good catalytic activity. The probable explanation for the catalytic activity of FSNPs might be due to silver nanoparticles which facilitate the electron relay from the donor to the acceptor. Owing to their large surface area silver nanoparticles act as substrate for the electron transfer reaction. Prior to electron transfer both the reactants are absorbed on the surface of the silver nanoparticles and subsequently reactant gains electron and gets reduced. Thus, in all catalytic dye degradation reactions FSNPs act as an efficient catalyst through the electron transfer process. Similar catalytic activity has been reported recently with gold nanoparticles supported on carbon tubes [37]. The in vitro antioxidant activity of FSNPs and FFE was assessed by DPPH assay in which the purple colored DPPH turns to yellow upon reduction by FSNPs and FFE. As shown in Fig. 7(A) the antioxidant activity of both FSNPs and FFE showed an increasing trend with increasing concentration. FSNPs induced 50% inhibition of DPPH at a concentration of 100 μg/ml, in which FFE was found to be more effective which exhibited 50% inhibition at a concentration of 30 μg/ml and its activity was comparable to that of standard antioxidant rutin. These activities may be attributed to the phytoconstituents present in FFE and that are attached to FSNPs. Similar antioxidant properties have been reported with gold nanoparticles derived from trolox a vitamin E analogue [38]. The total reducing power of FSNPs and FFE was determined by the standard ferric ion reducing assay where the Fe3+ ion gets reduced to Fe2+ by antioxidants. Results from this study indicated that FSNPs as well as FFE displayed potent reducing activity with increasing concentrations and interestingly FSNPs exhibited better reducing activity than FFE that was comparable with rutin. Similar observations were made with Piper longum silver nanoparticles [39], which showed reducing activity at high concentrations between 100 and 600 μg/ml while

Table 1 Zone of inhibition (mm) induced by FSNPs at different concentrations in two different bacterial species. Zone of inhibition in (mm) by FSNPs Test sample

Conc. (μg/ml)

E. coli

S. aureus

B. cereus

B. subtilis

FSNPs

100 150 200 250 300 400 350 400

– – 10 – 13 18 28 28

5 8 11 13 – – 23

3 6 8 11 – – 22

– – 8 – 12 16 20 20

Streptomycin

FSNPs in this study exhibited good reducing activity at a concentration range of 25–100 μg/ml (Fig. 7(B)). Superoxide radical scavenging activity of FSNPs and FFE was determined by using PMS-NBT reduction system that generated stable superoxide radicals. The superoxide radical scavenging activity of FSNPs, FFE and rutin is shown in Fig. 7(C), in which they exhibited 41.71, 75.94 and 80.49% maximum inhibition at a higher concentration (30 µg/ml) used in this study. The superoxide radical consumption capacity of FSNPs and FFE increased with increasing concentrations (5–30 μg/ml) and the superoxide radical quenching activity was more pronounced with FFE than FSNPs which may be attributed to its high content of phytoconstituents. Hydrogen peroxide radicals can rapidly cross the cell membrane and cause toxic effects by interacting directly with thiol groups (− SH groups) of enzymes and with essential Fe2 + and Cu2 + ions. The hydroxyl radical scavenging activity of FSNPs and FFE is presented in Fig. 7(D) which suggests that FSNPs exhibited better hydrogen peroxide radical scavenging activity than FFE and rutin. An inhibition of 91.80, 77.19 and 78.01% was observed at a concentration of 60 μg/ml respectively for FSNPs, FFE and rutin. Similar hydrogen peroxide radical scavenging activity was observed with silver nanoparticles from Morinda pubescens [40].

Fig. 8. Antibacterial activity of FSNPs against (A) Escherichia coli and (B) Bacillus subtilis, with standard drug streptomycin.

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Fig. 9. Growth kinetic study of FSNPs in Escherichia coli.

Nitric oxide is a potent inhibitor of smooth muscle relaxation, neuronal signaling and regulation of cell mediated toxicity. NO• Nitic oxide is less stable at high electronegativity that can easily accept electrons from FSNPs and FFE and get reduced. Fig. 7(E) illustrates the percentage inhibition of nitric oxide radicals by FSNPs, FFE and standard quercetin. The nitric oxide radical scavenging ability of FSNPs and FFE increased with increasing concentration. At a concentration of 60 μg/ml FSNPs, FFE and quercetin showed 51.35, 84.04 and 91.73% inhibition respectively. Similar nitric oxide radical scavenging activity has been reported for cerium nanoparticles through internal electron transfer [41]. Silver is well known for its antibacterial activity and has been used to eliminate microorganisms in water and air filtration [42]. The inhibitory effect of FSNPs against bacterial growth was evaluated by exposing

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different concentrations (200–400 μg/ml) of FSNPs to E. coli, and B. subtilis. Results indicated that FSNPs produced an effective inhibition zone of 10, 13 and 18 mm against E. coli and 8, 12 and 16 mm against B. subtilis respectively for 200, 300 and 400 µg/ml concentrations after 12 h incubation (Fig. 8(A and B) and Table 1). Where the standard antibiotic streptomycin (400 µg/ml) showed inhibition zone of 28 and 20 mm against E. coli and B. subtilis respectively after 12 h of incubation. The possible mechanism of antibacterial activity of FSNPs might be attributed to their interaction with the bacterial cell membranes and which may resulting in disturbing the respiratory functions culminating in bacterial cell death. Our findings were agreed with the earlier report on antibacterial activity of silver nanoparticles synthesized from citrus peel extract [43]. The potential antibacterial effect of FSNPs was confirmed by the growth kinetic studies in E. coli using different concentrations (200, 300 and 400 μg/ml). As shown in Fig. 9 the FSNPs caused disturbance in the growth pattern of bacteria by interfering in the log phase and caused a reduction in the number of viable cells. These results confirm the inhibitory effect of FSNPs in E. coli and the effect was more pronounced with increasing concentrations as compared with control. A similar observation was reported earlier with silver nanoparticles in E. coli and Staphylococcus S. aureus [44]. The FSNPs and FFE were tested for their anticancer effects in COLO 205 cells in a dose dependent and time dependent manner as shown in Fig. 10(A and B). Data obtained from the MTT assay indicated that the cell viability decreased with increasing concentrations of FSNPs with prolonged incubation time. The IC50 values were found at 5.5 and 4 μg/ml respectively after 24 and 48 h of incubation for FSNPs and on the other hand FFE produced a maximum of 21 and 24% respectively after 24 and 48 h of inhibition at 250 μg/ml. These observations suggest that FSNPs were more effective on COLO 205 cells even at very lower concentration than FFE. Our results corroborate with an earlier report on the cytotoxic effect of green synthesized silver nanoparticles on HeLa cells [45].

Fig. 10. Cytotoxic effects of FSNPs and FFE on COLO 205 for 24 and 48 h by MTT assay (data represent mean ± SD of three replicates (n=6).

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Fig. 11. Fluorescence microscopic images of AO/EB staining for apoptosis (A) COLO 205 control and (B) treated with 24 h IC50 concentration (5.5 μg/ml) of FSNPs (arrows indicate the orange colored apoptotic bodies).

AO/EB staining method was used to assess whether the FSNPs induced cell death in COLO 205 cells was associated with controlled apoptotic changes, chromatin condensation and membrane potential loss. AO/EB is a vital dye that differentially stains live and dead cells. AO can enter and stain live cells, which will appear green in color and EB will stain cells that have lost cell membrane integrity therefore stain orange out. In this study, COLO 205 cells treated with FSNPs at appropriate IC50 concentrations (5.5 µg/ml) showed an increased number of apoptotic cells that appeared orange after 24 h of incubation while control cells showed a negligible number of apoptotic cells (Fig. 11(A and B)). These findings confirm the cytotoxic potential of FSNPs which mediates its activity through induction of apoptosis. Our AO/EB results were also supported by the earlier report on biogenic silver nanoparticles from Abutilon indicum leaf extract which emphasize the apoptosis mediated cytotoxicity in COLO 205 cells [46]. 4. Conclusion In conclusion green silver nanoparticles were successfully synthesized from frangipani flower extract by simple methodology and they were complemented with catalytic and biological activities. The spherical shaped FSNPs with an average size of 36.19 nm had associated phytocompounds from FFE. FTIR analysis confirmed the presence of phytocompounds that were involved in stabilization of nanoparticles. The FSNPs displayed remarkable free radical scavenging activities and significant antibacterial activity. The green synthesized FSNPs also displayed immensely powerful catalytic activity through efficient conversion of 4-nitrophenol to 4-amino phenol. The pronounced cytotoxic effect of FSNPs observed in COLO 205 cells through MTT assay and AO/ EB staining exemplifies its anticancer potential and provides a lead for further investigations with other cancer cell lines. These simple ecofriendly silver nanoparticles can be considered in nanotechnology with promising catalytic and biomedical applications. Acknowledgments Authors are pleased to acknowledge the DST-FIST and DBT BUILDER programs, Government of India, for equipment support. The authors

also thank the Central Instrumentation Facility (CIF), Pondicherry University. References [1] J. Nam, N. Won, H. Jin, H. Chung, S. Kim, pH-induced aggregation of gold nanoparticles for photothermal cancer therapy, J. Am. Chem. Soc. 131 (2009) 13639–13645. [2] K.B. Narayanan, N. Sakthivel, Synthesis and characterization of nano-gold composite using Cylindrocladium floridanum and its heterogeneous catalysis in the degradation of 4-nitrophenol, J. Hazard. Mater. 189 (2011) 519–525. [3] J. Li, X. Chen, N. Ai, J. Hao, Q. Chen, S. Strauf, et al., Silver nanoparticle doped TiO2 nanofiber dye sensitized solar cells, Chem. Phys. Lett. 514 (2011) 141–145. [4] A.M. Fayaza, M. Girilal, S.A. Mahdy, S.S. Somsundar, R. Venkatesan, P.T. Kalaichelvan, Vancomycin bound biogenic gold nanoparticles: a different perspective for development of anti VRSA agents, Process Biochem. 46 (2011) 636–641. [5] M. Darroudi, A.M. Zak, M.R. Muhamad, N.M. Huang, M. Hakimi, Green synthesis of colloidal silver nanoparticles by sonochemical method, Mater. Lett. 66 (2012) 117–120. [6] S. Sinha, I. Pan, P. Chanda, S.K. Sen, Nanoparticles fabrication using ambient biological resources, J. Appl. Biosci. 19 (2009) 1113–1130. [7] N. Vigneshwaran, A.A. Kathe, P.V.V. Aradarajan, R.P. Nachane, R.H. Balsubra-Manya, Synthesis of ecofriendly silver nanoparticle from plant latex used as an important taxonomic tool for phylogenetic interrelationship, Colloids Surf. B: Biointerfaces 53 (2006) 55–59. [8] M. Sathishkumar, K. Sneha, S.W. Won, C.W. Cho, S. Kim, Y.S. Yun, Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-crystalline silver particles and its bactericidal activity, Colloids Surf. B: Biointerfaces 73 (2009) 332–338. [9] V. Kumar, S.K. Yadav, Plant-mediated synthesis of silver and gold nanoparticles and their applications, J. Chem. Technol. Biotechnol. 84 (2009) 151–157. [10] D. Gangadharan, K. Harshvardan, G. Gnanasekar, D. Dixit, K.M. Popat, P.S. Anand, Polymeric microspheres containing silver nanoparticles as a bactericidal agent for water disinfection, Water Res. 44 (2010) 5481–5548. [11] Sampath Marimuthu, Abdul Abdul Rahuman, Govindasamy Rajakumar, Thirunavukkarasu Santhoshkumar, Arivarasan Vishnu Kirthi, Chidambaram Jayaseelan, Asokan Bagavan, Abdul Abduz Zahir, Gandhi Elango, Chinnaperumal Kamaraj, Evaluation of green synthesized silver nanoparticles against parasites, Parasitol. Res. 108 (2011) 1541–1549. [12] Shaswat Barua, Rocktotpal Konwarh, Satya Sundar Bhattacharya, Pallabi Das, K. Sanjana P. Devi, Tapas K. Maiti, Manabendra Mandal, Niranjan Karak, Nonhazardous anticancerous and antibacterial colloidal ‘green’ silver nanoparticles, Colloids Surf. B: Biointerfaces 105 (2013) 37–42. [13] N. Ahmad, S. Sharma, M.K. Alam, V.N. Singh, S.F. Shamsi, B.R. Mehta, et al., Rapid synthesis of silver nanoparticles using dried medicinal plant of basil, Colloids Surf. B: Biointerfaces 81 (1) (2010) 81–86. [14] A.R. Vilchis-Nestor, V. Sanchez-Mendieta, M.A. Camacho-Lopez, R.M. GomezEspinosa, M.A. Camacho-Lopez, J.A. Arenas-Alatorre, Solventless synthesis and optical properties of Au and Ag nanoparticles using Camellia sinensis extract, Mater. Lett. 62 (2008) 3103–3105.

R. Mata et al. / Materials Science and Engineering C 51 (2015) 216–225 [15] S.P. Chandran, M. Chaudhary, R. Pasricha, A. Ahmad, M. Sastry, Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract, Biotechnol. Prog. 22 (2006) 577–583. [16] H. Bar, D.Kr. Bhui, G.P. Sahoo, P. Sarkar, S.P. De, A. Misra, Green synthesis of silver nanoparticles using latex of Jatropha curcas, Colloids Surf. A Physicochem. Eng. Asp. 339 (2009) 134–139. [17] S.S. Shankar, A. Rai, A. Ahmad, M. Sastry, Controlling the optical properties of lemongrass extract synthesized gold nanotriangles and potential application in infraredabsorbing optical coatings, Chem. Mater. 17 (2005) 566–572. [18] S. Velavan, P. Arivoli, K. Mahadevan, Biological reduction of silver nanoparticles using Cassia auriculata flower extract and evaluation of their in vitro antioxidant activities, Nanosci. Nanotechnol. Int. J. 2 (2012) 30–35. [19] Ghassan Mohammad Sulaiman, Wasnaa Hatif Mohammed, Thorria Radam Marzoog, Ahmed Abdul Amir Al-Amiery, Abdul Amir H. Kadhum, Abu Bakar Mohamad, Green synthesis, antimicrobial and cytotoxic effects of silver nanoparticles using Eucalyptus chapmaniana leaves extract, Asian Pac. J. Trop. Biomed. 3 (1) (2013) 58–63. [20] JAMA. H.H. Comly, Supporting palladium metal on gold nanoparticles improves its catalysis for nitrite reduction, J. Am. Med. Assoc. 257 (1987) 2788–2792. [21] P. Kumar, M. Govindaraju, S. Senthamilselvi, K. Premkumar, Photocatalytic degradation of methyl orange dye using silver (Ag) nanoparticles synthesized from Ulva lactuca, Colloids Surf. B: Biointerfaces 103 (2013) 658–661. [22] Edward F. Gilman, Dennis G. Watson, Plumeria alba White Frangipani, October 1994. [23] B. Nagaraj, Barasa Malakar, T.K. Divya, N.B. Krishnamurthy, P. Liny, R. Dinesh, Environmental benign synthesis of gold nanoparticles from the flower extracts of Plumeria alba Linn. (frangipani) and evaluation of their biological activities, Int. J. Drug Dev. Res. 4 (1) (2012) (ISSN 0975–9344). [24] T. Jebakumar Immanuel Edison, M.G. Sethuraman, Instant green synthesis of silver nanoparticles using Terminalia chebula fruit extract and evaluation of their catalytic activity on reduction of methylene blue, Process Biochem. 47 (2012) 1351–1357. [25] Rakhi Majumdar, Braja Gopal Bag, Soma Rana, Azadirachta indica (neem) bark extract mediated rapid synthesis of gold nanoparticles and study of its catalytic activity, J. Res. Chem. Environ. 3 (3) (2013) 144–152. [26] N. Nenadis, I. Zafiropoulou, M. Tsimidou, Commonly used food antioxidants: a comparative study in dispersed systems, Food Chem. 82 (2003) 403–407. [27] G.C. Yen, H.Y. Chen, Antioxidant activity of various tea extracts in relation to their antimutagenicity, J. Agric. Food Chem. 43 (1) (1995) 27–32. [28] M. Nishikimi, N.A. Rao, K. Yagi, The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen, Biochem. Biophys. Res. Commun. 46 (1992) 849–854. [29] S.M. Nabavi, M.A. Ebrahimzadeh, S.F. Nabavi, A. Hamidinia, A.R. Bekhradnia, Determination of antioxidant activity, phenol and flavonoids content of Parrotia persica Mey, Pharmacol. Online 2 (2008) 560–567. [30] M.A. Ebrahimzadeh, S.F. Nabavi, S.M. Nabavi, Antioxidant activities of methanol extract of Sambucus ebulus L. flower, Pak. J. Biol. Sci. 12 (5) (2009) 447–450. [31] Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim, J.O. Kim, A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus, J. Biomed. Mater. 52 (4) (2000) 662–668.

225

[32] K.G. Stamplecoskie, J.C. Scaiano, Light emitting diode can control the morphology and optical properties of silver nanoparticles, J. Am. Chem. Soc. 132 (2010) 1825–1827. [33] Raman Sukirtha, Kandula Manasa Priyanka, Jacob Joe Antony, Soundararajan Kamalakkannan, Ramar Thangam, Palani Gunasekaran, Muthukalingan Krishnan, Shanmugam Achiraman, Cytotoxic effect of Green synthesized silver nanoparticles using Melia azedarach against in vitro HeLa cell lines and lymphoma mice model, Process Biochem. 47 (2012) 273–279. [34] S.P. Dubey, M. Lahtinen, M. Sillanpaa, Tansy fruit mediated greener synthesis of silver and gold nanoparticles, Process Biochem. 45 (2010) 1065–1071. [35] Uthirappan Mani, Sujatha Dhanasingh, Rajeswari Arunachalam, Edwin Paul, Ponnusamy Shanmugam, Chellan Rose, Asit Baran Mandal, A simple and green method for synthesis of silver nanoparticles using Ricinus communis leaf extract, Prog. Nanotechnol. Nanomater. 2 (1) (2013) 21–25. [36] J. Huang, C. Chen, N. He, J. Hong, Y. Lu, L. Qingbiao, et al., Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf, Nanotechnology 18 (2007) 104–105. [37] Tarek M. Abdel-Fattah, Alex Wixtrom, Catalytic reduction of 4-nitrophenol using gold nanoparticles supported on carbon nanotubes, ECS J. Solid State Sci. Technol. 3 (4) (2014) M18–M20. [38] Z. Nie, K.J. Liu, C.J. Zhong, L.F. Wang, Y. Yang, Q. Tian, Y. Liu, Enhanced radical scavenging activity by antioxidant-functionalized gold nanoparticles: a novel inspiration for development of new artificial antioxidants, Free Radic. Biol. Med. 43 (2007) 1243–1254. [39] N. Jayachandra Reddy, D. Nagoor Vali, M. Rani, S. Sudha Rani, Evaluation of antioxidant, antibacterial and cytotoxic effects of green synthesized silver nanoparticles by Piper longum fruit, Mater. Sci. Eng. C 34 (2014) 115–122. [40] L. Inbathamizh, T. Mekalai Ponnu, E. Jancy Mary, In vitro evaluation of antioxidant and anticancer potential of Morinda pubescens synthesized silver nanoparticles, J. Pharm. Res. 6 (2013) 32–38. [41] A. Chaudhary, Ayurvedic bhasma: nanomedicine of ancient India—its global contemporary perspective, J. Biomed. Nanotechnol. 7 (2011) 68–69. [42] Q. Chen, L. Yue, F. Xie, M. Zhou, Y. Fu, Y. Zhang, et al., J. Phys. Chem. C 112 (2008) 10004. [43] S. Kaviya, J. Santhanalakshmi, B. Viswanathan, J. Muthumary, K. Srinivasan, Biosynthesis of silver nanoparticles using Citrus sinensis peel extract and its antibacterial activity, Spectrochim. Acta A 79 (2011) 594–598. [44] Soo-Hwan Kim, Hyeong-Seon Lee, Deok-Seon Ryu, Soo-Jae Choi, Dong-Seok Lee, Antibacterial activity of silver-nanoparticles against Staphylococcus aureus and Escherichia coli, Korean J. Microbiol. Biotechnol. 39 (1) (2011) 77–85. [45] M. Jeyaraj, M. Rajesha, R. Arunb, D. MubarakAlic, G. Sathishkumara, G. Sivanandhana, et al., An investigation on the cytotoxicity and caspase-mediated apoptotic effect of biologically synthesized silver nanoparticles using Podophyllum hexandrum on human cervical carcinoma cells, Colloids Surf. B: Biointerfaces 102 (2013) 708–717. [46] R. Mata, J.R. Nakkala, S.R. Sadras, Biogenic silver nanoparticles from Abutilon indicum: Theirantioxidant, antibacterial and cytotoxic effects in vitro, Colloids Surf B, Biointerfaces, 2015.http://dx.doi.org/10.1016/j.colsurfb.2015.01.052.