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Asian Pac J Trop Med 2014; 7(Suppl 1): S294-S300
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Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm
Document heading
doi: 10.1016/S1995-7645(14)60249-2
Rapid synthesis of biocompatible silver nanoparticles using aqueous of Rosa damascena petals and evaluation of their anticancer activity
extract
Balaji Venkatesan, Vimala Subramanian, Anusha Tumala, Elangovan Vellaichamy* Department of Biochemistry, University of Madras, Guindy Campus, Chennai-600 025, India
ARTICLE INFO Article history:
Received 5 May 2014 Received in revised form 27 May 2014 Accepted 13 Jun 2014 Available online 2 Aug 2014
Keywords: Green synthesis Silver nanoparticles Rosa damascena Hemolytic activity assay Human lung cancer cell line Anticancer activity
ABSTRACT Objective: To optimize the process parameters involved in the green synthesis of silver nanoparticles (G-SNPs) by aqueous extract of Rosa damascena petals and to evaluate the biocompatibility and anti cancer activity of the synthesized silver nanoparticles against human lung adenocarcinoma (A549). Methods: The process variables that include concentration of extract, mixing ratio of reactants, silver salt concentration and interaction time were analyzed. The compatibility of the G-SNPs was verified by incubating with erythrocytes and the anticancer property of the G-SNPs against A549 cells was performed by MTT assay. Results: Formation of G-SNPs was confirmed by the visual change in the colour of the reaction mixture from pale yellow to brown yellow. Surface plasmon resonance of synthesized G-SNPs was observed at 420 nm; the size of G-SNPs were analyzed by DLS and found to be in the range of (84.00依10.08) nm. Field emission scanning electron microscope and high resolution transmission electron microscopy analysis confirmed that the G-SNPs were fairly spherical. Fourier transform infrared spectroscopy spectroscopy and X-ray diffraction revealed the characteristic peaks of G-SNPs. Energy dispersive X-ray analysis showed a signal of silver around 3 keV. The synthesized G-SNPs exhibited anticancer activity as evidenced by the MTT assay. IC50 value of G-SNPs was found to be 80 µg/mL. Conclusion: The results of the present study suggest that G-SNPs can be synthesized rapidly within first minute of the reaction; they are biocompatible and possess anticancer activity against human lung adenocarcinoma.
1. Introduction Synthesis of nanoparticles with variable shapes, sizes and chemical compositions has gained importance in nanotechnological applications. The metallic nanoparticles have received much attention due to their unique catalytic, optical and electrical properties[1]. The silver nanoparticles (SNPs) have been widely utilized in biology and medicine due to its attractive physiochemical properties[2]. SNPs have been reported to exhibit number of pharmacological activities, including antibacterial activity[3-5], antifungal activity[6], anticancer activity[7], antiviral activity[8,9] antiplasmodium activity and mosquito larvicidal activity[10,11], etc. Although *Corresponding author: Dr. Elangovan Vellaichamy, Associate Professor, Department of Biochemistry, University of Madras, Guindy Campus, Chennai- 600 025, India. E-mail: [email protected], [email protected] Tel: +91-044-22202734 Foundation Project: Supported by Indian Council of Medical Research-Grant no. 35/27/2010-BMS and University Grants Commission (Basic Scientific Research fellowship in sciences)-Grant no. F7-114/2007 (BSR).
the chemical methods of synthesizing SNP s promise to provide high purity and desired structure to the nanoparticles, they are quite expensive and toxic. Since most of these chemically synthesized nanoparticles are utilized for human benefits, they pose severe hazard to human health and to the environment[12,13]. Recently, ‘green synthesis’ of nanoparticles using extracts of plants and microbes have gained importance because it could solve the problem of toxicity imposed by chemical methods. Green synthesis of SNPs using plant extracts have been studied previously using several plants which includes Rhizophora mucronatas[14], Acalypha indica[3], Phyllanthus emblica[15], Panicum virgatum[16], Piper nigrum[17], Glycine max[18], Murraya Koengii[19], Rosa rugosa[20], Camellia sinensis[21]. These studies have shown that SNPs can be synthesized in environmental friendly and green method using plant extracts. Still, studies on green synthesis of SNPs continue by employing medicinal plants extract to explore the potentials of traditional and medicinal plants to synthesize biocompatible SNPs. Since, each traditional
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plant has specific medicinal properties, by employing theses plants the medicinal property of the synthesized SNPs may be enhanced. Previous studies elucidated the presence of several pharmacologically active compounds in the petals of Rosa damascena (R. damascena)[22,23]. Hence, we hypothesize that the nanoparticles synthesized using R. damascena could be biocompatible and can be used for biological applications. R. damascena belongs to the Rosaceae family of flowers. It is one of the important species in that family, which is used as an ornamental plant and its flowers are utilized as additives in food and medicine [24] . I t has gained great importance due to its pharmacological properties including anti-diabetic, anti-HIV, and its potential to cure cardiovascular diseases[22]. R. damascena petals have been reported to contain several important chemical compounds, which includes flavonoids, terpenes, antocyanins, glycosides, carboxylic acid, vitamin C, myrcene, kaempferol and quercetin[22]. These properties of this plant make it a suitable choice for synthesis of silver nanoparticles, which may lead to the synthesis of eco-friendly and non-toxic green-synthesized silver nanoparticles (G-SNPs) for medical and biological applications. H ere, we reported the green synthesis of silver nanoparticles using an aqueous extract of R. damascena and evaluated its anticancer activity against human lung adenocarcinoma ( A 549 ) . T he process variables such as concentration of extract, mixing ratio of reactants, silver salt concentration and interaction time were analyzed. The results of this study uncovered the potentials of R. damascena petal extracts to synthesize G-SNPs. G-SNPs can be synthesized rapidly by using aqueous extract of R. damascena petals and they are biocompatible and possess anticancer activity.
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cooled and stored at 4 °C for further experiments[25]. This solution serves as 100% extract concentration. 2.3. Green synthesis of silver nanoparticles In order to enhance the reproducibility of the experimental results and to optimize the process parameters, the factorial design of experiments, the “one-factor-at-atime” method was employed in this study. In this method of experimentation, only one experimental factor is varied at a time, while keeping all the other variables constant. Different concentrations of rose extracts (10%, 25%, 50%, 75% and 100%) were prepared by diluting the bioreductant (rose extract) with deionized water accordingly. They were added to 10 mmol/L silver nitrate solution at 2:3 ratio and incubated for 10 min in dark at room temperature. In another process parameter, the silver reduction reaction was carried out by varying the different mixing ratios of rose extract to 10 mmol/ L silver nitrate solution (1:4, 2:3, 1:1, 3:2, 4:1) at 100% extract concentration while keeping all the other variables constant. In order to determine the interaction time between the rose extract and silver nitrate solution, 10 mmol/L silver nitrate solution and 100% rose extract was added together in 2:3 ratio and incubated for different time points (0 min, 5 min, 10 min, 15 min and 25 min). To determine the optimum silver salt concentration, different concentrations of silver nitrate solution (1 mmol/L, 2 mmol/L, 3mmol/L, 5mmol/L, 10 mmol/L) were reacted to 100% rose extract at 2:3 ratio. T o obtain the dry powders of G - SNP s, the solution containing the silver nanoparticles were centrifuged at 10 000 r/min for 15 min. The pellet was resuspended in deionized water and the centrifugation process was repeated thrice to get rid of any non-reacted biological materials. The purified pellets were then freeze dried to get powder G-SNPs. This powder was further used for characterization studies.
2. Materials and methods
2.4. Characterization
2.1. Materials
The preliminary characterization of silver nanoparticles was carried out using UV - visible spectroscopy. T he reduction of silver nitrate to silver nanoparticles was analyzed by recording the spectra of silver nanoparticles from 340 to 700 nm (Shimadzu UV 150-02, Japan). The size distribution and zeta potential of the nanoparticles in the solution were determined using particle size and zeta analyzer (Malvern Nano ZS, UK). The G-SNP solution was diluted 10 times with deionised water prior to analysis. The powder X-ray diffraction pattern was obtained by using the lyophilized powder of silver nanoparticles (BYE FIEX 2002 model, Seifert, Germany) with Cu Kα radiation (λ=1.54 nm) in the 2θ range of 20° to 80° operated at a voltage of 40 kV and a current of 30 mA. The Fourier transform infrared spectroscopy (FT-IR) spectroscopic analysis was carried on lyophilized powders of 100% petal extract and silver nanoparticles. The measurements were performed using Nicolet Impact 400 FT-IR spectrophotometer with KBr pellet in the wave number region of 2 000 to 800 cm-1. For field
Fresh roses (R. damascena) were procured from the local market, Chennai, India. Silver nitrate, dimethylsulfoxide and 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St Louis, MO, USA) . D ulbecco’s modified E agle’s medium ( DMEM) , fetal bovine serum (FBS), Trypsin-EDTA and antibioticantimycotic solution were purchased from HiMedia (Mumbai, India). Deionized water was used throughout the study.
2.2. Preparation of R. damascena extract The rose petals were washed thoroughly with deionized water and shade dried at room temperature for 2 d. The extraction process was started by adding one gram of dried petals and 100 mL of deionized water in to a 250 mL Erlenmeyer flask. This mixture was slowly stirred using a magnetic stirrer at 80 °C for 1 h. This solution was filtered,
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emission scanning electron microscope (FE-SEM) and high resolution transmission electron microscopy ( HR - TEM ) analysis, the synthesized nanoparticle solution was diluted and a drop was placed on aluminum foil and on a carbon grid respectively and allowed to dry. The FE-SEM micrograph was obtained using Hitachi SU6600, Germany and HR-TEM analysis was carried out on FEI TECNAI G2. The purified and lyophilized samples were subjected to EDAX analysis (Hitachi SU6600, Germany). 2.5. Hemolytic activity assay The in vitro hemolytic activity of G-SNPs was evaluated by measuring the hemoglobin release from the erythrocytes on interaction with nanoparticles. The blood was collected from male Wistar albino rat in a tube containing EDTA and the tube was centrifuged at 1 500 r/min for 10 min. The supernatant and buffy coat was carefully removed and the pellet was washed thoroughly with phosphate buffered saline (pH 7.4) several times. Cells (5% v/v) in phosphate buffer solution (PBS) were added to each tube and the G-SNP at different concentration (25, 50, 75, 100, 125 µg) in PBS were added and the final volume was made up to 1 mL. Erythrocytes in PBS solution and in 1% triton X-100 were considered as negative and positive controls respectively. The reaction mixtures were incubated in shaker incubator at 37 °C with mild shaking for 1 h. Then the tubes were centrifuged at 1 500 r/min for 10 min and the supernatant were read at 540 nm against their blank. The percentage of hemolysis=[(Abs540 nm in the G-SNP solution-Abs540 nm in PBS)/(Abs540 nm in 1% Triton X-100-Abs540 nm in PBS)]伊 100[26].
2.6. Cell proliferation assay Human lung adenocarcinoma epithelial cell line (A549) was obtained from National Center for Cell Science, Pune, India. The cell lines were maintained in DMEM, supplemented with 10% FBS and antibiotic antimycotic solution. Cells were grown in a humidified chamber at 37 °C with 5% CO2 supply. To assess the anticancer activity of G-SNPs quantitatively, 3 MTT reduction assay was performed[27]. A549 cells (2伊10 cells/100 µL) were seeded on 96 well plate with 200 µL of complete DMEM media containing 10% FBS. The plate was incubated at 37 °C in a humidified chamber with 5% CO2. After 24 h of culturing, the spent media was removed and fresh DMEM media with predefined concentrations (20 to 120 µg/mL) of G-SNPs were added and incubated at 37 °C in a humidified chamber with 5% CO2. After 24 h of incubation with G-SNPs, the spent media was replaced with 10 µL of MTT solution to each well (5 mg/mL) and incubated for 4 h at 37 °C in a humidified chamber with 5% CO2. After completion of 4 h, the MTT was removed and 100 µL of dimethylsulfoxide was added to all the wells. The absorbance was read at 570 nm with microplate spectrophotometer. The cells which were not treated with nanoparticles were considered as control.
2.7. Statistical analysis Values of three independent experiments were expressed as mean 依 SD . T he data was evaluated using SPSS / 11 . 5
software.
3. Results The results of the present study demonstrate the successful green synthesis of G-SNPs using R. damascena petal extract. The G-SNPs were found to be biocompatible and exhibited cytotoxic activity against human lung adenocarcinoma (A549) cells. On addition of R. damascena extract to silver nitrate solution, the colour of the reaction mixture changed rapidly from pale yellow to brown yellow (Figure 1). The surface plasmon resonance of the silver nanoparticles reduced by R. damascena aqueous extract was obtained at 420 nm. (a)
(b)
(c)
Figure 1. Photographic representation of silver nanoparticle synthesis. a: Silver nitrate solution; b: Aqueous extract of R. damascena petal extract; c: Green synthesized silver nanoparticles.
Figure 2a shows the effect of different concentrations of petal extract on G-SNPs formation. The absorbance of the UV-Vis spectrum gradually increased with the increase in concentration of the extract from 10% to 100%. The highest absorption was recorded when 100% extract was used. Figure 2b shows the results of second process variable, in which the ratio of petal extract to silver nitrate solution (1:4, 2:3, 1:1, 3:2, 4:1) was analyzed. The results clearly revealed that 2:3 ratio of extract to silver nitrate solution was the perfect combination to get maximum yield. This was observed from the maximum absorbance in UV-Vis spectrum. Figure 2c shows the effect of incubation time on G-SNPs formation. The absorbance was observed even at 0 min of the reaction. As the incubation time was increased from 0 to 25 min (0, 5, 10, 15, 25 min) there was no significant change in the absorbance of the nanoparticles. Our results indicate that the formation of G-SNPs starts immediately after addition of extract to the silver nitrate solution, thereafter no marked increase in the absorbance was observed. To determine the exact silver salt concentration for effective G-SNP synthesis,
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10 5 T=25 1 2 3 T=15 T=0 T=5 T=10 Figure 2. UV-Vis spectroscopic analysis of process variables in silver nanoparticles synthesis. a: UV-Vis spectra of different concentration of R. damascena petal extract with 10 mmol/L silver nitrate solution; b: UV-Vis spectra of different mixing ratio of petal extract with 10 mmol/L silver nitrate solution; c: UV-Vis spectra of reaction time of petal extract with 10 mmol/L silver nitrate solution; d: UV-Vis spectra of different concentration of silver nitrate solution with petal extract.
different concentrations of silver nitrate solutions ranging from 1 to 10 mmol/L were reacted with extract in 2:3 ratio for 5 min. Figure 2d represents the effect of different silver nitrate solution for synthesizing the G-SNP. From the graph, it is observed that 10 mmol/L silver nitrate was identified as the optimum concentration for the effective G-SNP formation. 140
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Figure 3. XRD pattern of biosynthesized silver nanoparticles using 100% petal extract with 10 mmol/L silver nitrate for 5 min at room temperature.
From the dynamic light scattering measurement, the average size of the particles was found to be (84.00依10.08)
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nm. Zeta potential analysis of the synthesized G-SNP was found to be (-17.4依2.1). The crystalline nature of the silver nanoparticles was analyzed by X -ray diffraction ( XRD) (Figure 3). The results revealed four diffraction peaks [38.05 (111), 46.12 (200), 67.19 (220), 76.3 (311)] corresponding to four diffraction facets of silver. These results indicate that the synthesized nanoparticles were face centered cubic and crystalline in nature (JCPDS file No. 84-0713 and 04-0783).
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Figure 4. FT-IR spectra of aqueous petal extract of R. damascena and silver nanoparticles. a: Aqueous petal extract of R. damascena; b: Silver nanoparticles obtained by treating 100% petal extract with 10 mmol/L silver nitrate for 5 min at room temperature.
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(a)
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performed using different concentrations of G-SNPs (25, 50, 75, 100, 125 µg/mL) on erythrocytes. The absorbance values of erythrocytes in PBS and in 1 % triton X - 100 was used along with the values of erythrocytes treated with G-SNPs on the formula. It was found that G-SNPs do not exhibit hemolytic activity against erythrocytes on different tested concentrations (Table 1). Table 1 H emolytic activity of silver nanoparticles on erythrocytes at different concentration after incubation at 37 °C for 1 h. Sample (n=3)
hemolysis (%) 1.20依0.15 100.00依0.00 1.20依0.12 1.30依0.17
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Data are represented as mean依SD (n=3).
T he in vitro anticancer effect of G - SNP s was tested on A 549 cell lines. A dose dependent decrease in the viability of A549 cells were observed on treatment with G - SNP s, as shown in the F igure 6 . T he half maximum inhibitory concentration (IC50) was observed at 80 µg/mL. At this concentration, G-SNPs inhibited the growth of A549
cells by 50%. (a)
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FT-IR is reported as an appropriate technique to analyze chemical adsorption and functional groups in newly synthesized particles. The FT-IR transmission spectra of rose extract and silver nanoparticles are represented in Figure 4. Characteristic absorption bands at 1 719, 1 615, -1 1 351, 1 232 and 1 057 cm in the FT-IR spectrum of rose petal extracts were observed ( F igure 4 a ) . B and at 1 719 cm-1 which is attributed to the C=O stretching due to the carboxyl content in the rose petal extracts, which might reduced the silver ion to silver nanoparticles. Further, peaks assigned at 1 615, 1 351 and 1 057 cm-1, suggest that amine group and the hydroxyl groups of water/alcohol. T he FT-IR spectra of silver nanoparticles are shown in Figure 4b which shows comparatively similar absorption bands at 1 619, 1 384 and 1 058 cm-1 of rose petals extract. -1 The suppression of C=O stretching in the 1 719 cm region has been related to evidence of silver ion reduction by rose petals as consequence of silver nanoparticles formation. F igure 5 a and 5 b shows the FE - SEM and HR - TEM micrographs, which provides a clear idea on the shape of nanoparticles. Majority of the particles were spherical in shape with the size ranging from 15 to 27 nm. These particles were well distributed without any aggregation. T he presence of elemental silver was confirmed by EDAX spectroscopy analysis (Figure 5c), which indicated the reduction of silver ion to silver. M etallic silver nanocrystals generally display an optical absorption peak at 3 keV[28]. There was also a strong signal for Al in the EDAX data, which was due to the Al FE-SEM grid.
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Figure 5. Electron microscopic imaging of silver nanoparticles. a: FE-SEM micrograph; b: HR-EM analysis of silver nanoparticles; c: EDAX spectrum of silver nanoparticles synthesized from 10 mmol/L silver nitrate solution and 100% petal extract for 5 min at room temperature of silver nanoparticles.
H emolytic activity of G - SNP s was performed to evaluate the biocompatibility of the G-SNPs on normal cells, especially erythrocytes. The hemolytic assay was
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Figure 6. In vitro anticancer effect of G-SNPs. a: Cell proliferation assay using MTT for analyzing dose dependent effect of silver nanoparticles on A549 cells for 24 h. Fifty persents inhibition of cells was observed at 80 µg/mL silver nanoparticles concentration (10伊); b: Inverted microscopic view of A549 control cells without any treatment; c: Cells treated with 80 µg/mL silver nanoparticles for 24 h (10伊).
4. Discussion Green synthesis of nanoparticles has received much attention due to easy and eco-friendly nature of the
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synthesis [29] . O n addition of the petal extract to the silver nitrate solution, the colour of the reaction mixture changed from pale yellow to brown yellow. This change in colour of the mixture was due to the reduction of silver ion and excitation of surface plasmon resonance by the nanoparticles. This change in the colour of the reaction mixture indicated the formation of G - SNP [30]. I n the present study, the process parameters have been optimized, to reduce the time required for G-SNPs synthesis and to improve the yield. B y varying the concentration of the petal extract from 10% to 100%, the plasmon resonance band raised gradually. M aximum plasmon resonance band was attained at 100% of extract concentration, which shows that the reduction of silver ion was directly proportional to the concentration of petal extract. T here was no formation of plasmon resonance band when 10% of extract concentration was used, which indicated that there was not enough reducing agent in the extract to reduce the silver ion. Mixing ratio and silver salt concentration plays a vital role in the optimization of process parameters, which determines the yield of the nanoparticles. T he maximum P lasmon resonance was obtained for 2:3 mixing ratio and 10 mmol/L silver nitrate. D ubey et al. synthesized SNP s using leaves of Rosa rugosa and reported the synthesis of nanoparticles after incubating the extract with the silver nitrate solution for 10 min[20]. In the present study, the plasmon resonance band was observed even at the first minute of the reaction and there were no significant change even after 25 min of the reaction. This rapid synthesis might be due to the higher percentage of extract concentration (100%) in the reaction mixture. Similar to our observation, T ripathy et al. have optimized the process parameters ( concentration of extract, mixing ratio and interaction time) for synthesizing SNPs by employing aqueous extracts of Azadirachta indica [31] . H e also emphasized in his report that size and shape of biosynthesized nanoparticles strongly depends on the optimization of process parameter. F urther, the G - SNP s were characterized by various analytical techniques. I n the XRD , there were few unassigned peaks marked with asterisk, which might be raised due to the crystallization of bio-organic phase or protein on the surface of the G-SNP from petal extract. S imilar results were reported in the synthesis of SNPs by Coleus aromaticus leaf extract and by Citrus limon aqueous extract[4,32]. The hemocompatibiliy of the G - SNP s were analyzed on erythrocytes and the results obtained were found to be correlated with the previous study done by Ruden et al. who reported that SNPs did not show hemolytic activity against erythrocytes even at the higher concentrations (up to 1024 µg/mL)[33]. The G-SNPs was found to exhibit anticancer activity against human lung adenocarcinoma but, the mechanism by which the SNPs induced cytotoxic activity was not clear. H owever, several possible mechanisms for anticancer activity of silver nanoparticles can be proposed. Cytotoxic
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effects of silver nanoparticles might be due to the physiochemical interaction of silver nanoparticles with the intracellular proteins and DNA . S tudies have also suggested that anticancer activity might also be due to induction of apoptosis activated by caspase 3 enzyme[7]. I n addition, the presence of pharmacologically active anticancer compound which includes flavonoids such as quercetin, etc., in the petals of R. damascena could also be one of the reasons for the G-SNPs to exhibit anticancer activity. Further, studies are needed to unravel the exact mechanism behind the anticancer efficacy of G-SNP. T o conclude, we have demonstrated that G - SNP s were rapidly synthesized using aqueous extracts of R. damascena within the first minute of the reaction at room temperature. Various process parameters involved in the green synthesis were analyzed for the better yield of G-SNPs and the synthesized G-SNPs were found to be biocompatible. T he G - SNP s exhibited inhibitory effect against A549 cell line, which might be due to the synergistic effect of SNP s and the pharmacologically active compounds on the surface of G-SNPs from the R. damascena extract. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements T he authors would like to thank I ndian C ouncil of M edical R esearch- G rant N o. 35 / 27 / 2010 - BMS and University Grants Commission (Basic Scientific Research fellowship in sciences-G rant N o. F 7 - 114 / 2007 ( BSR) for the financial support. We also like to acknowledge the electron microscopy facilities provided by the National Center for Nanoscience and Nanotechnology, University of Madras, India.
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[5] Dattu S, Vandana R, Shivaraj N, Jyothi H, Ashish KS, Jasmine M. Optimization and characterization of silver nanoparticle by endophytic fungi Penicillium sp. isolated from Curcuma longa (Turmeric) and application studies against MDR E. coli and S. aureus. Bioinorg Chem Appl 2014; doi: 10.1155/2014/408021. [6] Singh A, Jain D, Upadhyay MK, Khandelwal N, Verma HN. G reen synthesis of silver nanoparticles using argemone mexicana leaf extract and evaluation of their antimicrobial activities. Dig J Nanomater Biostruct 2010; 5: 483-489. [7] S riram MI , K anth SBM , K alishwaralal K , G urunathan S . A ntitumor activity of silver nanoparticles in D alton’s lymphoma ascites tumor model. Int J Nanomed 2010; 5: 753762. [8] Lu L, Sun RWY, Chen R, Hui CK, Ho CM, Luk JM, et al. Silver nanoparticles inhibit hepatitis B virus replication. Antivir Ther 2008; 13: 253-262. [9] Lara HH, Ayala-Nuñez NV, Ixtepan-Turrent L, RodriguezP adilla C . M ode of antiviral action of silver nanoparticles against HIV-1. J Nanobiotechnology 2010; 8: 1-10. [10] Allahverdiyev AM, Abamor ES, Bagirova M, Ustundag CB, Kaya C, Kaya F, et al. Antileishmanial effect of silver nanoparticles and their enhanced antiparasitic activity under ultraviolet light. Int J Nanomed 2011; 6: 2705-2714. [11] Mondal NK, Chowdhury A, Dey U, Mukhopadhya P, Chatterjee S, Das K, et al. Green synthesis of silver nanoparticles and its application for mosquito control. Asian Pac J Trop Dis 2014; 4(Suppl 1): S204-S210. [12] S a s t r y M , A h m a d A , K h a n M I , K u m a r R . M i c r o b i a l nanoparticle production. In: Niemeyer CM, Mirkin CA, editors. Nanobiotechnology: concepts, applications and perspectives. Germany, Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA; 2005, p. 126-135. [13] Bhattacharya D, Gupta RK. Nanotechnology and potential of microorganisms. Crit Rev Biotechnol 2005; 25: 199-204. [14] G nanadesigan M , A nand M , R avikumar S , M aruthupandy M , V ijayakumar V , S elvam S , et al. B iosynthesis of silver nanoparticles by using mangrove plant extract and their potential mosquito larvicidal property. Asian Pac J Trop Med 2011; 4: 799-803. [15] F a t h i m a S R , V a d i v e l A , S a m u t h i r a N , S a n k a r a n M . A ntiproliferative effect of silver nanoparticles synthesized using amla on Hep2 cell line. Asian Pac J Tropical Med 2010; 6: 1-12. [16] Mason C, Vivekanandhan S, Misra M, Mohanty AK. Switchgrass (Panicum virgatum) extract mediated green synthesis of silver nanoparticles. World J Nano Sci Eng 2012; 2: 47-52. [17] P aulkumar K , G nanajobitha G , V anaja M , R ajeshkumar S , M alarkodi C , P andian K , et al. Piper nigrum leaf and stem assisted green synthesis of silver nanoparticles and evaluation of its antibacterial activity against agricultural plant pathogens. ScientificWorldJournal 2014; doi: 10.1155/2014/829894. [18] Vivekanandhan S, Misra M, Mohanty AK. Biological synthesis of silver nanoparticles using Glycine max ( soybean ) leaf extract: an investigation on different soybean varieties. J Nanosci Nanotechnol 2009; 9: 6828-6833. [19] C hristensen L , V ivekanandhan S , M isra M , M ohanty AK .
Biosynthesis of silver nanoparticles using Murraya koenigii leaf: an investigation on the effect of broth concentration in reduction mechanism and particle size. Adv Mater Lett 2011; 2: 429-434. [20] D ubey SP , L ahtinen M , S illanpää M . G reen synthesis and characterizations of silver and gold nanoparticles using leaf extract of Rosa rugosa. Colloids Surf A Physicochem Eng Asp 2010; 364: 34-41. [21] V ilchis- N estor AR , S anchez- M endieta V , C amacho- L opez MA , G omez- E spinosa RM , C amacho- L opez MA , A renasAlatorre J. Solventless synthesis and optical properties of Au and Ag nanoparticles using Camellia sinensis extract. Mater Lett 2008; 62: 3103-3105. [22] Boskabady MH, Shafei MN, Saberi Z, Amini S. Pharmacological effects of Rosa damascena. Iran J Basic Med Sci 2011; 14: 295307. [23] K ashani AD , R asooli I , R ezaee MB , O wlia P . A ntioxidative properties and toxicity of white rose extract. Iran J Toxicol 2011; 5(12 and 13): 415-425. [24] N ikbakht A , K afi M . A study on the relationships between I ranian people and damask rose ( Rosa damascena ) and its therapeutic and healing properties. Acta Hortic 2008; 790: 251254. [25] Noruzi M, Zare D, Khoshnevisan K, Davoodi D. Rapid green synthesis of gold nanoparticles using Rosa hybrida petal extract at room temperature. Spectrochim Acta A Mol Biomol Spectrosc 2011; 79: 1461-1465. [26] V edakumari WS , P rabu P , B abu SC , S astry TP . F ibrin nanoparticles-possible vehicles for drug delivery. Biochim Biophys Acta 2013; 1830(8): 4244-4253. [27] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65(1-2): 55-63. [28] Kohler JM, Csaki A, Reichert J, Moller R, Straube W, Fritzsche W . S elective labeling of oligonucleotide monolayers by metallic nanobeads for fast optical readout of DNA -chips. Sens Actuators B Chem 2001; 76(1-3): 166-172. [29] Sulaiman GM, Mohammed WH, Marzoog TR, Al-Amiery AAA, Kadhum AAH, Mohamad AB. Green synthesis, antimicrobial and cytotoxic effects of silver nanoparticles using Eucalyptus chapmaniana leaves extract. Asian Pac J Trop Biomed 2013; 3(1): 58-63. [30] Sanghi R, Verma P. Biomimetic synthesis and characterization of protein capped silver nanoparticles. Bioresour Technol 2009; 100: 501-504. [31] T ripathy A , R aichur AM , C handrasekaran N , P rathna TC , M ukherjee A . P rocess variables in biomimetic synthesis of silver nanoparticles by aqueous extract of Azadirachta indica (Neem) leaves. J Nanopart Res 2010; 12: 237-246. [32] Prathna TC, Chandrasekaran N, Raichur AM, Mukherjee A. Biomimetic synthesis of silver nanoparticles by Citrus limon (lemon) aqueous extract and theoretical prediction of particle size. Colloids Surf B Biointerfaces 2011; 82: 152-159. [33] R uden S , H ilpert K , B erditsch M , W adhwani P , U lrich AS . S ynergistic I nteraction between silver nanoparticles and membrane-permeabilizing antimicrobial peptides. Antimicrob agents chemother 2009; 53(8): 3538-3540.
Asian Pac J Trop Med 2014; 7(Suppl 1): S294-S300
Contents lists available at ScienceDirect
Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm
Document heading
doi: 10.1016/S1995-7645(14)60249-2
Rapid synthesis of biocompatible silver nanoparticles using aqueous of Rosa damascena petals and evaluation of their anticancer activity
extract
Balaji Venkatesan, Vimala Subramanian, Anusha Tumala, Elangovan Vellaichamy* Department of Biochemistry, University of Madras, Guindy Campus, Chennai-600 025, India
ARTICLE INFO Article history:
Received 5 May 2014 Received in revised form 27 May 2014 Accepted 13 Jun 2014 Available online 2 Aug 2014
Keywords: Green synthesis Silver nanoparticles Rosa damascena Hemolytic activity assay Human lung cancer cell line Anticancer activity
ABSTRACT Objective: To optimize the process parameters involved in the green synthesis of silver nanoparticles (G-SNPs) by aqueous extract of Rosa damascena petals and to evaluate the biocompatibility and anti cancer activity of the synthesized silver nanoparticles against human lung adenocarcinoma (A549). Methods: The process variables that include concentration of extract, mixing ratio of reactants, silver salt concentration and interaction time were analyzed. The compatibility of the G-SNPs was verified by incubating with erythrocytes and the anticancer property of the G-SNPs against A549 cells was performed by MTT assay. Results: Formation of G-SNPs was confirmed by the visual change in the colour of the reaction mixture from pale yellow to brown yellow. Surface plasmon resonance of synthesized G-SNPs was observed at 420 nm; the size of G-SNPs were analyzed by DLS and found to be in the range of (84.00依10.08) nm. Field emission scanning electron microscope and high resolution transmission electron microscopy analysis confirmed that the G-SNPs were fairly spherical. Fourier transform infrared spectroscopy spectroscopy and X-ray diffraction revealed the characteristic peaks of G-SNPs. Energy dispersive X-ray analysis showed a signal of silver around 3 keV. The synthesized G-SNPs exhibited anticancer activity as evidenced by the MTT assay. IC50 value of G-SNPs was found to be 80 µg/mL. Conclusion: The results of the present study suggest that G-SNPs can be synthesized rapidly within first minute of the reaction; they are biocompatible and possess anticancer activity against human lung adenocarcinoma.
1. Introduction Synthesis of nanoparticles with variable shapes, sizes and chemical compositions has gained importance in nanotechnological applications. The metallic nanoparticles have received much attention due to their unique catalytic, optical and electrical properties[1]. The silver nanoparticles (SNPs) have been widely utilized in biology and medicine due to its attractive physiochemical properties[2]. SNPs have been reported to exhibit number of pharmacological activities, including antibacterial activity[3-5], antifungal activity[6], anticancer activity[7], antiviral activity[8,9] antiplasmodium activity and mosquito larvicidal activity[10,11], etc. Although *Corresponding author: Dr. Elangovan Vellaichamy, Associate Professor, Department of Biochemistry, University of Madras, Guindy Campus, Chennai- 600 025, India. E-mail: [email protected], [email protected] Tel: +91-044-22202734 Foundation Project: Supported by Indian Council of Medical Research-Grant no. 35/27/2010-BMS and University Grants Commission (Basic Scientific Research fellowship in sciences)-Grant no. F7-114/2007 (BSR).
the chemical methods of synthesizing SNP s promise to provide high purity and desired structure to the nanoparticles, they are quite expensive and toxic. Since most of these chemically synthesized nanoparticles are utilized for human benefits, they pose severe hazard to human health and to the environment[12,13]. Recently, ‘green synthesis’ of nanoparticles using extracts of plants and microbes have gained importance because it could solve the problem of toxicity imposed by chemical methods. Green synthesis of SNPs using plant extracts have been studied previously using several plants which includes Rhizophora mucronatas[14], Acalypha indica[3], Phyllanthus emblica[15], Panicum virgatum[16], Piper nigrum[17], Glycine max[18], Murraya Koengii[19], Rosa rugosa[20], Camellia sinensis[21]. These studies have shown that SNPs can be synthesized in environmental friendly and green method using plant extracts. Still, studies on green synthesis of SNPs continue by employing medicinal plants extract to explore the potentials of traditional and medicinal plants to synthesize biocompatible SNPs. Since, each traditional
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plant has specific medicinal properties, by employing theses plants the medicinal property of the synthesized SNPs may be enhanced. Previous studies elucidated the presence of several pharmacologically active compounds in the petals of Rosa damascena (R. damascena)[22,23]. Hence, we hypothesize that the nanoparticles synthesized using R. damascena could be biocompatible and can be used for biological applications. R. damascena belongs to the Rosaceae family of flowers. It is one of the important species in that family, which is used as an ornamental plant and its flowers are utilized as additives in food and medicine [24] . I t has gained great importance due to its pharmacological properties including anti-diabetic, anti-HIV, and its potential to cure cardiovascular diseases[22]. R. damascena petals have been reported to contain several important chemical compounds, which includes flavonoids, terpenes, antocyanins, glycosides, carboxylic acid, vitamin C, myrcene, kaempferol and quercetin[22]. These properties of this plant make it a suitable choice for synthesis of silver nanoparticles, which may lead to the synthesis of eco-friendly and non-toxic green-synthesized silver nanoparticles (G-SNPs) for medical and biological applications. H ere, we reported the green synthesis of silver nanoparticles using an aqueous extract of R. damascena and evaluated its anticancer activity against human lung adenocarcinoma ( A 549 ) . T he process variables such as concentration of extract, mixing ratio of reactants, silver salt concentration and interaction time were analyzed. The results of this study uncovered the potentials of R. damascena petal extracts to synthesize G-SNPs. G-SNPs can be synthesized rapidly by using aqueous extract of R. damascena petals and they are biocompatible and possess anticancer activity.
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cooled and stored at 4 °C for further experiments[25]. This solution serves as 100% extract concentration. 2.3. Green synthesis of silver nanoparticles In order to enhance the reproducibility of the experimental results and to optimize the process parameters, the factorial design of experiments, the “one-factor-at-atime” method was employed in this study. In this method of experimentation, only one experimental factor is varied at a time, while keeping all the other variables constant. Different concentrations of rose extracts (10%, 25%, 50%, 75% and 100%) were prepared by diluting the bioreductant (rose extract) with deionized water accordingly. They were added to 10 mmol/L silver nitrate solution at 2:3 ratio and incubated for 10 min in dark at room temperature. In another process parameter, the silver reduction reaction was carried out by varying the different mixing ratios of rose extract to 10 mmol/ L silver nitrate solution (1:4, 2:3, 1:1, 3:2, 4:1) at 100% extract concentration while keeping all the other variables constant. In order to determine the interaction time between the rose extract and silver nitrate solution, 10 mmol/L silver nitrate solution and 100% rose extract was added together in 2:3 ratio and incubated for different time points (0 min, 5 min, 10 min, 15 min and 25 min). To determine the optimum silver salt concentration, different concentrations of silver nitrate solution (1 mmol/L, 2 mmol/L, 3mmol/L, 5mmol/L, 10 mmol/L) were reacted to 100% rose extract at 2:3 ratio. T o obtain the dry powders of G - SNP s, the solution containing the silver nanoparticles were centrifuged at 10 000 r/min for 15 min. The pellet was resuspended in deionized water and the centrifugation process was repeated thrice to get rid of any non-reacted biological materials. The purified pellets were then freeze dried to get powder G-SNPs. This powder was further used for characterization studies.
2. Materials and methods
2.4. Characterization
2.1. Materials
The preliminary characterization of silver nanoparticles was carried out using UV - visible spectroscopy. T he reduction of silver nitrate to silver nanoparticles was analyzed by recording the spectra of silver nanoparticles from 340 to 700 nm (Shimadzu UV 150-02, Japan). The size distribution and zeta potential of the nanoparticles in the solution were determined using particle size and zeta analyzer (Malvern Nano ZS, UK). The G-SNP solution was diluted 10 times with deionised water prior to analysis. The powder X-ray diffraction pattern was obtained by using the lyophilized powder of silver nanoparticles (BYE FIEX 2002 model, Seifert, Germany) with Cu Kα radiation (λ=1.54 nm) in the 2θ range of 20° to 80° operated at a voltage of 40 kV and a current of 30 mA. The Fourier transform infrared spectroscopy (FT-IR) spectroscopic analysis was carried on lyophilized powders of 100% petal extract and silver nanoparticles. The measurements were performed using Nicolet Impact 400 FT-IR spectrophotometer with KBr pellet in the wave number region of 2 000 to 800 cm-1. For field
Fresh roses (R. damascena) were procured from the local market, Chennai, India. Silver nitrate, dimethylsulfoxide and 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St Louis, MO, USA) . D ulbecco’s modified E agle’s medium ( DMEM) , fetal bovine serum (FBS), Trypsin-EDTA and antibioticantimycotic solution were purchased from HiMedia (Mumbai, India). Deionized water was used throughout the study.
2.2. Preparation of R. damascena extract The rose petals were washed thoroughly with deionized water and shade dried at room temperature for 2 d. The extraction process was started by adding one gram of dried petals and 100 mL of deionized water in to a 250 mL Erlenmeyer flask. This mixture was slowly stirred using a magnetic stirrer at 80 °C for 1 h. This solution was filtered,
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emission scanning electron microscope (FE-SEM) and high resolution transmission electron microscopy ( HR - TEM ) analysis, the synthesized nanoparticle solution was diluted and a drop was placed on aluminum foil and on a carbon grid respectively and allowed to dry. The FE-SEM micrograph was obtained using Hitachi SU6600, Germany and HR-TEM analysis was carried out on FEI TECNAI G2. The purified and lyophilized samples were subjected to EDAX analysis (Hitachi SU6600, Germany). 2.5. Hemolytic activity assay The in vitro hemolytic activity of G-SNPs was evaluated by measuring the hemoglobin release from the erythrocytes on interaction with nanoparticles. The blood was collected from male Wistar albino rat in a tube containing EDTA and the tube was centrifuged at 1 500 r/min for 10 min. The supernatant and buffy coat was carefully removed and the pellet was washed thoroughly with phosphate buffered saline (pH 7.4) several times. Cells (5% v/v) in phosphate buffer solution (PBS) were added to each tube and the G-SNP at different concentration (25, 50, 75, 100, 125 µg) in PBS were added and the final volume was made up to 1 mL. Erythrocytes in PBS solution and in 1% triton X-100 were considered as negative and positive controls respectively. The reaction mixtures were incubated in shaker incubator at 37 °C with mild shaking for 1 h. Then the tubes were centrifuged at 1 500 r/min for 10 min and the supernatant were read at 540 nm against their blank. The percentage of hemolysis=[(Abs540 nm in the G-SNP solution-Abs540 nm in PBS)/(Abs540 nm in 1% Triton X-100-Abs540 nm in PBS)]伊 100[26].
2.6. Cell proliferation assay Human lung adenocarcinoma epithelial cell line (A549) was obtained from National Center for Cell Science, Pune, India. The cell lines were maintained in DMEM, supplemented with 10% FBS and antibiotic antimycotic solution. Cells were grown in a humidified chamber at 37 °C with 5% CO2 supply. To assess the anticancer activity of G-SNPs quantitatively, 3 MTT reduction assay was performed[27]. A549 cells (2伊10 cells/100 µL) were seeded on 96 well plate with 200 µL of complete DMEM media containing 10% FBS. The plate was incubated at 37 °C in a humidified chamber with 5% CO2. After 24 h of culturing, the spent media was removed and fresh DMEM media with predefined concentrations (20 to 120 µg/mL) of G-SNPs were added and incubated at 37 °C in a humidified chamber with 5% CO2. After 24 h of incubation with G-SNPs, the spent media was replaced with 10 µL of MTT solution to each well (5 mg/mL) and incubated for 4 h at 37 °C in a humidified chamber with 5% CO2. After completion of 4 h, the MTT was removed and 100 µL of dimethylsulfoxide was added to all the wells. The absorbance was read at 570 nm with microplate spectrophotometer. The cells which were not treated with nanoparticles were considered as control.
2.7. Statistical analysis Values of three independent experiments were expressed as mean 依 SD . T he data was evaluated using SPSS / 11 . 5
software.
3. Results The results of the present study demonstrate the successful green synthesis of G-SNPs using R. damascena petal extract. The G-SNPs were found to be biocompatible and exhibited cytotoxic activity against human lung adenocarcinoma (A549) cells. On addition of R. damascena extract to silver nitrate solution, the colour of the reaction mixture changed rapidly from pale yellow to brown yellow (Figure 1). The surface plasmon resonance of the silver nanoparticles reduced by R. damascena aqueous extract was obtained at 420 nm. (a)
(b)
(c)
Figure 1. Photographic representation of silver nanoparticle synthesis. a: Silver nitrate solution; b: Aqueous extract of R. damascena petal extract; c: Green synthesized silver nanoparticles.
Figure 2a shows the effect of different concentrations of petal extract on G-SNPs formation. The absorbance of the UV-Vis spectrum gradually increased with the increase in concentration of the extract from 10% to 100%. The highest absorption was recorded when 100% extract was used. Figure 2b shows the results of second process variable, in which the ratio of petal extract to silver nitrate solution (1:4, 2:3, 1:1, 3:2, 4:1) was analyzed. The results clearly revealed that 2:3 ratio of extract to silver nitrate solution was the perfect combination to get maximum yield. This was observed from the maximum absorbance in UV-Vis spectrum. Figure 2c shows the effect of incubation time on G-SNPs formation. The absorbance was observed even at 0 min of the reaction. As the incubation time was increased from 0 to 25 min (0, 5, 10, 15, 25 min) there was no significant change in the absorbance of the nanoparticles. Our results indicate that the formation of G-SNPs starts immediately after addition of extract to the silver nitrate solution, thereafter no marked increase in the absorbance was observed. To determine the exact silver salt concentration for effective G-SNP synthesis,
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10 5 T=25 1 2 3 T=15 T=0 T=5 T=10 Figure 2. UV-Vis spectroscopic analysis of process variables in silver nanoparticles synthesis. a: UV-Vis spectra of different concentration of R. damascena petal extract with 10 mmol/L silver nitrate solution; b: UV-Vis spectra of different mixing ratio of petal extract with 10 mmol/L silver nitrate solution; c: UV-Vis spectra of reaction time of petal extract with 10 mmol/L silver nitrate solution; d: UV-Vis spectra of different concentration of silver nitrate solution with petal extract.
different concentrations of silver nitrate solutions ranging from 1 to 10 mmol/L were reacted with extract in 2:3 ratio for 5 min. Figure 2d represents the effect of different silver nitrate solution for synthesizing the G-SNP. From the graph, it is observed that 10 mmol/L silver nitrate was identified as the optimum concentration for the effective G-SNP formation. 140
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From the dynamic light scattering measurement, the average size of the particles was found to be (84.00依10.08)
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nm. Zeta potential analysis of the synthesized G-SNP was found to be (-17.4依2.1). The crystalline nature of the silver nanoparticles was analyzed by X -ray diffraction ( XRD) (Figure 3). The results revealed four diffraction peaks [38.05 (111), 46.12 (200), 67.19 (220), 76.3 (311)] corresponding to four diffraction facets of silver. These results indicate that the synthesized nanoparticles were face centered cubic and crystalline in nature (JCPDS file No. 84-0713 and 04-0783).
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Figure 4. FT-IR spectra of aqueous petal extract of R. damascena and silver nanoparticles. a: Aqueous petal extract of R. damascena; b: Silver nanoparticles obtained by treating 100% petal extract with 10 mmol/L silver nitrate for 5 min at room temperature.
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(a)
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performed using different concentrations of G-SNPs (25, 50, 75, 100, 125 µg/mL) on erythrocytes. The absorbance values of erythrocytes in PBS and in 1 % triton X - 100 was used along with the values of erythrocytes treated with G-SNPs on the formula. It was found that G-SNPs do not exhibit hemolytic activity against erythrocytes on different tested concentrations (Table 1). Table 1 H emolytic activity of silver nanoparticles on erythrocytes at different concentration after incubation at 37 °C for 1 h. Sample (n=3)
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T he in vitro anticancer effect of G - SNP s was tested on A 549 cell lines. A dose dependent decrease in the viability of A549 cells were observed on treatment with G - SNP s, as shown in the F igure 6 . T he half maximum inhibitory concentration (IC50) was observed at 80 µg/mL. At this concentration, G-SNPs inhibited the growth of A549
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FT-IR is reported as an appropriate technique to analyze chemical adsorption and functional groups in newly synthesized particles. The FT-IR transmission spectra of rose extract and silver nanoparticles are represented in Figure 4. Characteristic absorption bands at 1 719, 1 615, -1 1 351, 1 232 and 1 057 cm in the FT-IR spectrum of rose petal extracts were observed ( F igure 4 a ) . B and at 1 719 cm-1 which is attributed to the C=O stretching due to the carboxyl content in the rose petal extracts, which might reduced the silver ion to silver nanoparticles. Further, peaks assigned at 1 615, 1 351 and 1 057 cm-1, suggest that amine group and the hydroxyl groups of water/alcohol. T he FT-IR spectra of silver nanoparticles are shown in Figure 4b which shows comparatively similar absorption bands at 1 619, 1 384 and 1 058 cm-1 of rose petals extract. -1 The suppression of C=O stretching in the 1 719 cm region has been related to evidence of silver ion reduction by rose petals as consequence of silver nanoparticles formation. F igure 5 a and 5 b shows the FE - SEM and HR - TEM micrographs, which provides a clear idea on the shape of nanoparticles. Majority of the particles were spherical in shape with the size ranging from 15 to 27 nm. These particles were well distributed without any aggregation. T he presence of elemental silver was confirmed by EDAX spectroscopy analysis (Figure 5c), which indicated the reduction of silver ion to silver. M etallic silver nanocrystals generally display an optical absorption peak at 3 keV[28]. There was also a strong signal for Al in the EDAX data, which was due to the Al FE-SEM grid.
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H emolytic activity of G - SNP s was performed to evaluate the biocompatibility of the G-SNPs on normal cells, especially erythrocytes. The hemolytic assay was
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Figure 6. In vitro anticancer effect of G-SNPs. a: Cell proliferation assay using MTT for analyzing dose dependent effect of silver nanoparticles on A549 cells for 24 h. Fifty persents inhibition of cells was observed at 80 µg/mL silver nanoparticles concentration (10伊); b: Inverted microscopic view of A549 control cells without any treatment; c: Cells treated with 80 µg/mL silver nanoparticles for 24 h (10伊).
4. Discussion Green synthesis of nanoparticles has received much attention due to easy and eco-friendly nature of the
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synthesis [29] . O n addition of the petal extract to the silver nitrate solution, the colour of the reaction mixture changed from pale yellow to brown yellow. This change in colour of the mixture was due to the reduction of silver ion and excitation of surface plasmon resonance by the nanoparticles. This change in the colour of the reaction mixture indicated the formation of G - SNP [30]. I n the present study, the process parameters have been optimized, to reduce the time required for G-SNPs synthesis and to improve the yield. B y varying the concentration of the petal extract from 10% to 100%, the plasmon resonance band raised gradually. M aximum plasmon resonance band was attained at 100% of extract concentration, which shows that the reduction of silver ion was directly proportional to the concentration of petal extract. T here was no formation of plasmon resonance band when 10% of extract concentration was used, which indicated that there was not enough reducing agent in the extract to reduce the silver ion. Mixing ratio and silver salt concentration plays a vital role in the optimization of process parameters, which determines the yield of the nanoparticles. T he maximum P lasmon resonance was obtained for 2:3 mixing ratio and 10 mmol/L silver nitrate. D ubey et al. synthesized SNP s using leaves of Rosa rugosa and reported the synthesis of nanoparticles after incubating the extract with the silver nitrate solution for 10 min[20]. In the present study, the plasmon resonance band was observed even at the first minute of the reaction and there were no significant change even after 25 min of the reaction. This rapid synthesis might be due to the higher percentage of extract concentration (100%) in the reaction mixture. Similar to our observation, T ripathy et al. have optimized the process parameters ( concentration of extract, mixing ratio and interaction time) for synthesizing SNPs by employing aqueous extracts of Azadirachta indica [31] . H e also emphasized in his report that size and shape of biosynthesized nanoparticles strongly depends on the optimization of process parameter. F urther, the G - SNP s were characterized by various analytical techniques. I n the XRD , there were few unassigned peaks marked with asterisk, which might be raised due to the crystallization of bio-organic phase or protein on the surface of the G-SNP from petal extract. S imilar results were reported in the synthesis of SNPs by Coleus aromaticus leaf extract and by Citrus limon aqueous extract[4,32]. The hemocompatibiliy of the G - SNP s were analyzed on erythrocytes and the results obtained were found to be correlated with the previous study done by Ruden et al. who reported that SNPs did not show hemolytic activity against erythrocytes even at the higher concentrations (up to 1024 µg/mL)[33]. The G-SNPs was found to exhibit anticancer activity against human lung adenocarcinoma but, the mechanism by which the SNPs induced cytotoxic activity was not clear. H owever, several possible mechanisms for anticancer activity of silver nanoparticles can be proposed. Cytotoxic
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effects of silver nanoparticles might be due to the physiochemical interaction of silver nanoparticles with the intracellular proteins and DNA . S tudies have also suggested that anticancer activity might also be due to induction of apoptosis activated by caspase 3 enzyme[7]. I n addition, the presence of pharmacologically active anticancer compound which includes flavonoids such as quercetin, etc., in the petals of R. damascena could also be one of the reasons for the G-SNPs to exhibit anticancer activity. Further, studies are needed to unravel the exact mechanism behind the anticancer efficacy of G-SNP. T o conclude, we have demonstrated that G - SNP s were rapidly synthesized using aqueous extracts of R. damascena within the first minute of the reaction at room temperature. Various process parameters involved in the green synthesis were analyzed for the better yield of G-SNPs and the synthesized G-SNPs were found to be biocompatible. T he G - SNP s exhibited inhibitory effect against A549 cell line, which might be due to the synergistic effect of SNP s and the pharmacologically active compounds on the surface of G-SNPs from the R. damascena extract. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements T he authors would like to thank I ndian C ouncil of M edical R esearch- G rant N o. 35 / 27 / 2010 - BMS and University Grants Commission (Basic Scientific Research fellowship in sciences-G rant N o. F 7 - 114 / 2007 ( BSR) for the financial support. We also like to acknowledge the electron microscopy facilities provided by the National Center for Nanoscience and Nanotechnology, University of Madras, India.
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