Materials Science and Engineering C 53 (2015) 120–127
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Bio-synthesis of silver nanoparticles using Potentilla fulgens Wall. ex Hook. and its therapeutic evaluation as anticancer and antimicrobial agent Amit Kumar Mittal a, Debabrata Tripathy b, Alka Choudhary c, Pavan Kumar Aili a, Anupam Chatterjee b, Inder Pal Singh c, Uttam Chand Banerjee a,⁎ a b c
Department of Pharmaceutical Technology Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, 160062 Punjab, India Department of Biotechnology and Bioinformatics, North Eastern Hill University, Shillong, 793002 Meghalaya, India Department of Natural Products, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, 160062 Punjab, India
a r t i c l e
i n f o
Article history: Received 27 December 2014 Received in revised form 22 March 2015 Accepted 21 April 2015 Available online 22 April 2015 Keywords: Potentilla fulgens Antimicrobial Flavonoids Anticancer Silver nanoparticles
a b s t r a c t The present study aims to develop an easy and eco-friendly method for the synthesis of silver nanoparticles using extracts from the medicinal plant, Potentilla fulgens and evaluation of its anticancer and antimicrobial properties. The various parts of P. fulgens were screened and the root extract was found to have the highest potential for the synthesis of nanoparticles. The root extracts were able to quickly reduce Ag+ to Ag0 and stabilized the nanoparticles. The synthesis of nanoparticles was confirmed by UV–Visible spectrophotometry and further characterized using Zeta sizer, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscope (TEM) and X-ray diffraction (XRD). Electron microscopic study showed that the size of the nanoparticle was in the range of 10 to 15 nm and spherical in shape. The studies of phytochemical analysis of nanoparticles indicated that the adsorbed components on the surface of nanoparticles were mainly flavonoid in nature. Furthermore, nanoparticles were evaluated as cytotoxic against various cancer cell lines and 0.2 to 12 μg/mL nanoparticles showed good toxicity. The IC50 value of nanoparticles was found to be 4.91 and 8.23 μg/mL against MCF-7 and U-87 cell lines, respectively. Additionally, the apoptotic effect of synthesized nanoparticles on normal and cancer cells was studied using trypan blue assay and flow-cytometric analysis. The results indicate the synthesized nanoparticle ability to kill cancer cells compared to normal cells. The nanoparticles also exhibited comparable antimicrobial activity against both Gram-positive and Gram-negative bacteria. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nanomaterials are considered as basic fundamental building blocks in the nanotechnology area [1]. A variety of physical and chemical methods have been reported to synthesize nanoparticles [2]. Physical methods are cost effective and do not produce uniform size nanoparticles while the chemical methods are not environmentally friendly and require harsh conditions [2]. Syntheses of various metal nanoparticles (Ag, Au, Pt, Cu and Se) by biological means are also reported [2–4] in literature. Among various metal nanoparticles, silver nanoparticles (AgNPs) play a significant role in the field of biology and medicine [4]. It possesses higher levels of antibacterial activity and is well known antimicrobial agent. Metallic silver has been used as anti-infective agent from the last thousands of years in the form of ornaments and utensils. However, silver ions have only limited microbicidal activity against limited microbes with higher toxicity in comparison to silver nanoparticles [5,6]. The antiviral and antimicrobial actions of the different forms of the ⁎ Corresponding author. E-mail address: [email protected] (U.C. Banerjee).
http://dx.doi.org/10.1016/j.msec.2015.04.038 0928-4931/© 2015 Elsevier B.V. All rights reserved.
nano-silver were also reported by various researchers [4,7]. It is well known that biological methods are much simpler and single step process for the production of metal nanoparticles from metal salts [4]. The process can proceed at room temperature, can be easily scaled up and is environmentally friendly [4,8]. The biomolecules present in the biological sources having reducing properties that can reduce metal ions (M+) to zero-valent (M0) metals form and stabilize them [9]. The efficacy of biologically functionalized nanoparticles with a biologicalshell and metal-core is used for the treatment and diagnosis of various life threatening diseases [10,11]. They are reported to demonstrate bactericidal synergistic effect against various types of pathogenic microbes when used in combination with antibiotics [10]. These biosynthesized nanoparticles, capped or functionalized with various biomolecules are becoming important tool for developing next generation therapies and diagnosis [3]. Surface functionalization is possible due to the presence of various active groups (\\COOH, \\NH2, \\OH, etc.) of the capped phytochemicals. The nanoparticles synthesized by plant extracts might be conjugated with different bio-moieties like hormones, enzymes, antibody, proteins, antibiotics, etc. to achieve the targeted drug delivery. The plants of genus Potentilla have been used for a long time
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National Center for Cell Science (Pune, India). The bacterial strains and culture medium components were procured from Microbial Type Culture Collection Center, Institute of Microbial Technology, Chandigarh, India and M/S Himedia, Mumbai, India, respectively.
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2.2. Preparation of the plant extract
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Various plant parts of P. fulgens were air-dried in shade and crushed to powder form. The roots (25 g) were dried and grounded to coarse powder. It was further macerated with methanol at 37 °C for 72 h. The extract was filtered and methanol was evaporated using a rotary vacuum evaporator (Buchi, Switzerland) to yield the extract.
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2.3. Synthesis of silver nanoparticles
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Fig. 1. UV–vis absorption spectrum of silver nanoparticles produced by the root extract of Potentilla fulgens.
for the treatment of inflammation, wounds, microbial infections, diabetes mellitus and other human ailments [12–15]. Potentilla sps. contain various types of compounds such as polyphenols and flavonoids in the roots and rhizomes [14,16]. Recently the presence of stereoisomeric antioxidant compounds in the root extracts of Potentilla fulgens is also reported [17]. P. fulgens Wall. ex Hook. is a vital medicinal plant of Himalayan region and commonly known as Himalayan Cinquefoil [18]. P. fulgens root-stock and whole herb is used as astringent and tonic for curing gum and tooth ailments, diabetes, diarrhea and other varieties of diseases [12–16]. The aim of the present study was to synthesize the metallic nanoparticles by P. fulgens extracts and their applications as therapeutic and diagnostic agents. The nanoparticles were characterized using various techniques and therapeutic potentials were evaluated as anticancer and antimicrobial agents. Furthermore, the cell cycle analysis and trypan blue exclusion assays were performed on various types of cells to know apoptotic mechanism. 2. Materials and methods
Nanoparticles were synthesized following the previously reported method with a little modification [19]. To synthesize metallic nanoparticles, P. fulgens extracts were suspended in 50 mL deionized water, silver salt (1 mM) was added to the solution and reaction mixture was incubated at 35 °C (200 rpm) in dark. Reduction of metal ions was initially confirmed by visual inspection of color change and then by spectrophotometric absorption at 400–420 nm. Various reaction parameters (concentrations of plant extract and metal ions, pH, temperature and reaction time) were optimized to enhance the yield of nanoparticle. The nanoparticles were harvested by centrifugation (Eppendorf, Germany) to remove un-reacted phytochemicals from the reaction medium at 15,000 ×g for 30 min and the pellet was thoroughly washed with deionized water. The resulting suspension was dried in a lyophilizer (Allied Frost, New Delhi, India) and then further characterized. 2.4. Characterization of nanoparticles 2.4.1. UV–vis spectra analysis The UV–vis absorbance spectra were used for the preliminary identification of silver nanoparticle formation. An absorption band near 400–450 nm indicated the formation of nanoparticles [20]. The reduction of Ag+ in the reaction mixture was periodically monitored after 6 h of reaction, in a quartz cuvette with plant extract used as baseline. Sample absorbance was taken in the range of 300–700 nm using U2900 Hitachi spectrophotometer.
2.1. Chemicals P. fulgens roots were obtained from the Shillong Peak Forest area, Meghalaya, India. All of the reagents and solvents were procured either from Himedia laboratories or Rankem, Mumbai, India. Silver nitrate (AgNO3), concanavalin-A (Con-A), quercetin dihydrate, gallic acid and Folin–Ciocalteu reagents were purchased from Sigma-aldrich (Bangalore, India). Dulbecco's MEM, fetal bovine serum (FCS), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine were procured from Invitrogen—GIBCO, USA. MCF-7 (human breast cancer) and U-87 (human glioblastoma cancer) cell lines were purchased from the
2.4.2. Zeta-sizer and zeta potential analysis Dynamic light scattering (DLS) or zeta-sizer is a technique used to measure the size and size distribution of very small particles dispersed in a liquid. To estimate the particle size and zeta potential, a dilute suspension of nanoparticles was prepared in deionized water and sonicated (for removing aggregation) at 35 °C for 30 min and subjected to DLS analysis. The measurements were taken at 532 nm (35 °C) with a detection angle of 90°. The average particle size and zeta potential of nanoparticles were estimated by Zetasizer Nano ZS (Malvern Instruments, Malvern, UK).
Fig. 2. Transmission electron microscopy (TEM) micrograph of silver nanoparticles produced by the root extract of Potentilla fulgens.
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Fig. 3. (a) Energy dispersive X-ray spectroscopy (EDX) micrograph and (b) X-ray diffraction (XRD) spectra of silver nanoparticle synthesized by the root extract of Potentilla fulgens.
2.4.3. Electron microscopy analysis HR-TEM measurements were taken on a FEI HR-TEM instrument operated at an accelerating voltage at 100–200 keV. Samples were prepared by drop coating of colloidal solution onto carbon coated copper TEM grids. The drop on the TEM grids was allowed to leave for 2 min and the grid was allowed to dry. The synthesized silver nanoparticles were analyzed for their size. The elemental content of nanoparticle was determined using energy dispersive X-ray analysis (EDX) attached with TEM instrument.
65 °C under shaking condition (200 rpm). Similarly, reaction mixtures having initial pH in the range of 3 to 13 were incubated at optimized temperature under shaking condition (200 rpm) in dark. To optimize reaction time, in terms of yield and properties of nanoparticles, samples were collected at various time intervals (6, 12, 24, 36, 48 and 72 h) and subjected to characterization. The nanoparticle colloidal solution and their lyophilized form (nanopowder) were kept in dark condition at
2.4.4. FTIR analysis of Ag nanoparticles FTIR spectroscopy was performed to identify the functional groups capped on the surface of nanoparticles. The functional groups of the synthesized nanoparticles were identified using FTIR (Perkin Elmer) spectroscopy. The bio-reduced colloidal nanoparticle solution was centrifuged at 15,000 ×g for 30 min and the pellets were thoroughly washed with deionized water. The resulting suspension was completely dried in a freeze dryer and analyzed by FTIR spectroscopy. Plant extract was also dried in a freeze dryer and subjected to comparable FTIR analysis. 2.4.5. X-ray diffraction analysis X-ray diffraction (XRD) analysis, carried out on a XRD instrument operating at 45 kV and a current of 40 mA with CuKα radiation, was performed to determine the nature of nanoparticles. The instrument was operated over the 2θ range of 30–80° (Phillips, USA) [11]. 2.5. Optimization of physico-chemical parameters for the synthesis of nanoparticles Various physico-chemical parameters such as concentrations of plant extract and metal ions, pH of the reaction mixture, incubation temperature and reaction time were optimized to increase the yield of nanoparticles. Ratio of metal ions to reducing agent affects the rate of synthesis as well as the size, shape and yield of nanoparticles. The different amounts (1 to 200 mg) of plant extract were added in water (50 mL). Metal salt was added to these dilutions and reaction mixtures were incubated at 35 °C (200 rpm) in dark. The absorbance of the resultant solutions was measured by UV–vis spectrophotometer (Hitachi U2900) periodically. To optimize the metal ion concentration, various flasks were incubated at 35 °C (200 rpm) containing different concentrations of silver nitrate (0.5 to 5 mM) with a fixed concentration of plant extract. In order to have temperature optimization studies, reaction flasks were incubated at various temperatures ranging from 25 to
Fig. 4. Effect of various reaction parameters (concentrations of plant extract and metal ions, pH, temperature and reaction time) on silver nanoparticle synthesis by Potentilla fulgens extract.
A.K. Mittal et al. / Materials Science and Engineering C 53 (2015) 120–127 Table 1 Total phenolic and flavonoid contents of Potentilla fulgens silver nanoparticles and standard compounds. Phenolics/flavonoids
Content (%/100 mg standard)
Potentilla fulgens silver nanoparticles % (for phenolic content) Control gallic acid % (for phenolic content) Potentilla fulgens silver nanoparticles % (for flavonoid content) Control quercetin % (for flavonoid content)
7.7 ± 0.68 86 ± 0.23 30.604 ± 0.16 99 ± 0.43
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group, lymphocyte cultures were set up in RPMI 1640 medium supplemented with 10% heat inactivated FCS and stimulated with PHA using addition. Penicillin (100 U/mL) and streptomycin (100 mg/mL) and 2 mM L-glutamine were also added to the medium. In another group, lymphocytes were kept in RPMI 1640 medium with all the supplements without PHA. After 6 h, lymphocytes of both the groups were treated with AgNPs. The cells were incubated at 37 °C for 24 h and harvested. The study was performed with the compliance of the “Ethical Guidelines for Biomedical Research on Human Subjects” formulated by the Indian Council of Medical Research, India. 2.8. Cytotoxicity assay
the room temperature for a year to check their stability. The stability of the particles was periodically checked by UV–vis spectral analysis. 2.6. Determination of total phenolics and flavonoid content A sample of nanoparticle (20 μL, 50–500 μg/mL) was mixed with deionized water (1.58 mL) followed by the addition of 2 N Folin– Ciocalteu's phenol reagent. The resulting mixture was vortexed and incubated at room temperature for 8 min followed by the addition of aqueous sodium carbonate solution (20%, 300 μL). The resultant mixture was vortexed again and kept at room temperature for 2 h. Absorbance of the blue-colored solution was recorded at 725 nm [21,22]. The gallic acid was used as a standard for the calculation of total phenolic content and expressed as gallic acid equivalents (GAE %). Total flavonoid content was determined using colorimetric aluminium chloride method and quercetin dihydrate was used as a standard. The samples and standards were individually dissolved in water. The sample solution (100 μL) was then mixed with an aqueous solution of anhydrous aluminium chloride (2%, 100 μL). After 10 min incubation at room temperature the absorbance of the supernatant was measured at 435 nm. The absorbances of samples in 96 well plates were taken on a multi-well micro-plate reader (Multiskan EX; Thermo Scientific, USA). The total flavonoid content was expressed in quercetin dihydrate equivalents. All the experiments were performed in triplicate [21,22]. 2.7. Cell line cultivation and treatment MCF-7 and U-87 cells were grown in Dulbecco's MEM high glucose medium and in Dulbecco's MEM low glucose medium, respectively. Both the media were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine. Freshly heparinized peripheral blood was collected from two healthy male donors (HPBL) and peripheral blood mononuclear cells were isolated by Ficoll-Hypaque (Sigma Diagnostics, St. Louis, MO) density gradient centrifugation (specific gravity 1.077 g/mL) for 30 min at 400 ×g. Lymphocytes were divided into two groups. In one
(a)
The cytotoxicity and cell viability was calculated using a clonogenic cell survival assay in MCF-7 and U-87 cancer cell lines. This method for measuring cytotoxicity using colony-forming assay is considered as the most reliable method [17]. Cells were plated in triplicate at a cell density of 1000 cells onto 25 cm2 tissue culture flask while for the untreated controls; four flasks were plated at a cell density of 500 cells per culture flask. 5 h after the plating, cells were exposed for 24 h with 2 to 12 μg/mL of the Potentilla root extract-silver nanoparticles or 0.2 to 1 μg/mL of only silver nanoparticles. Cells were washed twice with the DMEM medium and finally flasks were incubated in a CO2 incubator with DMEM containing 10% fetal calf serum for 8 to 10 days. The colonies were fixed and stained with 0.2% crystal violet in 70% ethanol. Colonies with a minimum of 50 cells were counted as the progeny of a viable cell. All survival points were checked in triplicate and experiments were repeated three times. Results were expressed as surviving fraction. The plating efficiency (PE) and survival fraction (SF) were calculated as follows: Plating efficiencyðPEÞ% ¼ ðNo: of colonies formed=No: of cells seededÞ 100 Survival fractionðSF Þ% ¼ ðPE of treated=PE of controlÞ 100:
2.9. Trypan blue exclusion assay Cytotoxicity of synthesized nanoparticles was determined by trypan blue (TB) exclusion assay also. Various types of cells were incubated with AgNPs for 24 h and washed with DMEM. Finally, the cells were incubated for 10 min at room temperature with 0.4% trypan blue dye solution. The trypan blue dye stains dead cells for blue color, while the live cells remain unstained. 2.10. Flow-cytometric analysis of cells Various types of cells were grown in a respective culture medium, treated for 24 h with and without AgNPs and fixed with 70% ethanol. In case of MCF-7 and U-87 cells, cells were fixed with 70% ethanol
(b)
Fig. 5. Dose dependent reduction in the cloning efficiency of (a) MCF-7 and (b) U-87 cancer cell lines treated with synthesized silver nanoparticles.
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Table 2 Trypan blue exclusion assay of various normal/cancer cells treated with Potentilla fulgens silver nanoparticles. Cell type
Percentage of dead cells ± S.D.
Primary normal cell lines Untreated lymphocytes (PHA−) AgNPs treated lymphocytes (PHA−) Untreated lymphocytes (PHA+) AgNPs treated lymphocytes (PHA+)
9.4 ± 1.0 17.8 ± 0.8⁎ p = 0.001 6.4 ± 0.8 19.7 ± 0.8⁎ p = 0.004
Cancer cell lines Untreated MCF-7 cells AgNPs treated MCF-7 cells Untreated U87 cells AgNPs treated U87 cells
8.5 ± 0.9 42.4 ± 2.4⁎p = 0.0007 6.7 ± 0.9 40.6 ± 2.6⁎p = 0.002
⁎ Student's t-test with respect to respective control value.
soon after 24 h treatment while lymphocytes were fixed with ethanol after additional 6 h of stimulation period. The fixed cells were washed with PBS and resuspended in 500 μL propidium iodide solution (50 μg/mL propidium iodide, 0.2 mg/mL RNase) and incubated for 1 h at room temperature in dark. A large number of cells (10,000) were acquired for each sample and analyzed with FACS Calibur (Becton-Dickinson) instrument. CELLQ-uest Pro-software was used to quantify cell cycle compartments to estimate the percentage of cells distributed in the different phases of cell cycle.
2.11. Antibacterial assays In vitro antibacterial activities were evaluated using Escherichia coli and Bacillus subtilis cells as susceptible test organisms using broth microdilution and agar well diffusion methods [8,23]. Bacterial suspension equivalent to 0.5 McFarland's standard was pipetted into a 96 well plate at 37 °C and treated with a varied concentration of nanoparticles (25, 50 and 100 μg/mL), after 24 h incubation at 37 °C, the optical density was recorded using automated microplate reader (Multiskan EX; Thermo Scientific, USA) at 595 nm. The standard antibiotic (chloramphenicol) and silver nitrate were used as positive controls and cells without any treatment were used as negative control. All the experiments were performed in triplicate. For agar well diffusion assay, the fresh overnight cultures (100 μL each) of E. coli and B. subtilis, approximately 108 CFU/mL were lawned onto MH agar plates. The wells in the plates were made by cork borer and filled with 50 μL nanoparticle solution having its varied concentration (25, 50 and 100 μg/mL). The other wells consisting each of 100 μg/mL chloramphenicol and silver nitrate were used as positive controls and plant extract as negative control. The plates were incubated overnight (24 to 36 h) at 37 °C. The diameter of zone of inhibition around the well was observed and recorded. All experiments were conducted in triplicate and average inhibitory zone diameters were measured.
3. Results and discussion Initially the synthesized nanoparticles were identified by observing the color change of the reaction mixture as reported [24] earlier. After the screening of plant extracts from the various parts of P. fulgens, only the root extract was capable to reduce the silver ions to silver nanoparticles. Before the reaction, both the AgNO3 and plant extract solution were colorless and after the reaction, the yellowish brown color was clearly observed (Figure S-1 in the electronic supplementary material). The color change in the reaction mixture indicated the formation of nanoparticles. The reaction color was due to the excitation of surface plasmon vibrations (essentially the vibration of the group conducting electrons) in the colloidal solution of nanoparticles [19].
3.1. Characterization of nanoparticles UV–vis absorbance spectra were used for the preliminary characterization of the nanoparticle synthesis. Fig. 1 shows the UV–vis spectra of synthesized nanoparticles using P. fulgens root extract. Using DLS, the average hydrodynamic size and zeta potential of silver nanoparticles in aqueous solution was found to be 39.04 nm and −18 mV, respectively. PDI of 0.25 indicates the good mono-dispersity and -ve zeta potential indicates good stability with capping by negatively charged groups of nanoparticles (Figure S-2 in the electronic supplementary material). Fig. 2 shows the TEM micrograph of the silver nanoparticles synthesized by P. fulgens root extract indicating the spherical shape of the particles in the order of 10–15 nm. The revealing of strong signal by EDX confirmed the presence of silver nanoparticles. The arrival of another hafnium peak was due to the carbon coated cooper grid used for this analysis (Fig. 3a). Other elemental signals recorded were possibly due to elements from phytochemicals or proteins present in the plant extract. FTIR spectra were analyzed to investigate the possible reducing functional groups present in the P. fulgens root extract. Figure S-3 (In the electronic supplementary material) shows the FTIR spectra overlay of plant extract and synthesized silver nanoparticles. The nanoparticles capped with phytochemicals of P. fulgens showed the IR peaks at 3366, 1653, 1384 and 744 cm−1 and plant extract showed the IR peaks at 3365, 1614, 1519, 1448, 1354, 1235, 1144, 1063 and 822 cm−1 regions. The FTIR spectrum of nanoparticles showed the involvement of O\\H stretching, involvement of C_N in plane vibrations of amino acids, corresponding to amides I, II and III, aromatic rings and ether linkages were found commonly present in the nanoparticles synthesized by root extract of P. fulgens [25]. These representative IR peaks suggested the presence of flavonoid, phenolics and terpenoids capped on nanoparticles. The XRD pattern of silver nanoparticles illustrated that the synthesized nanoparticles are crystalline in nature (Fig. 3b). There are four intense peaks in the whole spectrum of 2θ values ranging from 24 to 80° at 38, 44, 65 and 78°, the pattern of reflection according to Bragg's equation of silver nanoparticles showing crystalline nature of nanomaterials [26]. These peaks of nanoparticles were matched with JCPDS databases of the standard silver (file No. 04-0783). It confirms that the resultant particles are face centered cubic (FCC) in shape. 3.2. Optimization of nanoparticle synthesis The nanoparticles were primarily characterized by UV–vis spectroscopy. Based on this, various physico-chemical parameters (concentrations of plant extract and metal ions, reaction temperature, pH and reaction time) were optimized to enhance the yield of nanoparticle synthesis (Fig. 4). The reaction mixture containing 4 mg extract in 50 mL water was able to produce the maximum concentration of silver nanoparticles. Peak with 4 mg root extract was sharp as compared to other peaks, indicating the formation of relatively better sized nanoparticles. Effect of AgNO3 concentration on the synthesis of silver nanoparticles (AgNPs) was studied by varying its concentration from 0.5 to 5 mM using 4 mg root extract. The yield of silver nanoparticles increased with the increase of metal salt concentration from 0.5 to 1 mM, beyond which, there was again a fall in the absorbance. The synthesis of nanoparticles to the desired size and shape by optimizing the plant extract and metal ions is also reported in literature [27–29]. Increase in the absorbance of reaction mixture with the increase in incubation temperature evidently depicted the higher rate of synthesis of nanoparticles at elevated temperatures. Moreover, nanoparticles synthesized at higher temperatures exhibited surface plasmon resonance at narrow absorption range indicating monodispersity. At higher temperature, the rate of formation of smaller size nanoparticles increased. The maximum absorbance was obtained at 45 °C (Fig. 4). It may be due to the reduction of aggregation in the growing nanoparticles at that
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Fig. 6. Cell cycle analysis of MCF-7, U87, PHA-stimulated and unstimulated human lymphocyte cells using flow cytometry when cells were treated with Potentilla fulgens silver nanoparticles.
temperature and further increasing the temperature increased the crystal formation around the nucleus which caused the reduction in absorption. Similar results have been reported by Kaviya et al. [30]. Effect of pH on the synthesis of nanoparticles by the root extract and organic compounds was examined over a wide range of pH staring from pH 3 to 13 (Fig. 4). At acidic pH, larger size nanoparticles were formed, whereas at alkaline pH, smaller size nanoparticles were observed. This was concluded on the basis of UV–vis absorption spectra (narrow or broad peak). Effect of reaction pH on the synthesis and stabilization of metal nanoparticles as well as on their size and shape was also reported by various investigators [31,32]. Nanoparticle aggregation seems to outdo the nucleation process in acidic condition, whereas at alkaline pH, the large number of nuclei formation, instead of aggregation, led to the synthesis of higher amount of nanoparticles with smaller diameter. Blue shift in the absorption pattern confirmed the formation of relatively smaller size nanoparticles. Gericke and Pinches reported the various manipulation methods to control the size and shape of gold nanoparticles synthesized by Verticillium luteoalbum [33]. They found that aggregation of NPs was observed after 36 h reaction, which is a sign of instability. The optimum time required for the synthesis of maximum nanoparticles by P. fulgens was found to be 18 h. The stability results showed no alteration in the absorption peak at 420 nm up to 60 days, indicating good stability of synthesized silver nanoparticles. Lyophilized form of nanoparticle powder remained stable more than a year when stored at room temperature. The total phenolic content on nanoparticles was determined by Folin– Ciocalteu's method using gallic acid (GA) as a standard. It demonstrated lower quantities (7.7 ± 0.68% of GA/100 mg) of phenolics capping on nanoparticles (Table 1). The total flavonoid content in nanoparticle samples was expressed as quercetin dihydrate (QD) equivalent and it was found to be 30.604 ± 0.16% of QD/100 mg (Table 1). The flavonoid content in P. fulgens mediated nanoparticles was four-fold higher than phenolic, indicating the role of flavonoids in the nanoparticle stabilization. These assays are usually used for
the screening of antioxidant properties of food products and dietary supplements [34]. 3.3. Evaluation of cell survival, cell death and cell cycle analysis A dose-dependent reduction in the cloning efficiency was observed on Potentilla extract-silver nanoparticle-treated MCF-7 and U-87 cell lines. However, the degree of reduction in cloning efficiencies was negligible when only silver ion treatment was used. The minimum inhibitory concentration (IC50 ) values, calculated from the sigmoidal graph, were 4.91 and 8.23 μg/mL for MCF-7 and U87 cell lines, respectively (Fig. 5). The IC 50 values of the various plant extract fractions of P. fulgens were reported to be in the range of 45 to 75 μg/mL [17]. The lower IC50 values indicate the possibility of silver nanoparticles (AgNPs) to be a potent anticancer agent. Trypan blue exclusion assay concluded that the frequencies of dead cells are higher, when treated with AgNPs (Table 2). It was also found that the frequencies of dead cells are significantly higher in cancer cells than the normal primary cells. The trypan blue staining studies confirmed that AgNPs at 6 μg/ml killed more cancer cells than the normal cells. The data shown in Table 2 indicates the exclusion data of various cells, when treated with AgNPs. The synthesized AgNPs killed both types of human lymphocytes, however, the extent of killing was more for the cancer cells. Flow cytometric analysis of cancer cells after the treatment with nanoparticles showed the decline of G1 phase and increase of sub-G1 phase as shown in Fig. 6. However, for human lymphocytes, increase frequency of sub-G1 was marginal on nanoparticletreated samples. This observation was unswerving with the data observed in clonogenic and trypan blue staining assays. The FACS analysis showed the increase in percentage of cells in sub-G1 phase in nanoparticle-treated cancer cells. The observed growth inhibition of these cell lines in clonogenic-assay studies might have resulted from the cell death. The flow cytometric analysis
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demonstrated a significant rise in the number of cells in sub-G1 after the treatment indicating cell death which could be due to apoptosis.
Similarly, Abdel-Raouf et al. studied the antibacterial activity of AuNPs synthesized by Gara elongata ethanolic extract with the maximum zones of inhibition (16–17 mm) against E. coli, Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus, followed by S. aureus and Pseudomonas aeruginosa (13 mm) [36].
3.4. Evaluation of antibacterial activity To evaluate the effect of silver nanoparticles on the bacterial growth, the growth inhibition was measured both by broth microdilution and zone of inhibition methods. In broth microdilution method, 30–40% growth inhibition was observed with 25 μg/mL silver nanoparticles and it was more (50–70%) when its concentration was increased to 100 μg/mL (Fig. 7). Control experiment (chloramphenicol) showed 60% inhibition at 50 μg/mL level while it was 40% with silver nitrate. It was found that silver nanoparticles at 45 μg/mL level were very effective as bacteriostatic, where bacterial growth showed insignificant optical absorption. Similarly, Saxena et al. synthesized silver nanoparticles using Ficus benghalensis leaf extract and its bactericidal properties against E. coli were evaluated by broth microdilution method [35]. The antibacterial effect of silver nanoparticles at 100 μg/mL was measured on the basis of the zone of inhibition and compared with negative and positive controls (Table 3 and Figure S-4 in the electronic supplementary material). The antimicrobial activity of synthesized silver nanoparticles against B. subtilis is higher than that of E. coli. It might be due to the variation in the cell wall composition of Gram-positive and Gram-negative bacteria. The nanoparticles showed the maximum bactericidal effect against B. subtilis with a zone of inhibition of 9.7 ± 0.6 mm. The effect of nanoparticles with standard antibiotics (chloramphenicol) and silver salt showed comparable antimicrobial activity. It was found that the silver nanoparticles and silver nitrate showed similar bactericidal activity against the selected bacterial strains. Silver salt is highly toxic and not bio-compatible, however, the nanoparticles synthesized by P. fulgens are highly biocompatible due to the capping of nanoparticles by phytochemicals. At the same concentration, plant extract did not show any activity against selected strains. These studies have shown that silver nanoparticle formulations have good antibacterial activity against Gram-positive and Gram-negative microbes.
4. Conclusions he reduction potential of root extract of the P. fulgens was explored for the synthesis of silver nanoparticles. The nanoparticles synthesized using P. fulgens showed the activity against various cancer cell lines and bacterial species. Capping of plant constituents on the surface of nanoparticles might be one of the reasons for this attribute. The approach can also be used for the large-scale synthesis of nanoparticles from other inorganic materials. The green process of synthesizing metal nanoparticles is always preferred and it needs to be scaled up in terms of quality and types of biocatalysts. The effect of environmental parameters (pH, temperature, concentrations of catalyst and metal salts etc.) on the reaction conditions needs to be studied more. In other words, the synthesis of metal nanoparticles by plant extracts needs to be thoroughly optimized so that nanoparticles of desired quality in terms of size, shape and monodispersity, can be produced. It is especially suited for making nanoparticles free of toxic contaminants as required in therapeutic applications. The properties of nanoparticles are dependent on size, shape and their synthesis methods. Applications in targeted drug delivery and clinical diagnostics are required in future. The environmental biocompatibility and the specific cytotoxicity against tumor cells make them a powerful tool in the field of nanomedicine. Furthermore, a lot of further studies are needed to be done to obtain additional clear evidences of nanoparticle-mediated cell apoptosis in cancer cells. In medicine, silver nanoparticles are being used as antimicrobial agents in bandages. Application of such eco-friendly nanoparticles in bactericidal, wound healing, medical and electronic fields makes this method potentially stimulating for large-scale synthesis of other metallic nanomaterials.
Fig. 7. Growth inhibition of microbial strains (Escherichia coli and Bacillus subtilis) in the presence of synthesized nanoparticles.
A.K. Mittal et al. / Materials Science and Engineering C 53 (2015) 120–127 Table 3 Antimicrobial activity of Potentilla fulgens silver nanoparticles against different microbial strains. The activity was calculated in terms of zone of incubation (mm). Escherichia coli (MTCC 433) and Bacillus subtilis (MTCC 441) were incubated with silver nanoparticles, silver nitrate, plant extract and standard chloramphenicol. Bacterial strains
Zone of inhibition (mm) a
Zone of inhibition (mm) b
Zone of inhibition (mm) c
E. coli MTCC 433 B. subtilis MTCC 441
9.5 ± 0.2 9.7 ± 0.6
7.2 ± 0.6 7.6 ± 0.4
18.2 ± 0.7 20.8 ± 0.5
a b c
P. fulgens silver nanoparticles; Silver nitrate. Standard drug chloramphenicol at 100 μg/mL concentration.
Acknowledgments This work was supported by DBT, Govt. of India (BT/59/NE/TBP/ 2010). AKM gratefully acknowledges the financial support from CSIR, New Delhi, India for providing CSIR-NET-JRF fellowship. Conflict of interest All the authors have no conflict of interest about this research work. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.04.038. References [1] J.A. Eastman, S.U.S. Choi, S. Li, W. Yu, L.J. Thompson, Anomalously increased effective thermal conductivities of ethylene glycol-based nanofluids containing copper nanoparticles, Appl. Phys. Lett. 78 (2001) 718–720. [2] S. Iravani, Green synthesis of metal nanoparticles using plants, Green Chem. 13 (2011) 2638–2650. [3] A.K. Mittal, Y. Chisti, U.C. Banerjee, Synthesis of metallic nanoparticles using plant extracts, Biotechnol. Adv. 31 (2013) 346–356. [4] M. Rai, A. Yadav, A. Gade, Silver nanoparticles as a new generation of antimicrobials, Biotechnol. Adv. 27 (2009) 76–83. [5] J.S. Kim, E. Kuk, K.N. Yu, J.H. Kim, S.J. Park, H.J. Lee, S.H. Kim, Y.K. Park, Y.H. Park, C.Y. Huang, Y.K. Kim, Y.S. Lee, D.H. Jeong, M.H. Cho, Antimicrobial effects of silver nanoparticles, Nanomed. Nanotechnol. Biol. Med. 3 (2007) 95–101. [6] N. Basavegowda, A. Idhayadhulla, Y.R. Lee, Preparation of Au and Ag nanoparticles using Artemisia annua and their in vitro antibacterial and tyrosinase inhibitory activities, Mater. Sci. Eng. C 43 (2014) 58–64. [7] N.J. Reddy, D. Nagoor Vali, M. Rani, S.S. 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. [8] R. Yuvakkumar, J. Suresh, A.J. Nathanael, M. Sundrarajan, S.I. Hong, Novel green synthetic strategy to prepare ZnO nanocrystals using rambutan (Nephelium lappaceum L.) peel extract and its antibacterial applications, Mater. Sci. Eng. C 41 (2014) 17–27. [9] K.N. Thakkar, S.S. Mhatre, R.Y. Parikh, Biological synthesis of metallic nanoparticles, Nanomed. Nanotechnol. Biol. Med. 6 (2010) 257–262. [10] A.M. Fayaz, K. Balaji, M. Girilal, R. Yadav, P.T. Kalaichelvan, R. Venketesan, Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive and gram-negative bacteria, Nanomed. Nanotechnol. Biol. Med. 6 (2010) 103–109. [11] B. Felice, M.P. Prabhakaran, A.P. Rodríguez, S. Ramakrishna, Drug delivery vehicles on a nano-engineering perspective, Mater. Sci. Eng. C 41 (2014) 178–195. [12] D.R. Chhetri, P. Parajuli, G.C. Subba, Antidiabetic plants used by Sikkim and Darjeeling Himalayan tribes, India, J. Ethnopharmacol. 99 (2005) 199–202.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Bio-synthesis of silver nanoparticles using Potentilla fulgens Wall. ex Hook. and its therapeutic evaluation as anticancer and antimicrobial agent Amit Kumar Mittal a, Debabrata Tripathy b, Alka Choudhary c, Pavan Kumar Aili a, Anupam Chatterjee b, Inder Pal Singh c, Uttam Chand Banerjee a,⁎ a b c
Department of Pharmaceutical Technology Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, 160062 Punjab, India Department of Biotechnology and Bioinformatics, North Eastern Hill University, Shillong, 793002 Meghalaya, India Department of Natural Products, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, 160062 Punjab, India
a r t i c l e
i n f o
Article history: Received 27 December 2014 Received in revised form 22 March 2015 Accepted 21 April 2015 Available online 22 April 2015 Keywords: Potentilla fulgens Antimicrobial Flavonoids Anticancer Silver nanoparticles
a b s t r a c t The present study aims to develop an easy and eco-friendly method for the synthesis of silver nanoparticles using extracts from the medicinal plant, Potentilla fulgens and evaluation of its anticancer and antimicrobial properties. The various parts of P. fulgens were screened and the root extract was found to have the highest potential for the synthesis of nanoparticles. The root extracts were able to quickly reduce Ag+ to Ag0 and stabilized the nanoparticles. The synthesis of nanoparticles was confirmed by UV–Visible spectrophotometry and further characterized using Zeta sizer, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscope (TEM) and X-ray diffraction (XRD). Electron microscopic study showed that the size of the nanoparticle was in the range of 10 to 15 nm and spherical in shape. The studies of phytochemical analysis of nanoparticles indicated that the adsorbed components on the surface of nanoparticles were mainly flavonoid in nature. Furthermore, nanoparticles were evaluated as cytotoxic against various cancer cell lines and 0.2 to 12 μg/mL nanoparticles showed good toxicity. The IC50 value of nanoparticles was found to be 4.91 and 8.23 μg/mL against MCF-7 and U-87 cell lines, respectively. Additionally, the apoptotic effect of synthesized nanoparticles on normal and cancer cells was studied using trypan blue assay and flow-cytometric analysis. The results indicate the synthesized nanoparticle ability to kill cancer cells compared to normal cells. The nanoparticles also exhibited comparable antimicrobial activity against both Gram-positive and Gram-negative bacteria. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nanomaterials are considered as basic fundamental building blocks in the nanotechnology area [1]. A variety of physical and chemical methods have been reported to synthesize nanoparticles [2]. Physical methods are cost effective and do not produce uniform size nanoparticles while the chemical methods are not environmentally friendly and require harsh conditions [2]. Syntheses of various metal nanoparticles (Ag, Au, Pt, Cu and Se) by biological means are also reported [2–4] in literature. Among various metal nanoparticles, silver nanoparticles (AgNPs) play a significant role in the field of biology and medicine [4]. It possesses higher levels of antibacterial activity and is well known antimicrobial agent. Metallic silver has been used as anti-infective agent from the last thousands of years in the form of ornaments and utensils. However, silver ions have only limited microbicidal activity against limited microbes with higher toxicity in comparison to silver nanoparticles [5,6]. The antiviral and antimicrobial actions of the different forms of the ⁎ Corresponding author. E-mail address: [email protected] (U.C. Banerjee).
http://dx.doi.org/10.1016/j.msec.2015.04.038 0928-4931/© 2015 Elsevier B.V. All rights reserved.
nano-silver were also reported by various researchers [4,7]. It is well known that biological methods are much simpler and single step process for the production of metal nanoparticles from metal salts [4]. The process can proceed at room temperature, can be easily scaled up and is environmentally friendly [4,8]. The biomolecules present in the biological sources having reducing properties that can reduce metal ions (M+) to zero-valent (M0) metals form and stabilize them [9]. The efficacy of biologically functionalized nanoparticles with a biologicalshell and metal-core is used for the treatment and diagnosis of various life threatening diseases [10,11]. They are reported to demonstrate bactericidal synergistic effect against various types of pathogenic microbes when used in combination with antibiotics [10]. These biosynthesized nanoparticles, capped or functionalized with various biomolecules are becoming important tool for developing next generation therapies and diagnosis [3]. Surface functionalization is possible due to the presence of various active groups (\\COOH, \\NH2, \\OH, etc.) of the capped phytochemicals. The nanoparticles synthesized by plant extracts might be conjugated with different bio-moieties like hormones, enzymes, antibody, proteins, antibiotics, etc. to achieve the targeted drug delivery. The plants of genus Potentilla have been used for a long time
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National Center for Cell Science (Pune, India). The bacterial strains and culture medium components were procured from Microbial Type Culture Collection Center, Institute of Microbial Technology, Chandigarh, India and M/S Himedia, Mumbai, India, respectively.
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2.2. Preparation of the plant extract
0.3
Various plant parts of P. fulgens were air-dried in shade and crushed to powder form. The roots (25 g) were dried and grounded to coarse powder. It was further macerated with methanol at 37 °C for 72 h. The extract was filtered and methanol was evaporated using a rotary vacuum evaporator (Buchi, Switzerland) to yield the extract.
0.2 0.1 0.0 300
2.3. Synthesis of silver nanoparticles
400
500
600
nm 700
Fig. 1. UV–vis absorption spectrum of silver nanoparticles produced by the root extract of Potentilla fulgens.
for the treatment of inflammation, wounds, microbial infections, diabetes mellitus and other human ailments [12–15]. Potentilla sps. contain various types of compounds such as polyphenols and flavonoids in the roots and rhizomes [14,16]. Recently the presence of stereoisomeric antioxidant compounds in the root extracts of Potentilla fulgens is also reported [17]. P. fulgens Wall. ex Hook. is a vital medicinal plant of Himalayan region and commonly known as Himalayan Cinquefoil [18]. P. fulgens root-stock and whole herb is used as astringent and tonic for curing gum and tooth ailments, diabetes, diarrhea and other varieties of diseases [12–16]. The aim of the present study was to synthesize the metallic nanoparticles by P. fulgens extracts and their applications as therapeutic and diagnostic agents. The nanoparticles were characterized using various techniques and therapeutic potentials were evaluated as anticancer and antimicrobial agents. Furthermore, the cell cycle analysis and trypan blue exclusion assays were performed on various types of cells to know apoptotic mechanism. 2. Materials and methods
Nanoparticles were synthesized following the previously reported method with a little modification [19]. To synthesize metallic nanoparticles, P. fulgens extracts were suspended in 50 mL deionized water, silver salt (1 mM) was added to the solution and reaction mixture was incubated at 35 °C (200 rpm) in dark. Reduction of metal ions was initially confirmed by visual inspection of color change and then by spectrophotometric absorption at 400–420 nm. Various reaction parameters (concentrations of plant extract and metal ions, pH, temperature and reaction time) were optimized to enhance the yield of nanoparticle. The nanoparticles were harvested by centrifugation (Eppendorf, Germany) to remove un-reacted phytochemicals from the reaction medium at 15,000 ×g for 30 min and the pellet was thoroughly washed with deionized water. The resulting suspension was dried in a lyophilizer (Allied Frost, New Delhi, India) and then further characterized. 2.4. Characterization of nanoparticles 2.4.1. UV–vis spectra analysis The UV–vis absorbance spectra were used for the preliminary identification of silver nanoparticle formation. An absorption band near 400–450 nm indicated the formation of nanoparticles [20]. The reduction of Ag+ in the reaction mixture was periodically monitored after 6 h of reaction, in a quartz cuvette with plant extract used as baseline. Sample absorbance was taken in the range of 300–700 nm using U2900 Hitachi spectrophotometer.
2.1. Chemicals P. fulgens roots were obtained from the Shillong Peak Forest area, Meghalaya, India. All of the reagents and solvents were procured either from Himedia laboratories or Rankem, Mumbai, India. Silver nitrate (AgNO3), concanavalin-A (Con-A), quercetin dihydrate, gallic acid and Folin–Ciocalteu reagents were purchased from Sigma-aldrich (Bangalore, India). Dulbecco's MEM, fetal bovine serum (FCS), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine were procured from Invitrogen—GIBCO, USA. MCF-7 (human breast cancer) and U-87 (human glioblastoma cancer) cell lines were purchased from the
2.4.2. Zeta-sizer and zeta potential analysis Dynamic light scattering (DLS) or zeta-sizer is a technique used to measure the size and size distribution of very small particles dispersed in a liquid. To estimate the particle size and zeta potential, a dilute suspension of nanoparticles was prepared in deionized water and sonicated (for removing aggregation) at 35 °C for 30 min and subjected to DLS analysis. The measurements were taken at 532 nm (35 °C) with a detection angle of 90°. The average particle size and zeta potential of nanoparticles were estimated by Zetasizer Nano ZS (Malvern Instruments, Malvern, UK).
Fig. 2. Transmission electron microscopy (TEM) micrograph of silver nanoparticles produced by the root extract of Potentilla fulgens.
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Fig. 3. (a) Energy dispersive X-ray spectroscopy (EDX) micrograph and (b) X-ray diffraction (XRD) spectra of silver nanoparticle synthesized by the root extract of Potentilla fulgens.
2.4.3. Electron microscopy analysis HR-TEM measurements were taken on a FEI HR-TEM instrument operated at an accelerating voltage at 100–200 keV. Samples were prepared by drop coating of colloidal solution onto carbon coated copper TEM grids. The drop on the TEM grids was allowed to leave for 2 min and the grid was allowed to dry. The synthesized silver nanoparticles were analyzed for their size. The elemental content of nanoparticle was determined using energy dispersive X-ray analysis (EDX) attached with TEM instrument.
65 °C under shaking condition (200 rpm). Similarly, reaction mixtures having initial pH in the range of 3 to 13 were incubated at optimized temperature under shaking condition (200 rpm) in dark. To optimize reaction time, in terms of yield and properties of nanoparticles, samples were collected at various time intervals (6, 12, 24, 36, 48 and 72 h) and subjected to characterization. The nanoparticle colloidal solution and their lyophilized form (nanopowder) were kept in dark condition at
2.4.4. FTIR analysis of Ag nanoparticles FTIR spectroscopy was performed to identify the functional groups capped on the surface of nanoparticles. The functional groups of the synthesized nanoparticles were identified using FTIR (Perkin Elmer) spectroscopy. The bio-reduced colloidal nanoparticle solution was centrifuged at 15,000 ×g for 30 min and the pellets were thoroughly washed with deionized water. The resulting suspension was completely dried in a freeze dryer and analyzed by FTIR spectroscopy. Plant extract was also dried in a freeze dryer and subjected to comparable FTIR analysis. 2.4.5. X-ray diffraction analysis X-ray diffraction (XRD) analysis, carried out on a XRD instrument operating at 45 kV and a current of 40 mA with CuKα radiation, was performed to determine the nature of nanoparticles. The instrument was operated over the 2θ range of 30–80° (Phillips, USA) [11]. 2.5. Optimization of physico-chemical parameters for the synthesis of nanoparticles Various physico-chemical parameters such as concentrations of plant extract and metal ions, pH of the reaction mixture, incubation temperature and reaction time were optimized to increase the yield of nanoparticles. Ratio of metal ions to reducing agent affects the rate of synthesis as well as the size, shape and yield of nanoparticles. The different amounts (1 to 200 mg) of plant extract were added in water (50 mL). Metal salt was added to these dilutions and reaction mixtures were incubated at 35 °C (200 rpm) in dark. The absorbance of the resultant solutions was measured by UV–vis spectrophotometer (Hitachi U2900) periodically. To optimize the metal ion concentration, various flasks were incubated at 35 °C (200 rpm) containing different concentrations of silver nitrate (0.5 to 5 mM) with a fixed concentration of plant extract. In order to have temperature optimization studies, reaction flasks were incubated at various temperatures ranging from 25 to
Fig. 4. Effect of various reaction parameters (concentrations of plant extract and metal ions, pH, temperature and reaction time) on silver nanoparticle synthesis by Potentilla fulgens extract.
A.K. Mittal et al. / Materials Science and Engineering C 53 (2015) 120–127 Table 1 Total phenolic and flavonoid contents of Potentilla fulgens silver nanoparticles and standard compounds. Phenolics/flavonoids
Content (%/100 mg standard)
Potentilla fulgens silver nanoparticles % (for phenolic content) Control gallic acid % (for phenolic content) Potentilla fulgens silver nanoparticles % (for flavonoid content) Control quercetin % (for flavonoid content)
7.7 ± 0.68 86 ± 0.23 30.604 ± 0.16 99 ± 0.43
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group, lymphocyte cultures were set up in RPMI 1640 medium supplemented with 10% heat inactivated FCS and stimulated with PHA using addition. Penicillin (100 U/mL) and streptomycin (100 mg/mL) and 2 mM L-glutamine were also added to the medium. In another group, lymphocytes were kept in RPMI 1640 medium with all the supplements without PHA. After 6 h, lymphocytes of both the groups were treated with AgNPs. The cells were incubated at 37 °C for 24 h and harvested. The study was performed with the compliance of the “Ethical Guidelines for Biomedical Research on Human Subjects” formulated by the Indian Council of Medical Research, India. 2.8. Cytotoxicity assay
the room temperature for a year to check their stability. The stability of the particles was periodically checked by UV–vis spectral analysis. 2.6. Determination of total phenolics and flavonoid content A sample of nanoparticle (20 μL, 50–500 μg/mL) was mixed with deionized water (1.58 mL) followed by the addition of 2 N Folin– Ciocalteu's phenol reagent. The resulting mixture was vortexed and incubated at room temperature for 8 min followed by the addition of aqueous sodium carbonate solution (20%, 300 μL). The resultant mixture was vortexed again and kept at room temperature for 2 h. Absorbance of the blue-colored solution was recorded at 725 nm [21,22]. The gallic acid was used as a standard for the calculation of total phenolic content and expressed as gallic acid equivalents (GAE %). Total flavonoid content was determined using colorimetric aluminium chloride method and quercetin dihydrate was used as a standard. The samples and standards were individually dissolved in water. The sample solution (100 μL) was then mixed with an aqueous solution of anhydrous aluminium chloride (2%, 100 μL). After 10 min incubation at room temperature the absorbance of the supernatant was measured at 435 nm. The absorbances of samples in 96 well plates were taken on a multi-well micro-plate reader (Multiskan EX; Thermo Scientific, USA). The total flavonoid content was expressed in quercetin dihydrate equivalents. All the experiments were performed in triplicate [21,22]. 2.7. Cell line cultivation and treatment MCF-7 and U-87 cells were grown in Dulbecco's MEM high glucose medium and in Dulbecco's MEM low glucose medium, respectively. Both the media were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine. Freshly heparinized peripheral blood was collected from two healthy male donors (HPBL) and peripheral blood mononuclear cells were isolated by Ficoll-Hypaque (Sigma Diagnostics, St. Louis, MO) density gradient centrifugation (specific gravity 1.077 g/mL) for 30 min at 400 ×g. Lymphocytes were divided into two groups. In one
(a)
The cytotoxicity and cell viability was calculated using a clonogenic cell survival assay in MCF-7 and U-87 cancer cell lines. This method for measuring cytotoxicity using colony-forming assay is considered as the most reliable method [17]. Cells were plated in triplicate at a cell density of 1000 cells onto 25 cm2 tissue culture flask while for the untreated controls; four flasks were plated at a cell density of 500 cells per culture flask. 5 h after the plating, cells were exposed for 24 h with 2 to 12 μg/mL of the Potentilla root extract-silver nanoparticles or 0.2 to 1 μg/mL of only silver nanoparticles. Cells were washed twice with the DMEM medium and finally flasks were incubated in a CO2 incubator with DMEM containing 10% fetal calf serum for 8 to 10 days. The colonies were fixed and stained with 0.2% crystal violet in 70% ethanol. Colonies with a minimum of 50 cells were counted as the progeny of a viable cell. All survival points were checked in triplicate and experiments were repeated three times. Results were expressed as surviving fraction. The plating efficiency (PE) and survival fraction (SF) were calculated as follows: Plating efficiencyðPEÞ% ¼ ðNo: of colonies formed=No: of cells seededÞ 100 Survival fractionðSF Þ% ¼ ðPE of treated=PE of controlÞ 100:
2.9. Trypan blue exclusion assay Cytotoxicity of synthesized nanoparticles was determined by trypan blue (TB) exclusion assay also. Various types of cells were incubated with AgNPs for 24 h and washed with DMEM. Finally, the cells were incubated for 10 min at room temperature with 0.4% trypan blue dye solution. The trypan blue dye stains dead cells for blue color, while the live cells remain unstained. 2.10. Flow-cytometric analysis of cells Various types of cells were grown in a respective culture medium, treated for 24 h with and without AgNPs and fixed with 70% ethanol. In case of MCF-7 and U-87 cells, cells were fixed with 70% ethanol
(b)
Fig. 5. Dose dependent reduction in the cloning efficiency of (a) MCF-7 and (b) U-87 cancer cell lines treated with synthesized silver nanoparticles.
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Table 2 Trypan blue exclusion assay of various normal/cancer cells treated with Potentilla fulgens silver nanoparticles. Cell type
Percentage of dead cells ± S.D.
Primary normal cell lines Untreated lymphocytes (PHA−) AgNPs treated lymphocytes (PHA−) Untreated lymphocytes (PHA+) AgNPs treated lymphocytes (PHA+)
9.4 ± 1.0 17.8 ± 0.8⁎ p = 0.001 6.4 ± 0.8 19.7 ± 0.8⁎ p = 0.004
Cancer cell lines Untreated MCF-7 cells AgNPs treated MCF-7 cells Untreated U87 cells AgNPs treated U87 cells
8.5 ± 0.9 42.4 ± 2.4⁎p = 0.0007 6.7 ± 0.9 40.6 ± 2.6⁎p = 0.002
⁎ Student's t-test with respect to respective control value.
soon after 24 h treatment while lymphocytes were fixed with ethanol after additional 6 h of stimulation period. The fixed cells were washed with PBS and resuspended in 500 μL propidium iodide solution (50 μg/mL propidium iodide, 0.2 mg/mL RNase) and incubated for 1 h at room temperature in dark. A large number of cells (10,000) were acquired for each sample and analyzed with FACS Calibur (Becton-Dickinson) instrument. CELLQ-uest Pro-software was used to quantify cell cycle compartments to estimate the percentage of cells distributed in the different phases of cell cycle.
2.11. Antibacterial assays In vitro antibacterial activities were evaluated using Escherichia coli and Bacillus subtilis cells as susceptible test organisms using broth microdilution and agar well diffusion methods [8,23]. Bacterial suspension equivalent to 0.5 McFarland's standard was pipetted into a 96 well plate at 37 °C and treated with a varied concentration of nanoparticles (25, 50 and 100 μg/mL), after 24 h incubation at 37 °C, the optical density was recorded using automated microplate reader (Multiskan EX; Thermo Scientific, USA) at 595 nm. The standard antibiotic (chloramphenicol) and silver nitrate were used as positive controls and cells without any treatment were used as negative control. All the experiments were performed in triplicate. For agar well diffusion assay, the fresh overnight cultures (100 μL each) of E. coli and B. subtilis, approximately 108 CFU/mL were lawned onto MH agar plates. The wells in the plates were made by cork borer and filled with 50 μL nanoparticle solution having its varied concentration (25, 50 and 100 μg/mL). The other wells consisting each of 100 μg/mL chloramphenicol and silver nitrate were used as positive controls and plant extract as negative control. The plates were incubated overnight (24 to 36 h) at 37 °C. The diameter of zone of inhibition around the well was observed and recorded. All experiments were conducted in triplicate and average inhibitory zone diameters were measured.
3. Results and discussion Initially the synthesized nanoparticles were identified by observing the color change of the reaction mixture as reported [24] earlier. After the screening of plant extracts from the various parts of P. fulgens, only the root extract was capable to reduce the silver ions to silver nanoparticles. Before the reaction, both the AgNO3 and plant extract solution were colorless and after the reaction, the yellowish brown color was clearly observed (Figure S-1 in the electronic supplementary material). The color change in the reaction mixture indicated the formation of nanoparticles. The reaction color was due to the excitation of surface plasmon vibrations (essentially the vibration of the group conducting electrons) in the colloidal solution of nanoparticles [19].
3.1. Characterization of nanoparticles UV–vis absorbance spectra were used for the preliminary characterization of the nanoparticle synthesis. Fig. 1 shows the UV–vis spectra of synthesized nanoparticles using P. fulgens root extract. Using DLS, the average hydrodynamic size and zeta potential of silver nanoparticles in aqueous solution was found to be 39.04 nm and −18 mV, respectively. PDI of 0.25 indicates the good mono-dispersity and -ve zeta potential indicates good stability with capping by negatively charged groups of nanoparticles (Figure S-2 in the electronic supplementary material). Fig. 2 shows the TEM micrograph of the silver nanoparticles synthesized by P. fulgens root extract indicating the spherical shape of the particles in the order of 10–15 nm. The revealing of strong signal by EDX confirmed the presence of silver nanoparticles. The arrival of another hafnium peak was due to the carbon coated cooper grid used for this analysis (Fig. 3a). Other elemental signals recorded were possibly due to elements from phytochemicals or proteins present in the plant extract. FTIR spectra were analyzed to investigate the possible reducing functional groups present in the P. fulgens root extract. Figure S-3 (In the electronic supplementary material) shows the FTIR spectra overlay of plant extract and synthesized silver nanoparticles. The nanoparticles capped with phytochemicals of P. fulgens showed the IR peaks at 3366, 1653, 1384 and 744 cm−1 and plant extract showed the IR peaks at 3365, 1614, 1519, 1448, 1354, 1235, 1144, 1063 and 822 cm−1 regions. The FTIR spectrum of nanoparticles showed the involvement of O\\H stretching, involvement of C_N in plane vibrations of amino acids, corresponding to amides I, II and III, aromatic rings and ether linkages were found commonly present in the nanoparticles synthesized by root extract of P. fulgens [25]. These representative IR peaks suggested the presence of flavonoid, phenolics and terpenoids capped on nanoparticles. The XRD pattern of silver nanoparticles illustrated that the synthesized nanoparticles are crystalline in nature (Fig. 3b). There are four intense peaks in the whole spectrum of 2θ values ranging from 24 to 80° at 38, 44, 65 and 78°, the pattern of reflection according to Bragg's equation of silver nanoparticles showing crystalline nature of nanomaterials [26]. These peaks of nanoparticles were matched with JCPDS databases of the standard silver (file No. 04-0783). It confirms that the resultant particles are face centered cubic (FCC) in shape. 3.2. Optimization of nanoparticle synthesis The nanoparticles were primarily characterized by UV–vis spectroscopy. Based on this, various physico-chemical parameters (concentrations of plant extract and metal ions, reaction temperature, pH and reaction time) were optimized to enhance the yield of nanoparticle synthesis (Fig. 4). The reaction mixture containing 4 mg extract in 50 mL water was able to produce the maximum concentration of silver nanoparticles. Peak with 4 mg root extract was sharp as compared to other peaks, indicating the formation of relatively better sized nanoparticles. Effect of AgNO3 concentration on the synthesis of silver nanoparticles (AgNPs) was studied by varying its concentration from 0.5 to 5 mM using 4 mg root extract. The yield of silver nanoparticles increased with the increase of metal salt concentration from 0.5 to 1 mM, beyond which, there was again a fall in the absorbance. The synthesis of nanoparticles to the desired size and shape by optimizing the plant extract and metal ions is also reported in literature [27–29]. Increase in the absorbance of reaction mixture with the increase in incubation temperature evidently depicted the higher rate of synthesis of nanoparticles at elevated temperatures. Moreover, nanoparticles synthesized at higher temperatures exhibited surface plasmon resonance at narrow absorption range indicating monodispersity. At higher temperature, the rate of formation of smaller size nanoparticles increased. The maximum absorbance was obtained at 45 °C (Fig. 4). It may be due to the reduction of aggregation in the growing nanoparticles at that
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Fig. 6. Cell cycle analysis of MCF-7, U87, PHA-stimulated and unstimulated human lymphocyte cells using flow cytometry when cells were treated with Potentilla fulgens silver nanoparticles.
temperature and further increasing the temperature increased the crystal formation around the nucleus which caused the reduction in absorption. Similar results have been reported by Kaviya et al. [30]. Effect of pH on the synthesis of nanoparticles by the root extract and organic compounds was examined over a wide range of pH staring from pH 3 to 13 (Fig. 4). At acidic pH, larger size nanoparticles were formed, whereas at alkaline pH, smaller size nanoparticles were observed. This was concluded on the basis of UV–vis absorption spectra (narrow or broad peak). Effect of reaction pH on the synthesis and stabilization of metal nanoparticles as well as on their size and shape was also reported by various investigators [31,32]. Nanoparticle aggregation seems to outdo the nucleation process in acidic condition, whereas at alkaline pH, the large number of nuclei formation, instead of aggregation, led to the synthesis of higher amount of nanoparticles with smaller diameter. Blue shift in the absorption pattern confirmed the formation of relatively smaller size nanoparticles. Gericke and Pinches reported the various manipulation methods to control the size and shape of gold nanoparticles synthesized by Verticillium luteoalbum [33]. They found that aggregation of NPs was observed after 36 h reaction, which is a sign of instability. The optimum time required for the synthesis of maximum nanoparticles by P. fulgens was found to be 18 h. The stability results showed no alteration in the absorption peak at 420 nm up to 60 days, indicating good stability of synthesized silver nanoparticles. Lyophilized form of nanoparticle powder remained stable more than a year when stored at room temperature. The total phenolic content on nanoparticles was determined by Folin– Ciocalteu's method using gallic acid (GA) as a standard. It demonstrated lower quantities (7.7 ± 0.68% of GA/100 mg) of phenolics capping on nanoparticles (Table 1). The total flavonoid content in nanoparticle samples was expressed as quercetin dihydrate (QD) equivalent and it was found to be 30.604 ± 0.16% of QD/100 mg (Table 1). The flavonoid content in P. fulgens mediated nanoparticles was four-fold higher than phenolic, indicating the role of flavonoids in the nanoparticle stabilization. These assays are usually used for
the screening of antioxidant properties of food products and dietary supplements [34]. 3.3. Evaluation of cell survival, cell death and cell cycle analysis A dose-dependent reduction in the cloning efficiency was observed on Potentilla extract-silver nanoparticle-treated MCF-7 and U-87 cell lines. However, the degree of reduction in cloning efficiencies was negligible when only silver ion treatment was used. The minimum inhibitory concentration (IC50 ) values, calculated from the sigmoidal graph, were 4.91 and 8.23 μg/mL for MCF-7 and U87 cell lines, respectively (Fig. 5). The IC 50 values of the various plant extract fractions of P. fulgens were reported to be in the range of 45 to 75 μg/mL [17]. The lower IC50 values indicate the possibility of silver nanoparticles (AgNPs) to be a potent anticancer agent. Trypan blue exclusion assay concluded that the frequencies of dead cells are higher, when treated with AgNPs (Table 2). It was also found that the frequencies of dead cells are significantly higher in cancer cells than the normal primary cells. The trypan blue staining studies confirmed that AgNPs at 6 μg/ml killed more cancer cells than the normal cells. The data shown in Table 2 indicates the exclusion data of various cells, when treated with AgNPs. The synthesized AgNPs killed both types of human lymphocytes, however, the extent of killing was more for the cancer cells. Flow cytometric analysis of cancer cells after the treatment with nanoparticles showed the decline of G1 phase and increase of sub-G1 phase as shown in Fig. 6. However, for human lymphocytes, increase frequency of sub-G1 was marginal on nanoparticletreated samples. This observation was unswerving with the data observed in clonogenic and trypan blue staining assays. The FACS analysis showed the increase in percentage of cells in sub-G1 phase in nanoparticle-treated cancer cells. The observed growth inhibition of these cell lines in clonogenic-assay studies might have resulted from the cell death. The flow cytometric analysis
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demonstrated a significant rise in the number of cells in sub-G1 after the treatment indicating cell death which could be due to apoptosis.
Similarly, Abdel-Raouf et al. studied the antibacterial activity of AuNPs synthesized by Gara elongata ethanolic extract with the maximum zones of inhibition (16–17 mm) against E. coli, Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus, followed by S. aureus and Pseudomonas aeruginosa (13 mm) [36].
3.4. Evaluation of antibacterial activity To evaluate the effect of silver nanoparticles on the bacterial growth, the growth inhibition was measured both by broth microdilution and zone of inhibition methods. In broth microdilution method, 30–40% growth inhibition was observed with 25 μg/mL silver nanoparticles and it was more (50–70%) when its concentration was increased to 100 μg/mL (Fig. 7). Control experiment (chloramphenicol) showed 60% inhibition at 50 μg/mL level while it was 40% with silver nitrate. It was found that silver nanoparticles at 45 μg/mL level were very effective as bacteriostatic, where bacterial growth showed insignificant optical absorption. Similarly, Saxena et al. synthesized silver nanoparticles using Ficus benghalensis leaf extract and its bactericidal properties against E. coli were evaluated by broth microdilution method [35]. The antibacterial effect of silver nanoparticles at 100 μg/mL was measured on the basis of the zone of inhibition and compared with negative and positive controls (Table 3 and Figure S-4 in the electronic supplementary material). The antimicrobial activity of synthesized silver nanoparticles against B. subtilis is higher than that of E. coli. It might be due to the variation in the cell wall composition of Gram-positive and Gram-negative bacteria. The nanoparticles showed the maximum bactericidal effect against B. subtilis with a zone of inhibition of 9.7 ± 0.6 mm. The effect of nanoparticles with standard antibiotics (chloramphenicol) and silver salt showed comparable antimicrobial activity. It was found that the silver nanoparticles and silver nitrate showed similar bactericidal activity against the selected bacterial strains. Silver salt is highly toxic and not bio-compatible, however, the nanoparticles synthesized by P. fulgens are highly biocompatible due to the capping of nanoparticles by phytochemicals. At the same concentration, plant extract did not show any activity against selected strains. These studies have shown that silver nanoparticle formulations have good antibacterial activity against Gram-positive and Gram-negative microbes.
4. Conclusions he reduction potential of root extract of the P. fulgens was explored for the synthesis of silver nanoparticles. The nanoparticles synthesized using P. fulgens showed the activity against various cancer cell lines and bacterial species. Capping of plant constituents on the surface of nanoparticles might be one of the reasons for this attribute. The approach can also be used for the large-scale synthesis of nanoparticles from other inorganic materials. The green process of synthesizing metal nanoparticles is always preferred and it needs to be scaled up in terms of quality and types of biocatalysts. The effect of environmental parameters (pH, temperature, concentrations of catalyst and metal salts etc.) on the reaction conditions needs to be studied more. In other words, the synthesis of metal nanoparticles by plant extracts needs to be thoroughly optimized so that nanoparticles of desired quality in terms of size, shape and monodispersity, can be produced. It is especially suited for making nanoparticles free of toxic contaminants as required in therapeutic applications. The properties of nanoparticles are dependent on size, shape and their synthesis methods. Applications in targeted drug delivery and clinical diagnostics are required in future. The environmental biocompatibility and the specific cytotoxicity against tumor cells make them a powerful tool in the field of nanomedicine. Furthermore, a lot of further studies are needed to be done to obtain additional clear evidences of nanoparticle-mediated cell apoptosis in cancer cells. In medicine, silver nanoparticles are being used as antimicrobial agents in bandages. Application of such eco-friendly nanoparticles in bactericidal, wound healing, medical and electronic fields makes this method potentially stimulating for large-scale synthesis of other metallic nanomaterials.
Fig. 7. Growth inhibition of microbial strains (Escherichia coli and Bacillus subtilis) in the presence of synthesized nanoparticles.
A.K. Mittal et al. / Materials Science and Engineering C 53 (2015) 120–127 Table 3 Antimicrobial activity of Potentilla fulgens silver nanoparticles against different microbial strains. The activity was calculated in terms of zone of incubation (mm). Escherichia coli (MTCC 433) and Bacillus subtilis (MTCC 441) were incubated with silver nanoparticles, silver nitrate, plant extract and standard chloramphenicol. Bacterial strains
Zone of inhibition (mm) a
Zone of inhibition (mm) b
Zone of inhibition (mm) c
E. coli MTCC 433 B. subtilis MTCC 441
9.5 ± 0.2 9.7 ± 0.6
7.2 ± 0.6 7.6 ± 0.4
18.2 ± 0.7 20.8 ± 0.5
a b c
P. fulgens silver nanoparticles; Silver nitrate. Standard drug chloramphenicol at 100 μg/mL concentration.
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