Synthesis and characterization of silver nanoparticles using Caesalpinia pulcherrima flower extract and assessment of their in vitro antimicrobial, antioxidant, cytotoxic, and genotoxic activities

Abstract

Caesalpinia pulcherrima flower extract mediated synthesis of silver nanoparticles was attempted in the present work including optimization of some procedure parameters. Characterization of synthesized silver nanoparticles was done by various spectral analyses. The size of synthesized silver nanoparticles was 12 nm and they were spherical in shape. The green synthesized silver nanoparticles were further evaluated for antimicrobial, antioxidant, cytotoxic, and genotoxic activities; they showed good antimicrobial, antioxidant, and cytotoxic effects. Genotoxic study revealed non-toxic nature at lower concentration. Overall results suggest that the synthesized silver nanoparticles have pronounced applicability in pharmaceutical and biomedical field.

Keywords:

• Silver nanoparticles
• characterization
• antimicrobial activity
• antioxidant activity
• cytotoxicity
• genotoxicity

Introduction

Nanotechnology is a novel area of science that combines biology, chemistry, and physics. Nanoparticles possess more surface atoms than microparticles which enhance their functional capabilities. Different types of metals such as Ag, Au, Pt, Zn, Cu, and Pd have been used for the synthesis of nanoparticles. Metal nanoparticles have multifunctional properties and very interesting applications in various fields such as medicine, catalysis, sensors, optoelectronics, food industries, agriculture, and textile industry. Nanoparticles have unique size-dependent crystalline structures (1–100 nm), greater surface to volume ratios, drug encapsulation, surface structure, and biocompatibility due to their material composition. Among the several noble metal nanoparticles, silver nanoparticles (AgNPs) have attained special focus because of their unique distinctive properties, like good chemical stability, surface plasmon resonance properties, and electrical conductivity and they have wide application in pharmaceutical, industrial, and medical applications (Chanda 2013).

Antibiotic resistance and various micro-organisms infection has become a major bottleneck in public healthcare sectors. Almost every variant of microorganism has developed resistance to antibiotics mainly due to gene transfer. Another problem is oxidative damage created by free radical generation, is a critical factor implicated in several chronic human diseases such as cancer, atherosclerosis, arthritis, and neurodegenerative diseases. Cancer is one of the most common leading causes of mortality worldwide. Chemotherapy and radiation therapy are available for the treatment of cancer but they are not free from adverse effects. Nanomaterials are expected hopefully to revolutionize and prevent the spread of infections and various cancers.

The nanoparticles can be prepared by physical, chemical, and biological methods. Physical and chemical methods have low yields and the chemical methods cause contamination due to precursor chemicals, the use of toxic solvents, and the generation of hazardous by products (Wang et al. 2007). The most preferred method is synthesis using biological entities like bacteria, yeast, fungi, and plants; however, plant extracts mediated synthesis is potentially advantageous over microorganisms due to the ease of scale up, safe, simple, and environment friendly. Plant extracts have a broad variety of metabolites that can be used in the reduction of silver ions, capping and stabilizing agent for the nanoparticles synthesis (Moteriya et al. 2016).

On one hand, flowers are cheap, readily available, and are generally used in garlands for social and religious purposes in most of the countries; however, they generate huge amount of waste daily. On the other hand, plant waste is a “biofactory” of different organic compounds such as polyphenols, flavonoids, carotenoids and vitamins. Caesalpinia pulcherrima (CP) is an ornamental plant belonging to Caesalpiniaceae family. Caesalpinia pulcherrima flower is known for is antiviral, antimicrobial, antioxidant, analgesic, anti inflammatory, anthelmintic activities (Chiang et al. 2003; Dhaked et al. 2011; Patel et al. 2010; Pawar et al. 2009; Sudhakar et al. 2006).

In the present study, a novel, one-step biosynthesis of AgNPs using the flower extract of C. pulcherrima at room temperature is reported perhaps for the first time. Synthesized AgNPs were characterized using various spectral analyses like ultraviolet–visible (UV–Vis) spectroscopy, Fourier Transform Infrared spectroscopy (FTIR), Thermogravimetric analysis (TGA), X-ray diffraction analysis (XRD), and Transmission Electron Microscopy (TEM). In addition, an attempt was made to evaluate its antimicrobial, antioxidant, and anticancer potential by elucidating cytotoxic effects on HeLa cells and genotoxicity on normal human peripheral blood lymphocyte culture.

Materials and methods

Chemicals

The fresh flowers of Caesalpinia pulcherrima (CP) DC were collected from Saurashtra University campus, Rajkot, Gujarat, India. All the chemicals were obtained from Hi Media Laboratories and Sisco Research Laboratories Pvt. Limited, Mumbai, India. Ultra purified water was used for all the experiments.

Preparation of the extract

Fresh flowers of C. pulcherrima were thoroughly washed with tap water, followed by double distilled water, and cut into small pieces. About 5 g of cut flowers were boiled in 100 ml ultra pure water and filtered through Whatmann No. 1 filter paper. The filtrate was cooled to room temperature and used as bioreductant for the synthesis of AgNPs.

Optimization of different parameters

The filtered C. pulcherrima flower extract was used for the synthesis of AgNPs. The synthesis of AgNPs was initially confirmed by UV–Vis absorption spectrophotometer. The reduction of the Ag⁺ions in solution was monitored by UV–Vis spectrophotometer (Shimadzu UV-1601, Shimadzu Corporation, Kyoto, Japan) in 350–700 nm range operated at an interval of 10 nm. Influence of various parameters such as boiling time of plant extract (5 min, 10 min, and 15 min), extract amount (1.5 ml, 3 ml, 6 ml, 9 ml, and 12 ml), concentration of silver nitrate (0.5 mM, 1 mM, 1.5 mM, and 2 mM), pH (6–10), and incubating the reaction mixtures with optimum composition (0 min–24 h).

Characterization of the synthesized silver nanoparticles

FTIR analysis

Possible functional groups involved in the synthesis and stabilization of AgNPs was studied by FTIR spectroscopy. The FTIR spectrum was recorded in the range of 400–4000 cm⁻¹ Nicolet IS10 (Thermo Scientific, Waltham, MA). Various modes of vibrations were identified and assigned to determine the different functional groups present in the C. pulcherrima flower extract.

XRD analysis

The structure and composition of synthesizd AgNPs was analyzed by XRD. The formation of AgNPs was determined by an X’Pert Pro X-ray diffractometer (PAN analytical BV) operated at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation in θ–2θ configurations. The crystallite domain size was calculated from the width of the XRD peaks, assuming that they are free from non-uniform strains, using the Scherrer formula. D = 0.94 λ/β cos θ, where D is the average crystallite domain size perpendicular to the reflecting planes, λ is the X-ray wavelength, β is the full width at half maximum (FWHM), and θ is the diffraction angle.

Thermogravimetric analysis

Thermal stability and surface weight loss of AgNPs was analyzed by DTG-60H (Shimadzu Corporation, Kyoto, Japan). Instruments, under a nitrogen gas flow of 100.0 ml min⁻¹ and heating rate of 10 ∘C min⁻¹ to 1000 ∘C.

TEM analysis

TEM analysis was done to visualize the shape as well as to measure the diameter of the biologically synthesized AgNPs. The sample was dispersed in double distilled water. A drop of thin dispersion was placed on a “staining mat”. Carbon-coated copper grid was inserted into the drop with the coated side upwards. After about 10 min, the grid was removed and air dried. Then screened in JEOL JEM 2100 Transmission Electron Microscope (JOEL Corp, Tokyo, Japan).

Antimicrobial activity

Antimicrobial activity of AgNPs was tested by resazurin microtiter (REMA) plate method with some modifications (Palomino et al. 2002) against four Gram-positive bacteria (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, and Corallium rubrum) and four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, and Klebsiella pneumoniae) and three fungi (Candida albicans, Candida glabrata, and Cryptococcus neoformans). The inoculums of the test micro-organism were prepared using the colony suspension method (EUCAST 2003). Ninety-six-well culture plates (Tarsons Products Pvt. Ltd., Kolkata, India) were used, and serial two-fold dilutions of the nanoparticles (10–0.019 mg/ml) were dispensed into the plate wells. Two-fold dilutions of Chloramphenicol and Amphotericin B (10–0.019 mg/ml) were used as a positive control. The volume of dispensing nanoparticles was 20 μl per well along with 150 μl of Mueller Hinton Broth (MHB)/Sabouraud Dextrose Broth (SDB) for bacteria and fungi. About 30 μl of microbial culture at a density of 6 × 10⁵ CFU/ml was added to the wells. Three control wells were maintained for each test batch: the positive control (antibiotic, MHB/SDB and test organism), sterility control (MHB/SDB and water), and negative control (MHB/SDB, test organism, and water). The plates were incubated at 37 °C and 28 °C for 24 h and 48 h for bacteria and fungi, respectively. The microbial activity in the test wells was detected by adding 50 μl of 0.01% w/v of Resazurin (HiMedia, Mumbai, India) solution dissolved in sterile distilled water to each well. The plates were incubated for further 2 h, and estimated visually for any change in blue to pink indicating reduction of the dye due to bacterial growth. The lowest concentration (highest dilution) of the nanoparticles required to inhibit visible growth of the tested microorganism was designated as the MIC. The experiment was done in triplicate and the average values were calculated for minimum inhibitory concentration value. Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were determined from all wells showing no growth as well as from the lowest concentration showing MIC assay for all the samples. Bacterial or fungal cells from the MIC plates were sub-cultured on freshly prepared solid nutrient agar plates by making streaks on the surface of agar. The plates were incubated at 37 °C and 28 °C for 24 h and 48 h for bacteria and fungi, respectively. Plates that did not show growth were considered to be the MBC/MFC for AgNPs used (Akinyemi et al. 2005).

Antioxidant activity

The antioxidant activity of C. pulcherrima flower extract synthesized AgNPs was measured by five in vitro antioxidant assays. The antioxidant assays evaluated were 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay (DPPH), superoxide anion radical scavenging assay (SO), 2,2′-Azino-bis-(3-ethyl) benzothiazoline-6-sulfonic acid radical cation scavenging assay (ABTS), reducing capacity assessment (RCA), and ferric reducing antioxidant power assay (FRAP). The details of the method followed are as described earlier (Kaneria et al. 2012).

In vitro cytotoxic activity

Cancer cell viability of the AgNPs was evaluated by the MTT assay (Labieniec and Gabryelak 2003). About 150 μl of HeLa cells were seeded in 96-well plates (Becton Dickinson (BD), Franklin Lakes, NJ) at a density of 1.1 × 10⁴ viable cells/well (HeLa) and incubated 24 h to allow cell attachment. Following attachment, the medium was replaced with complete medium (150 μl/well) containing AgNPs (concentrations ranging from 2 to 200 μg/ml) for 72 h. Following treatment, the cells were washed with PBS and incubated with 100 μl/well fresh medium containing 0.5 mg/ml MTT. The MTT-containing medium was removed after 3 h incubation in dark condition. The MTT formazan was dissolved in 100 μl/well DMSO and the optical density was determined at 570 nm using an ELISA plate reader (Bio-Tek, Winooski, VT). Cell viability was calculated by the following equation: $\mathrm{Cell}\hspace{1em}\mathrm{viability}\hspace{1em}\left(\%\right)=(A_S/A_{\mathrm{Control}})\times 100$ where A_s is the absorbance of the cells incubated with the AgNP_S and A_control is the absorbance of the cells incubated with the culture medium only.

Comet assay (single cell gel electrophoresis)

The genotoxicity of AgNPS was determined using comet assay, based on the measurement of DNA migration under electrophoresis as per slightly modified technique given by Singh et al. (1988). In brief, 180 μl of 1% normal melting point agarose (NMP) was gelled on fully frosted slide (75 × 25 mm) and 100 μl 0.5% low melting point agarose (LMP)-containing cell suspension (20 μL) was layered on the top of the NMP agarose. After formation of cell suspension containing layer, additional 100 μl of LMP agarose was added to fill the residual hole and to form an additional layer to increase the distance between the cells and the gel surface. After agarose gel solidification, the slides were placed in a lysis solution [2.5 M NaCl, 100 mM EDTA and 10 mM Tris HCl (pH 10), plus 1% Triton X-100 and 10% DMSO were added just before use] for 24 h at 4 °C, incubated in an alkaline electrophoresis buffer (300 mM NaOH/1 mM EDTA, pH >13) for 40 min and electrophoresed (Midi-Submarine electrophoresis Unit-7050, Tarsons, Kolkata, India) for 35 min. After the comets were formed, the alkaline gel was neutralized by rinsing the slides with a suitable buffer (0.4 M Tris, pH 7.5) three times. The slides were then stained with the fluorescent dye SYBR Green I (SG) and the comet images were captured under fluorescence microscope. The proportion of DNA damaged was scrutinized by scoring 100 comets for each group (Tri-Tek Comet-Score™ V1.5 software, Tri-Tek, Bremen Germany).

Chromosomal aberration assay

Lymphocyte cultures were setup by Hungerford (1965) with slight modifications. Heparinized whole blood (0.5 ml) was added to mixture containing 5 ml of culture medium RPMI 1640, 0.1 ml phytohemagglutinin (Lectin), and 0.05 ml heparin. Then the culture vials were put in incubator for 72 h, at 37 °C. Doses of AgNPs (2 μg/ml, 50 μg/ml, and 200 μg/ml) positive controls and internal controls were exposed/added at 72th h of incubation. After exposure, vials were put in incubator for 96 h. Then 0.1 ml demecolcine solution was added at last 2 h of incubation period to arrest cells at metaphase. The cells were collected by centrifugation, resuspended in a pre-warmed hypotonic solution (KCl, 0.075 M) for 20–25 min and fixed in chilled methanol/acetic acid (3:1, v/v) solution (Carnoy’s fixative). Suspensions of cells were prepared after several changes of carnoy’s fixative washes. Using the cell suspension slides were prepared and air dried on a hot plate at 50–60 °C. After air dry preparation, slides were stained with 2% Giemsa’s stain for 10 min. From each slide, 100-well spread metaphase chromosomes were scored for chromosome and chromatid type of aberrations.

Results and discussion

Optimization of different parameters

Optimization of various procedure parameters is essential in order to achieve optimum conditions for AgNPs formation. It will yield smaller particles and best activity as also reported by Ibrahim (2015). UV–Vis spectroscopy was used to monitor the formation of nanoparticles during the reaction. When the plant extract is added to silver nitrate solution, there is a color change from pink to dark brown indicating the formation of AgNPs in solution. They exhibit brown color in aqueous media owing to SPR bands of synthesized AgNPs (Moteriya and Chanda 2014a). The characteristic absorption maxima are invisible region in the range of 350–700 nm.

It is well known that broader absorption peak indicates larger particle size and narrow absorption peak indicates smaller particle size (Jin et al. 2001); while the absorption intensity indicates the number of nanoparticles formed (Ahmad et al. 2013). This criterion was used for the selection of correct parameter for the synthesis of AgNPs. The parameters optimized were effect of boiling time of plant extract preparation, plant extract concentration, silver ion concentration, pH of the reaction medium, and time of synthesis reaction. The results are presented in Figure 1. In general, the absorption peak width and intensity changed when the level of variable changed. A narrow peak with greater intensity was observed when plant extract was boiled for 5 min. Increasing the time of boiling leads to lower and broader absorption peak (Figure 1(A)). Similar results are reported by Moteriya and Chanda (2014b) for Cassia roxburghii stem-mediated synthesis of AgNPs.

Figure 1. UV–Vis spectroscopy shows the effects of variuos parameters for efficient of AgNps: (A) effect of boiling time, (B) effect of extra amount, (C) effect of silver nitrate concentration, (D) effect of pH, and (E) different incubation times of reaction mixture.

Increasing the amount of plant extract concentration from 1.5 ml to 12 ml, a steady increase in absorption peak was observed after which no change was found (Figure 1(B)). When the plant extract concentration is increased, the biomolecules involved in the reduction of silver ions to silver nanoparticles are more available which results in more AgNPs formation (Ibrahim 2015). Further, Kathiravan et al. (2014) reported that particle size decreased with increased plant extract.

On one hand, the silver ion concentration also had a tremendous effect on AgNPs formation. At 0.5 mM concentration, no particles were formed as evidenced by its absorption peak (no peak was found). On the other hand, at 1 mM silver ion concentration AgNPs formation was rapid and a maximum absorption peak was found at 410 nm (Figure 1(C)). At higher concentration (1.5 mM and 2.0 mM), the absorption peak broadened indicating larger particle size formation (Valodkar et al. 2011). Metal ion concentration also influences the AgNPs formation (Vimala et al. 2015).

pH of the reaction medium also affected the AgNPs formation. Both acidic pH (pH 6 and pH 7) induced less number and larger size AgNPs as evidenced by its broader absorption peak. Alkaline pH produced narrow peak with greater intensity but best was with pH 8 (Figure 1(D)). In acidic pH, the biomolecules responsible for AgNPs formation may be inactivated (Roopan et al. 2013). Generally, alkaline pH is utilized for AgNPs formation (Mittal et al. 2014).

Increasing the synthesis reaction time from 30 min to 24 h caused a steady increase in the number of AgNPs formed (Figure 1(E)). After 24 h, no change in absorption peak was observed. In optimal condition, the absorption maxima peak was observed at 410 nm which is due to surface plasmon vibrations. The AgNPs synthesized using C. haematocephala extract showed an absorption peak at 414 nm (De Aragão et al. 2016).

Thus, it was evident that all the parameters definitely contributed in AgNPs formation. Therefore, optimum conditions for green synthesis of AgNPs by C. pulcherrima flower extract was 5 min boiling time for flower extract preparation, 12 ml flower extract addition to reaction medium, 1 mM silver nitrate concentration, pH of reaction medium is 8, and reaction time for synthesis of AgNPs is 24 h.

Green synthesis of silver nanoparticles at optimum conditions and characterization

Using the above-optimized conditions, AgNPs were synthesized using C. pulcherrima flower extract and characterization was done using various spectral analyses like FTIR, XRD, TGA, and TEM.

FTIR analysis

FTIR analysis is carried out to identify the possible functional groups responsible for the reduction of silver ions to silver nanoparticles. The FTIR spectrum was recorded in the range of 500–4000 cm⁻¹. FTIR spectrum of AgNPs showed peaks at 2916.37, 2848.86, 1741.72, 1602.85, 1357.89, 1205.51, 1033.85, and 669.30 cm⁻¹ in Figure 2(A). The peaks at 2916.37 cm⁻¹ and 2848.86 cm⁻¹ correspond to C–H stretching of the alkanes group and the aldehyde group, respectively. Peak at 1741.72 cm⁻¹ indicates the C = O stretching of the ester group. Peak at 1602.85 cm⁻¹ is due to N–H bend of primary amines. A value of 1357.89 corresponds to N–O symmetric stretch of nitro compounds and 669.30 corresponds to C–H bend of the alkanes group. A value of 1205.51 cm⁻¹ assigned to the C–N stretching of aliphatic amines, 1033.85 cm⁻¹ indicate the C–O stretching of the alcohols group. A value of 1750–1735 cm⁻¹ showed carbonyl C = O stretch of esters also found by Augustine et al. (2014) in P. nigrum leaves biosynthesized AgNPs. Aliphatic amines and alcohols group as also reported by Mittal et al. (2014) in Syzygium cumini fruit extract synthesized AgNPs. Huang et al. (2011) reported that –C–O group was responsible for reductants and protecting agents. The amine group also plays a reducing and controlling role during the formation of AgNPs (Li et al. 2007).

Figure 2. (A) FTIR spectrum of AgNPs, (B) XRD spectrum of AgNPs, and (C) TG curve of AgNPs.

X-ray diffraction analysis

The crystalline nature of AgNPs synthesized by C. pulcherrima flower extract was studied by XRD analysis (Figure 2(B)). It is apparent that the characteristic peaks at 38.10, 44.59, 64.64, and 77.35 in the 2θ values correspond to the lattice planes (111), (200), (220), and (311), respectively (JCPDS no. 4.0783). The strong and narrow diffraction peaks confirmed the crystalline structure of synthesized AgNPs. All the different peaks confirm the face-centered-cubic (fcc) structure of AgNPs. Padalia et al. (2015) and Arthanari et al. (2016) reported similar lattice planes (111), (200), (220), and (311).

Thermogravimetric analysis

The flower extract responsible for the capping action on the surface of biomolecules was further confirmed by TGA (Figure 2(C)). The first weight loss was observed at 100 °C due to the loss of adsorbed water molecules present in the samples. Also, there is a steady weight loss in temperature range of 0–800 °C. The total weight loss up to 800 °C for AgNPs is about 71.68%. It indicates that a bioorganic compound present in the nanoparticle powder is responsible for the reduction and stabilizing of the particles (Mittal et al. 2014).

TEM analysis

TEM is used to determine the morphology, shape, and size of AgNPs. TEM images reveal that the particles are spherical in shape and are uniformly distributed without agglomeration (Figure 3(A)–(C)). The AgNPs size ranges from 2 to 22 nm and possess an average size of 12 nm. Average particle size of 14 nm is reported in Mustard seed synthesized AgNPs (Khatami et al. 2015). The selected area electron diffraction (SAED) pattern of AgNPs prepared by C. pulcherrima flower extract suggests the crystalline nature of AgNPs which is in good agreement with the X-ray diffraction (XRD) results (Figure 3(D)).

Figure 3. TEM images (A–C) of AgNPs in low and high magnification and (D) SAED patterns of the AgNPs.

Antimicrobial activity

The MIC and the MBC of synthesized AgNPs were performed against four Gram-positive bacteria, four Gram-negative bacteria, and three fungi using different concentrations (0.019–10 mg/ml) by resazurin assay (Figure 4). Resazurin is a simple method used to detect viable cells in the medium. Different organisms represented different levels of MIC values. The MIC value of Gram-positive bacteria S. aureus and fungi C. glabrata had the lowest MIC value (2.5 mg/ml), B. cereus, E. coli, S. typhimurium, C. albicans, and C. neoformans exhibited slightly higher MIC value (5 mg/ml) and B. subtilis and C. rubrum exhibited the highest MIC value (10 mg/ml). Pseudomonas aeruginosa and K. pneumoniae were resistant strains against AgNPs. MBC/MFC of all the organisms was >10 (mg/ml) except C. glabrata (10 mg/ml). The green synthesized AgNPs exhibited broad spectrum antimicrobial activity but best activity was shown against Gram-positive bacteria and fungi. The most susceptible strains were S. aureus and C. glabrata while resistant strains were P. aeruginosa and K. pneumonia. In accordance with the present results, Barapatre et al. (2016) also reported that S. aureus was most sensitive and P. aeruginosa was resistant to AgNPs biosynthesized by lignin degrading fungus.

Figure 4. Antimicrobial activity of AgNPs.

Islam et al. (2016) reported greater zone of inhibition against Gram-positive bacteria S. aureus as compared with Gram-negative strains E. coli and P. aeruginosa by P. armeniaca fresh gum extract synthesized AgNPs. According to Ciobanu et al. (2013a) and Ciobanu et al. (2013b), Ag:HAp showed good antibacterial and antibiofilm activity against both Gram-positive and Gram-negative bacteria. Bonilla et al. (2015) found good antifungal activity of AgNPs against fluconazole-resistant Candida species while Groza et al. (2016) found good antibiofilm activity against C. albicans using Ag:HAp-PDMS. In general in the present work, AgNPs inhibited Gram-positive bacteria more than Gram-negative bacteria.

Rajkuberan et al. (2015) reported good antibacterial activity against B. cereus, Enterococci sp, Shigella sp, P. aeruginosa, K. pneumonia, S. aureus, and E. coli. They (AgNPs) generally show antibacterial activity more against Gram-negative bacteria than Gram-positive bacteria (He et al. 2013). This differential activity may be due to the difference found in their cell wall composition. The Gram-negative bacteria possess a thin peptidoglycan layer as compared with cell wall of Gram-positive bacteria which is composed of thick peptidoglycan layer which facilitates easier penetration of AgNPs into cell wall of Gram-negative bacteria, while Gram-positive bacterial cell wall forms a tough, formidable barrier for AgNPs penetration. The differential response of synthesized AgNPs against multi-drug-resistant micro-organisms can be attributed to size and shape of AgNPs, secondary metabolites present in the plant extract and also the micro-organism under study. Uddin and Akrema (2016) found similar types of relationship between the size of the nanoparticles and antibacterial activity in C. viminalis leaves-mediated AgNPs. The efficient antimicrobial property AgNPs is because they have extremely large surface area providing better contact with cell wall of micro-organisms (Ibrahim 2015). AgNPs interfere with the thiol group of bacterial cell which affects respiratory chain reaction, cell division, and leads to death (Rajkuberan et al. 2015). Positively charged silver ions interact with negatively charged bio-macromolecule components (phosphate and disulfide or sulfhydride of enzymes and nucleic acids), which cause structural changes in bacterial cell walls and disruption of the metabolic process followed by cell death (Khan et al. 2016). The AgNPs get attached to cell surface of bacteria and alter physical and chemical properties of cell membrane (Marambio-Jones and Hoek 2010). This further disturbs membrane permeability, osmoregulation, electron transport, and respiration.

Antioxidant potential

The colorimetric assay of AgNPs synthesized from C. pulcherrima flower extract was done to confirm the antioxidant potential of synthesized AgNPs. The antioxidant activity was tested using DPPH, superoxide anion radical, ABTS cation radical scavenging activity, and reducing capacity assessment. In general, the antioxidant activity increased with an increase in the concentration of AgNPs.

The results of effect of different concentrations of AgNPs on DPPH radical scavenging activity is given in Figure 5(A). At concentrations 20–120 μg/ml, AgNPs showed scavenging rate ranging from 15% to 70%. The DPPH radical scavenging activity was evident but it was less than that of standard since the IC₅₀ value of AgNPs was 70 μg/ml while that of standard ascorbic acid was 11.5 μg/ml. Mittal et al. (2014) reported similar results in S. cumimi fruit extract synthesized AgNPs. The IC₅₀ value of synthesized AgNPs was 31 μg/ml while that of standard was 22.19 μg/ml.

Figure 5. Antioxidant activity of AgNPs (A) DPPH-free radical scavenging activity, (B) superoxide anion radical scavenging activity, (C) ABTS radical scavenging activity, and (D) reducing capacity assessment.

The antioxidant potential was further assessed by their ability to quench superoxide ions by PMS-NBT reduction system using gallic acid as a positive control. The AgNPs showed an increasing trend of quenching super oxide radicals with increase in the concentration of AgNPs (Figure 5(B)). In fact, at a concentration of 60 μg/ml, 72% inhibition was found. The IC₅₀ value of AgNPs was 38.5 μg/ml while that of standard gallic acid was 185 μg/ml. The super oxide quenching ability of AgNPs was many folds better than even the standard. Mata et al. (2015) reported 52% inhibition at a concentration of 200 μg/ml as compared with standard rutin which exhibited 78% inhibition in Abutilon indicum silver nanoparticles.

The results of effect of different concentration of AgNPs on ABTS cation radical scavenging activity are given in Figure 5(C). There was a steady increase in the ABTS cation radical scavenging activity by increasing the concentration of synthesized AgNPs. In the concentration range of 20–120 μg/ml, an inhibition of 23–95% was envisaged. Mittal et al. (2014) found 63% inhibition at 50 μg/ml, while in the present work, it was 57% inhibition is found at a concentration of 60 μg/ml. The reducing capacity of AgNPs showed a steady increase in the absorbance of the reaction mixture with an increase in the concentration of AgNPs (Figure 5(D)). Similar results are reported by Muniyappan and Nagarajan (2014). In the ferric reducing power assay, the presence of antioxidants in sample would result in the reduction of Fe³⁺to Fe²⁺and the amount of Fe²⁺complex monitored by measuring the formation of blue color at 700 nm. Ferric reducing antioxidant power (FRAP) of AgNPs was 8.8 (Mg⁻¹). FRAP activity of green synthesized AgNPs is also reported by Nayak et al. (2016).

In living organisms, thermal oxidation and reactive oxygen species stimulation oxidation of membrane lipids lead to the formation of super oxide radical, hydroxyl radical, nitrous oxide radical, hydrogen peroxide radical, singlet oxygen radical, etc. which cause many degenerative human diseases such as cancer, glaucoma, atherosclerosis, Alzheimer’s disease, Parkinson’s disease, and diabetes (Barhe and Feuya Tchouya 2016). In the present work, the antioxidant activity of C. pulcherrima flower extract-synthesized AgNPs as determined by DPPH-free radical, super oxide anion radical, ABTS cation radical scavenging activity, FRAP, and reducing power assessment suggest that they can be a good source of natural antioxidants and AgNPs have a great potential to scavenge various reactive oxygen species. Among all the free radicals, most dangerous are super oxide radicals since although it itself is weak oxidant, it gives rise to other powerful and dangerous hydroxyl radicals as well as singlet oxygen both of which contribute significantly to oxidative stress and also if they are not scavenged they could damage DNA and protein in living cells. In the present study, among the various antioxidant activities tested, superoxide radical scavenging activity was best by the synthesized AgNPs. Thus, they can be fruitfully used as drug candidates to treat against oxidative stress-related diseases.

Cytotoxicity study

The cell viability assay is one of the important methods for toxicology analysis which explain the cellular response to toxic materials, and it can provide information on cell death, survival, and metabolic activities (Rani et al. 2009). The cytotoxicity of the nanoparticles synthesized from C. pulcherrima flower extract on HeLa cells was examined by the MTT assay. The cytotoxic effect of HeLa cell line was dose dependent and cell viability decreased with an increased concentration of Ag NPs. The HeLa cells showed a decrease in the viability of cells with an increase in the concentration of Ag NPs (Figure 6). The highest inhibition (18%) was found at a concentration of 200 μg/ml of AgNPs, while at 50 μg/ml concentration, 23% cells were viable (Figure 6). Vasanth et al. (2014) found 6% cell viability at 250 μg/ml concentration, while Li et al. (2015) reported 83.3 ± 3.5% cell viability at 30 μg/ml concentration against HeLa cell line. The cytotoxicity exhibited by AgNPs may be due to physical and chemical interactions of silver ions with functional groups of intracellular proteins, nitrogen bases, and phosphate groups. Gurunathan et al. (2013) suggested that nanoparticles induce generation of reactive oxygen species, activate Caspase 3, induced DNA fragmentation and membrane leakage which results in cell death.

Figure 6. Demonstration of the cytotoxicity of biosynthesized AgNPs against HeLa cell line.

Genotoxicity study

The normal human peripheral blood lymphocyte culture was used for single cell gel electrophoresis (comet assay) and chromosomal aberration for the evaluation of DNA damage assay.

Comet assay

The comet assay is a promising technique which is applied mainly to the study of single-strand DNA breaks induced by a variety of toxic agents, including chemical compounds, ionizing radiation, and nanoparticles in cells (Collins 2004). The genotoxicity of AgNPs to damage DNA in the lymphocyte culture was evaluated by alkaline comet assay. DNA damage (according to tail length) in lymphocyte culture exposed to three different concentrations of AgNPs is given in Figure 7. In the control, halo surrounding cell nuclei was clearly visible (Figure 7(A)). In the positive control, cells were treated with mitomycin C drug (Figure 7(B)). Microscopic images confirmed that 2 μg and 50 μg AgNPs-treated cells showed that nuclei were intact and round without any fragmented DNA (Figure 7(C) and (D)). In 200 μg-treated cells, fragmented DNA was found (Figure 7(E)). The comet length was increased with an increasing concentration of AgNPs (Figure 8). Out of three different concentrations, a maximum comet length was found at 200 μg concentration, but it was less than positive control. Thus, dose-dependent genotoxicity was found in C. pulcherrima flower-synthesized AgNPs as also reported in MCF-7 cells (Farah et al. 2016).

Figure 7. DNA damage by cornet assay: (A) Cointrol, (B) positive control, (C) 2 μg/ml of AgNPs, (D) 50 μg/ml of AgNPs, and (E) 200 μg/ml of AgNPs (magnification – 40×, 10×).

Figure 8. DNA damage in cell after exposure of AgNPs.

Chromosomal aberration assay

The metaphase chromosomal aberration is given in Figure 9. The control sample where no AgNPs are added show clean metaphase (Figure 9(A)) while cells treated with mitomycin C drug as a positive control is given in Figure 9(B) which showed break and interchange in metaphase. The cells treated with three different concentrations of AgNPs (2 μg/ml, 50 μg/ml, and 200 μg/ml) are given in Figure 9(C)–(E)). Lower concentration of AgNPs treatment showed no appearance of aberrations (Figure 9(C) and (D)) while cells treated with higher concentration of AgNPs showed chromatid gap in chromosome (Figure 9(E)). Dose-dependant genotoxic effect of AgNPs is also reported by Patlolla et al. (2015).

Figure 9. Chromosomal aberration at different concentration: (A) control, (B) positive control, (C) 2 μg/ml of AgNPs, (D) 50 μg/ml of AgNPs, and (E) 200 μg/ml of AgNPs (magnification 0).

Conclusion

The use of greener and safer method for synthesis of silver nanoparticles was applied in the present study by using flower extracts of C. pulcherrima. The synthesis of AgNPs depends on pH, reaction time, plant extract, and silver nitrate concentration. The reaction rate is optimum in alkaline pH. The synthesized AgNPs with optimized reaction parameters showed maximum absorption peak at 410 nm. The particle size was 12 nm and was highly crystalline in nature. FTIR analysis revealed as various phytoconstituents of C. pulcherrima flower extract which were responsible for reducing silver ions to silver nanoparticles and also acted as capping agents. TGA analysis revealed its stability. The synthesized AgNPs showed broad spectrum antimicrobial activity with greater efficacy against Gram-positive bacteria and fungi, excellent antioxidant activity, and effective anticancer activity evidenced through MTT assay against HeLa cell line. In vivo genotoxic study demonstrated that AgNPs do not cause any toxic effects at lower concentration. The outcome of this study illustrates a broad range of applications of biosynthesized silver nanoparticles from C. pulcherrima flower extract. Hence, the flower-mediated synthesis of nanoparticles can be used as good therapeutic agent against human pathogens and cancer. They can be used for successful development of drug delivery in future. Therefore, further studies are needed to fully characterize the toxicity and the molecular mechanisms involved in antimicrobial and anticancer activity of these nanoparticles.

Acknowledgements

The authors thank Department of Biosciences (UGC-CAS) for providing excellent research facilities.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Funding

One of the authors Ms. Pooja Moteiya is thankful to UGC, New Delhi, for providing meritorious Junior Research Fellowship.