Studies on green synthesized silver nanoparticles using Abelmoschus esculentus (L.) pulp extract having anticancer (in vitro) and antimicrobial applications

2.1 Materials

Analytical grade silver nitrate (AgNO₃) was purchased from Merck Mumbai, India, and used as received without further purification. The A. esculentus (L.) was collected from the local market of our institute.

2.2 Culture media and chemicals

RPMI (Roswell Park Memorial Institute) medium 1640, N-acetyl-L-cysteine (NAC, ⩾99%), N-(1 naphthyl)-1,2-ethylenediamine dihydrochloride (>98%), sulfanilamide (⩾99%), penicillin, and streptomycin were procured from Sigma, St. Louis, MO. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from GIBCO/Invitrogen. Commercially available dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), Rhodamin 123, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) reagent were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. All other chemicals were from Merck Ltd. and SRL Pvt. Ltd. Mumbai (India) and were of the highest purity grade available. Solvents were dried according to the standard procedure and distilled prior to use (Perrin et al., 1980).

2.3 Synthesis of silver nanoparticles

A fixed amount of small slices of fresh A. esculentus (L.) pulp was put into triple distilled water and left for 2 h in order to maximize the diffusion of the materials present in the A. esculentus (L.) pulp into triple distilled water. After that, the pulps were taken out from the medium and the solution was subjected to centrifugation to remove any solid impurities present in the stock. The supernatant was carefully collected and used as the A. esculentus (L.) pulp extract. The extract was mixed with the aqueous solution of 1 mM silver nitrate (4:1 v:v) at room temperature with continuous stirring for approximately 9 h. Then, the colloidal solution was left undisturbed for 27 h at room temperature. The formation of Ag NPs was confirmed by observing the color change of this colloid solution from colorless to light yellow.

2.4 Cell lines culture and maintenance

Jurkat cell line (Human T-cell lymphoma) was obtained from the National Centre for Cell Science (NCCS), Pune, India. This cell line was cultivated and maintained in RPMI medium 1640 complete media supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 4 mM L-glutamine under 5% CO₂ and a 95% humidified atmosphere at 37 °C in a CO₂ incubator. Cells were used for different experiments until the number of cells reaches at 1.0 × 10⁶ cells/ml.

2.5 Drug preparation

One milligram per milliliter stock of Ag NPs was prepared by concentrating the prepared Ag NPs containing solution using a rotary evaporator. It was then serially diluted with RPMI media to prepare working concentrations.

2.6 Experimental design

The Jurkat cells were divided into 11 groups. Each group contained 6 petri dishes (2 × 10⁴ cells in each). The following groups were considered for the experiment and cultured for 24 h:

Group I: Control i.e., Cells + culture media, Group II: Cells + 1 μg/ml Ag NPs in culture media, Group III: Cells + 5 μg/ml Ag NPs in culture media, Group IV: Cells + 10 μg/ml Ag NPs in culture media, Group V: Cells + 25 μg/ml Ag NPs in culture media, and Group VI: Cells + 50 μg/ml Ag NPs in culture media.

After the treatment schedule, the cells were collected from the petri dishes separately and centrifuged at 2,200 rpm for 10 min at 4 °C to separate cells and supernatant (Dash et al., 2013). Cells were washed twice with 50 mM PBS at pH 7.4. Intact cells were used for mitochondrial membrane potential and ROS measurement.

2.7 In vitro cell viability assay (MTT assay)

The Jurkat cells were maintained in RPMI-1640 supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) in a humidified atmosphere of 5% CO₂ at 37 °C. The cytotoxicity of synthesized Ag NPs on Jurkat cells was determined by the MTT assay (Dash et al., 2013). Cells (2 × 10⁴/well) were plated in 100 μl of medium/well in 96-well plates. After 48 h incubation, the cell reached confluence. Then, the cells were incubated in the presence of various concentrations of NPs suspended in RPMI medium for 24 h at 37 °C. After the treatment period, cells were centrifuged to remove the supernatant and subsequently washed with phosphate buffered saline (PBS). Then the cell were stained with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) at 20 μl/well (5 mg/ml) in each well of 96 well plates. After 4 h incubation, 0.04 M HCl/isopropanol was added to each well. Viable cells were determined by absorbance at 570 nm with reference at 655 nm. Measurements were performed 3 times. The absorbance at 570 nm was measured with a UV–Vis spectrophotometer using wells without sample containing cells as blanks. All experiments were performed in triplicate. The effect of the Ag NPs on the proliferation of Jurkat cells was expressed as the % cell viability using the following formula: $$\%\text{Proliferation}=\left[{\text{OD}}_{\text{sample}}-{\text{OD}}_{\text{control}}\right]\times 100/{\text{OD}}_{\text{control}}$$

2.8 Assay for antimicrobial activity of silver nanoparticles against microorganisms

Silver nanoparticles in sterilized deionized water were tested for their antibacterial activity by the agar diffusion method. Five bacterial strains, Bacillus subtilis [MTCC 736], Bacillus cereus [MTCC 306], Pseudomonas aeruginosa [MTCC 8158], Micrococcus luteus [MTCC 1538] and Escherichia coli [MTCC 68] were used for this analysis. These bacteria were grown in liquid nutrient agar media (HiMedia Laboratories Pvt. Ltd., Mumbai, India) for 24 h prior to the experiment and were seeded in agar plates by the pour plate technique. A cup was made using a cork borer (10 mm diameter) and was filled with the Ag NPs (0.2 mg/ml) solution and then incubated at 37 °C for 24 h. Simultaneously, the control sets (only plant extract and sterilized deionized water respectively) were also examined against the same five bacterial strains. Every experiment was repeated three times.

2.9 Characterization

3.1 UV–Vis absorption spectroscopy analysis

The optical properties of the as-synthesized Ag NPs using UV–Vis spectroscopy were studied and the recorded spectra are shown in Fig. 1. We have already mentioned that the A. esculentus (L.) pulp extract reduces the silver ion present in the aqueous solution resulting in the change of color of the solution from colorless to light yellow due to the formation of Ag NPs. The absorption behavior shown in Fig. 1 arises due to surface plasmon resonance (SPR), which is the result of the coherent oscillation of the electrons in the conduction band of the Ag NPs induced by the electromagnetic field. It is well known that Ag NPs exhibit yellowish color in aqueous solution due to the excitation of surface plasmon vibrations in Ag NPs (Shankar et al., 2004).

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Figure 1

UV–Vis absorption spectra of (a) Ag NPs synthesized by treating 0.001 M aqueous silver nitrate solution with Abelmoschus esculentus (L.) pulp extract at room temperature and (b) UV–Vis absorption spectra of Ag NPs stabilized with Abelmoschus esculentus (L.) pulp extract (after reaction termination; after 8 months storage at room temperature; after re-dispersion of Ag NPs in the triple distilled water.

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Fig. 1a shows the UV–Vis spectra of the synthesized Ag NPs, which give a sharp absorbance band at around 403 nm at room temperature. To examine the stability of Ag NPs, one part of the colloidal solution of Ag NPs was left undisturbed for 8 months at room temperature and the other part was subjected to ultra-centrifugation. The obtained residue after ultra-centrifugation is homogeneously dispersed in triple distilled water by constant stirring followed by ultra-sonication. UV–Vis study has been carried out with both the colloidal solutions of Ag NPs, which were being restored before ultra-centrifugation and the dispersed after ultra-centrifugation (Fig. 1b). In both cases, it has been observed that there is no significant change in SPR band position or the spectral shape; only absorption intensities of Ag NPs change slightly. The above result revealed that the synthesized Ag NPs are very stable at room temperature with negligible aggregation for several months. Similar results are reported in the literature (Mollick et al., 2014a,b; Zhao et al., 2014).

3.2 X-ray diffraction (XRD) measurement

The crystalline nature of Ag NPs synthesized by reduction with A. esculentus (L.) pulp extract is studied by XRD analysis and given in Fig. 2. It is apparent from Fig. 2 that the characteristic peaks at 27.2°, 31.63°, 45.66°, 54.24°, and 57.04° in the 2θ region correspond to the lattice planes (2 2 0), (3 1 1), (4 2 0), (5 1 1) and (4 4 0) respectively (JCPDS No. 01-1167 and 01-1164). These results confirm the face-centered-cubic (fcc) structure of Ag NPs.

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Figure 2

XRD peaks corresponding to the diffraction from (2 2 0), (3 1 1), (4 2 0), (5 1 1), and (4 4 0) planes of fcc lattice of Ag NPs.

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3.3 Dynamic light scattering (DLS) measurement

The dynamic light scattering (DLS) technique is used to determine the average particle size and particle size distribution profile of small particles in suspension. Fig. 3 shows the average particles size distribution of Ag NPs having a mean particle size of ∼21.29 nm.

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Figure 3

DLS showing particles size distribution of Ag NPs prepared by treating 0.001 M aqueous silver nitrate solution with Abelmoschus esculentus (L.) pulp extract at room temperature.

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3.4 Transmission electron microscopy (TEM) measurement

TEM is employed to determine the morphology, shape and size of NPs. TEM micrographs (Fig. 4a) reveal that the particles are spherical in shape and are uniformly distributed without significant agglomeration. The selected area electron diffraction (SAED) pattern of Ag NPs prepared by A. esculentus (L.) pulp extract (Fig. 4b) suggests the crystalline nature of Ag NPs which is in good agreement with the X-ray diffraction (XRD) results. The NPs particle size ranges from 3 to 11 nm as seen from the particle size histogram (Fig. 4c) and possess an average size of 6.7 nm. The particle size determined by TEM is an actual diameter of the NPs as it is measured at the dried state of the sample, whereas the size measured by the dynamic light scattering method (DLS) is a hydrodynamic diameter (hydrated state). Therefore, the NPs will have a larger hydrodynamic volume due to the solvent effect in the hydrated state (Gao et al., 2008). Thus, the size of the synthesized Ag NPs observed by the TEM method is less than the size measured by the DLS method. The energy dispersive X-ray analysis (EDX) reveals a strong signal at 3 keV (Fig. 4d) which is generally shown by metallic silver nanocrystals due to surface Plasmon resonance (Magudapatty et al., 2001). There is also a strong signal for Cu in the EDX data, which originates from the carbon coated copper grid.

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Figure 4

(a) HRTEM images of Ag NPs, (b) the selected area electron diffraction image of Ag NPs, (c) particle size distribution histogram of Ag NPs determined from HRTEM micrograph prepared at room temperature, and (d) EDS spectrum of Ag NPs from HRTEM.

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3.5 Fourier transform infrared (FTIR) spectroscopy analysis

The A. esculentus (L.) pulp extract contains different phytochemicals such as vitamins, protein, etc. which play an important role in the reduction of metal ions and the stabilization of the nanoparticles. The A. esculentus (L.) pulp extract shows several characteristic bands in the FTIR spectra given in Fig. 5a. The peak at 3402 cm⁻¹ is due to the N–H stretch vibrations of the peptide linkages (Kannan and John, 2008; Whiteman et al., 2008), and the peak at 2934.5 cm⁻¹ corresponds to the stretching vibration of methyl groups (Li et al., 2007). The peaks at 1741.1, 1427, and 1631 cm⁻¹ are attributed to carbonyl stretching vibration (Bar et al., 2009b; Luo et al., 2005), germinal methyl group (Tripathy et al., 2010), and amide I group of proteins (Whiteman et al., 2008), respectively, and the peak at 1079.7 cm⁻¹ is responsible for the bending vibration of C–OH groups and the anti-symmetric stretching band of C–O–C groups of polysaccharides and/or chlorophyll (Jain et al., 2011). The FTIR spectra of Ag NPs synthesized from A. esculentus (L.) pulp extract are given in Fig. 5b. The slight shift in peak position from 3402, 2934.5, 1741.1, 1427, 1631, and 1079.7 cm⁻¹ to 3421.3, 2921.06, 1730.9, 1648, 1020.47 cm⁻¹ respectively of the synthesized Ag NPs indicates that the different phytochemicals present in the A. esculentus (L.) pulp extract are involved in reducing and stabilizing the nanoparticles.

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Figure 5

FTIR spectra of (a) pure Abelmoschus esculentus (L.) pulp extract, and (b) extract stabilized Ag NPs.

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3.6 In vitro cell proliferation assay

Jurkat cells are exposed to Ag NPs at the concentrations of 0, 1, 10, 25 and 50 μg/ml for 24 h, and cytotoxicity is determined using a MTT assay (Fig. 6). Results have shown that Ag NPs up to the concentration of 50 μg/ ml produced a significant reduction in viability of Jurkat cells (P > 0.05 for each). The reduction in viability of Ag NPs treated cancer cells occurs in a dose-dependent fashion. The Ag NPs exposed Jurkat cell viability is significantly decreased by 52.6%, 85.4%, and 91.6%, at 10, 25, and 50 μg/ml doses, respectively. As shown in our previous study (Mollick et al., 2014a), the pulp extract has no anticancer components for killing the anticancer cell and it is solely caused by the Ag NPs only.

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Figure 6

In vitro cell proliferation assay of Ag NPs treated Jurkat cell line. Values are expressed as mean ± SEM of three experiments; ^∗ indicates significant difference (p < 0.05) compared with the control group.

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The IC₅₀ value of Ag NPs against Jurkat cells is determined using the Statistica version 5.0 (Statsoft, India) software package. From this statistical calculation, it is determined that the IC₅₀ value of Ag NPs is 16.15 μg/ml. As the IC₅₀ value is found to be a 16.15 μg/ml concentration of Ag NPs, further experiments are carried out using this concentration to see the effect of Ag NPs against the leukemic cell line in vitro.

3.7 Nitric oxide (NO) release level

In the present study, Ag NPs are able to significantly (p < 0.05) increase the reactive nitrogen species (RNS) through NO level in Jurkat cells by 67.9% at 16.15 μg/ml dose as compared to the control (Fig. 7). It has been well established that NO reacts with superoxide to form more toxic peroxynitrite (ONOO⁻), which has an important microbicidal and tumoricidal function. In our study, the elevated level of NO in leukemic cells may be due to severe oxidative injury, which in turn helps in cell killing ability due to Ag NPs exposure (Dash et al., 2013).

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Figure 7

Nitric oxide (NO) release levels of Ag NPs treated Jurkat cell lines. The levels of NO were expressed as μmol mg⁻¹ protein. Values are expressed as mean ± SEM of three experiments; * indicate significant differences (p < 0.05) compared with the control group.

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3.8 Cellular ROS level

The H₂DCFDA staining method measures intracellular generation of hydrogen peroxide, an indirect procedure for estimating ROS. In the present study, the intracellular ROS concentration is significantly (p < 0.05) higher in Ag NPs treated Jurkat cells when compared to the untreated control. In Jurkat cells, ROS fluorescence intensity is elevated by 60.9% at 16.15 μg/ml NPs concentration (Fig. 8a–c). ROS are chemically reactive molecules (free radicals) that play crucial functions in living organisms. In a biological system, ROS play a dual role. A moderate increase in ROS can promote cell proliferation and differentiation, whereas excessive amounts of ROS can cause oxidative damage to lipids, proteins and DNA. Therefore, maintaining cellular ROS homeostasis is crucial for normal cell growth and survival (Trachootham et al., 2009). Estimation of intracellular ROS level explores the potential role of oxidative stress as a mechanism for Ag NPs induced toxicity. Studies show that the generation or external addition of ROS can cause cell death by two distinct pathways, vis-a-vis apoptosis or necrosis (Jeyaraj et al., 2013). In our study, significant elevation of ROS due to Ag NPs exposure may be assumed as the contribution of the oxidative attack in Jurkat cell death (Foldbjerg et al., 2011).

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Figure 8

(a) Measurement of ROS level in Ag NPs treated Jurkat cells. DCF fluorescence intensity was expressed in term of ROS production. Values are expressed as mean ± SEM of three experiments; ∗ indicates significant difference (p < 0.05) compared with the control group. Intensity of control cells was set to 100. Data are represented as the percentage of the ROS level in the control group. (b and c) Qualitative characterization of reactive oxygen species formation by DCFH₂-DA staining using fluorescence microscopy. Here, (b) untreated Jurkat cells, and (c) Jurkat cells treated with 16.15 μg/ml Ag NPs.

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3.9 Alterations of mitochondrial membrane potential (MMP)

Rhodamin 123 is used to measure mitochondrial membrane potential (MMP). Live cells contain mitochondria capable of retaining this dye due to higher polarity, whereas dead or apoptotic cells lose this ability because of the loss of mitochondrial membrane integrity and, thus, exhibit an extrusion of the dye (Tan et al., 2003). Because mitochondria are known to be involved in the process of programmed cell death, we investigated changes in mitochondrial membrane potential in terms of Rh 123 fluorescence intensity. In this study, Ag NPs cause a significant depletion of MMP in Jurkat cells treated with Ag NPs for 24 h (Fig. 9a) at an IC₅₀ dose. The percentage of MMP decreased significantly (P < 0.05) in the Jurkat cell line with the increase of Ag NPs concentration and reaches a value of 68.7% when treated with the IC₅₀ dose. Depletion of MMP is also confirmed by florescence microscopic images (Fig. 9b and c). In our study, the strong dissipation in MMP in the Ag NPs exposed cancer cell line suggests a possible disruption of cellular mitochondrial membrane after Ag NPs treatment. The increased ROS levels and subsequent loss of mitochondrial membrane potential might be the reason for the involvement of apoptosis in the Ag NPs treated cells (Jeyaraj et al., 2013).

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Figure 9

(a) Measurement of mitochondrial membrane potential (MMP) Ag NPs in pre-treated Jurkat cells. Values are expressed as mean ± SEM of three experiments; ∗ indicates significant difference (p < 0.05) compared with the control group. (b and c) Qualitative estimation of MMP by Rh-123 staining using fluorescence microscopy. Here (b) untreated Jurkat cells, and (c) Jurkat cells treated with 16.15 μg ml Ag NPs.

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3.10 Pre-treatment with NAC

This is to confirm the main contribution of ROS in Ag NPs-induced leukemic cell death. The Jurkat cells are pre-treated with N-acetyl-L-cysteine (NAC). After these pre-treatments, NPs exposure is done at 16.15 μg/ml for 24 h, followed by experiments where cell viability is estimated using the MTT method. We find that pre-treatment with NAC (ROS scavenger) protects the cells from the cytotoxic effects of NPs. In these cases, >85% of cell viability suggests that Ag NPs selectively kill the leukemic cells by generation of ROS (Fig. 10).

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Figure 10

Quenching of ROS rescues Jurkat cells from Ag NPs induced cytotoxicity. Jurkat cells were pre-treated with 5 mM N-acetyl cysteine (NAC) for 4–6 h and then subsequently exposed to Ag NPs at IC 50 dose. Cell viability was estimated by MTT assay.

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3.11 Induction of caspase-3 activity and chromosome condensation by Ag NPs

The disruption of cellular metabolic activity of Jurkat cells followed by Ag NPs exposure seems to be affected by apoptosis phenomenon. Thus, involvement of apoptosis was investigated by caspase-3 assay. Caspase-3, considered to be the most significant of the executioner caspases resulting in cellular apoptosis, was induced following exposure with Ag NPs (Fig. 11a). When Jurkat cells were treated with IC₅₀ concentrations of Ag NPs for 24 h, caspase-3 activity was significantly (p < 0.05) increased with compare to untreated cells. Chromatin condensation was also noted by DAPI staining with the addition to the caspase-3 activity. When Jurkat cells were treated with the IC₅₀ concentrations of Ag NPs for 24 h, chromatin condensation and fragmentation were observed in the treated group (Fig. 11b and c). Caspase-3 activation and chromatin condensation/fragmentation in Jurkat cells suggest that Ag NPs caused cell death was mainly due to apoptotic process (Gurunathan et al., 2013; Mollick et al., 2014a).

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Figure 11

Increase of chromosome condensation and caspase-3 activity in Jurkat cells after exposure of Ag NPs for 24 h. (a) Caspase-3 activity; (b) control; (c) exposed to 16.15 μg/ml Ag NPs. Values are expressed as mean ± SEM of three experiments; ∗ indicates significant difference (p < 0.05) compared with the control group.

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3.12 Antimicrobial activity of silver nanoparticles against microorganisms

The Ag NPs solution exhibits excellent antibacterial activity against the bacteria, B. subtilis, B. cereus, E. coli, M. luteus and P. aeruginosa, by showing the clearing zone around the holes with bacterial growth on petri plates by the cup plate method. The radial diameter of the inhibiting zones of B. subtilis, B. cereus, E. coli, M. luteus, and P. aeruginosa is 33, 28, 19, 40, and 26 mm, respectively. The Ag NPs at the concentration of 0.2 mg/ml show a range of specificity toward their antimicrobial activity (Fig. 12). Fig. 13 represents the clear inhibition zone made by the Ag NPs solution against only the strain of B. subtilis. The clear zone indicates bacterial growth restriction by the diffused Ag NPs solution. At the same time, the control sets did not show any inhibition. Results are the mean of three separate experiments, each in triplicate. Thus, the synthesized Ag NPs have great potential due to their antibacterial activity.

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Figure 12

Histogram shows the radial diameter of inhibitory zone by Ag NPs at the same concentration (0.2 mg/ml) against different microorganisms.

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Figure 13

Antibacterial activity of Ag NPs assayed by the agar diffusion method in petri plate. Ag NPs, poured in the circular well, showed the zone of inhibition against Bacillus subtilis. The cavity of the petri plate was filled with Ag NPs solution (0.2 mg/ml).

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