Copperpod Plant Synthesized AgNPs Enhance Cytotoxic and Apoptotic Effect in Cancer Cell Lines

: The utilization of biological resources on the manufacture of nano silver has attracted the interest of researchers to develop an eco-friendly, cost-effective technology in nanomaterials production. In the present study, plant-mediated silver nanoparticles (AgNPs) were synthesized using aqueous leaf extracts of the Copperpod plant, which was well characterized. The ultraviolet-visible spectrophotometric study showed a maximum absorbance peak at 425 nm, and the observation of transmission electron microscopic features revealed that the nanoparticles size ranged between 20 and 70 nm. The synthesized AgNPs were tested for in vitro cytotoxic effects against cancerous cells, such as HepG2, A549 and MCF-7 cells. The ﬁndings showed that the IC50 values of AgNPs against cancerous cells viz., HepG2, MCF-7 and A549 cells, were observed to be 69 µ g/mL, 62 µ g/mL and 53 µ g/mL, respectively. In addition, the apoptosis property was analysed using propidium iodide and acridine orange-ethidium bromide via the DNA fragmentation technique. Thus, the outcomes of the current analysis presume that the plant mediated AgNPs obtained from a synthesized Copperpod plant possess signiﬁcant anti-cancer properties against various cancerous cells. mixture of AgNO 3 . The increase in the colour intensity with time showed the enhanced synthesis of AgNPs. In addition, the development of AgNPs was con-firmed by UV-visible spectrophotometric analysis showing a maximum absorption spectrum of 425 nm, while AgNO 3 showed no peaks between 300 nm and 700 nm (Figure 1). The characteristic peak in the absorption range between 400 nm and 450 nm is a validation of the presence of surface plasma resonance in AgNPs. The present study demonstrates a single plasma resonance peak at 425 nm, which presented the evidence of biosynthesis in AgNPs [35]. have investigated the apoptotic property of AgNPs using a DNA fragmentation assay [78,84].


Introduction
Nanoscience is a division in science that deals with the report on the manipulation of materials at the atomic and molecular level. In general, nanostructures are materials considered to have a diameter size in the range of 1 to 100 nm. Recently, several forms of nanomaterials have been investigated as it is considered to be an emerging area with promising prospective applications in diverse fields [1,2]. The major reason behind the vast applications of nanostructures is their reduced size and high-level surface to volume proportion [2][3][4]. Several nanoparticles have been found to have different applications in various fields. Nano-based structures provide excellent benefits to the environment as well as in the field of biomedical applications, such as drug delivery, diagnostic tools, treatment of cancer, biosensors, photothermal therapy, antibacterial and as antifungal agents, etc. [5,6]. Among numerous nanostructures, the application of metal-based nanoparticles, especially silver and silver-based nanomaterials, have provided a breakthrough in the field of biomedicine [7,8]. Among the various forms of nanostructures that are established and characterized, AgNPs have been found to have significant applications due to their inherent properties in the solid states that show exceptional physicochemical properties and good stability [9][10][11][12]. Although the significance of silver and silver-based compounds was recognized much earlier, these materials were not well exploited before, and they were only applied for antimicrobial activity, as antioxidants, for wound healing, for anticancer and anti-inflammatory properties, etc. This suggests that AgNPs are a promising and the filtrate was screened for further processing of AgNPs [40,42]. The collected mature leaves were washed in distilled water and air-dried under shade. Then, the leaves were pulverized into a powder using a blender and stored in the sealed bottle. Five grams of dried leaf power was combined with 100 mL of glass-distilled water. It was kept at 55 • C for 15 min in a water bath and then cool down to room temperature. The extract was then filtered using Whatman's No.1 filter paper, and the filtrate was stored at 4 • C for further study.

Synthesis and Characterization of AgNPs
Then, 1 mM of silver nitrate was added separately to 1 mL of leaf extract in test tubes, which were incubated at room temperature in dark conditions for 24 h. During preparation, each extract with silver nitrate was continuously monitored for colour change, and the observations were recorded. The aqueous leaf extracts of the respective plants were kept as a control. The test tube that showed a change in the colour of the reaction mixture was recorded and further subjected to a UV-visible spectrophotometer, and the maximum absorption spectra was determined [42].

Characterization of Synthesized AgNPs
The plant biogenic AgNPs were characterized by various techniques, such as UVvisible spectroscopy (U2900, Hitachi, Tokyo, Japan), X-ray diffraction (XRD) (Philips X-ray diffractometer, London, UK), and transmission electron microscopy (TEM) (FEI Tecnai, Oregon, USA) equipped with an energy-dispersive X-ray (EDX). The XRD study of AgNPs produced using the Copperpod leaf extract was performed to investigate the crystalline nature of the nanoparticle formed. For this study, the AgNPs formed using Copperpod leaf extract were made into dried powder and then coated onto the XRD grid. The grid was then analysed using an XRD spectrum at a high voltage of 40 kV with Cu-Ka radiations by a Philips X-ray diffractometer [43]. In addition, the concentration of AgNPs synthesized from the aqueous leaf extract of Copperpod was determined using Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES, Elementar, Frankfurt, Germany). The surface properties of Biogenic AgNPs formed using leaf extracts were investigated using an atomic force microscope (XE-70 Park system, Mannheim, Germany) [44]. The quantitative analyses of Biosynthesized AgNPs were performed using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES, Elementar, Frankfurt, Germany). The elemental concentration of the silver was obtained from the synthesized AgNPs stabilized at room temperature for two months. The two-month-old AgNPs samples were evaluated by ICP-OES Perkin Elmer optima 5300 DV.

Cell Culture and Maintenance
The following cell lines, that is, Vero (African monkey kidney), HepG2 (hepatocellular carcinoma), A549 (lung cancer) and MCF-7 (breast cancer) cells, were procured from the National Centre for Cell Science (NCCS), in Pune, India, and cultured in Dulbecco's modified Eagle's medium (DMEM) accompanied with necessary growth factors. The subculture was performed after reaching 95% confluence by the trypsinization process, and the cells were replenished with fresh growth medium once every 3 days.

Cell Viability Assay
The comparative study on the assessment of nanotoxicity was evaluated between the normal-control cells (Vero cells) and cancer cells with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described by Mosmann [45], albeit with some slight modifications. The cell lines of Vero cells (control-normal, non-cancerous cells) and 3 different cancer cells (HepG2, A549 and MCF-7) were separately seeded in a sterile 96 well flat bottom plates. They were then developed at 37 • C in a moistened incubator with 5% CO 2 supply and allowed to reach 95% confluence. After achieving 95% confluence, the cells were replenished with a fresh medium and treated with different concentrations of biogenic AgNPs (10 to 100 µg/mL), separately. The treated cells were then incubated in a humidified incubator with 5% CO 2 at 37 • C for 24 h. After incubation, the treated and untreated cells were washed in phosphate-buffered saline (pH 7.4). Each well of the 96 plates was loaded with 20 µL of MTT reagent (5 mg/mL in PBS) and allowed to incubate for 4 h. The wells were then loaded with 100 µL of dimethyl sulfoxide (DMSO), and observations were made for the formation of the purple-coloured formazan crystals [13,46]. The plates were then subjected to absorbance reading at 570 nm using a microplate ELISA reader. The IC 50 values were plotted by using a software program (Origin pro-9.0).

Propidium Iodide (PI) Staining
For PI staining, the tumour cells, HepG2, A549 and MCF-7, were separately grown in 24 well plates (1 × 10 5 cells per well) as described above, and the cells were treated using IC50 concentration of biogenic AgNPs. The treated cells were then washed in phosphatebuffered saline and fixed in methanol:acetic acid (3:1, v/v) for 10 min. The fixed cells were then stained with propidium iodide (50 µg/mL in PBS) and incubated for another 20 min [47]. The cells were then observed for morphology of apoptotic cells under a FLoid ® cell imaging station (Life Technologies)

Acridine Orange-Ethidium Bromide Dual Staining
The extent of apoptosis in the cells treated with biogenic AgNPs was analysed microscopically using an acridine orange/ethidium bromide (AO/EB) dual staining method. Dual staining was performed to detect the chromatin condensation of dead apoptotic cells by staining them with a fluorescence stain. The tumour cells, including HepG2, A549 and MCF-7, were separately grown in 24 well plates (1 × 10 5 cells per well) as described above, and the cells were treated with an IC50 concentration of biogenic AgNPs. The treated cells were washed with phosphate-buffered saline, and the dual stain (AO/EB) (1 mg/mL AO and 1 mg/mL EB in PBS) was then added, and the mixture incubated for 5 min. The cells were again washed with PBS buffer, and the excess stain was removed and then visualized under a FLoid ® cell imaging station (Life Technologies) with an excitation filter of 482 nm [47].

DNA Fragmentation Analysis
The DNA fragmentation analysis is considered as an important feature of cell apoptosis. The effect of apoptosis in the cells treated with biogenic AgNPs was analyzed using a DNA fragmentation technique, where the characteristic ladder of fragmented genomic DNA was visualized using agarose gel electrophoresis [48]. For this analysis, the IC50 concentration of biogenic AgNPs was used for HepG2, A549 and MCF-7 cells. After incubation, the cells treated with lysis buffer and extracted with phenol/chloroform/isoamyl alcohol mixture were further precipitated with ethanol and re-suspended using TE buffer containing RNase A. The extracted DNA was electrophoresed with 1% Agarose electrophoresis, and the untreated cells were used as controls for the experiments. The electrogram was stained with ethidium bromide and visualized under UV light. The gel was then photographed with gel documentation equipment. The separation of the DNA fragments was described using a standard molecular weight marker ladder.

Synthesis and Characterization of AgNPs
The plant leaf extracts obtained from Copperpod were employed for AgNPs synthesis. The optimal situation for producing AgNPs was observed to be 1 mg/mL plant concentration, 7 pH, 1 mM silver nitrate concatenation and an incubation temperature of 37 • C. The stability of produced AgNPs found to be even up to 3 months was also proved [40]. The formation of AgNPs was visually confirmed by recording the colour change from the initial one to the brown colour. The colour change was because of the addition of plant extracts to the aqueous Processes 2021, 9, 888 5 of 17 mixture of AgNO 3 . The increase in the colour intensity with time showed the enhanced synthesis of AgNPs. In addition, the development of AgNPs was con-firmed by UV-visible spectrophotometric analysis showing a maximum absorption spectrum of 425 nm, while AgNO 3 showed no peaks between 300 nm and 700 nm ( Figure 1). The characteristic peak in the absorption range between 400 nm and 450 nm is a validation of the presence of surface plasma resonance in AgNPs. The present study demonstrates a single plasma resonance peak at 425 nm, which presented the evidence of biosynthesis in AgNPs [35]. [40]. The formation of AgNPs was visually confirmed by recording the colo the initial one to the brown colour. The colour change was because of the ad extracts to the aqueous mixture of AgNO3. The increase in the colour inten showed the enhanced synthesis of AgNPs. In addition, the development con-firmed by UV-visible spectrophotometric analysis showing a maxim spectrum of 425 nm, while AgNO3 showed no peaks between 300 nm and 1). The characteristic peak in the absorption range between 400 nm and 45 dation of the presence of surface plasma resonance in AgNPs. The present strates a single plasma resonance peak at 425 nm, which presented the ev synthesis in AgNPs [35]. , (4 2 0) and (4 2 2) planes, respectivel shape of the biogenic AgNPs were determined using TEM analysis. The revealed the presence of AgNPs, which were globular in structure with a varying from 20 to 60 nm; in addition, the presence of silver peaks in the confirms the formation of AgNPs (Figure 3a,b). The surface properties of t biogenic AgNPs were analysed using an atomic force microscope. The re three-dimensional morphology of the synthesized AgNPs having a part proximately 40 nm, with a spherical shape ( Figure 4).  2 0) and (4 2 2) planes, respectively. These distinct peaks corresponding to the face-centered cubic (fcc) shape of the tested samples confirm the crystalline nature of the biogenic AgNPs (Figure 2), which was consistent with the database of the Joint Committee on Powder Diffraction Standards (JCPDS) (file No. 04-0783). The results of this study were supported by an earlier report [49] highlighting that the XRD spectrum of silver chloride nanoparticles displays solid peaks at 2θ values of 27.9 • , 32.  2 0) and (4 2 2) planes, respectively. The size and shape of the biogenic AgNPs were determined using TEM analysis. The TEM analysis revealed the presence of AgNPs, which were globular in structure with an median size varying from 20 to 60 nm; in addition, the presence of silver peaks in the EDX spectrum confirms the formation of AgNPs (Figure 3a,b). The surface properties of the synthesized biogenic AgNPs were analysed using an atomic force microscope. The results showed a threedimensional morphology of the synthesized AgNPs having a particle size of approximately 40 nm, with a spherical shape ( Figure 4).      In the present report, Copperpod was investigated for the synthesis of AgNP was found to be stable for up to 3 months. The utilization of plant and plant-base ments for the manufacture of AgNPs has been exploited by several researchers. How the mechanism of AgNPs synthesis is not clear; however, most researchers have sug that plant metabolites, such as alkaloids, phenols, tannins, glycosides, enzymes, po charides, proteins and vitamins, may play a major role in the reduction of silver nit AgNPs with good stability [50,51]. Such kinds of phytochemical compounds, incl phenolic compounds, flavonoids, xanthoproteins, tannins, saponins, carboxylic coumarins and carbohydrates, which are obtained from the Copperpod plant, a ported to have a high potential in regard to antimicrobial activity [39]. The biogeni thesis of AgNPs was performed using an aqueous leaf extract of Copperpod, whic then treated with a 1mM solution of silver nitrate to induce the reduction proces visible colour change to a brown colour is attributed to the excitation phenomenon o bands, which is a characteristic feature of synthesized AgNPs. Carmona et al. [5 Banerjee et al. [53] observed similar colour changes during the particle-formation p when the leaf extract was treated with silver nitrate. Thus, the reduction process silver nitrate to AgNPs is coupled with the shift in the pigment of the liquid, esp due to the phenomenon of surface plasmon absorption of AgNPs. Moteriya and C also suggested that the colour change from a colourless solution to a brown colour d the formation of AgNPs using the extract of C. pulcherrima was mainly due to a s plasmon resonance phenomenon [54].
The inductively coupled plasma optical emission spectrometry (ICP-OES) w lized to establish the concentration of the synthesized AgNPs [55]. The results sh In the present report, Copperpod was investigated for the synthesis of AgNPs and was found to be stable for up to 3 months. The utilization of plant and plant-based elements for the manufacture of AgNPs has been exploited by several researchers. However, the mechanism of AgNPs synthesis is not clear; however, most researchers have suggested that plant metabolites, such as alkaloids, phenols, tannins, glycosides, enzymes, polysaccharides, proteins and vitamins, may play a major role in the reduction of silver nitrate to AgNPs with good stability [50,51]. Such kinds of phytochemical compounds, including phenolic compounds, flavonoids, xanthoproteins, tannins, saponins, carboxylic acids, coumarins and carbohydrates, which are obtained from the Copperpod plant, are reported to have a high potential in regard to antimicrobial activity [39]. The biogenic synthesis of AgNPs was performed using an aqueous leaf extract of Copperpod, which was then treated with a 1mM solution of silver nitrate to induce the reduction process. The visible colour change to a brown colour is attributed to the excitation phenomenon of SPR bands, which is a characteristic feature of synthesized AgNPs. Carmona et al. [52] and Banerjee et al. [53] observed similar colour changes during the particle-formation process when the leaf extract was treated with silver nitrate. Thus, the reduction process from silver nitrate to AgNPs is coupled with the shift in the pigment of the liquid, especially due to the phenomenon of surface plasmon absorption of AgNPs. Moteriya and Chanda also suggested that the colour change from a colourless solution to a brown colour during the formation of AgNPs using the extract of C. pulcherrima was mainly due to a surface plasmon resonance phenomenon [54].
The inductively coupled plasma optical emission spectrometry (ICP-OES) was utilized to establish the concentration of the synthesized AgNPs [55]. The results showed that the concentration of AgNPs was 280.5 mg/L. The elemental wavelength of AgNPs was 328.068. Mukherjee et al. [56] reported that the concentrations of a silver solution of bio-synthesized (b-AgNPs) and chemically produced (c-AgNPs) nanoparticles can be quantitatively verified by ICP-OES analysis [57,58]. They showed that the concentration of b-AgNPs and c-AgNPs was 332.8 mg/mL and 383.7 mg/mL, respectively. However, in the present study, the concentration of AgNPs was found to be 280.5 mg/L. Similar findings were reported by other researchers who determined the concentration of AgNPs synthesized using an aqueous extract via the ICP-OES technique [59,60]. The crystalline nature of biogenic AgNPs synthesized from the aqueous leaf extract of Copperpod was analysed by XRD studies, revealing three distinct peaks of 1 1 1, 2 0 0 and 2 2 0, representing Bragg reflections that are reliable with the JCPDS database file No. 04-0783. In consistence with our results, Mishra et al. [61], Khatami et al. [62] and Nasar et al. [63] also recorded similar results of an XRD pattern showing a face-centred cubic (fcc) shape of silver ions.
The biogenic AgNPs synthesized from the aqueous leaf extracts of Copperpod were further characterized by TEM analysis. In the present study, HRTEM analysis confirmed that the shape of the synthesized AgNPs was spherical by an median size varying between 20 nm and 60 nm. The energy dispersive X-ray analysis confirmed the presence of elemental silver ions in the AgNPs synthesized using the aqueous leaf extracts of Copperpod. These results are consistent with the earlier studies performed by Amin et al. [64] and Khalil et al. [65] who also noticed the main peaks representing silver ions via the EDX analysis, thus confirming the synthesis of AgNPs for different plant extracts.
The surface properties of biogenic AgNPs were analysed using an atomic force microscope. The results revealed the presence of spherical nanoparticles with an approximate size of 40 nm. Daphedar and Taranath [66] studied the particle structure and dimension of the AgNPs synthesized using an aqueous leaf extract of Getonia floribunda by AFM. They reported that the synthesized AgNPs were found to be monodispersed spherical-shaped particles with an average size ranging between 10 and 25 nm. Similar findings were similarly described by several scholars who have previously explored the morphology and size of the AgNPs using AFM analysis [67,68].

In Vitro Cytotoxicity Analysis of Biogenic AgNPs by MTT Assay
The in vitro cytotoxic impact of AgNPs on normal Vero cells and cancerous cells, such as HepG2, A549 and MCF-7 cells, was performed with an MTT assay. The IC50 value of AgNPs on Vero cells was found to be 90 µg/mL ( Figure 5). The morphological changes in the cells treated with AgNPs are indicated in Figure 4, where the cells are observed to be shrunken and rounded compared to normal untreated cells. Similarly, the cancerous cells treated with AgNPs showed significant morphological modifications via a phase contrast microscope, as in Figure 6. The IC50 values of AgNPs against cancerous cells, viz., HepG2, A549 and MCF-7 cells, were observed to be 47 µg/mL, 40 µg/mL and 44 µg/mL, respectively.
Recently, cancer-based research associated with nanoparticles has emerged as one of the potential areas of research among the scientific community. Several researchers have focused their studies towards validating the impacts of the various forms of nanoparticles on various cancerous cells [69,70].
Moldovan et al. [71] studied the in vitro cytotoxic properties of the biologically synthesized AgNPs from a Ligustrum ovalifolium extract against ovarian carcinoma cells, i.e., A2780 and A2780-Cis, in a dose-dependent manner. They found that when the concentration of AgNPs is increased from 0.01 to 180 µg/mL, the cells' viability gradually decreases. The IC50 value of biologically synthesized AgNPs against A2780 and A2780-Cis was determined as 7 µg/mL and 14.04 µg/mL, respectively. In the present study, the in vitro cytotoxic effect of AgNPs synthesized from an aqueous leaf extract showing a monodisperse property, which was tested against normal Vero cells and cancerous cells, such as HepG2, A549 and MCF-7 cells, via an MTT assay. The results showed that the biogenic AgNPs have a potential cytotoxic property against all the tested cells. The treated cells showed apparent morphological changes, such as rounded cells, plasma membrane, and chromatin condensation, as compared to normal cells. The IC50 values of AgNPs against cancerous cells were recorded and found to be 69 µg/mL, 62 µg/mL and 53 µg/mL against HepG2, MCF-7 and A549 cells, respectively. The MTT viability assay can enhance knowledge on cell metabolic actions, survival and death. In addition, it is an essential technique used to analyse cytotoxicity, which clarifies the cellular reactions to toxic substances [72]. The AgNPs toxicity results clearly showed a dose-dependent response with exposure time in all the tested cancer cells. This demonstrates a significant increase in cell death with increased dosage of the nanoparticle treatment. The results reveal that the phytosynthesized AgNPs had a higher cytotoxicity on cancer cells as compared to the control.
Processes 2021, 9, x FOR PEER REVIEW 9 of 17 knowledge on cell metabolic actions, survival and death. In addition, it is an essential technique used to analyse cytotoxicity, which clarifies the cellular reactions to toxic substances [72]. The AgNPs toxicity results clearly showed a dose-dependent response with exposure time in all the tested cancer cells. This demonstrates a significant increase in cell death with increased dosage of the nanoparticle treatment. The results reveal that the phytosynthesized AgNPs had a higher cytotoxicity on cancer cells as compared to the control.   Similar findings were observed by Venugopal et al. [73] who synthesized AgNPs using Piper nigrum extract and tested the cytotoxic effectiveness against MCF-7 and HEP-2 cancer cells. They evaluated the cytotoxic activity of AgNPs in a dose-dependent approach using concentrations ranging from 10 to 100 μg and found significant activities against both cancerous cells, i.e., MCF-7 and Hep-2 cells, when related to Piper nigrum extract. Satpathy et al. [74] studied the in vitro cytotoxic effect of AgNPs synthesized using Pueraria tuberosa aqueous extract. A few other reports also suggested that AgNPs had the tendency to reduce the cell's sustainability in a dose-dependent approach, whereby it suppresses the cell proliferation of selective cells via arrest of the G2/M phase [47,75].

Propidium Iodide and Acridine Orange-Ethidium Bromide (AO-EB) Fluorescent Staining
The changes in the morphologic characteristics of cancerous cells, i.e., HepG2, A549 and MCF-7 cells, that were treated with AgNPs were observed under a fluorescent microscope. The AgNPs-treated cells stained with PI revealed the presence of condensation and fragmentation of dead cells (Figure 7). The extent of the apoptotic properties of AgNPs against cancerous cells, such as HepG2, A549 and MCF-7 cells, was analysed with AO-EB Similar findings were observed by Venugopal et al. [73] who synthesized AgNPs using Piper nigrum extract and tested the cytotoxic effectiveness against MCF-7 and HEP-2 cancer cells. They evaluated the cytotoxic activity of AgNPs in a dose-dependent approach using concentrations ranging from 10 to 100 µg and found significant activities against both cancerous cells, i.e., MCF-7 and Hep-2 cells, when related to Piper nigrum extract. Satpathy et al. [74] studied the in vitro cytotoxic effect of AgNPs synthesized using Pueraria tuberosa aqueous extract. A few other reports also suggested that AgNPs had the tendency to reduce the cell's sustainability in a dose-dependent approach, whereby it suppresses the cell proliferation of selective cells via arrest of the G2/M phase [47,75].

Propidium Iodide and Acridine Orange-Ethidium Bromide (AO-EB) Fluorescent Staining
The changes in the morphologic characteristics of cancerous cells, i.e., HepG2, A549 and MCF-7 cells, that were treated with AgNPs were observed under a fluorescent microscope. The AgNPs-treated cells stained with PI revealed the presence of condensation and fragmentation of dead cells (Figure 7). The extent of the apoptotic properties of AgNPs against cancerous cells, such as HepG2, A549 and MCF-7 cells, was analysed with AO-EB dual staining. The fluorescent images of AgNPs-treated cancerous cells, i.e., HepG2, A549, and MCF-7 cells, with corresponding IC50 concentration was analysed using dual staining (AO-EB), which clearly show early apoptotic cells and shrinkage of cells. The presence of orange-coloured cells indicates late apoptotic cells due to the condensation of nuclei, thus confirming the cell's apoptotic property. The untreated (control) cells showed a characteristic bright green colour when stained with AO-EB, confirming the viability of the cells (Figure 7).

DNA Fragmentation Analysis
The apoptosis property of biogenic AgNPs against cancerous cells was analysed by the genomic DNA fragmentation technique, where the genomic DNA of treated cells was extracted and observed by agarose electrophoresis. The IC50 concentration of biogenic AgNPs was used to treat against cancerous cells, such as HepG2, A549 and MCF-7 cells, and the genomic DNA was extracted via electrophoresis using 2% agarose gel. The results showed that the biogenic AgNPs induced DNA fragmentation of the treated cells ( Figure  8) compared to the DNA ladder control cells, which did not show any fragmentation, and this was well-evidenced in our earlier publication [80]. The ladder pattern of DNA damage in the treated cells showed the development of a long smear, whereas the control showed the least damage with no ladder pattern. The early study on the DNA fragmentation in control experiments on HepG2 also confirmed that DNA fragmentation is an important functional key which initiates the process of apoptosis [80,81]. The cells treated with AgNPs showed apoptosis due to the reduction in the percentage of cells in the G1 phase, followed by cell cycle arrest at G2 during mitosis. The occurrence of apoptosis in cancer cells generally occurs with the generation of ROS and mitochondrial disfunction Mittal et al. [13] reviewed the antiproliferative impact of AgNPs made from a medicinal plant (Potentilla fulgens leaf extract) against MCF-7 and U-87 cell lines. The synthesized AgNPs were found to have good activity against both cells, i.e., MCF-7 and U-87, showing an IC50 value of 4.91 and 8.23 µg/mL, respectively. Recently, Chokkalingam et al. [76] synthesized AgNPs from Lycium chinense extract and tested for its cytotoxicity property against the MCF7 cell line. The apoptotic property of biogenic AgNPs synthesized from aqueous leaf extract of Copperpod against normal HepG2, A549 and MCF-7 cells was assessed by propidium iodide and AO-EB dual staining. The AgNPs-treated cancerous cells stained with PI showed the characteristic presence of condensation and fragmentations of dead cells. Similarly, the AgNPs-treated cells stained with an AO-EB dual stain revealed the characteristic appearance of chromatin condensation, a bright green nuclei dot. The presence of late apoptotic cells was observed in the cells treated with AgNPs, showing a characteristic observation of cell shrinkage with membrane blabbing when stained with acridine orange and ethidium bromide; thus, confirming the apoptotic activity of synthesized AgNPs. These observations are consistent with the earlier findings [77][78][79].

DNA Fragmentation Analysis
The apoptosis property of biogenic AgNPs against cancerous cells was analysed by the genomic DNA fragmentation technique, where the genomic DNA of treated cells was extracted and observed by agarose electrophoresis. The IC50 concentration of biogenic AgNPs was used to treat against cancerous cells, such as HepG2, A549 and MCF-7 cells, and the genomic DNA was extracted via electrophoresis using 2% agarose gel. The results showed that the biogenic AgNPs induced DNA fragmentation of the treated cells (Figure 8) compared to the DNA ladder control cells, which did not show any fragmentation, and this was wellevidenced in our earlier publication [80]. The ladder pattern of DNA damage in the treated cells showed the development of a long smear, whereas the control showed the least damage with no ladder pattern. The early study on the DNA fragmentation in control experiments on HepG2 also confirmed that DNA fragmentation is an important functional key which initiates the process of apoptosis [80,81]. The cells treated with AgNPs showed apoptosis due to the reduction in the percentage of cells in the G1 phase, followed by cell cycle arrest at G2 during mitosis. The occurrence of apoptosis in cancer cells generally occurs with the generation of ROS and mitochondrial disfunction which was induced by the cytotoxicity of AgNPs [82]. The DNA damage activity relates to ROS, which act as a signal molecule in the support of cell cycle development. In the progression of DNA damage, the cell is shrunken due to its irregular reduction in size, which finally results in apoptosis. The present results are consistent with studies conducted by Fard et al. [83] who studied the apoptotic property of biogenic AgNPs synthesized using Artemisia oliveriana extract via DNA fragmentation techniques. Similar results were likewise stated by many investigators who have investigated the apoptotic property of AgNPs using a DNA fragmentation assay [78,84].  Mittal et al. [13] reviewed the antiproliferative impact of AgNPs made from a medicinal plant (Potentilla fulgens leaf extract) against MCF-7 and U-87 cell lines. The synthesized AgNPs were found to have good activity against both cells, i.e., MCF-7 and U-87,

Conclusions
The interest in AgNPs has significantly grown due to their tuneable properties and potential applications. Plant-based green synthesis approaches present a non-toxic, costeffective and ecologically safe technology for the large-scale development of AgNPs. Thus, in the current report, the biogenic AgNPs were synthesized employing an aqueous extract of the Copperpod plant. The selected plant extract is known for its high productivity as a reducing and stabilizing agent for the biogenesis of highly stable AgNPs. The analytical method of characterization demonstrated that the synthesized biogenic AgNPs were evenly distributed with no accumulation. The unusual physio-chemical properties of the biogenic AgNPs occurred due to the presence of a broad range of bio-functional compounds in the plant extract which was utilized as a strong reductant during nanoparticle synthesis. In addition, phyto-extracts are the most valuable resource of bioactive elements for the evolution of novel chemotherapeutic agents. The biogenic particles also showed a promising cell cytotoxic activity against the tested cancerous cells, such as HepG2, A549, and MCF-7 cells. The present results indicate that the synthesized biogenic AgNPs are likely to have significant cytotoxic properties, which can be used in various bio-medical-related applications soon.

Conflicts of Interest:
The authors declare no conflict of interest.