Antibacterial, Antibioﬁlm and Anticancer Activity of Biologically Synthesized Silver Nanoparticles Using Seed Extract of Nigella sativa

: Silver nanoparticle (AgNP) based approaches using plant materials have been accepted as biomedical applications. The current study aimed to test the antibacterial, antibioﬁlm, and anticancer activity of silver nanoparticles synthesized by seed extract of Nigella sativa (Ns) as stabilizing and reducing agents. Characterization was done through UV–visible spectroscopy, X-ray di ﬀ raction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electronic microscopy (SEM), and transmission electronic microscopy (TEM) analyses. UV-Vis spectroscopy showed a speciﬁc silver plasmon peak at 400 nm and a quick color change was observed in the bio-reaction medium. Electron microscopic images of Ns-AgNPs identiﬁed as spherical in shape with varied size ranged between 8 and 80 nm and zeta potential analysis evidenced the particles stability and polydisperity. Antibioﬁlm activity of Ns-AgNPs was evident as at 12.5 µ g / mL Ns-AgNps restricted the bioﬁlm formation by 88.42% for Enterococcus faecalis , 84.92% for E. coli , 81.86% for Klebsiella pneumonia , 82.84% for Staphylococcus aureus , and 49.9% for Pseudomonas aeruginosa , respectively. Furthermore, biologically synthesized AgNPs showed the signiﬁcant bacteriostatic and bactericidal activity. Even the lowest concentration of Ns-AgNps restricted the highest rate of inhibition against S. aureus (6.5 and 15 µ g / mL) and E. faecalis (6.5 and 15 µ g / mL). Antimicrobial activity of S. aureus and E. fecalis was more prominent than E. coli (15 and 30 µ g / mL), K. pneumonia (15 and 30 µ g / mL) and P. aeruginosa (30 and 60 µ g / mL) respectively. Moreover, Ns-AgNPs revealed signiﬁcant cytotoxic ability and substantially killed human breast cancer cell (HCC-712) viability. The results of current study advocate that Ns-AgNps may be considered as a potential option in biomedical applications, alternative therapy, designing anti-bioﬁlm agents, treating multi drug resistance bacterial infection, and anti-cancer therapy. The assay ﬁlter and the growth inhibition rates were calculated as a percentage. Growth inhibition A570 treated cells A570 of control AgNPs seed Nigella sativa , , the of (111), (200), (220), (240) crystalline cubic silver di ﬀ raction pattern on Nigella sativa nano in size, crystalline and stable in nature. In this with biosynthesized nanoparticles demonstrated XRD data and The Scherrer equation used to calculate the average size of Ag nanoparticles from extract [30]. e ect S. aureus , and E. faecalis . The current study ﬁndings revealed that Ns-AgNPs showed a signiﬁcant cytotoxic e ﬀ ect on HCC712 breast cancer cell lines. In summary, our results a suggest cost-e ﬀ ective and eco-friendly approach for Ns-AgNps synthesis which may support the advancement of potential biomedical applications and alternative therapy involving the design of novel antibioﬁlm agents and multi drug resistance bacterial infection, and further could be developed as a template for other anti-cancer actions.


Introduction
Currently, modern research in the nanoscience and technology fields are developing rapidly and attracting attention worldwide. In the interest of bio-nanomaterial synthesis, such as gold, copper, platinum, and silver, due to its peculiar design and synthesis process, those metal

Preparation of Aqueous Nigella Sativa Seed Extract
Briefly, 2.5 g of Nigella Sativa seed powder was dissolved in 100 mL of distilled water in a conical flask. The solution was boiled at 70-80 • C for 15-20 min. in a water bath. Solution was filtered at room temperature and stored at 4 • C for further analysis as previously done by Kuppuswamy et al. [18].

Biosynthesis of Silver Nanoparticles from Nigella Sativa
Biogenic AgNps were amalgamated by adding 10 mL of seed extract to 90 mL of 0.1 mM AgNO 3 . The solution obtained was kept in dark condition to avoid auto-oxidation of silver.

Isolation and Purification of AgNPs
After synthesis the reaction mixture was sequentially centrifuged at 8000 rpm and 5000 rpm for 10 min by replacing supernatant with distilled water every time. The pellet form of silver nanoparticles was dried up at 60 • C in hot air oven and was finally diluted in 1% dimethyl sulfoxide (DMSO). The biosynthesized AgNps was stored and employed for further characterization process.

Characterization of Synthesized Ns-AgNps
The reduction of Ag+ ions was observed and recorded by using UV-Vis spectrophotometer (Lambda 35, Perkin-Elmer, USA) at various time intervals in the range of 300 to 800 nm. Fourier transform infrared spectroscopy (FTIR) was utilized to identify the presence of possible biomolecules or functional groups in the aqueous seed extract of Nigella sativa, which are accountable for the reduction of the Ag+ ions and capping of AgNPin the range of 400 to 4000 cm −1 at a resolution of 4 cm −1 at 25 • C using FTIR-Perkin Elmer Spectrum Two model, UK. The X-ray diffraction patterns of the AgNP samples were examined by using XRD-Smart Lab (9kW)-RIKAGU, Japan). The size and morphology of the prepared Ag NPs from the seed extracts were examined by transmission electron microscopy (TEM) JEOL JEM2100 and Scanning electron microscopy JEOL JSM 5500. Dynamic light scattering (DLS) (Zetasizer Nano ZS, Japan) were used to examine the nanoparticle size distribution with measuring and recording the zeta potential.
MIC values of synthesized AgNPs against different bacteria strains were examined by using protocol described by Ansari et al [19]. Briefly, all the bacterial strains were cultured firstly on blood agar plates at 37 • C for overnight and then next day single colony was inoculated into the tryptic soy broth(TSB) for 5-6 h at 37 • C on rotor shaker with 100 rpm to get the bacterial growth of 10 5 to 10 6 (CFU S /mL) for each standard strains. The Ns-AgNPs were serially diluted with different concentrations (6.5-100 µg/mL). Then, 20 µL of bacterial culture of each strain was added into 180 µL TSB and then kept incubator at 37 • C for 24 h, and optical density was measured at 550 nm. The lowest concentration of AgNPs which restricted the 99 % of bacterial growth was considered as MIC. Further to determine the MBC value, 100 µL bacterial inoculums were streaked on the agar plate from the MIC tubes and incubated at 37 • C for overnight. The lowest concentration of Ns-AgNps at which no bacterial growth was seen on inoculated agar plate has been considered as MBC.

Detection of Anti-Biofilm Activity
The anti-biofilm activity of biosynthesized Ns-AgNPs was observed against the biofilm producing standard bacterial strains with or without Ns-AgNPs. Biofilm production of the concerned pathogenic bacteria was carried out using the tissue culture plate (TCP) method as explained by Balasamy et al. [20], which is commonly applied worldwide and considered as standard test for detection of biofilm production with some modifications. MTCC bacterial strains of K. pneumonia, E. coli, S. aureus, P. aeruginosa, and E. faecalis were grown on blood agar plate for overnight at 37 • C and the next day a single colony of all bacteria were inoculated into different conical flasks containing the 100 mL TSB at 37 • C for 6-7 h with shaking at 100 rpm until to obtain approximately 2.5 × 10 8 CFUs/mL. These bacterial inoculums were further diluted (1:100) with fresh TSB medium to approximately 10 6 CFUs/mL and transferred it to 96 wells flat bottom TCPs and incubated at 37 • C for overnight. After completing the incubation, the old culture medium was changed with a fresh TSB medium including different concentrations of Ns-AgNPs (6.5-100 µg/mL), without disrupting the biofilm. Samples with AgNPs were incubated further at 37 • C for 24 h. Thereafter, the medium was discarded; each well were gently washed two times with sterile 1x Phosphate buffer saline (PBS) to get rid of the planktonic state or free floating bacteria and dried it at room temperature for 20 min. 0.1% crystal violet solution was added to each well for 15 min to stain the biofilms. The surplus stain was eliminated by washing three times with sterile 1% PBS, and dried at room temperature for 30 min. 100 mL of 95% ethanol was then added to each well. Optical densities (OD) of stained acquired biofilm were observed by a micro ELISA reader at wavelength 595 nm. Experiments were repeated in triplicate. Average OD values of sterile medium were measured and deducted from the all test values. [21]. The OD of sample was converted to percentage of biofilm inhibition, calculated as follows: Percentage of biofilm = OD of the test/OD of the control × 100 2.8. Determination of In Vitro Anticancer Activity of Synthesized AgNPs 2.8.1. Cell Culture and Cell Line Maintenance Breast cancer cell line (HCC712) was acquired from National Centre for Cell Science (NCCS), Pune, India. The HCC712 cells were freshly cultivated as monolayer in Dulbecco's Modified Eagles's Medium (DMEM), supplemented with 10% FBS, 1% glutamine, and 100 U/mL penicillin/streptomycin and incubated at 37 • C in 5% CO 2 atmosphere. It was grown in a 75 cm 2 tissue culture flask.

Cell Viability Test
The colorimetric MTT assay was applied to scrutinize the cytotoxic effects. The activity of Ns-AgNPs was examined against HCC712 cell lines (1 × 10 6 cells/mL) and the culture was seeded in 96 flat-bottom well plate that was suitable for high throughput screening. Different concentrations (25 to 200 µg/mL) of silver nanoparticles were added to the cultures and incubated for 24 h in 5% CO 2 atmosphere. After 24 h, the cells were washed with PBS followed by mixing of 100 µL of MTT, and the cells were again incubated for 3-4 hr at 37 • C in 5% CO 2 atmosphere. The formazan crystals were suspended in 200 µL of DMSO and optical density of each well was observed. The quantity of formazan product as measured in the calorimetric assay at 570 nm with a reference filter at 630 nm determined and the growth inhibition rates were calculated as a percentage. Growth inhibition = A570 of treated cells/A570 of control cells × 100.

Measurement of Cytomorphological Changes in HCC712
After the adequate growth of HCC712 cells in tissue culture flask, it was treated with different concentrations of Ns-AgNPs and then incubated for 24 h at 37 • C in 5% CO 2 atmosphere. After the incubation, the gross morphological changes in the cells were examined by bright field microscopy.

Statistical Analysis
All antibacterial and cytotoxic activities were performed with minimum of three replicates. Means, standard deviations, error bars were calculated using Microsoft excel. The error bars are given for each bar showing a 95% confidence interval.

Ethics Statement
As this experiment was totally based on in-vitro study using bacterial isolates to evaluate the anti-microbial and anti-biofilm activity of black seed silver nanoparticle, ethical review is not required.

Results and Discussion
The synthesis of Ns-AgNPs was primarily authenticated visually and then by using UV-visible spectroscopy which has been commonly applied to characterize the synthesized metal nanoparticles. After mixing the silver nitrate into the homogenized extract, a change in colour from pale yellow to tan-brownish colour was observed due to a surface plasmon resonance phenomenon, which confirmed the formation of AgNPs in the reaction mixture.

UV-Vis Spectrum
UV-Vis spectroscopy is one of the best applicable tools to confirm the formation of AgNPs. Moreover, surface plasmon resonance (SPR) patterns are frequently utilized as suggestive tools for metal nanoparticles formation as SPR depends on a number of parameters, including size and medium dielectric constant [22]. The maximum absorption spectrum was determined between 300-800 nm ( Figure 1). Moreover, characteristic peak at 400 nm in the UV-Vis spectrum was observed and this study specifically provides evidence of formation of biosynthesized silver nanoparticles from Nigella sativa seeds extract ( Figure 1). This result is in consistent with the previous study, which exhibited that the synthesis of silver nanoparticles from Phoenix dactylifera (date palm) seeds and recorded the plasma resonance peak at 459 nm [11]. In this context, another study result revealed that a reduction of silver ions was observed between 300 and 600 nm and peak absorption was monitored at 440 nm by UV-Vis spectroscopy [23].

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The synthesis of Ns-AgNPs was primarily authenticated visually and then by using UV-visible 193 spectroscopy which has been commonly applied to characterize the synthesized metal nanoparticles.

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After mixing the silver nitrate into the homogenized extract, a change in colour from pale yellow to 195 tan-brownish colour was observed due to a surface plasmon resonance phenomenon, which 196 confirmed the formation of AgNPs in the reaction mixture.

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UV-Vis spectroscopy is one of the best applicable tools to confirm the formation of AgNPs.

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Moreover, surface plasmon resonance (SPR) patterns are frequently utilized as suggestive tools for 200 metal nanoparticles formation as SPR depends on a number of parameters, including size and 201 medium dielectric constant [22]. The maximum absorption spectrum was determined between 300-202 800 nm ( Figure 1). Moreover, characteristic peak at 400 nm in the UV-Vis spectrum was observed and 203 this study specifically provides evidence of formation of biosynthesized silver nanoparticles from 204 Nigella sativa seeds extract ( Figure 1). This result is in consistent with the previous study, which 205 exhibited that the synthesis of silver nanoparticles from Phoenix dactylifera (date palm) seeds and 206 recorded the plasma resonance peak at 459 nm [11]. In this context, another study result revealed that 207 a reduction of silver ions was observed between 300 and 600 nm and peak absorption was monitored 208 at 440 nm by UV-Vis spectroscopy [23].

FTIR Spectroscopy
FTIR spectroscopy, an analytical technique, was employed to discover the biomolecules of Nigella sativa seeds extract that were bound particularly on the synthesized AgNPs. FTIR results (Figure 2A) for Nigella sativa showed the compounds responsible for the synthesis of AgNPs. FTIR spectroscopy was scanned at a range 400-4000 cm −1 of resolution to ensure the formation of silver nanoparticles. Bands were observed at 3421. 30 This finding was supported by the previous findings that were conducted on different plant extract [24][25][26]. In this regard, silver nanoparticle using garlic extract reported stretching peaks at 3270 and 2930 cm −1 , probably belonging to -O-H and -C-H peaks, correspondingly, which match to sugars in the garlic extract. Furthermore, it is identified that garlic hold sugars including sucrose and fructose, which play role as reducing and stabilizing agents [27]. So, based on finding FTIR results confirmed that biomolecules and some proteins are present in the extract sample to protect it from further changes and responsible for capping, stabilization and reduction of Ag + to AgNPs.

X-ray Diffraction Analysis
XRD analysis was performed to examine the crystalline characteristics of the biosynthesized AgNPs produced by the seed of Nigella sativa seeds extract. The X-ray diffraction pattern of the biosynthesized AgNPs procured by the seed of Nigella sativa extract was determined at 29.18 • , 34.18 • , 39.45 • , 47.18 • and can be indexed to the angle values of (111), (200), (220), (240) crystalline planes of cubic silver ( Figure 2C).The diffraction pattern result based on Nigella sativa extract established the formation of particles was nano in size, crystalline and stable in nature. In this sense, similar observations with biosynthesized nanoparticles have been demonstrated [28,29]. XRD data and The Scherrer equation was used to calculate the average size of Ag nanoparticles from extract [30].   observations with biosynthesized nanoparticles have been demonstrated [28,29]. XRD data and The

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Scherrer equation was used to calculate the average size of Ag nanoparticles from extract [30].

SEM and TEM Analysis
SEM analysis was done to check the dimensions and shape of the nanoparticles. The AgNPs synthesized from Nigella sativa were of uniform size disseminated evenly and spherical in shape ( Figure 3A,B). The result of SEM analysis of biosynthesized silver nanoparticles from extract was obviously differentiable due to their size difference. The current results were in accordance with that of Otunola et al. justified the similar range of Ag NPs from pepper and garlic [31]. EDX technique was used to observe the elemental composition of biosynthesized Ag nanoparticles in solution. In X-ray spectrum, all the elements showed a unique set of peaks with different atomic structure as shown in ( Figure 3C). Meanwhile, silver metal exhibited a strong signal in EDX image and weight percent 40% clearly indicated the presence of metallic silver nanocrystals ( Figure 3C,D).
The most appropriate microscopic technique to investigate the size and morphology of AgNPs is TEM [32]. TEM was used to determine the biosynthesized AgNPs (Appendix A) in nanoscale from extract. TEM analysis confirmed the presence of AgNPs in the sample of N. sativa average size ranging between 8 and 80 nm, smaller size of particle good indicator for effective performance and quality of AgNPs ( Figure 4A,B). The previous report also showed that the AgNPs obtained from seed extract of Phoenix dactylifera were mostly spherical in shape and similar sizes ranging [33]. Another pioneer study result revealed that TEM image of AgNPs were found similar shape and with narrow distribution [34]. DLS was also used to investigate the particle size distribution, and revealed the particle size 8-80 nm with average particle size 55 nm, as shown in ( Figure 4C). Similar size distribution studies have been reported previously [33].

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The most appropriate microscopic technique to investigate the size and morphology of AgNPs 257 is TEM [32]. TEM was used to determine the biosynthesized AgNPs (Appendix A) in nanoscale from 258 extract. TEM analysis confirmed the presence of AgNPs in the sample of N. sativa average size 259 ranging between 8 and 80 nm, smaller size of particle good indicator for effective performance and 260 quality of AgNPs ( Figure 4A, B). The previous report also showed that the AgNPs obtained from 261 seed extract of Phoenix dactylifera were mostly spherical in shape and similar sizes ranging [33].   (Table 1). In addition, Ns-AgNps concentration against 276 above mentioned bacterial strains showed 6.5-30 µg/ml concentration for MIC and MBC values were 277 15-60 µg/ml. Our above-mentioned results revealed that Ns-AgNps exhibited excellent antibacterial 278 activity and was in accordance with the findings of a previous study [35]. Silver nanoparticle play an 279 important role in bactericidal, bacteriostatic and also kill the other types of pathogens. Sadeghi et al.

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evaluated the bactericidal effect of silver nanoparticles and chlorhexidine against S. mutans to observe 281 that silver nanoparticles showed a statistically significant bactericidal effect as compared to Enterococcus faecalis (MTCC 439) 6 . 5 1 5

Assessment of MIC and MBC Activity
Initially, the effect of Ns-AgNps was investigated against E. coli, K. pneumonia, P. aeruginosa, S. aureus and E. faecalis bacterialstrains. Ns-AgNps showed the significant bacteriostatic and bactericidal activity. Even the lowest concentration of Ns-AgNps restricted the highest rate of inhibition against S. aureus (6.5 and 15 µg/mL) and E. faecalis (6.5 and 15 µg/mL). Antimicrobial activity of S. aureus and E. fecalis was more pronounced than E. coli (15 and 30 µg/mL), K. pneumonia (15 and 30 µg/mL), and P. aeruginosa (30 and 60 µg/mL) respectively (Table 1). In addition, Ns-AgNps concentration against above mentioned bacterial strains showed 6.5-30 µg/mL concentration for MIC and MBC values were 15-60 µg/mL. Our above-mentioned results revealed that Ns-AgNps exhibited excellent antibacterial activity and was in accordance with the findings of a previous study [35]. Silver nanoparticle play an important role in bactericidal, bacteriostatic and also kill the other types of pathogens. Sadeghi et al. evaluated the bactericidal effect of silver nanoparticles and chlorhexidine against S. mutans to observe that silver nanoparticles showed a statistically significant bactericidal effect as compared to chlorhexidine [36]. Staphylococcus aureus (MTCC 3160) 6.5 15 5 Enterococcus faecalis (MTCC 439) 6.5 15

Anti-Biofilm Activity
The results of tissue culture plate method demonstrated that antibiofilm activity of the biosynthesized AgNPs from N. sativa seed extract was 88.42% for E. faecalis, 84.92% for E. coli, 81.86% for K. pneumonia, and 82.84% for S. aureus, respectively at 12.5 µg/mL, while with the same concentration, the biofilm formation was reduced by 49.9% in the case of P. aeruginosa ( Figure 5). Moreover, the evidence for screening the biofilm inhibition in the presence of Ns-AgNPs is shown in Figure 6. These findings suggest that the uptake of the biosynthesised Ns-AgNPs could diminish the biofilm forming abilities of tested pathogens. Similar studies were documented as results of biofilm inhibition at 100 µg/mL AgNPs [37]. Moreover, another study based on anti-biofilm demonstrated that the AgNPs synthesized from Momordica charantia extract were exhibited improved anti-biofilm activity against E.faecalis and A. hydrophilia. Additionally, the antibiofilm activity of AgNPs was attributed to the diffusion through the biofilms imparting antimicrobial actions [38].

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for K. pneumonia, and 82.84% for S. aureus, respectively at 12.5 µg/ml, while with the same 289 concentration, the biofilm formation was reduced by 49.9% in the case of P. aeruginosa ( Figure 5).

Effect of Ns-AgNPs Against Human (HCC712) Cell Lines
The cytotoxic effect of Ns-AgNPs in the current study was examined against HCC712 (human breast cancer cells) using MTT assay. The toxicities were examined at different concentrations (25 to 200 µg/mL) and compared to control. The percent inhibition of cell growth was increased with the concentration of the nanoparticles (Figure 7). The inhibition effect was time and dose dependent and the IC 50 value of 150 µg/mL supports the levels of the inhibition.    It was observed that a high concentration of Ns-AgNPs showed promising activity against breast cancer cell lines and cell proliferation decreased after 24 h. Moreover, findings exhibited that 150 µg/mL concentration of AgNPs is able to provoke the approximately 50% of cell mortality at different time intervals. The cell viability in the presence of AgNPs at different concentrations is illustrated in (Figure 8 A-F). The calculated IC50 value is approximately 175 µg/mL concentrations of Ns-AgNPs.
Previously, similar findings were reported by Khateef et al. [39] in the form of a cytotoxicity effect in human breast cancer cells (MCF-7) against green synthesized AgNPs procured from Buchanania axillaris extracts. The morphological observation of the HCC712 cancer cells exhibited significant morphological changes and the cell density was decreased, which is a predictable mark of apoptotic cells (Figure 9). cancer cell lines and cell proliferation decreased after 24 hours. Moreover, findings exhibited that 150 318 µg/mL concentration of AgNPs is able to provoke the approximately 50% of cell mortality at different    It was observed that a high concentration of Ns-AgNPs showed promising activity against breast 317 cancer cell lines and cell proliferation decreased after 24 hours. Moreover, findings exhibited that 150 318 µg/mL concentration of AgNPs is able to provoke the approximately 50% of cell mortality at different

Conclusions
The study provides another example of an easy, non-toxic, and low cost, preparation of nanoparticles by Nigella sativa seed extract without any involvement of toxic chemical reducing agents. The biologically synthesized particles exhibited significant levels of activity such as an antimicrobial effect and the inhibition of biofilm formation against E. coli, K. pneumonia, P. aeruginosa, S. aureus, and E. faecalis. The current study findings revealed that Ns-AgNPs showed a significant cytotoxic effect on HCC712 breast cancer cell lines. In summary, our results a suggest cost-effective and eco-friendly approach for Ns-AgNps synthesis which may support the advancement of potential biomedical applications and alternative therapy involving the design of novel antibiofilm agents and multi drug resistance bacterial infection, and further could be developed as a template for other anti-cancer actions.