processes

Article
Antibacterial, Antibiofilm and Anticancer Activity of
Biologically Synthesized Silver Nanoparticles Using
Seed Extract of Nigella sativa
Ahmad Almatroudi 1, *, Habeeb Khadri 1 , Mohd Azam 1 , Arshad Husain Rahmani 1 ,
Fahd Khaleefah Al Khaleefah 2 , Riazunnisa Khateef 3, *, Mohammad Azam Ansari 4 and
Khaled S. Allemailem 1
1 Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Qassim 51452,
Saudi Arabia; kdry@qu.edu.sa (H.K.); m.aftab@qu.edu.sa (M.A.); ah.rahmani@qu.edu.sa (A.H.R.);
k.allemailem@qu.edu.sa (K.S.A.)
2 Infection Prevention and control Department, Al Rass General Hospital, Al Qassim 58867, Saudi Arabia;
fkalkhaleefah@moh.gov.sa
3 Departments of Biotechnology and Bioinformatics, Yogi Vemana University, Kadapa 516005, AP, India
4 Department of Epidemic Disease Research, Institutes of Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, Dammam 31441, Saudi Arabia; maansari@iau.edu.sa
* Correspondence: aamtrody@qu.edu.sa (A.A.); krbtbi@yogivemanauniversity.ac.in (R.K.)




Received: 2 March 2020; Accepted: 20 March 2020; Published: 26 March 2020


Abstract: 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 diffraction
(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 µg/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 µg/mL)
and E. faecalis (6.5 and 15 µg/mL). Antimicrobial activity of S. aureus and E. fecalis was more prominent
than E. coli (15 and 30 µg/mL), K. pneumonia (15 and 30 µg/mL) and P. aeruginosa (30 and 60 µg/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.

Keywords: anti-biofilm; anti-cancer; HCC-712 cell lines; Nigella sativa; silver nanoparticle

1. 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

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Processes 2020, 8, 388 2 of 14

nanoparticles ranging between 1 and 100 nanometers have drawn great attention towards potential
applications in multi-disciplinary areas such as biomedicine, environment, biotechnology, drug delivery,
and nanomedicine [1]. Physical and chemical methods for the synthesis of metal nanoparticles are limited
because of its costly equipment’s, toxicity, and environmental damage [2]. Currently, green-approach
technology is attracting consideration due to its being a biogenic, non-toxic, and cheap approach that
has become a central focusing area of research, leading to explore various types of bio-reducing agents.
Many researchers reported that microbes, plants and their products, such as Ananas comosus (L.), Cynara
scolymus, Oscillatoria limnetica, and Phoenix sylvestris L., have been used as bio-reducing agents [3–6] to
design and manipulate the metal particles for acquiring unique shape and size.
The constantly increasing antibiotic resistance in clinical strains is a serious concern to global
health sector that needs an urgent action. Therefore, discovering the new substitutes to the presently
applied antibiotics has become an essential task. Nayak et al synthesized crystalline and spherical
AgNPs using bark extracts showedantibacterial activity against gram-positive and -negative bacteria [7].
However, silver nanoparticles have been utilized in various sectors mainly in health and medicine
industry because of its excellent antimicrobial properties [8]. The biofilm formation is a critical method
applied by different pathogenic bacteria to stay lively in environment. Biofilm forming microbes can
cause numerous serious infections such as eye, skin, wound urinary and heart leading high mortality
and morbidity due to biomedical devices [9]. Traditional treatment for bacterial infection is commonly
based on bactericidal or bacterial static agents. Some pathogens showed protection against drugs due
to the development of biofilm. Furthermore, in the virulence of various pathogenic microbes, biofilms
play a vital role as well. In these circumstances, the bioscience and pharmaceutical applications have
been revolutionized due to the discovery of new biocide targeting biofilm [10]. According to the
National Institute of Health (NIH), approximately sixty percent of microbial infections have been found
to be association of biofilm. Various biofilm associated bacteria have proven ineffective to chemical
disinfectants, germicides, and antibiotics [11].
All over the world, women breast cancer has grown an immense concern, particularly with
treatment procedures which can cause adverse side effects [12]. Nowadays, different types of cancer
are spreading day by day, so there is an immediate requirement to discover and identify new anticancer
drugs with less side effects on the immune system [13]. In spite of various attempts, chemotherapy of
cancerous cells is not fully successful due to multi drug resistance [14]. However, silver nanoparticles
have been demonstrated for potential role in anticancer activity [15].
Black seed is indigenous to North Africa, Southwest Asia, and Southern Europe and it is farmed
in many countries like Saudi Arabia, Middle Eastern Mediterranean region, India, Pakistan, South
Europe, Turkey, Syria. Nigella sativa posesses therapeutic potential and biological activities and it
acquires broad spectrum of activities such as antioxidant, diuretic, antidiabetic, antihypertensive,
anticancer, antimicrobial, immunomodulatory, anti-schistosomiasis, anti-inflammatory, analgesics,
bronchodilator, spasmolytic, hepatoprotective, gastro protective, and renal protective [16]. Nigella
sativa oil is multipurpose product with nutritional and medicinal properties [17]. Several studies have
shown the application of AgNPs in different fields, but scanty reports are available to exhibit the role
of AgNPs as anti-biofilm activity as well as anti-cancerous activity. The aim of the current study was (i)
biosynthesis of AgNps using seed extract of Nigella sativa, (ii) characterization of prepared AgNPs
by different sophisticated techniques, (iii) investigation of antibacterial and anti-biofilm potential of
synthesized AgNPs against different bacterial strains for biomedical application, and (iv) evaluation of
the cytotoxic effect of biosynthesized AgNPs on human breast cancer (HCC-712) cell line for therapeutic
application in the treatment of breast cancer.
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2. Material and Methods

2.1. Bacterial Strains, Culture Media, Seeds and Chemicals

Nigella sativa seeds (Ns) were obtained from a local market, Qassim, Saudi Arabia. Analytical
grade Silver nitrate (AgNO3 ), were procured from Sigma- Aldrich Company. Breast cancer cell line
(HCC712) was acquired from National Centre for Cell Science (NCCS), Pune, India. Other reagents
and culture Medias were bought from Hi-Media, India. Bacterial cultures of K. pneumoniae (MTCC
618), E. coli (MTCC 40), S. aureus (MTCC 3160), P. aeruginosa (MTCC 1688) and E. faecalis (MTCC 439)
were obtained from Institute of microbial technology, IMTECH, Chandigarh, India.

2.2. 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].

2.3. 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 AgNO3 .
The solution obtained was kept in dark condition to avoid auto-oxidation of silver.

2.4. 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.

2.5. 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.

2.6. Assessment of Minimum Inhibitory Concentrations (MICs) and Minimum Biocidal Concentration
(MBCs) Activity
To evaluate the antibacterial activity of Ns-AgNps, it was tested against the bacterial strains such
as K. pneumoniae (MTCC 618), E. coli (MTCC 40), S. aureus (MTCC 3160), P. aeruginosa (MTCC 1688),
and E. faecalis (MTCC 439).
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
105 to 106 (CFUS /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
Processes 2020, 8, 388 4 of 14

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.

2.7. 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 × 108 CFUs/mL. These
bacterial inoculums were further diluted (1:100) with fresh TSB medium to approximately 106 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% CO2 atmosphere. It was grown in a 75 cm2 tissue culture flask.

2.8.2. 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 × 106 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%
CO2 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% CO2 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.
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2.8.3. 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% CO2 atmosphere. After the
incubation, the gross morphological changes in the cells were examined by bright field microscopy.

2.9. 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
Processes 2019, 7, x FOR PEER REVIEW 5 of 14
bar showing a 95% confidence interval.
184 All antibacterial and cytotoxic activities were performed with minimum of three replicates.
2.10.
185Ethics Statement
Means, standard deviations, error bars were calculated using Microsoft excel. The error bars are given
186 for each bar showing a 95% confidence interval.
187As 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.
188 2.10. Ethics Statement
3. 189
Results and As Discussion
this experiment was totally based on in-vitro study using bacterial isolates to evaluate the
190 anti-microbial and anti-biofilm activity of black seed silver nanoparticle, ethical review is not
The synthesis of Ns-AgNPs was primarily authenticated visually and then by using UV–visible
191 required.
spectroscopy which has been commonly applied to characterize the synthesized metal nanoparticles.
192mixing
After 3. Results and Discussion
the silver nitrate into the homogenized extract, a change in colour from pale yellow to
193
tan-brownish colour
The wasofobserved
synthesis Ns-AgNPsdue wasto a surface
primarily plasmonvisually
authenticated resonance phenomenon,
and then which confirmed
by using UV–visible
the194 spectroscopy
formation of AgNPs whichin has been
the commonly
reaction applied to characterize the synthesized metal nanoparticles.
mixture.
195 After mixing the silver nitrate into the homogenized extract, a change in colour from pale yellow to
196
3.1. UV-Vistan-brownish
Spectrum colour was observed due to a surface plasmon resonance phenomenon, which
197 confirmed the formation of AgNPs in the reaction mixture.
UV–Vis spectroscopy is one of the best applicable tools to confirm the formation of AgNPs.
198 3.1. UV-Vis Spectrum
Moreover, surface plasmon resonance (SPR) patterns are frequently utilized as suggestive tools for metal
199
nanoparticles UV–Vis
formation spectroscopy is one of on
as SPR depends the abest applicable
number tools to confirm
of parameters, the formation
including size and of medium
AgNPs. dielectric
200 Moreover, surface plasmon resonance (SPR) patterns are frequently utilized as suggestive tools for
constant
201 [22]. The maximum absorption spectrum was determined between 300–800 nm (Figure 1).
metal nanoparticles formation as SPR depends on a number of parameters, including size and
Moreover,
202 characteristic
medium dielectricpeak at 400
constant [22].nm
The in the UV-Vis
maximum spectrum
absorption waswas
spectrum observed andbetween
determined this study
300- specifically
203
provides evidence
800 nm (Figure of formation
1). Moreover,of biosynthesized
characteristic peak at silver
400 nm nanoparticles from Nigella
in the UV-Vis spectrum sativaand
was observed seeds extract
204 1).
(Figure thisThis
studyresult
specifically
is in provides
consistentevidence
with oftheformation
previous of biosynthesized
study, whichsilver nanoparticles
exhibited fromsynthesis of
that the
205 Nigella sativa seeds extract (Figure 1). This result is in consistent with the previous study, which
silver
206nanoparticles from Phoenix dactylifera (date palm) seeds and recorded the plasma resonance peak at
exhibited that the synthesis of silver nanoparticles from Phoenix dactylifera (date palm) seeds and
459207
nm [11]. In this
recorded context,
the plasma another
resonance study
peak at 459result revealed
nm [11]. that aanother
In this context, reduction
studyof silver
result ionsthat
revealed was observed
208 300
between and 600
a reduction nm and
of silver ions peak absorption
was observed betweenwas300monitored
and 600 nm atand440 nm
peak by UV-Vis
absorption was spectroscopy
monitored [23].
209 at 440 nm by UV-Vis spectroscopy [23].

210
211 Figure 1. UV absorption spectrum of the AgNPs synthesized from Nigella sativa seed extract (Inset:
UV absorption
212Figure 1.Change of color fromspectrum of light
dark brownto the golden
AgNPs synthesized from Nigella sativa seed extract (Inset:
yellow).
Change of color from dark brownto light golden yellow).
213 3.2. FTIR Spectroscopy
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3.2. FTIR Spectroscopy

214 FTIR spectroscopy, an analytical technique, was employed to discover the biomolecules of
215 NigellaFTIR
sativaspectroscopy,
seeds extract an analytical
that weretechnique, was employed
bound particularly on to discover
the the biomolecules
synthesized AgNPs. FTIR of Nigella
results
216 sativa
(Figure 2A) for Nigella sativa showed the compounds responsible for the synthesis of AgNPs.2A)
seeds extract that were bound particularly on the synthesized AgNPs. FTIR results (Figure FTIR
217 for Nigella sativa
spectroscopy wasshowed
scannedthe atcompounds responsible
a range 400–4000 cm-1 for the synthesis
of resolution to of AgNPs.
ensure theFTIR spectroscopy
formation of silver
−1 of resolution to ensure the formation of silver nanoparticles.
218 was scanned Bands
nanoparticles. at a rangewere400–4000
observed cmat 3421.30, 2923.92, 2350.08, 1019.06, 666.76, and 2926 cm-1. The
−1 . The bands at 3421.30
219 Bands were observed at 3421.30, 2923.92,
bands at 3421.30 and 2923.92 cm-1 demonstrated 2350.08, 1019.06, 666.76, and
the reduction of 2926 cmions
silver in NPs. The band at
and 2923.92 cm−1 demonstrated the reduction of silver ions in NPs. The band at 1019.06 cm−1 showed
220 1019.06 cm-1 showed the N-H bonding vibrate of amides. The band in seed extract at−1 1019.06 to 666.76
the N-H bonding vibrate of amides. The band in seed extract at 1019.06 to 666.76 cm after the bio
221 cm after the bio reduction of AgNP’s pointed out the C=C stretching mode in the aromatic
-1
reduction of AgNP’s pointed out the C=C stretching mode in the aromatic compounds which validated
222 compounds which validated the presence of aromatic compounds like flavonoids. The peaks
the presence of aromatic compounds like flavonoids. The peaks observed at the 605 cm−1 confirms the
223 observed at the 605 cm confirms the C-S stretch (Figure 2B).
-1
C-S stretch (Figure 2B).
224 This finding was supported by the previous findings that were conducted on different plant
This finding was supported by the previous findings that were conducted on different plant
225 extract [24-26]. In this regard, silver nanoparticle using garlic extract reported stretching peaks at 3270
extract [24–26]. In this regard, silver nanoparticle using garlic extract reported stretching peaks at
226 and
3270 andcm
2930 2930, cm
−1 probably belonging to –O–H and –C–H peaks, correspondingly, which match to
−1 , probably belonging to –O–H and –C–H peaks, correspondingly, which match to
227 sugars
sugarsininthe
thegarlic
garlicextract.
extract.Furthermore,
Furthermore, itit is
is identified thatgarlic
identified that garlichold
holdsugars
sugarsincluding
including sucrose
sucrose andand
228 fructose, which play role as reducing and stabilizing agents [27]. So, based on
fructose, which play role as reducing and stabilizing agents [27]. So, based on finding FTIR resultsfinding FTIR results
229 confirmed
confirmedthat thatbiomolecules
biomoleculesand and some
some proteins are present
proteins are presentininthe
theextract
extractsample
sample toto protect
protect it from
it from
230 further changes and responsible for capping, stabilization and reduction
further changes and responsible for capping, stabilization and reduction of Ag to AgNPs. of +
Ag+ to AgNPs.

231 3.3.
3.3. X-ray
X-ray DiffractionAnalysis
Diffraction Analysis

232 XRDXRDanalysis
analysiswas
wasperformed
performed to
to examine
examine thethe crystalline
crystallinecharacteristics
characteristicsofof
thethe
biosynthesized
biosynthesized
233 AgNPs produced by the seed of Nigella sativa seeds extract. The X-ray diffraction
AgNPs produced by the seed of Nigella sativa seeds extract. The X-ray diffraction pattern pattern of of
thethe
◦ , 34.18◦ ,
234 biosynthesized AgNPs procured by the seed of Nigella sativa extract was determined at 29.18
biosynthesized AgNPs procured by the seed of Nigella sativa extract was determined at 29.18o, 34.18o,
◦ , 47.18◦ and can be indexed to the angle values of (111), (200), (220), (240) crystalline planes
235 39.45
39.45 o, 47.18 o and can be indexed to the angle values of (111), (200), (220), (240) crystalline planes of
of cubic silver (Figure 2C).The diffraction pattern result based on Nigella sativa extract established
236 cubic silver (Figure 2C).The diffraction pattern result based on Nigella sativa extract established the
the formation of particles was nano in size, crystalline and stable in nature. In this sense, similar
237 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
238 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].
239 Scherrer equation was used to calculate the average size of Ag nanoparticles from extract [30].

240 Figure 2. (A) FTIR spectrum of Nigella sativa seed extract (B) FTIR spectrum of AgNp’s (C) XRD
241 spectrum
Figure of AgNp’s
2. (A) of Nigellaof
FTIR spectrum Sativa.
Nigella sativa seed extract (B) FTIR spectrum of AgNp’s (C) XRD
242 spectrum of AgNp’s of Nigella Sativa.

243 3.4. SEM and TEM Analysis

244 SEM analysis was done to check the dimensions and shape of the nanoparticles. The AgNPs
245 synthesized from Nigella sativa were of uniform size disseminated evenly and spherical in shape
 Processes 2020, 8, 388 7 of 14

3.4. 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).
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The most appropriate microscopic technique to investigate the size and morphology of AgNPs is
246 TEM [32].
(Figure 3A, TEM
B). The was usedof
result toSEM
determine theofbiosynthesized
analysis biosynthesizedAgNPssilver (Appendix
nanoparticlesA) from
in nanoscale from
extract was
247 extract. TEM analysis confirmed the presence of AgNPs in the sample of N. sativa average
obviously differentiable due to their size difference. The current results were in accordance with that size ranging
248 ofbetween
Otunola8 et and al.80 nm, smaller
justified size ofrange
the similar particle
of good
Ag NPsindicator for effective
from pepper performance
and garlic [31]. EDXandtechnique
quality of
249 AgNPs
was used(Figure
to observe 4A,B).
theThe previous
elemental report alsoof
composition showed that the AgNPs
biosynthesized obtained from
Ag nanoparticles seed extract
in solution. In X-of
250 Phoenix dactylifera were mostly spherical in shape and similar sizes ranging [33]. Another
ray spectrum, all the elements showed a unique set of peaks with different atomic structure as shown pioneer study
251 result revealed that TEM image of AgNPs were found similar shape and with
in (Figure 3C). Meanwhile, silver metal exhibited a strong signal in EDX image and weight percentnarrow distribution [34].
252 DLSclearly
40% was also used tothe
indicated investigate
presencethe particle silver
of metallic size distribution,
nanocrystals and revealed
(Figure the particle size 8–80 nm
3C,D).
with average particle size 55 nm, as shown in (Figure 4C). Similar size distribution studies have been
reported previously [33].

Figure 3. SEM and EDX images of synthesized AgNPs. (A,B) SEM micrograph of AgNPs at lower
(X3000), and higher magnification (X12000), (C,D) represent EDX images and weight % of elements.
253
254 Figure 3. SEM and EDX images of synthesized AgNPs. (A) and (B) SEM micrograph of AgNPs at
255 lower (X3000) ,and higher magnification (X12000), (C, D) represent EDX images and weight % of
256 elements.

257 The most appropriate microscopic technique to investigate the size and morphology of AgNPs
258 is TEM [32]. TEM was used to determine the biosynthesized AgNPs (Appendix A) in nanoscale from
259 extract. TEM analysis confirmed the presence of AgNPs in the sample of N. sativa average size
260 ranging between 8 and 80 nm, smaller size of particle good indicator for effective performance and
261 quality of AgNPs (Figure 4A, B). The previous report also showed that the AgNPs obtained from
262 seed extract of Phoenix dactylifera were mostly spherical in shape and similar sizes ranging [33].
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267
268 TEM images
Figure 4. TEM images of
of Ns-AgNPs
Ns-AgNPs scale
scale bar
bar correspond
correspond to
to 0.5
0.5 µm
µm (A) 100 nm (B) particle size
269 distribution by DLS (C).

3.5. Assessment of MIC and MBC Activity

270 3.5. Assessment of MIC and MBC activity
Initially, the effect of Ns-AgNps was investigated against E. coli, K. pneumonia, P. aeruginosa, S. aureus
271 Initially, the effect of Ns-AgNps was investigated against E. coli, K. pneumonia, P. aeruginosa, S.
and E. faecalis bacterialstrains. Ns-AgNps showed the significant bacteriostatic and bactericidal activity.
272 aureus and E. faecalis bacterialstrains. Ns-AgNps showed the significant bacteriostatic and bactericidal
Even the lowest concentration of Ns-AgNps restricted the highest rate of inhibition against S. aureus
273 activity. Even the lowest concentration of Ns-AgNps restricted the highest rate of inhibition against
(6.5 and 15 µg/mL) and E. faecalis (6.5 and 15 µg/mL). Antimicrobial activity of S. aureus and E. fecalis
274 S. aureus (6.5 and 15 µg/ml) and E. faecalis (6.5 and 15 µg/ml). Antimicrobial activity of S. aureus and
was more pronounced than E. coli (15 and 30 µg/mL), K. pneumonia (15 and 30 µg/mL), and P. aeruginosa
275 E. fecalis was more pronounced than E. coli (15 and 30 µg/ml), K. pneumonia (15 and 30 µg/ml), and
(30 and 60 µg/mL) respectively (Table 1). In addition, Ns-AgNps concentration against above mentioned
276 P. aeruginosa (30 and 60 µg/ml) respectively (Table 1). In addition, Ns-AgNps concentration against
bacterial strains showed 6.5–30 µg/mL concentration for MIC and MBC values were 15–60 µg/mL. Our
277 above mentioned bacterial strains showed 6.5–30 µg/ml concentration for MIC and MBC values were
above-mentioned results revealed that Ns-AgNps exhibited excellent antibacterial activity and was
278 15-60 µg/ml. Our above-mentioned results revealed that Ns-AgNps exhibited excellent antibacterial
in accordance with the findings of a previous study [35]. Silver nanoparticle play an important role
279 activity and was in accordance with the findings of a previous study [35]. Silver nanoparticle play an
in bactericidal, bacteriostatic and also kill the other types of pathogens. Sadeghi et al. evaluated the
280 important role in bactericidal, bacteriostatic and also kill the other types of pathogens. Sadeghi et al.
bactericidal effect of silver nanoparticles and chlorhexidine against S. mutans to observe that silver
281 evaluated the bactericidal effect of silver nanoparticles and chlorhexidine against S. mutans to observe
nanoparticles showed a statistically significant bactericidal effect as compared to chlorhexidine [36].
282 that silver nanoparticles showed a statistically significant bactericidal effect as compared to
283 chlorhexidine [36].
Table 1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
activity of Ns-AgNps.
284 Table 1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
285 activity of Ns-AgNps. Synthesized Ns-AgNps
S. No Bacterial Strains
S. No Synthesized Ns-AgNps
MIC (µg/mL) MBC (µg/mL)
Bacterial strains
1 Klebsiella pneumonia (MTCC 618) MIC
15 (µg/ml) MBC(µg/ml)
30
21 Klebsiella pneumonia
Pseudomonas (MTCC
aeruginosa (MTCC 618)
1688) 30 15 30 60
32 Pseudomonas aeruginosa
Escherichia coli (MTCC (MTCC
40) 1688) 15 30 60 30
43 Escherichia
Staphylococcus coli (MTCC
aureus (MTCC3160)
40) 6.5 15 30 15
54 Staphylococcus aureus
Enterococcus faecalis (MTCC
(MTCC 439)3160) 6.5 6.5 15 15
5 Enterococcus faecalis (MTCC 439) 6.5 15

286 3.6. Anti-Biofilm Activity

 Processes 2020, 8, 388 9 of 14

3.6. Anti-Biofilm Activity

Processes 2019, 7, x FOR PEER REVIEW 9 of 14
The results of tissue culture plate method demonstrated that antibiofilm activity of the
287 The resultsAgNPs
biosynthesized of tissue
fromculture plate
N. sativa seedmethod
extractdemonstrated
was 88.42% for thatE.antibiofilm activity
faecalis, 84.92% for ofE. the
coli,
288 biosynthesized AgNPs from N. sativa seed extract was 88.42% for E. faecalis, 84.92%
81.86% for K. pneumonia, and 82.84% for S. aureus, respectively at 12.5 µg/mL, while with the same for E. coli, 81.86%
289 for K. pneumonia,
concentration, and 82.84%
the biofilm for S.was
formation aureus, respectively
reduced by 49.9%atin 12.5 µg/ml,
the case of P.while with the
aeruginosa same5).
(Figure
290 concentration,
Moreover, the biofilm
the evidence formation the
for screening wasbiofilm
reduced by 49.9%
inhibition in in
thethe case ofof
presence P.Ns-AgNPs
aeruginosa is(Figure
shown5).in
291 Moreover,
Figure the evidence
6. These findings for screening
suggest theuptake
that the biofilmof
inhibition in the presence
the biosynthesised of Ns-AgNPs
Ns-AgNPs is shown the
could diminish in
292 Figure forming
biofilm 6. These findings suggest
abilities of testedthat the uptake
pathogens. of thestudies
Similar biosynthesised Ns-AgNPs as
were documented could diminish
results the
of biofilm
293 biofilm forming
inhibition abilities
at 100 µg/mL of tested
AgNPs [37].pathogens.
Moreover,Similar
anotherstudies were documented
study based on anti-biofilm as demonstrated
results of biofilmthat
294 inhibition at 100 µg/ml AgNPs [37]. Moreover, another study based on anti-biofilm
the AgNPs synthesized from Momordica charantia extract were exhibited improved anti-biofilm activity demonstrated
295 that theE.faecalis
against AgNPs and synthesized from Momordica
A. hydrophilia. charantia
Additionally, extract were
the antibiofilm exhibited
activity improved
of AgNPs was anti-biofilm
attributed to
296 activity against E.faecalis and A. hydrophilia. Additionally,
the diffusion through the biofilms imparting antimicrobial actions [38].the antibiofilm activity of AgNPs was
297 attributed to the diffusion through the biofilms imparting antimicrobial actions [38].

K. pneumoniae

E. fecalis
100
Ag-NPs (µg/ml)

50

25 P. aeruginosa

12.5

S. aureus

E.coli

0 20 40 60 80 100 120
Biofilm inhibition (%)
298
299 Figure5.5. Anti-biofilm
Figure Anti-biofilm activity
activity by using
using different concentrations (12.5, 25,
concentrations (12.5, 25, 50,
50, 100
100 µg/ml)
µg/mL)ofofsilver
silver
300 nanoparticles
nanoparticlessynthesized
synthesizedfrom
from Black
Black Seed
Seed extract
extract against different standard bacterial strains.
strains.
 Processes 2019, 7, x FOR PEER REVIEW 10 of 14
Processes 2020, 8, 388 10 of 14
Processes 2019, 7, x FOR PEER REVIEW 10 of 14

301
302
301 Figure 6. Image showing the screening of biofilm inhibition in the presence of Ns-AgNPs.
Figure 6. Image showing the screening of biofilm inhibition in the presence of Ns-AgNPs. Row
302
303 Row A & Figure 6. Image
B- Negative showing
control (N.C.): the screening
Culture mediaof biofilm
only, inhibition
No bacterial in the
colony andpresence of Ns-AgNPs.
no Ns-AgNPs;
A&B-Negative control (N.C.): Culture media only, No bacterial colony and no Ns-AgNPs; Row
Row C & D-
304 Positive control (P.C.): Culture media plus 5 different bacterial colonies in different wells accordingly and no
C&D-Positive control (P.C.): Culture media plus 5 different bacterial colonies in different wells
303
305 Row A & B-Row
Ns-AgNPs; Negative control
E to H- (N.C.):
Culture Culture
media plus 5media only,
different No bacterial
bacterial colony
colonies and nowells
in different Ns-AgNPs; Rowand
accordingly
accordingly and no Ns-AgNPs; Row E to H-Culture media plus 5 different bacterial colonies in different
C & D-
306
304 various concentrations (100 to 12.5 µg/ml) of AgNPs synthesized from Nigella sativa extract. E.C.- E.
Positive control (P.C.): Culture media plus 5 different bacterial colonies in different wells accordingly and coli; S.A.- S. no
wells accordingly and various concentrations (100 to 12.5 µg/mL) of AgNPs synthesized from Nigella
307
305 aureus; P.A.-P.
Ns-AgNPs; Rowaeruginosa; E.F.-E. faecalis;
E to H- Culture K.A.-K.
media plus pneumoniae
5 different bacterial colonies in different wells accordingly and
sativa extract. E.C.-E. coli; S.A.-S. aureus; P.A.-P. aeruginosa; E.F.-E. faecalis; K.A.-K. pneumoniae
306 various concentrations (100 to 12.5 µg/ml) of AgNPs synthesized from Nigella sativa extract. E.C.- E. coli; S.A.- S.
308
307 3.7. Effect
3.7. Effect
aureus; Ns-AgNPs
of Ns-AgNPs
P.A.-P. Against
E.F.-E.Human
Against
aeruginosa; Human (HCC712)
faecalis;(HCC712) Cell Lines
Cell Lines
K.A.-K. pneumoniae
309 The cytotoxic
The cytotoxic effect
effect of
of Ns-AgNPs
Ns-AgNPsininthe thecurrent
currentstudy
studywas
wasexamined
examined against HCC712
against (human
HCC712 (human
308
310 3.7. Effect
breast of Ns-AgNPs
cancer cells) Against
using MTT HumanThe
assay. (HCC712)
toxicitiesCell Lines
were examined at different concentrations (25 to
breast cancer cells) using MTT assay. The toxicities were examined at different concentrations (25 to
311
309 200 µg/ml) and
The cytotoxic
200 µg/mL) compared to
effect of to
and compared control.
Ns-AgNPs The percent
in the
control. The inhibition
current
percent of
study was
inhibition cell growth
examined
of cell was increased
growthagainst with
HCC712with
was increased the
(humanthe
312
310 concentration of the nanoparticles (Figure 7). The inhibition effect was time and dose dependent and
breast cancer cells)
concentration of theusing MTT assay.
nanoparticles The toxicities
(Figure were examined
7). The inhibition at different
effect was time andconcentrations
dose dependent (25 to
and
313
311 the IC50 value of 150 µg/ml supports the levels of the inhibition.
200 IC
the µg/ml)
valueandofcompared
150 µg/mL to control.
supports The
the percent
levels of inhibition
the of
inhibition. cell growth was increased with the
50
312 concentration of the nanoparticles (Figure 7). The inhibition effect was time and dose dependent and
313 the IC50 value of 150 µg/ml supports the levels of the inhibition.

314 Figure 7. MTT assay results confirming the in-vitro cytotoxicity of the Ns-AgNPsagainst HCC 712
human breast cancer cell-lines.
315 Figure 7. MTT assay results confirming the in-vitro cytotoxicity of the Ns-AgNPsagainst
316 ItHCC
was712
observed that acancer
human breast high cell-lines.
concentration of Ns-AgNPs showed promising activity against breast
314 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
315 Figure 7. MTT assay results confirming the in-vitro cytotoxicity of the Ns-AgNPsagainst
intervals. The cell viability in the presence of AgNPs at different concentrations is illustrated in
316 HCC 712 human breast cancer cell-lines.
(Figure 8 A–F). The calculated IC50 value is approximately 175 µg/mL concentrations of Ns-AgNPs.
 317 It 2019,
Processes was 7,observed that
x FOR PEER a high concentration of Ns-AgNPs showed promising activity against11breast
REVIEW of 14
318 cancer cell lines and cell proliferation decreased after 24 hours. Moreover, findings exhibited that 150
319
317 µg/mL concentration
It was observed that of AgNPs is able to provoke
a high concentration the approximately
of Ns-AgNPs 50% of cell
showed promising mortality
activity at different
against breast
320
318 time intervals.
cancer cell lines The
and cell
cell viability in the
proliferation presenceafter
decreased of AgNPs at different
24 hours. Moreover,concentrations is illustrated
findings exhibited that 150 in
321
319 (Figureconcentration
µg/mL
Processes 8 A,8, B,
2020, 388C, D, of
E& F). The
AgNPs is calculated IC50 value
able to provoke is approximately
the approximately 50% of175cell
µg/ml concentrations
mortality at different of
11 of 14
322
320 Ns-AgNPs.
time intervals.Previously, similarinfindings
The cell viability wereofreported
the presence AgNPs atby Khateefconcentrations
different et al. [39] inisthe form ofina
illustrated
323
321 cytotoxicity
(Figure 8 A, B,effect
C, D,inEhuman
& F). Thebreast cancer cells
calculated IC50 (MCF-7) against green synthesized
value is approximately AgNPs procured
175 µg/ml concentrations of
Previously, similar findings were reported by Khateef et al. [39] in the form of a cytotoxicity effect
324
322 from Buchanania
Ns-AgNPs. axillaris
Previously, extracts.
similar The morphological
findings were reported observation
by Khateef et of al.
the[39]
HCC712
in the cancer
form of cells
a
in human breast cancer cells (MCF-7) against green synthesized AgNPs procured from Buchanania
325
323 exhibited significant
cytotoxicity morphological
effect in human breast cancerchanges and theagainst
cells (MCF-7) cell density was decreased,
green synthesized AgNPswhich is a
procured
axillaris extracts. The morphological observation of the HCC712 cancer cells exhibited significant
326
324 predictable
from mark axillaris
Buchanania of apoptotic cells (Figure
extracts. 9).
The morphological observation of the HCC712 cancer cells
morphological changes and the cell density was decreased, which is a predictable mark of apoptotic
325 exhibited significant morphological changes and the cell density was decreased, which is a
cells (Figure 9).
326 predictable mark of apoptotic cells (Figure 9).

327
328 Figure 8. Morphology of the control and Ns-AgNPs treated HCC 712 human breast cancer cell-lines.
327
329 Scale bar:
Figure 50 μm.
8. Morphology of the control and Ns-AgNPs treated HCC 712 human breast cancer cell-lines.
328 Scale bar:
Figure 50 µm.
8. Morphology of the control and Ns-AgNPs treated HCC 712 human breast cancer cell-lines.
329 Scale bar: 50 μm.

330
Figure 9. Morphology of control cells and 200 µg/mL silver nanoparticles treated HCC 712 human
331
330 Figurecancer
breast 9. Morphology of control
cell-lines. Scale cells
bar: 30 µm.and 200 µg/ml silver nanoparticles treated HCC 712 human
332 breast cancer cell-lines. Scale bar: 30 μm.
331 4. Conclusions
Figure 9. Morphology of control cells and 200 µg/ml silver nanoparticles treated HCC 712 human
332 breast cancer provides
The study cell-lines. Scale bar: 30
another μm.
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,
Processes 2020, 8, 388 12 of 14

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.

Author Contributions: A.A. design the conceptualized research study. H.K. and M.A. executed the detailed
analysis and drafted manuscript F.K.A.K. and M.A.A. drafted manuscript. K.S.A. and A.H.R. given potential input
in results interpretation. R.K. supervised the whole process. All authors have read and agreed to the published
version of the manuscript.
Funding: This research work was financially supported by Deanship of scientific research grant, Qassim University,
KSA. Grant number: 3970-cams1-2018-1-14-S.
Acknowledgments: Authors like to thanks to their respective institutions for sophisticated analytical
instrumentation facilities. The authors acknowledge deanship of scientific research (DSR/grant number/year)
Qassim University, kingdom of Saudi Arabia, providing finance support for research project fund.
Conflicts of Interest: The authors declare no conflict of interest.

Appendix A

Ns Nigella sativa
AgNPs Silver nanoparticles
DLS Dynamic light scattering
UV-Vis Ultraviolet visible
FTIR Fourier-Transform Infrared Radiometer
SEM Scanning light microscope
EDX Energy dispersed X-ray

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