Materials Chemistry and Physics 249 (2020) 123007

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Materials Chemistry and Physics

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An eco-friendly plant-mediated synthesis of silver nanoparticles:

Characterization, pharmaceutical and biomedical applications
Taghrid S. Alomar a, Najla AlMasoud a, **, Manal A. Awad b, *, Maha F. El-Tohamy c,
Dina A. Soliman d
a
Department of Chemistry, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia
b
King Abdullah Institute for Nanotechnology, King Saud University, Riyadh, 11451, Saudi Arabia
c
Department of Chemistry, College of Science, King Saud University, Riyadh, Saudi Arabia
d
Department of Microbiology, Faculty of Science, King Saud University, Riyadh, 11459, Saudi Arabia

H I G H L I G H T S G R A P H I C A L A B S T R A C T

� An ecofriendly synthesis of silver nano­

particles (AgNPs) is described.
� The reducing agent and stabilizer is the
peganum hermala leaf extract.
� AgNPs have a potential against different
strains of microbes.
� A spectrophotometric method using
synthesized AgNPs has high sensitivity
for the quantification of the antibiotic
Rifaximin.
� The synthesized NPs are promising for
application in the pharmaceutical and
biomedical fields.

A R T I C L E I N F O A B S T R A C T

Keywords: The current study focused on green chemistry approach to synthesize eco-friendly AgNPs using an aqueous
Green synthesis extract of peganum harmala leaves. The formed AgNPs were characterized using different spectroscopic and
Silver nanoparticles microscopic analyses: ultraviolet–visible spectrophotometry (UV–Vis), Fluorolog 3 spectrometry, transmission
Peganum harmala
electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform infrared spectroscopy (FTIR),
Antimicrobial activity
Rifaximin
techniques using a Zetasizer. The resulted nanoparticles were screened for their biomedical and pharmaceutical
properties. They investigated for antimicrobial activity against various strains of bacteria and fungi. The syn­
thesized AgNPs showed a higher antibacterial potential against Gram negative pathogen E. coli with inhibition
zone of 65 mm rather than both Gram positive pathogens S. aurous and B. cereus of inhibition zone 50 mm.
Meanwhile, no inhibition zone was observed for E. faecalis. Furthermore, the formed AgNPs were applied to
enhance the sensitivity and selectivity of the spectrophotometric determination of the antibiotic Rifaximin in bulk
powder or tablet form with a λmax of 340 nm. The proposed spectrophotometric technique for determining
Rifaximin in the presence of silver nanoparticles showed a linear relationship in the concentration ranges of 5–80
μg/mL and followed the linear regression equation A ¼ 0.039C-0.166 (r ¼ 0.9997), with low limits of detection
and quantification of 1.75 and 5.0 μg mL 1, respectively. According to the ICH guidelines, the proposed tech­
nique was validated.

* Corresponding author.
** Corresponding author.
E-mail addresses: tsalomar@pnu.edu.sa (T.S. Alomar), nsalmasoud@pnu.edu.sa (N. AlMasoud), mawad@ksu.edu.sa (M.A. Awad), star2000star@gmail.com,
moraby@ksu.edu.sa (M.F. El-Tohamy), dsoliman@ksu.edu.sa (D.A. Soliman).

https://doi.org/10.1016/j.matchemphys.2020.123007
Received 23 January 2020; Received in revised form 17 March 2020; Accepted 30 March 2020
Available online 4 April 2020
0254-0584/© 2020 Elsevier B.V. All rights reserved.
T.S. Alomar et al.
1. Introduction
Medicinal plants are considered as the most modern resources for
providing agents with fewer side effects that could candidate as alter­
natives to chemical drugs [1,2]. For long history, use of plants in the
treatment of many diseases has been well known such as Falcaria vul­
garis, Stevia rebaudiana, Allium saralicum, Alyssum meniocoides and Gly­
cyrrhiza glabra L. Traditionally, these medicinal plants have been the
basis of prevention, control, and treatment of various diseases including
microbial and viral infections, inflammatory diseases, gastritis; diabetes,
in hepatitis, immunostimulatory and nephroprotective etc. [3–5].
The shrub harmal (Peganum harmala) is a glabrous plant that is widely
grown and distributed in the eastern Mediterranean region. It belongs to
the family Zygophyllaceae and grows spontaneously in semisoiled areas.
In central Asia and North Africa, this plant is widely recommended as a
medicinal plant, and all of its components, including the roots, bark and
fruit, have been long used in folk medicines in Asia [6].
Currently, green chemistry is rapidly growing with a clear worldwide
impact. Medicinal plants have been widely employed to produce the
nanoparticles. A lengthy list of sources are used for the biological pro­
duction of metal nanoparticles, such as plants and plant extracts [7,8].
Due to the significant role of green chemistry as eco-friendly, safer,
clean, nontoxic, and biocompatible, methods, synthesized nanoparticles
with different nano-sizes, composition, novel shape, physiochemical,
and pharmacological properties [9].
Nanotechnology has recently developed rapidly and its applications
have extended to many different scientific fields [10]. This growing
interest is due to the potential applications of this technology for
different purposes such as drug delivery [11], tissue engineering [12],
chemistry [13], environmental studies [14], catalysis [15] and cancer
[16]. Recently, many noble metals have been used to synthesize nano­
particles, including Au, Ag, Pt, and Pd [17].
Metallic nanoparticles are generally used in functions that require
direct contact with the human body. Consequentially, there is a need to
develop a safe method that involves an ecofriendly process rather than
toxic chemicals; thus, the green synthesis of AgNPs has acquired an
extensive interest because of this need to develop environmentally
friendly technologies for material synthesis [18,19].
Since ancient times, metals such as gold, copper and silver have been
widely employed for preventing and treating the infectious diseases.
Silver is the essential metal that has been recognized as antimicrobial
agent [20]. Additionally, metallic nanoparticles have antibacterial
properties. Previous studies addressed that the green synthesized metal
nanoparticles exhibit strong activities against microbial diseases [21].
The possible mechanism of metal nanoparticles as antimicrobial agents
can be attributed to cause cell death by inducing membrane damage,
oxidative stress, and leakage of cell contents [22]. The green synthesis of
AgNPs from extracts of the leaves of Peganum harmala have been re­
ported, and there are many studies on the aqueous green synthesis of
AgNPs from the seed extracts [23] and methanolic extract [24]. Also
examination and comparison of the biosynthesis of AgNPs from leaf and
seed extract broths of Peganum harmala have been reported [25].
Recently, several silver nanoparticles have been reported for the
spectrophotometric quantification of different drugs [26,27], due to
their high extinction coefficient and cost benefit analysis. They also have
a specific surface plasmon resonance (SPR) peak, which incorporated
with another detected drug could be shifted to higher wavelength
accompanied by a color change of the solution tested.
Rifaximin (Fig. 1) is a semisynthetic member of a broad spectrum of
antibacterial drugs and are in a class of medications recommended for
the treatment of hepatic eccephalopathy, traveler’s diarrhea and irri­
table bowel syndrome, and there is a unique previously reported spec­
trophotometric technique for determining the Rifaximin concentration
[28].
In this study, silver nanoparticles were prepared via an ecofriendly
green chemistry technique using the leaf extract of P. harmala as a
2
Materials Chemistry and Physics 249 (2020) 123007
reducing and capping agent. This study also aimed to approve and
characterize the synthesis of nanoparticles using a different spectro­
scopic and microscopic techniques. Furthermore, the objective of this
study was to screen the as-prepared, environmentally friendly silver
nanoparticles activities against different strains of pathogens. Addi­
tionally, the synthesized nanoparticles were exploited to identify
Rifaximin in its bulk powder and pharmaceutical dosage forms. To our
knowledge, the present study is the first to focus on exploiting silver
nanoparticles synthesized via green chemistry in enhancing the deter­
mination of Rifaximin using spectrophotometry technique.
2. Methods and materials
2.1. Reagents and materials
Rifaximin in pure form and as gastrobiotic tablets (550 mg/tablet)
were supplied by the Al Andalous Medical Company (Cairo, Egypt).
Three different acids, boric acid (99.5%), citric acid (99.5%) and acetic
acid (99.7%), were acquired from Winlab (East Midland, UK). Sodium
tetraborate decahydrate (99.0%), sodium acetate (99.0%), sodium
monobasic phosphate (99.0%), sodium citrate dehydrate (99.0%), so­
dium dibasic phosphate (99.0%), silver nitrate (99.0%) and ethanol
(99.8%) were acquired from Sigma-Aldrich (Hamburg, Germany).
2.2. Preparation of AgNPs using an aqueous peganum harmala Leaf
extract
The leaves of the Peganum harmala plant were collected from
Almuzahemiah in Riyadh, Saudi Arabia in February 2019. The collected
leaves were washed and set aside to dry. The air-dried Peganum harmala
leaves were macerated three times in boiling, double-distilled water (at
a ratio 1:10) under constant shaking at ambient temperature overnight,
the extract was filtered, and approximately 5 mL of the aqueous extract
of leaves was then added to 50 mL of silver nitrate (1.0 mM), to drive
nanoparticle formation the reaction mixtures were under constant stir­
ring at 60 � C, until the color changed from colorless to brown (within 1
h). Once color intensities of the solution reached a maximum, the mixing
container was removed from stirrer and stored in darkness at room
temperature to prevent agglomeration of the nanoparticles. The change
in color indicated silver nanoparticles formation.
2.3. Characterization of the green synthesis of AgNPs
Ultraviolet–visible (UV–Vis) spectral analysis was carried out using a
UV 2450 Spectrophotometer (Shimadzu Corporation, Kyoto, Japan) in
the range of 100–800 nm. A Fluorolog 3 Spectrofluorometer (FL-3-11,
Horiba JobinYvon, USA) was used to measure the excitation and emis­
sion spectra of the green synthesized AgNPs. The average size of these
AgNPs was determined by DLS procedure using a Zetasizer (HT Laser,
ZEN3600 Malvern, Nano series, Instruments, Malvern, and UK). Fourier-
Fig. 1. Chemical structure of Rifaximin.
T.S. Alomar et al. Materials Chemistry and Physics 249 (2020) 123007

Transform Infrared spectroscopy (FTIR) was carried out on a Spectrum
BX spectrometer (PerkinElmer, Waltham, USA) to distinguish the
possible functional groups in the biomolecules that were in the extracts
of Peganum harmala and the as-prepared AgNPs. The surface
morphology and the resulting nanoparticle sizes were studied by
transmission electron microscopy with a 200 kV accelerating voltage
(TEM, JEM-2100F, JEOL Ltd, USA).
Using a JEM-2100F transmission electron microscope, energy-
dispersive X-ray spectroscopy (EDX) analysis was performed to verify
the existence of silver in the suspension and detect other fundamental
components of the particles.
2.4. Antibacterial screening
The antibacterial screening of AgNPs synthesized using the aqueous
extract of Peganum harmala was carried out by using the disk diffusion
method. Nutrient agar was prepared by dissolving 14 g of agar powder
in 500 mL of distilled water and was then autoclaved. Approximately 20
mL of the agar was poured into each Petri dish and left for 15 min to
solidify. Next, the plates were swabbed with human pathogens and
cultured overnight: Escherichia coli ATCC35218, Spahylococcus aureus
ATCC 43300, Bacillus cereus ATCC 11778 (clinical isolate) and Entero­
coccus faecalis ATCC 29212. The human pathogens were obtained from
King Khalid University Hospital, Riyadh, Saudi Arabia. The solid me­
dium was gently punctured with a sterile cork borer to make 4 wells in
each plate which were equidistant from the center of the dish. Then,
three different concentrations of synthesized AgNPs (1000 μL, 500 μL
and 250 μL) were added gradually until each hole was saturated with the
synthesized AgNPs and were incubated for approximately 24 h at 37 � C.
The Peganum harmala aqueous leaf extract was used as a positive control.
The inhibition zones were measured after incubation and expressed as
millimeter (mm) in diameter.
2.5. Antifungal activity of the green synthesized AgNPs
The antifungal activity of the green synthesized AgNPs was deter­
mined using a diffusion assay method against three different kinds of
fungi: Fusarium oxysporum, Alternaria alternata, and Tricoderma. These
fungal organisms were grown on potato dextrose agar plates at 28 ᵒC.
The agar media were made by dissolving 19 g of potato dextrose agar in
500 mL of distilled water, which was then autoclaved. Two different
concentrations (1000 μL and 500 μL) of AgNPs were incubated with the
fungi for 7 days at 28 ᵒC and the aqueous Peganum harmala leaf extract
was used as a positive control. The antifungal activity was evaluated by
determining the colony diameter, and the antifungal activity was
expressed as the percentage inhibition of mycelia growth.
buffer type and amount. The absorbance of each test solution was
recorded compared to a blank solvent at λmax of 340 nm.
3. Discussion and results
3.1. UV–visible spectroscopy of the synthesized silver nanoparticles
The components in the plant extract work as reductants that reduced
the silver ions (Agþ) to silver atoms (Ag0) [29]. The changing color was
observed after adding the aqueous leaf extract to silver nitrate solution.
The solution color changed from pale yellow to brown as a result of
excitation of the surface plasmon on metal nanoparticles [30,31]. The
presence of the AgNPs in the solution was characterized by the UV ab­
sorption spectrum, which a semi-sharp peak for the AgNPs was observed
in the spectrum at � 350 nm (Fig. 2), suggesting the formation of pol­
ydispersed nanoparticles because of the spectrum of SPR for AgNPs [32].
3.2. DLS analysis
DLS was used to determine the mean Z-average diameter (nm) of the
AgNPs. As shown in Fig. 3, the mean average size of the resulting
nanoparticles was found to be 184 nm. The size distribution profile of
the AgNPs showed two significant peaks at 240 nm (94.8%) and 30.41
nm (5.2%). The polydispersity index (PDI) of the AgNP suspension was
found to be 0.304, indicating that the synthesized particles varied in size
and showed an agglomeration.
3.3. Photoluminescence property of the AgNPs
The luminescence of noble metals such as silver is related to elec­
tronic conversions between both the conduction sp band and the upper
d band [33]. The luminescence spectrum of the AgNPs showed an
emission peak at λem 447.7 nm during photoexcitation at λex 320 nm,
and as shown in Fig. 4, the intensity increased sharply. This clearly
revealed that the reduction of silver using the plant extract greatly
improved the photoluminescence property of the AgNPs.
3.4. FT-IR analysis of the silver nanoparticles
The FT-IR spectrum displayed the functional groups of the green
synthesized AgNPs and the aqueous Peganum harmala leaf extract
(Fig. 5A–B, respectively). The FT-IR spectra of the leaf extract and the
produced AgNPs presented highly similar peaks with a small shift in
both spectra, indicating that the produced AgNPs encompass com­
pounds from the aqueous leaf extract. Therefore, we can reference the

2.6. Analytical applications

2.6.1. Standard solution preparation

To prepare a standard Rifaximin solution of 100 μg mL 1, 10 mg of
the selected dosage form was dissolved in 100 mL of ethanol. Serial
dilutions using ethanol were carried out to prepare working solutions
between 5 and 80 μg mL 1.

2.6.2. Preparation of Gastrobiotic®Tablets

To quantify the gastrobiotic tablets of Rifaximin, contents of not less
than 10 tablets were ground into a fine powder and mixed well, and then
an average of approximately 10 mg was dissolved in 50 mL of ethanol.
The filtrate pH was identified and adjusted to 9 using a borate buffer.
The final volume was modified to 100 mL using similar buffer solution.

2.6.3. Experimental procedure Fig. 2. UV–Visible absorption spectra of the green synthesized AgNPs using an
The overall spectrophotometric study was performed after opti­ aqueous Peganum harmala leaf extract. (For interpretation of the references to
mizing all of the relevant conditions, including the amount of silver color in this figure legend, the reader is referred to the Web version of
nanoparticles added, the pH value, processing time, temperature, and this article.)

3
T.S. Alomar et al.
Fig. 3. Recorded DLS data of the green synthesized silver nanoparticles using an aqueous Peganum harmala leaf extract. (For interpretation of the references to color
in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Photoluminescence spectrum of the synthesized AgNPs, using an
aqueous Peganum harmala leaf extract at λex 320 nm.
stability of the AgNPs with the appearance of compounds holding the
nanoparticles together [34,35]. At 3396.12 cm 1 and 3427.48 cm 1, the
bands were related to OH-stretching groups, which may be attributed to
phenols, alcohols, or the NH functional group of amides or amines. The
absorption band at 2926.35 cm 1 (Fig. 5B) revealed CH-stretching. The
bands at 1623.45 cm 1 and 1605.45 cm 1 were assigned to the C– –O
group of carboxylic acid, and the bands at 1384.91 cm 1 and 1423.43
cm 1 were assigned to the C– – C group of alkenes (Fig. 5A–B, respec­
tively). The absorption from 1050 to 1200 cm 1 is related to the CO
group of ethers, alcohols, and esters.
3.5. Transmission electron Microscopy and EDX analysis
The transmission electron microscopy image (Fig. 6A) shows that the
shape of the produced AgNPs shape is spherical with less agglomeration
and various sizes. The agglomeration was as a result of the aggregation
and adsorption of the compounds found onto the surface of the AgNPs
extract [36].
4
Materials Chemistry and Physics 249 (2020) 123007
The finding results are in line with the UV–vis spectrophotometric
results and DLS measurements, UV spectrum showed a broad SPR band
because of the aggregation and adsorption of compounds in the extract
of leaves on the surface of AgNPs and the DLS showed the polydispersity
index of the AgNP was 0.304, indicating that the synthesized particles
varied in size and showed an agglomeration. This result agreed with
many studies, the researchers reported that AgNPs are surrounded by a
faint thin layer of other materials considered as capping organic mate­
rials in addition to less agglomerated particles [37,38].
Energy dispersive X-ray spectrometry gives both quantitative and
qualitative information about the elements utilized in the formation of
the nanoparticles. Generally, metallic silver nanocrystals presented an
ideal optical absorption peak at approximately 3 keV because of their
surface plasmon resonance [39]. The elemental profile spectrum of the
synthesized nanoparticles showed greater counts at 3 keV because of the
silver presence, which confirmed the foundation of the AgNPs. However,
signals of carbon, sodium, copper and oxygen atoms were seen in
Fig. 6B. The obtained results indicated the presence of the plant extract
(as a capping agent) on the surfaces of the AgNPs. These results are in
line with the results of the FT-IR analysis that was presented in Fig. 5.
3.6. Biomedical applications
3.6.1. Antibacterial and antifungal analysis
The synthesized AgNPs exhibited promising antibacterial efficacy
against pathogenic bacteria in a dose-dependent manner [40]. In the
present study, the inhibition zones displayed by the synthesis of AgNPs
against various pathogenic bacteria were due to the different concen­
trations of nanoparticles (AgH1, AgH2, AgH3). The synthesis of AgNPs
showed observable antibacterial activity against Gram positive bacteria
such as Staph aurous (ATCC 43300) and Bacillus cereus (ATCC 11778),
with inhibition zones of 50 mm for both strains under high concentra­
tions of AgNPs (AgH3). Meanwhile, no inhibition zone was observed for
Enterococcus faecalis (ATCC 29212), but the synthesized AgNPs exhibi­
ted a large inhibition zone of 65 mm against the Gram negative bacteria
E. coli (ATCC35218) (Fig. 7A and B). A similar results were observed and
T.S. Alomar et al. Materials Chemistry and Physics 249 (2020) 123007

Fig. 5. FT-IR spectra of (A) the synthesized AgNPs, using an aqueous extract of Peganum harmala leaves and (B) the Peganum harmala leaf extract.

Fig. 6. (A): TEM image and (B) EDX shape of the green synthesis of AgNPs using an aqueous extract of Peganum harmala leaves. (For interpretation of the references
to color in this figure legend, the reader is referred to the Web version of this article.)

obtained with the AgNPs produced using P. harmala [24,25] which
exhibit a good antibacterial activity against both Gram-positive and
Gram-negative bacteria.
Futhermore, the synthesis of AgNPs showed antifungal activity
against pathogenic fungi, including Fusarium oxysporum, Alternaria
alternata, and Tricoderma, as shown in Table 1 and Fig. 8. Under
experimental conditions, the extracellular aqueous extract did not
exhibit antimicrobial activity against the tested microbes, except in the
case of E. coli, which had observable antibacterial activity with an in­
hibition zone of 20 mm. This result can be attributed to the lack of any
active antimicrobial compounds in the extracellular extract.
To our knowledge, there are many factors that affect the antimi­
crobial activity of AgNPs, including the shape, size, and surface charge
of the AgNPs, tolerance to AgNPs, species sensitivity, the type, genus and
species of the bacteria, the concentration of the nanoparticles, and the
exposure time to the pathogens [41]. All of these factors have a role in
the demonstrated antimicrobial activity of the AgNPs. There are two
previously reported possible mechanisms of action by which AgNPs
shows antimicrobial effects against bacterial cells:
(a) The membrane is damaged through the interaction and associa­
tion of the AgNPs with the DNA and other biomolecules, causing
the cell growth inhibition.
(b) Reactive Oxygen Species (ROS) are formed by interactions with
enzymes and/or biomolecules, causing cell damage [42].
Gram positive bacteria has a thick cell wall of peptidoglycan, consist
of linear polysaccharide chains and short peptides cross-linkages, which
is a firm structure that hinders the AgNPs from penetrating into the
bacterial cell wall. In contrast, in Gram negative bacteria, the cell wall
consists of a thin peptidoglycan layer [43]. Therefore, the results of the
present study suggested that the green synthesized AgNPs using the
Peganum harmala leaf extract have more potentially promising antimi­
crobial activities against various pathogenic microbes.
3.7. Pharmaceutical applications
3.7.1. Optimization of the analytical conditions
Metallic nanoparticles especially AgNPs possess their unique SPR
band, which strongly affected by the size, shape of the nanoparticles and

5
T.S. Alomar et al. Materials Chemistry and Physics 249 (2020) 123007

Fig. 7. (A) Measured values of inhibition zone of the synthesized AgNPs against pathogenic bacteria using different concentrations: 250 μL (AgH1), 500 μL (AgH2),
1000 μL (AgH3). The control (H) was an aqueous Peganum harmala leaf extract. (B) Antibacterial activity of the AgNPs against: (1) Escherichia coli ATCC35218, (2)
Staph aurous ATCC 43300, (3) Enterococcus faecalis ATCC 29212 and (4) Bacillus cereus ATCC 11778.

can be attributed to the neutralization of their surface charges producing

Table 1
their aggregation and changing in their SPR band [44]. The analytical
Antifungal activity of the silver nanoparticles against different fungal organisms.
solution was stabilized at the optimum pH value using 0.2 mol L 1 of
NO. Diameter of growth inhibition (mm) each acetate, citrate, phosphate and borate buffer solutions. A high
Control H AgH(1) AgH(2) absorbance value of the test solution in the presence of AgNPs was
1 Fusarium oxysporum 10 14 10 observed by using the 0.2 mol L 1 borate buffer (Fig. 9A). The effect of
2 Alternaria alternata 20 7 2 the different pH values on the absorbance of the as-prepared AgNPs was
3 Trichoderma 15 20 12 studied using the 0.2 mol L 1 borate buffer with pH values ranging from
1 to 10 (Fig. 9B). The maximum absorbance was recorded at pH 9. It was
noticed that below this value, the prepared nanoparticles aggregated
the analytical medium, therefore, in order to enhance the detection
and the absorbance declined. Additionally, the volume of the added
sensitivity of this spectrophotometric approach, factors causing the SPR
buffer was studied ranging from 0.1 to 1.0 mL (Fig. 9C).
change by the tested drug were investigated and optimized.
The results showed that the maximum absorbance of the AgNPs so­
The optimization of the pH of testing medium is greatly important
lution occurred with the addition of 0.5 mL of 0.2 mol L 1 borate buffer
because of not only enhancing the electrical charge of the tested mole­
solution. The quantity of the added AgNPs was adjusted using 0.1–0.6
cules but also affect the Nanoparticles stability [26]. As previously re­
mL of AgNPs solution (Fig. 9D). It was observed that the addition of 0.3
ported, the agglomeration of nanoparticles surface could be achieved at
mL of the AgNPs solution gave the maximum absorbance. The addition
pH less than 4 without any interaction with the investigated drug. This
of excess amounts of AgNPs may cause aggregation and result in a

Fig. 8. Antifungal activity of the synthesized AgNPs against (A) Fusarium oxysporum, (B) Alternaria alternata, and (C) Tricoderma.

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T.S. Alomar et al. Materials Chemistry and Physics 249 (2020) 123007

Fig. 9. (A) Effect of different kinds of buffers on the absorption of the AgNP solution. (B) The effect of the pH on the absorption spectrum of the AgNPs. (C) Effect of
the borate buffer volume on the AgNP absorbance spectrum, (D) Effect of the volume of AgNPs added. All tests were performed at λmax 340 nm and with 10 μg mL 1
of Rifaximin.

decrease in the sensitivity and absorbance. Finally, the influence of
temperature and reaction time were tested. No significant changes were
observed and the reaction seemed to be stable at room temperature even
after 12 h.
3.7.2. Spectral analysis and calibration
The spectral analysis of each Rifaximin, AgNPs and both Rifaximin
-AgNPs were studied. As shown in Fig. 10A, it was noticed that the
investigated drug displayed a wide absorption band at 280 nm. After
adding the AgNPs, the electrostatic attraction between the investigated
drug and the as-preapared nanoparticles caused the change in the sur­
face plasmon resonance and the detection band shifted to 340 nm [44].
The calibration curve of the tested drug in the presence of AgNPs was
graphed by plotting the absorbance of a series of standard solutions of
the tested drug as a function of their concentrations. The tested con­
centrations were ranged from 5 to 80 μg mL 1 (Fig. 10B). The critical
performance data for the spectrophotometric identification of the
selected drug in the presence of AgNPs was calculated and is summa­
rized in Table 2.
3.8. Method validation
The suitability of the spectroscopic method was validated using the
recommended ICH guidelines [45]. The proposed spectrophotometric

Fig. 10. (A) UV–Visible spectra of the synthesized AgNPs, Rifaximin -AgNPs and Rifaximin. (B) Calibration graph for determining the Rifaximin concentration using a
spectrophotometric technique in the presence of AgNPs.

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T.S. Alomar et al. Materials Chemistry and Physics 249 (2020) 123007

technique for determining Rifaximin in the presence of AgNPs was linear Table 3
over the concentration in the range of 5–80 μgmL 1 and followed the Summary of the validation data for the proposed spectrophotometric technique
linear regression equation A ¼ 0.039C-0.166, (r ¼ 0.9997), with lower using green synthesized AgNPs.
limits of detection and quantifications of 1.75 and 5.0 μg/mL, Parameter Value
respectively. Linearity 5–80 μg/mL
The accuracy of the proposed spectrophotometric technique was Lower Limit of Detection (LOD) 1.75 μg/mL
evaluated by using nine concentrations of authentic Rifaximin samples, Lower Limit of Quantification (LOQ) 5.0 μg/mL
and the % recovery was found to be 99.36 � 0.4%. Furthermore, the Accuracy (%Recovery, n ¼ 9) 99.36 � 0.4%
Precision (%RSD)
intermediate precision was tested by estimating the % RSD from the
Same day (n ¼ 3) 0.2, 0.4 and 0.6%
detection of three different Rifaximin concentrations from multi-day Multi-day (n ¼ 3) 0.3, 0.5 and 0.7%
assays. The %RSDs were found to be 0.2, 0.6 and 0.4% for the same Robustness 99.54 � 0.4%
day assays and 0.3, 0.7 and 0.5% for the multi-day assays. The obtained Ruggedness 99.47 � 0.6%
results were less than 2%, revealing the high accuracy of the proposed Specificity:
Magnesium stearate 800
technique. The robustness of the proposed technique were assessed by colloidal silicon dioxide 780
changing the pH of the analytical solutions to 9 � 0.1. The obtained % lactose 640
recovery was found to be 99.54 � 0.4%. sodium starch glycolate 520
The reproducibility of the developed method was examined using a
different spectrophotometer (Shimadzu-UV-1800 spectrophotometer,
Kyoto, Japan), where the % recovery was 99.47 � 0.6%. The recorded Table 4
results indicated the excellent robustness and ruggedness of the pro­ Spectrophotometric determination of Rifaximin in bulk powder in the presence
posed spectrophotometric technique. of AgNPs.
Finally, the specificity of the analytical technique towards the tested Sample Spectrophotometric determination of Rifaximin in the presence of
drug was evaluated by detecting concentrations of 10 μgmL 1 in the AgNPs
presence of 1.0 μgmL 1 of different additives such as magnesium stea­ Initial Concentration Recovered Concentration %
rate, colloidal silicon dioxide, lactose, and sodium starch glycolate. (μg/mL) (μg/mL) Recovery
No significant difference was observed in the obtained results, with
5 4.99 99.8
tolerable levels of 800, 780, 640 and 520, respectively. The calculated Bulk 10 9.98 99.8
data revealed the high specificity of the recommended spectrometric Powder 20 19.89 99.5
technique for determining Rifaximin in AgNPs. The validated data are 40 39.97 99.9
summarized in Table 3. 60 59.96 99.9
80 79.98 99.9

3.9. Quantification of the antibiotic Rifaximin mean � SD 99.80 � 0.15

The green synthesized AgNPs were used in the quantification of
Rifaximin in both tablet forms and bulk powder. The proposed spectro­
photometric technique displayed high sensitivity for the identification
of the drug in its bulk power form, with a mean recovery percentage of
99.80% and percentage relative standard deviation (% RSD) of 0.15%
(Table 4).
The excellent results obtained of the bulk powder form were prom­
ising for the determination of Rifaximin in its pharmaceutical tablets.
The obtained data are summarized in Table 5. Statistical assessments in
terms of the t-test and F-test [46] were used to compare the results ob­
tained by using AgNPs with the results obtained from a previously
established spectrophotometric technique for drug detection in an
ethanol solvent at λmax 290 nm [28]. The results indicated no significant
differences between the two methods at a 95% confidence level with
high accuracy and precision.
Table 2
Optical characterization and regression data of the proposed spectrophotometric
technique in the presence of green synthesized AgNPs.
Parameter Result
n 6
Variance (v) 0.02
%SE 0.06
%RSD 0.15
4. Conclusion
This study investigated the use of a green, ecofriendly method for
synthesizing AgNPs using Peganum harmala aqueous leaf extract and
screened their biomedical and pharmaceutical applications. The applied
method provided excellent synthesized AgNPs, which was confirmed by
spectroscopic and microscopic analyses such as UV–Vis, FT-IR, DLS,
TEM and EDX. The antimicrobial activity of the as-prepared nano­
particles was screened against different strains of bacteria and fungi. The
obtained results demonstrated that the green synthesized AgNPs have
higher effectiveness against different strains of microbes. Additionally,
the green synthesized AgNPs were employed in the quantification of the
antibiotic Rifaximin in its bulk powder and tablet forms, with a mean
recovery percentage of 99.80 � 0.15% and 99.66 � 0.41%, respectively.
The recorded data confirmed that the proposed spectrophotometric
technique using the green synthesized AgNPs has high sensitivity for the
quantification of the investigated drug.
Authors contributions

Λmax (nm) 340 All co-authors did contribute to this work and are aware of this
Limitation to Beer’s law (μg/mL) 5–80
submission.
Lower Limit of Detection (LoD) 1.75
Lower Limit of Quantification (LoQ) 5.0
Molar Absorptivity (L mol 1 cm 1) 3.06 � 10 4 Declaration of competing interest
Sandell Sensitivity (S) (μg/cm2/0.001 absorbance unit) 0.125
Regression equation [Y ¼ aþ(bc)] A ¼ 0.039C-0.166
No conflicts of interest are declared by the authors.
Correlation coefficient (R) 0.9997
Standard deviation of slope (Sb) 0.000357
Standard deviation of intercept (Sa) 0.017003
Standard Error of Estimation 0.026762

8
T.S. Alomar et al. Materials Chemistry and Physics 249 (2020) 123007

Table 5
Two spectrophotometric techniques to determine the amount of Rifaximin in gastrobiotic tablets.
Sample Spectrophotometric determination of Rifaximin in the presence of AgNPs Previously reported spectrophotometric technique20

Initial Concentration (μg/ Recovered Concentration (μg/ % Initial Concentration (μg/ Recovered Concentration (μg/ %
mL) mL) Recovery mL) mL) Recovery

Gastrobiotic 5 4.95 99.0 10 9.78 97.8

tablets 10 9.93 99.3 12 11.95 99.5
20 19.99 99.9 15 14.96 99.7
40 40.00 100.0 20 19.97 99.9
60 59.99 99.9 25 24.89 99.6
80 79.95 99.9 30 29.68 98.9

mean � SD 99.66 � 0.41 99.23 � 0.78

n 6 6
Variance (v) 0.16 0.61
%SE 0.16 0.31
%RSD 0.41 0.78
t-test 1.232 (2.228)a
F-test 3.81 (5.05)a
a
The values in parentheses are the theoretical values for the F- and t-tests at p¼0.0533.

CRediT authorship contribution statement
Taghrid S. Alomar: Project administration, Resources, Methodol­
ogy, Funding acquisition, Visualization, Writing - original draft, Writing
- review & editing, Resources. Najla AlMasoud: Project administration,
Resources, Methodology, Funding acquisition, Visualization, Writing -
original draft, Writing - review & editing, Resources. Manal A. Awad:
Conceptualization, Validation, Methodology, Supervision, Formal
analysis, Investigation, Data curation, Visualization, Writing - original
draft, Writing - review & editing. Maha F. El-Tohamy: Validation,
Methodology, Investigation, Visualization, Writing - original draft,
Writing - review & editing. Dina A. Soliman: Validation, Methodology,
Writing - original draft.
Acknowledgements
This research was funded by the Deanship of Scientific Research at
Princess Nourah bint Abdulrahman University through the Fast–Track
Research Funding Program.
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