Green Processing and Synthesis 2024; 13: 20230229
Research Article
Aneesa Zia, Ayesha Shahzad, Nadia Riaz, Sara Khan, Umar Farooq, Syed Majid Bukhari,
Rizwana Sarwar*, Asaad Khalid, Hamdy Kashtoh*, Ajmal Khan*, and Ahmed Al-Harrasi*
Enhancement efficacy of omeprazole by
conjugation with silver nanoparticles as a urease
inhibitor
https://doi.org/10.1515/gps-2023-0229
received November 08, 2023; accepted February 20, 2024
Abstract: Omeprazole, a proton pump inhibitor, is used for
gastric and duodenal ulcers, gastroesophageal reflux dis-
ease, Helicobacter pylori infection, etc. Current research is
based on the loading of omeprazole on surface silver
nanoparticles by chemical method. The appearance of
an absorption peak at 421 nm confirmed the synthesis of
nanoparticles. The FT-IR further confirmed the conjuga-
tion of functional groups present in omeprazole moiety
with silver. The size and morphology were elucidated by
transmission electron microscopy and X-ray diffraction
which revealed a spherical shape with an average particle
size of 16–20 nm. To know enhancement in their efficacy,
the omeprazole-loaded nanoparticles were evaluated against
antibacterial, urease inhibition, and antioxidant activities.
Nanoparticles showed significant antibacterial potential
against Staphylococcus aureus and Escherichia coli with
12 ± 0.41 and 13.6 ± 1.02 mm zones of inhibition, respectively.
Almost 2.43 times enhanced urease inhibitory activity was

* Corresponding author: Rizwana Sarwar, Department of Chemistry,
COMSATS University Islamabad, Abbottabad Campus, 22060, KPK,
Pakistan, e-mail: rizwanasarwar@cuiatd.edu.pk
* Corresponding author: Hamdy Kashtoh, Department of
Biotechnology, Yeungnam University, Gyeongsan, 38541, Gyeongbuk,
Republic of Korea, e-mail: hamdy_kashtoh@ynu.ac.kr
* Corresponding author: Ajmal Khan, Natural and Medical Sciences
Research Center, University of Nizwa, Nizwa, 616, Sultanate of Oman,
e-mail: ajmalkhan@unizwa.edu.om
* Corresponding author: Ahmed Al-Harrasi, Natural and Medical
Sciences Research Center, University of Nizwa, Nizwa, 616, Sultanate of
Oman, e-mail: aharrasi@unizwa.edu.om
Aneesa Zia, Ayesha Shahzad, Sara Khan, Umar Farooq, Syed Majid
Bukhari: Department of Chemistry, COMSATS University Islamabad,
Abbottabad Campus, 22060, KPK, Pakistan
Nadia Riaz: Department of Environmental Sciences, COMSATS University
Islamabad, Abbottabad Campus, 22060, KPK, Pakistan
Asaad Khalid: Substance Abuse and Toxicology Research Center, Jazan
University, P.O. Box 114, Jazan, 45142, Saudi Arabia
Open Access. © 2024 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License.
found for nanoparticles (IC50 = 2.17 ± 0.10 µg·mL−1) as com-
pared to omeprazole (IC50 = 5.28 ± 0.14 µg·mL−1). The radical
scavenging activity of nanoparticles also increased signifi-
cantly. The synthesized nanoparticles were docked in the
active site of urease to investigate their binding mode.
Due to excellent urease and bacterial inhibition, these nano-
particles can be used for ulcers.
Keywords: omeprazole, nanoparticles, chemical synthesis
method, radical scavenging activity, urease inhibition
1 Introduction
Peptic ulcers are one of the most common gastrointestinal
conditions affecting approximately 10% of the population
across the globe. An imbalance between the gastro-protec-
tive factors such as mucus and antioxidant enzymes bicar-
bonate and secretion of acid and pepsin damages the
mucosal membrane of the stomach and duodenum and
results in life-threatening ulcers [1,2]. Approximately 15,000
fatalities are reported annually as a consequence of ulcers
[3]. It has become one of the most widespread problems
affecting 40% of developed countries and 80% of developing
countries due to the indiscriminate use of non-steroidal anti-
inflammatory disease and Helicobacter pylori infections [4].
Reactive oxygen species play a significant role in the etiology
of peptic ulcers, as the free radical released from the neu-
trophils and monocytes can cause oxidative damage to the
cells of gastric mucosa via lipid peroxidation and oxidation
of proteins [5]. Using medications with antioxidant proper-
ties can be effective in the treatment of ulcers [6]. Antiulcer
medications work by suppressing the secretion of gastric
acid and stimulating the immune system by enhancing the
secretion of mucin [7]. Antacids, H2 receptor blockers, mus-
carinic antagonists, and proton pump inhibitors are used in
the treatment of ulcers. However, the successful eradication
of H. pylori is essential for the treatment of peptic ulcers and
avoiding its recurrence. Triple therapy containing two
2  Aneesa Zia et al.
antibiotics clarithromycin and amoxicillin or metronidazole
along with proton pump inhibitors is currently being used as
a standard treatment. However, over the past 10–15 years,
due to the rising incidence of antibiotic resistance, notably
with clarithromycin, the effectiveness of this triple therapy
has considerably decreased. Consequently, there is a rising
need of developing potential antiulcer agents for effective
treatment of ulcers [8].
Omeprazole, a sulfinyl benzimidazole [9] is the first
clinically approved member of unique antisecretory drugs,
the proton pump inhibitors that control the secretion of
gastric acid via irreversible inhibition of enzyme H+/K+
-ATPase located in the parietal cells of stomach [10,11]. It
is found to be a potent inhibitor of gastric acid secretion for
the treatment of gastroesophageal reflux disease, gastritis,
Zollinger-Ellison syndrome, gastric and duodenal ulcers,
H. pylori infection, and other associated hypersecretory
disorders [12,13].
Omeprazole is susceptible to heat, light, and humidity
and undergoes rapid degradation in acidic solutions [14].
Hence, its exposure to the acidic contents of stomach
results in the inactivation of almost fifty percent of the
dose leading to its poor bioavailability [15]. The most effec-
tive approach to prevent its degradation via stomach acid
is to incorporate omeprazole into enteric-coated polymeric
nanoparticles and extend its half-life [1]. Due to their small
size, NPs can pass through the mucus layer and adhere to
the underlying epithelium or the mucus network directly.
Drug when administered in the form of mucoadhesive
nanoparticles can reside in the stomach for a longer period
of time and these adhesive interactions may increase
drug’s bioavailability via number of mechanism [16]. Var-
ious omeprazole-loaded nanoparticles have been reported
in literature including omeprazole-loaded cellulose acid
phthalate nanoparticles [1], omeprazole-loaded gliadin
nanoparticles [16], omeprazole containing eudragit chit-
osan nanoparticles [17], and omeprazole containing Bletilla
striata nanoparticles (OME-BSP NPs) [18]. The OMP-NPs
effectively prevented gastric mucosal lesions and have
enhanced antiulcer activity as compared to omeprazole.
The enteric-coated nanoparticles postpone the release of
the medication because it is insoluble in acidic environ-
ments. [19]. Enhanced gastric mucosal defense through
increased production of mucus and bicarbonate, decreased
gastric acid secretion volume, or simply neutralizing the
gastric acidity are some of the mechanisms that mediate
the protection of the gastric mucosa against acid produc-
tion [14].
It has been demonstrated that metallic nanoparticle
conjugation with antibiotics offers promising outcomes in
the treatment of infectious diseases. The effectiveness of
antibiotics could be increased by combining them with NPs
[20,21]. Among metallic NPs, silver nanoparticles are well
known for their broad spectrum activities [22]. The envir-
onmentally friendly manufacturing of silver nanoparticles
is emerging as a novel and effective approach to the treat-
ment of gastrointestinal ulcers [6]. Various sources were
used for the synthesis of silver nanoparticles for the anti-
ulcer activity. The AgNPs synthesized from Glycyrrhiza
glabra root extract were found to have antiulcer activity
[23]. Similarly, Solanum xanthocarpum extract-loaded silver
nanoparticles were proven to have antibacterial and urease
inhibitory properties against H. pylori [24].
The AgNPs synthesized from the seeds of Anethum
graveolens were tested against proton pump (H+/K+-ATPase)
inhibition and they were found to have inhibitory potential
against the enzyme establishing proton pump inhibition by
AgNPs [25].
Omeprazole and silver nanoparticles are well known
for their antiulcer activities. Literature has revealed that a
combination of drugs with nanoparticles may enhance their
therapeutic potential as compared to drugs and NPs alone.
Current research aims at synthesizing silver nanoparticles
of omeprazole by chemical reduction method. To the best of
our knowledge, this is the first study reporting the synthesis
of omeprazole-loaded silver nanoparticles and their urease
inhibition, antioxidant, and antibacterial activities against
Staphylococcus aureus and Escherichia coli.
2 Materials and methods
2.1 Materials
The chemicals and solvents including AgNO3, sodium hydro-
xide, HCl, sodium borohydride (NaBH4), and methanol used
for the synthesis were of analytical grade and purchased
from Sigma Aldrich; vendor Musaji, Abbottabad, Pakistan.
Nutrient agar was purchased from Oxoid Ltd, Basingstoke
Hampshire, UK. The DPPH powder and the urease enzyme
were purchased from Alfa Aesar and Sigma Aldrich, vendor
Musaji, Abbottabad, Pakistan, respectively. UV–Vis spectra
were recorded on Spectromax M2 (COMSATS University
Islamabad Abbottabad Campus). For pH adjustment, a
pH meter (Combo pH/EC/TDS Testers HANNA Instrument)
was used.
2.2 Synthesis of nanoparticles
Omeprazole-loaded silver nanoparticles were synthesized
via chemical reduction method. Around 16.9 mg of AgNO3
 Omeprazole conjugation with silver nanoparticles as a urease inhibitor
was dissolved in 100 mL of distilled water to prepare 1 mM
solution of silver nitrate and the solution was kept in dark
to prevent light exposure. Similarly, 34.54 mg of omepra-
zole was dissolved in 100 mL of methanol to prepare its
1 mM solution. After mixing the drug and salt solution no
significant color change was observed, so 2–3 drops of
NaBH4 (40 mM) were added to the solution which pro-
duced rapid variation in the color of solution indicating
the formation of silver nanoparticles which was further
confirmed by UV–Vis spectrophotometer.
2.3 Optimization of nanoparticles
The reaction was optimized by mixing different concentra-
tions of silver nitrate and omeprazole to achieve optimum
drug-to-metal ratio for the synthesis of nanoparticles. The
ratio at which the maximum formation of nanoparticles
occurs can be achieved by optimization. In this study, the
concentration of drug was fixed whereas the volume of
silver nitrate was varied. The ratio that gave the highest
absorption peak was selected for the synthesis of
nanoparticles.
2.4 Stability of NPs
After synthesis, the stability of NPs was tested against dif-
ferent pH (2–13), temperature range (25–100°C), and brine
solution (0.5–2 M). The NPs were kept at room temperature
and after 24 h, UV–Vis spectra were recorded; any change
in the intensity of absorption provides information about
the stability of nanoparticles.
2.4.1 pH stability test
The synthesized nanoparticles were exposed to different
pH ranges (2–14) to examine their stability in extremely
acidic, basic, and neutral conditions. The pH meter (Combo
pH/EC/TDS Testers HANNA Instrument) was used to measure
the different pH (2–14) of nanoparticle solution. The acidic
and basic conditions were developed by using 0.5 M solution
of HCl and NaOH in 10 mL of silver nanoparticle solutions.
The solutions were kept for 24 h and later, the stability was
confirmed through a decrease in absorbance by the UV–Vis
analysis.
 3
2.4.2 Heat stability test
The heat stability of omeprazole-loaded AgNPs was checked
by heating the reaction solutions at different temperatures,
i.e., 25°C, 50°C, 75°C, and 100°C for 30 min [26]. Around 15 mL
of freshly prepared nanoparticle solution was taken in four
separate beakers and heated at different temperatures for
30 min to check the heat stability of nanoparticles and
UV–Vis spectra were recorded. The change in the absorption
intensity provides data about the stability of synthesized
AgNPs at different temperatures.
2.4.3 Brine stability test
The stability of synthesized omeprazole-loaded silver nano-
particles was determined against different concentrations of
brine solutions. The 4 mL of freshly prepared Omp-AgNPs
were taken in four separate vials. Then, 2 mL of 0.5, 1, 1.5,
and 2 M solutions of NaCl were added to the solution of NPs.
The resulting mixture was thoroughly mixed and kept at
ambient temperature. UV–Vis spectra were recorded after
24 h. Change in the absorption give information about the
nanoparticles’ stability.
2.5 Characterization of NPs
2.5.1 FT-IR spectroscopy
FT-IR analysis was used to detect the functional moieties
present in drugs and their interaction with the metal sur-
face in drug-loaded silver nanoparticles. IR spectra of
samples were obtained by Cary630, Agilent Technologies,
USA.
2.5.2 X-ray diffraction (XRD) analysis
Crystallographic study of AgNPs was performed by the X-
ray diffractometer (JDX-3532, JEOL, Japan) operated at the
voltage of 20–40 kV having 2θ range 5–80°.
2.5.3 EDX spectroscopy
The compositional analysis of nanoparticles was carried
out by EDX (JSM-5910, JEOL, Japan) having 30 kV energy.
4  Aneesa Zia et al.
2.5.4 Transmission electron microscopy (TEM)
The size and shape of synthesized omeprazole-loaded silver
NPs were evaluated using a TEM (JEOL, Model JEM-1400)
operated at a voltage of 110 kV. Samples were prepared
for the TEM examination by coating the solution of silver
nanoparticles on copper grids coated with carbon.
2.6 Biological potential of NPs
2.6.1 Urease inhibition protocol
In vitro urease inhibition assay of omeprazole-loaded nano-
particles was performed in a 96-well plate by the Berthelot
method with few modifications. 25 µL phosphate buffer (pH
7), 10 µL enzyme solution of urease, and 10 µL of test com-
pounds were added to the wells and incubated at 37°C for
approximately 10 min. Then, 40 µL urea solution was added
to these wells and further incubated for 10 min. Afterwards,
the constituents were pre-read using ELISA microplate
reader at 625 nm. Then, 45 µL phenol reagent was added
to each well followed by the addition of 70 µL alkali
reagent. The incubation was continued for 10 min and
absorbance was measured. The % inhibition was calcu-
lated by using the following formula [27].
[Absorbance of test compopunds]
% Inhibition = 100 −
Absorbance of control
× 100.
Five dilutions of test samples were prepared and their
% inhibitions were determined. The data obtained were
used to calculate IC50 values using a graphpad prism.
2.6.2 Antioxidant activity
The radical scavenging potential of nanoparticles was eval-
uated by the DPPH method. The 100 mM DPPH solution was
prepared in HPLC-grade methanol. 50 µL of DPPH was
added to the 50 µL of test compound. The mixture was
incubated at room temperature for approximately half
an hour and the absorbance was measured at 517 nm
[28]. The t-BHA was used as a standard throughout the
experiment. The % scavenging activity was measured by
using the following formula:
[Absorbance of test compopunds]
% RSA = 100 − × 100.
Absorbance of control
Five dilutions of the test samples along with standard
were prepared and their % RSA was measured and data
were used to find IC50 values using graphpad prism [29].
2.6.3 Antibacterial screening
The antibacterial potential of drug-loaded nanoparticles
was screened against two clinically isolated bacterial strains
namely S. aureus (ATCC 6538) and E. coli (ATCC 10536) by
agar well diffusion method. The 100 µL of bacterial suspen-
sion having a concentration of 108 CFU·mL−1 was spread on
the agar petri plate. By using cork borer four wells of equal
size of approximately 6 mm and distance were made in the
Petri plate and 20 µL test compounds of the desired concen-
tration were added into each well along with positive con-
trol (Ciprofloxacin) and negative control (DMSO). The plates
were incubated for 24 h at 37°C. After incubation, the zone of
inhibition was measured [29].
2.6.4 Statistical analysis
One-way analysis of variance (ANOVA) with post hoc
Bonferroni test (Haris et al. 2023) was employed to deter-
mine significant differences between the ureases and anti-
oxidant activities of different test substances at the signifi-
cance level (α = 0.05). Independent samples t-test was used
to compare the anti-bacterial activity of two samples. The
statistical analyses were performed using SPSS 20 (IBM,
USA) computer software [30].
2.7 Molecular docking studies
A molecular operating environment (MOE) [31] was used to
investigate the binding interaction of synthesized com-
pounds with the active site of urease and xanthine oxidase.
X-ray crystal structure of urease (PDB ID: 4GOA [32])
retrieved from the Protein Data Bank was used as a
starting structure for docking. Co-crystalized ligands and
heteroatoms were removed and docking was carried out
using MOE software. The structures of the synthesized
compounds were drawn by using Chem Office 3D (2015)
and their energy was optimized using Gaussian 09 employing
the B3LYP density functional theory method with a 6-311G
basis set [33]. Initial optimization of the protein structure
was done using MOE software followed by molecular
docking studies. Ten of the lowest energy poses were
 Omeprazole conjugation with silver nanoparticles as a urease inhibitor  5

generated for compounds. A Discovery visualizer was used

3.0
to determine the 2D and 3D models of docked compounds.
Rxn Optimization
2.5 Drug:Metal
1:1

Absorbance (a.u)
3 Results and discussion 2.0
1:2
1:3
1:4
1.5
3.1 Synthesis of nanoparticles
1.0
A solution of AgNO3 (1 mM) and omeprazole was prepared
in distilled water and methanol separately. Equal quanti-
0.5
ties of both the solutions were mixed together and kept on
stirring at room temperature. No significant color change 0.0
was observed after mixing as apparent with the naked eye. 300 400 500 600 700
After 30 min, 2–3 drops of NaBH4 were added to the reac- Wavelength (nm)
tion mixture. A change in the color from transparent to
dark brown was observed immediately after the addition Figure 2: Optimization of Omp-AgNPs.

of NaBH4 indicating the reduction of silver. The stirring

was continued for up to 4 h to allow the complete reduction
of silver ions for the completion of reaction. The appear-
ance of peak at 421 nm confirmed the synthesis of omepra-
zole-loaded AgNPs. For the recovery of nanoparticles, the
reaction mixture was centrifuged at 8,000 rpm for approxi-
mately 30 min at room temperature and washed with dis-
tilled water twice to remove the excess ions and unreacted
drug. The UV–Vis spectrum (spectrophotometer BMS [UV-
1602] Canada) was recorded to confirm the formation of
nanoparticles (Figure 1).
their stability under the given conditions. The ratio 1:3
(Omp:AgNO3) corresponds to the highest absorption peak
in UV–Vis spectra as shown in Figure 2. Further increase in
the concentration of salt led to a decline in the intensity of
absorption which confirmed the decomposition and instability
of nanoparticles indicating that a certain amount of salt was
necessary for the optimum synthesis of NPs. The optimal ratio
for the large-scale production of nanoparticles showing max-
imum absorption in UV–Vis spectra was ratio 1:3.

3.5 3.3 pH stability of AgNPs

3.0 Omeprazole
Omp-AgNPs pH has a significant impact in controlling the size of nano-
2.5 particles by altering their surface charge and particle
Absorbance (a.u)

2.0

1.5 4.0
pH Effect
1.0
3.5 2-3
4-5
3.0
Transmittance (a.u)

0.5 6-7
2.5 8-9
0.0 10-11
2.0 12-13
200 300 400 500 600 700
1.5
Wavelength (nm)
1.0
Figure 1: UV–Vis spectra of Omeprazole loaded AgNPs.
0.5

0.0
3.2 Reaction optimization 300 400 500 600 700
Wavelength (nm)
The reaction mixture of Omp-AgNPs was optimized using
different drug-to-metal ratios and stored for 24 h to assess Figure 3: pH stability of AgNPs.
6  Aneesa Zia et al.
interaction [34]. The stability of synthesized Omp-AgNPs
was examined at different pH values ranging from 2 to 13
by using 0.5 M HCl and NaOH solutions as shown in Figure 3.
Omp-AgNPs were found to be slightly acidic in nature
having a pH in the range of 6–7. The nanoparticles were
stable at a slightly acidic, neutral and slightly basic medium.
The red shift in the wavelength was observed at pH 4–5 due
to an increase in particle size [35]. The pH 6–7 corresponds
to the highest absorption peak in the UV–Vis spectrum indi-
cating the maximum formation and stability of nanoparti-
cles having small size. However, AgNPs were unstable at
extremely acidic and basic conditions. The chloride ions
were responsible for the destabilization of nanoparticles
in highly acidic conditions having pH 2–3 [36], whereas
instability of AgNPs in the basic medium can be associated
with the destabilization caused by sodium ions, resulting in
the agglomeration of nanoparticles due to increase in par-
ticle size.
3.4 Heat stability test of NPs
The effect of heat on synthesized nanoparticles was exam-
ined at elevated temperatures to ascertain their thermal
stability and optimal temperature for nanoparticle synth-
esis. The AgNPs were heated at different temperatures, i.e.,
25°C, 50°C, 75°C, and 100°C for 30 min and their UV–Vis
spectra were recorded. The spectra of heated NPs indicate
a decrease in absorption intensity with an increase in tem-
perature confirming the destabilization of nanoparticles at
high temperatures as represented in Figure 4. This instability
of AgNPs is associated with the dephasing of electrons [37].
1.6
1.4
Heat Effect
1.2 Room Temperature
Absorbance (a.u)
50°C
1.0 75°C
0.8 100°C
0.6
0.4
0.2
0.0
300 400 500 600 700
Wavelength (nm)
Figure 4: Heat stability test of NPs.
0.8 Brine Stability Test
Omp-AgNPs
0.5 M
Absorbance (a.u)
0.6 1M
1.5 M
2M
0.4
0.2
0.0
300 400 500 600 700
Wavelength (nm)
Figure 5: Brine stability test of Omp-AgNPs.
The room temperature (25°C) is the optimum temperature
for the synthesis of Omp-AgNPs.
3.5 Brine stability test
The addition of an electrolyte causes the agglomeration of
nanoparticles, which makes them unstable [38]. The NaCl
solutions of varying concentrations (0.5, 1, 1.5, and 2 M)
were mixed with Omp-AgNPs to assess the effect of electro-
lyte solution on synthesized nanoparticles. The absorbance
of these mixtures was determined by using UV–Vis spectro-
meter as represented in (Figure 5). The findings indicate
that nanoparticles were unstable at all the concentrations
of brine solution. The instability of Omp-AgNPs in the pre-
sence of NaCl is due to the aggregation effect promoted by
chloride ions [37].
3.6 FT-IR spectroscopy
The FT-IR spectra of omeprazole and Omp-AgNPs were
compared to identify the functional groups involved in
the interaction with silver metal. The peak observed at
3,057 cm−1 corresponds to aromatic C–H, the absorption
band for stretching vibration of C]N was observed at
1,626 cm−1, the band at 1,406 cm−1 is due to the CH bending,
the peak at 1,205 cm−1 corresponds to the stretching of alkyl
aryl ether, and the strong absorption peak at 1,012 cm−1 is
assigned to the S]O stretching [39]. A decrease in the
intensity and shift in absorption position of bands corre-
sponding to C]N, OCH3, and S]O in the spectra of
 Omeprazole conjugation with silver nanoparticles as a urease inhibitor  7

100 nanoparticles (Omp-AgNPs) indicates the involvement of

Omp-AgNPs
90 Omeprazole these groups in bonding with silver. The FT-IR spectra of
omeprazole and Omp-AgNPs are represented in Figure 6.
80
1613
The mechanism of omeprazole’s interaction with AgNPs is
Transmittance (%)

70 1379 1152
presented in Figure 7. S]O and C]N groups of omepra-
1005
zole are involved in the structural stabilization of AgNPs
60
(Table 1).
50

40 1626
C=N
30 1406
C-H 3.7 XRD analysis
20 1205
OCH3
10 1012,S=O The phase, crystal structure, and crystallite size of synthe-
4000 3500 3000 2500 2000 1500 1000 500 sized nanoparticles were examined by XRD analysis. The
Wavenumber (cm-1) XRD spectrum of Omp-AgNPs showed diffraction peaks at
38.05, 44.04, 64.37, and 77.36° [40]. These peaks correspond
Figure 6: FT-IR spectra of omeprazole-loaded AgNPs.
to the (111), (200), (220), and (311) planes of cubic crystal
lattice (correlated to ICSD file # 064994). The average par-
ticle size of nanocrystal calculated using Debye Scherrer
O equation was found to be 17 nm (Figure 8).
H
O N
S N
N O
3.8 EDX spectroscopy

The elemental composition of chemically synthesized drug

loaded AgNPs was examined by EDX analysis. EDX profile
Ag of Omp-AgNPs displayed a very strong signal in silver
region which confirms the formation of omeprazole-loaded
AgNPs. The optical absorption peak for the nanocrystals of
metallic silver (72.97 wt%) was observed at about 3 keV due

N O
S N
90
O N
H
80
O Omp-AgNPs
70
Figure 7: Mechanism of the interaction between AgNPs and omeprazole.
Intensity (a.u)

60
(111)
50

Table 1: FT-IR data of omeprazole and Omp-AgNPs 40

(200)
(220) (311)
30
Omeprazole Omp-AgNPs
20
Frequency Allocated Frequency Allocated
(cm−1) groups (cm−1) groups 10
1,626 C]N 1,613 C]N 10 20 30 40 50 60 70 80
1,406 C–H 1,379 C–H
1,205 OCH3 1,152 OCH3
2T (degree)
1,012 S]O 1,005 S]O
Figure 8: XRD pattern of Omp-AgNPs.
8  Aneesa Zia et al.
Figure 9: EDX profile of Omp-AgNPs.
to SPR as shown in Figure 9. The additional peaks at 0.25, 0.5,
and 2.3 are the characteristic signals for carbon, oxygen, and
sulfur, respectively. The presence of these peaks in the spec-
trum of nanoparticles confirmed the presence of omepra-
zole moiety bound on the surface of AgNPs.
3.9 TEM
The shape and particle size of omeprazole-loaded silver
nanoparticles were determined by TEM analysis as shown
in Figure 10. TEM image was recorded at different magni-
fications of 50 and 100 nm. The Omp-AgNPs were polydis-
persed agglomerates, small in size, and predominantly
Figure 10: TEM image of synthesized Omp-AgNPs at different magnifications of 50 and 100 nm.
spherical in shape. The particle size of AgNPs was calcu-
lated using ImageJ software. The size of nanoparticles was
in the range of 12–24 nm with an average particle size of
17 nm (Figure 11). Results obtained from the TEM are in
agreement with XRD.
3.10 Urease inhibition assay
The Omp-AgNPs were evaluated for in vitro urease inhibi-
tion potential to examine their activity against enzyme
urease by following standard protocol. The formulation
of omeprazole in the form of nanoparticles significantly
enhanced its inhibitory potential against enzyme urease
 Omeprazole conjugation with silver nanoparticles as a urease inhibitor  9

Average Particle Size 17 nm Table 3: Antioxidant potential of Omp-AgNPs

3.0

Sample % RSA ± SD (500 µg·mL−1) IC50 ± SEM (µg·mL−1)

2.5
Omeprazole 72.21 ± 1.67a 18.7 ± 0.17
2.0 Omp-AgNPs 84.45 ± 2.16b 5.28 ± 0.14
t-BHA 89.56 ± 2.31c 58.76 ± 0.45
Count

1.5
Different small letters represent significant differences (ANOVA post hoc
Tukey test, P < 0.05) between the anti-oxidant activities of different
1.0 samples. SEM stands for standard error of the mean where n = 5.

0.5
by the degree of discoloration [29]. The results showed
0.0 higher scavenging activity for nanoparticles as compared
12 14 16 18 20 22 24
to omeprazole as shown in Table 3. % RSA of omeprazole
Particle Size Distribution(nm) was 72.21% whereas AgNPs were 84.45%. The IC50 value of
omeprazole is 18.7 ± 0.17 while that of Omp-AgNPs is 5.28 ±
Figure 11: Particle size distribution graph of Omp-AgNPs.
0.14 which is less than omeprazole. This shows that nano-
particles are more effective as compared to omeprazole.
Tertiary butylated hydroxy anisole (t-BHA) used as a refer-
Table 2: Urease inhibition assay of omeprazole and Omp-AgNPs
ence showed 89.56% RSA with an IC50 value of 58.76 ± 0.45.

Sample %Inhibition ± SD IC50 ± SEM (µg·mL−1)

Omeprazole 69.45 ± 1.24a 4.05 ± 0.15

Omp-AgNPs 80.23 ± 1.06b 2.17 ± 0.10
Thiourea 93.30 ± 1.24c 18.10 ± 0.22
Different small letters represent significant differences (ANOVA post hoc
Tukey test, P < 0.05) between the urease inhibition activity of different
substances. SEM stands for standard error of the mean where n = 5.
as compared to drug alone (Table 2). The percent inhibition
of nanoparticles was 80.23% while that of omeprazole is
69.45%, and IC50 values for Omp-AgNPs and omeprazole
are 2.17 ± 0.10 and 4.02 ± 0.15 respectively, which shows
nanoparticles are highly active against urease. Thiourea
was used as a standard inhibitor of urease during the
experiment. It showed 93.3% inhibition of enzyme with
an IC50 value of 18.1 ± 0.22.
3.12 Antibacterial activity
The antibacterial potential of omeprazole and its nanopar-
ticles (Omp-AgNPs) was evaluated against two bacterial
pathogens (e.g., S. aureus and E. coli) as shown in Table 4.
The results revealed that omeprazole is ineffective against
both the strains at conc. of 1 mg·mL−1 with no zone of inhibi-
tion as shown in Figures 12 and 13. The omeprazole-loaded
AgNPs showed immense antibacterial activity as compared
to drug. AgNPs were more effective against E. coli with
13.6 mm ± 1.02 zone of inhibition, whereas zone of inhibition
against S. aureus was 12 mm ± 0.41. The results indicate that
AgNPs can be a substitute for antibacterial agents as com-
pared to drugs.

Table 4: Antibacterial assay of omeprazole and Omp-AgNPs

3.11 Antioxidant activity
Sr. no. Conc. (1 mg·mL−1) Zone of inhibition (mm) ± SD
The radical scavenging potential of omeprazole and its
nanoparticles was performed by the DPPH method. The S. aureus E. coli
development of antioxidants for the treatment of oxidative 1. Omeprazole NA NA
stress-related disorders has been promoted by extensive 2. Omp-AgNPs 12.5 ± 0.41a 13.5 ± 1.02a
research on the protective role of antioxidants against 3. Ciprofloxacin 19.5 ± 1.50b 18.4 ± 0.86b
these disorders [41]. Antioxidants react with DPPH and
In the above table, NA stands for “not active”. Different small letters
convert it into diphenylpicrylhydrazine (DPPH-H) due to represent significant differences (t-test, P < 0.001) when data are com-
its hydrogen and electron-accepting capability. The radical pared within the column. SD stands for “Standard Deviation” where
scavenging potentials of the antioxidant can be determined n = 5.
10  Aneesa Zia et al.

Antibacterial Activity
Staphylococcus aureus Escherichia coli
25
20
15
10
5
0
Omeprazole Omp-AgNPs Ciprofloxacin

Figure 12: Antibacterial activity of omeprazole and Omp-AgNPs.

3.13 Hotspot mapping of active pocket

The crystal structure of urease reveals various key resi-

dues that are involved in the binding pocket and allow
the inhibitor to retain within the active site. These includes
Ni–metal atom, sheet S23 (residues 403–409 color-coded
Figure 14: 3D interaction mapping of active site residues of urease.
yellow) a small loop L30 (410–412 color coded blue), helix

Figure 13: Antibacterial assay of omeprazole and its AgNPs.

 Omeprazole conjugation with silver nanoparticles as a urease inhibitor
Figure 15: Interaction of nanoparticles in the active site of urease.
H14 (435–441 color coded brown), loop L37 (444–454 color-
coded red), helix H20 (580–593 color-coded green), loop L39
(594–598 color-coded cyan), helix H21 (599–610 color-coded
pink), and a loop L41 (632–638 color-coded orange) (Figure 14).
The complex of omeprazole interacts with the active
site along with the binding energy of −7.76 kcal·mol−1. It
binds into the active site of the protein by the interaction
of Ni–metal to form a complex with the oxygen atom of the
omeprazole complex. Hydrogen bond interactions were
observed between the complex and His492, CME592, ASP633,
Ala636, Glu525, His594, and His593 amino acid residues.
Asp494 interacts through pi–sulfur interaction with the
complex. Some pi–alkyl and pi–pi interactions were observed
between the complex for the retention of the complex mole-
cule in the active pocket of the urease (Figure 15).
4 Conclusion
The chemical approach is one of the simple, fast, low-cost,
and effective approaches for the synthesis of nanoparticles.
Omeprazole-loaded AgNPs were synthesized by using a
chemical method. Characterization of NPs was done by
advanced analytical techniques including UV–Vis, FT-IR,
EDX, XRD, and TEM analysis. The FT-IR studies confirmed
that S]O, C]N, and OCH3 groups of omeprazole are
responsible for the stabilization of silver nanoparticles.
XRD studies revealed that nanoparticles were crystalline in
nature with cubic crystal structure. Elemental composition
 11
and weight percent of AgNPs were studied by EDX analysis.
Silver was present as a major constituent in omeprazole-
loaded silver nanoparticles. The TEM analysis revealed that
Omp-AgNPs were spherical in shape having the particle size in
the range of 16–20 nm. The antibacterial potential of nanopar-
ticles was evaluated against two bacterial strains namely
S. aureus and E. coli by agar well diffusion method and NPs
showed significant activity against both the strains as com-
pared to omeprazole. The DPPH assay was used to evaluate
the antioxidant potential of nanoparticles, % RSA of Omp-
AgNPs is 84.45%, which is 1.16 times higher than omeprazole.
Urease inhibition of nanoparticles was determined by fol-
lowing standard protocol. The Omp-AgNPs showed 80.23%
inhibition against urease which is 1.15 times higher as com-
pared to omeprazole. The silver nanoparticles have shown
considerable potency when compared to the original drug.
Further optimization of the synthesized nanoparticles may
lead toward even better results which may lead toward the
initiation of preclinical trials on the synthesized nanoparticles.
Furthermore, conducting the toxicity studies of synthesized
products adds to the future perspective of this project. It can
safely be said that the urease inhibition potential of Omp-
AgNPs signifies them as potential lead compounds for the
development of relevant therapies to address damages caused
by H. pylori in clinically sick patients.
Acknowledgments: The authors would like to thank the
University of Nizwa for the generous support of this project.
We thank the analytical and technical staff for assistance.
12  Aneesa Zia et al.
This study was funded by the Deputyship for Research and
Innovation, Ministry of Education in Saudi Arabia (Project
number ISP23-81).
Funding information: This study was funded by the
Deputyship for Research and Innovation, Ministry of
Education in Saudi Arabia (Project number ISP23-81).
Funding was provided by the Research Council through
the Research Grant Program (BFP/RGP/HSS/23/037).
Author contributions: Aneesa Zia and Ayesha Shahzad:
methodology, formal analysis, writing – original draft;
Nadia Riaz and Sara Khan: software, data curation, writing
– original draft; Umar Farooq and Syed Majid Bukhari:
investigation, resources; Asaad Khalid: resources, visuali-
zation, data curation, funding acquisition; Hamdy Kashtoh:
writing – review and editing, resources, funding acquisi-
tion; Rizwana Sarwar, Ajmal Khan, and Ahmed Al-Harrasi:
conceptualization, writing – original draft. writing – review
and editing, supervision, funding acquisition.
Conflict of interest: Authors state no conflict of interest.
Data availability statement: All data generated or ana-
lyzed during this study are included in this published
article.
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