Journal of Molecular Liquids 347 (2022) 117954

Contents lists available at ScienceDirect

Journal of Molecular Liquids

journal homepage: www.elsevier.com/locate/molliq

Sequential microwave-ultrasound-assisted silver nanoparticles

synthesis: A swift approach, their antioxidant, antimicrobial, and in-silico
studies
Venkataraghavan Ragunathan, Chithra K ⇑
Nanomaterials and Environmental Research Laboratory, Department of Chemical Engineering, Alagappa College of Technology, Anna University, Chennai 600025, India

a r t i c l e i n f o a b s t r a c t

Article history: The present research work unravels the novel optimization of microwave-ultrasound-assisted rapid syn-
Received 24 June 2021 thesis of Olea europaea capped silver nanoparticles (O-AgNps) as a capping and stabilizing agent that is
Revised 1 September 2021 not previously studied. The synthesized O-AgNps were characterized using UV–Visible spectroscopy,
Accepted 24 October 2021
Fourier transform infrared spectroscopy (ATR-FTIR), proton nuclear magnetic resonance (1H NMR),
Available online 4 November 2021
dynamic light scattering-zeta potential (DLS-Zeta potential), high-resolution transmission electron
microscopy-energy dispersive X-ray spectroscopy (HR-TEM-EDAX), and atomic force microscopy (AFM)
Keywords:
techniques. The HR-TEM-EDAX revealed that O-AgNps were
10 nm in diameter with a polydispersity
Silver nanoparticles
Characterization
index of 0.191 and were spherical to elliptical. The nanoparticles were analysed for the in-vitro 2,2-
Olea europaea diphenyl-1-picrylhydrazyl (DPPH) antioxidant, agar-well diffusion antibacterial assay, minimum inhibi-
Antioxidant tory concentration (MIC), minimum bactericidal concentration (MBC) assays. The results were promising
Antibacterial assay with a half-maximal inhibitory concentration (IC50) of 155.08 mg/mL for the antioxidant assay. The
In-silico analysis antimicrobial agar-well diffusion assay showed maximum zones of 24.9 ± 0.14 mm diameters at 8 mg/
mL for Staphylococcus aureus (S. aureus) and 24.5 ± 0.7 mm at 8 mg/mL for Escherichia coli (E. coli). The
MICs and MBCs were 156.25 mg/mL and 625 mg/mL for the antimicrobial assays against S.
aureus and E. coli. Additionally, the research throws limelight on the in-silico molecular docking analysis
using AutoDock Vina software and arrives at a possible elucidation to support the experimental work.
Ó 2021 Elsevier B.V. All rights reserved.

1. Introduction

Due to their excellent chemical and physical properties, the use

Abbreviations: O-AgNps, Olea europaea capped silver nanoparticles; UV, Ultravi-
olet; ATR-FTIR, Attenuated total reflectance – Fourier transform infrared spec-
troscopy; 1H NMR, Proton nuclear magnetic resonance; DLS, Dynamic light
scattering; HR-TEM-EDAX, high-resolution transmission electron microscopy-
energy dispersive X-ray spectroscopy; AFM, Atomic force microscopy; BET,
Brunauer-Emmett-Teller; DPPH, 2,2-diphenyl-1-picrylhydrazyl; MIC, minimum
inhibitory concentration; MBC, Minimum bactericidal concentration; IC50, half-
maximal inhibitory concentration; S. aureus, Staphylococcus aureus; E. coli, Escher-
ichia coli; DNA, Deoxyribonucleic acid; BB, Box-Behnken design; RSM, Response
surface methodology; MTCC, Microbial Type Culture Collection and Gene Bank;
ANOVA, Analysis of variance; SAED, Selected area electron diffraction; EUCAST,
European Committee for Antimicrobial Susceptibility Testing; GC–MS, Gas
chromatography-mass spectrometry; RT, Retention time; min/mins, minute(s);
R2, Coefficient of determination; Adj-R2, Adjusted coefficient of determination; DF,
Degrees of freedom; Adj SS, Adjusted sum of squares; Adj MS, Adjusted mean
squares; P–Value, Probability value; F-Value, Fisher’s value Ag2+ Silver ions; Ag0,
Silver nanoparticles; PDB, Protein data bank.
⇑ Corresponding author.
E-mail address: kchithra@annauniv.edu (C. K).
of silver nanoparticles has considerably increased in the present
generation for various applications, including medical, food, phar-
maceutical, environmental, and industrial [1]. Nanoparticles range
between 1 and 100 nm in diameter in at least any dimensions with
enhanced properties than the bulk materials. Their remarkable
thermal, optical, electrical, catalytical, and conductivity properties
influence a lot to be used in every sector [2]. There is always a keen
on obtaining smaller particle sizes as the size influences their
physicochemical properties and applications, which has been an
incentive for developing novel technologies. Several methods for
synthesizing nanoparticles, including the three conventional meth-
ods, chemical, physical, photochemical, electrochemical, and bio-
logical [3], are practiced globally. Furthermore, the green or eco-
friendly synthesis of nanoparticles is gaining pace in the present
scenario since these are simple in synthesis and do not produce
any toxic or harmful by-products during the process [4].

https://doi.org/10.1016/j.molliq.2021.117954
0167-7322/Ó 2021 Elsevier B.V. All rights reserved.
V. Ragunathan and C. K
Moreover, green synthesis has several advantages in cost, sim-
plicity, effectiveness, viability, and stability [5]. The reducing
agents are often various parts of plants and microorganisms like
bacteria and fungi used through redox reactions between the
donor and acceptor atoms. It has been well known that silver
nanoparticles possess exceptional biological properties against
microorganisms and tumor cell lines, making them more attractive
towards biotechnological and biomedical applications worldwide
[6]. The underlying mechanism for these properties is that the sil-
ver nanoparticles are capped with various capping and reducing
agents that are capable of penetrating the cell membrane integrity,
causing leakage, generating free radicals causing oxidative stress,
DNA and protein rupture, inhibiting vital enzymes, interfering in
the electron transport chain, and so on. [7]. However, even though
the green synthesized silver nanoparticles are widely used, it suf-
fers from few drawbacks; one such drawback is that the time taken
for the synthesis in the ambient condition is contradictory to the
other methods such as chemical and physical. To overcome this,
the increase in reaction temperature would favor the rate at which
the nanoparticles are formed; this is due to rapid nucleation and
crystal growth [8–9]. Thus, here comes the idea of using an alter-
native energy source of heat by incorporating microwave irradia-
tion that causes uniform heating by penetrating swiftly; this is
attributed to the influence of microwaves’ electrical and magnetic
properties, resulting in narrow particle sizes [10].
Furthermore, during the microwave-assisted synthesis process,
there is a possibility for the aggregation of nanoparticles [11]
which could be overcome by using ultrasound to disperse the
aggregated particles. The significant advantage of using
microwave-ultrasound irradiations over other conventional routes
is energy efficiency up to 90% than the other methods [12], facile
approach, faster reaction kinetics, smaller particle size, amplified
physicochemical properties, and greater efficiency product yield
[13]. Many recent studies have also reported the reproducibility
and reliability of nanomaterials synthesized using microwave irra-
diations than nanomaterials synthesized using other physical,
chemical, and biological routes [14–15]. In addition, combining
bioactive compounds as reducing agents and microwave-
ultrasound irradiations would be an add-on benefit without releas-
ing any harmful by-products. Here comes the role of selecting the
appropriate plant source, a reservoir of plant secondary metabo-
lites for reducing bulk precursors to nanoparticles.
Olea europaea, commonly known as olive, which belongs to
the Oleaceae family, is a popular fruit commonly cultivated in
European countries and consumed as a Mediterranean diet. Olive
fruits are a rich source of bioactive compounds, fatty acids,
esters, etc., that are believed to lower the risk of various cardio-
vascular diseases, also act as effective antimicrobials, anti-tumor
agents, and antioxidants [16]. There have been several studies
that reported several bioactive compounds in olive leaf extract
[17]. The olive fruits are rich in bioactive constituents such as
polyphenolic flavonoids, phenolic acids, terpenoids, tannins,
saponins, glycosides, etc. Polyphenols like oleuropein, dimethyl-
oleuropein, ligstroside, nuzhenide, tyrosol, quercetin, rutin, caf-
feic acid, luteolin, gallic acid, and fatty acids like oleic acid, lino-
leic acid, myristic acid, hexadecanoic, stearic acid [18–19] are
the major components in olives responsible for reducing precur-
sor bulk salt to nanoparticles. The presence of these active com-
pounds facilitates the formation of nanoparticles. Till now, there
has been a focus on olive oil and olive fruit pulp. However, the
synthesis of silver nanoparticles from Olea europaea fruit extract
using microwave and ultrasound is not reported elsewhere.
Thus, we decided to use the sequential effect of microwave irra-
diation and ultrasound vibrations using Olea europaea as a
reducing and capping agent towards the practical synthesis of
silver nanoparticles for biological applications.
2
Journal of Molecular Liquids 347 (2022) 117954
The use of Box-Behnken design (BB) of Response Surface
Methodology (RSM) in the optimization of synthetic process condi-
tions helps in attaining the best results with statistical efforts [20].
BB design of experiments is a three-level effective factorial design
that gives many experimental trials and helps understand the
interactions among independent variables to predict the depen-
dent variable using multiple polynomial regression statistics.
Several sophisticated characterization instruments are used to
confirm the synthesis and study the essential properties of
nanoparticles. The most commonly used characterization includes
UV–Visible spectroscopy, FTIR spectroscopy, XRD, SEM, TEM,
EDAX, DLS, Zeta potential, NMR, BET, and AFM. Apart from these
characterization techniques, several other sophisticated instru-
mentations like UV-Diffused reflectance spectroscopy, vibrating
sample magnetometry, nanoparticle tracking analysis, and X-ray
photoelectron spectroscopic are also used depending on the appli-
cation of nanoparticles [21].
In-silico computational works have been constantly used as a
shred of supportive evidence to elucidate the chemical interactions
and bonding between the target and the ligand. They are accom-
plished using several high-accuracy software like AutoDock Vina,
AutoDock 4.2, GOLD, Glide, rDock, etc. [22] and arrive at a possible
mechanism using the in-built algorithms. However, the software is
supportive and is reinforced with in-vitro and in-silico experimen-
tal studies to conclude. Molecular dynamics simulations using
high-performance computers aids in further validation by assess-
ing the stability of the docked complexes using various algorithms
and force fields [23].
The main objectives of this research are to explore the Soxhlet
extraction of Olea europaea fruits and characterize the presence
of various phytochemicals using the conventional procedure for
phytochemical analyses, ATR-FTIR, GC–MS, and SEM. Secondly,
we statistically optimized the synthesis of silver nanoparticles
using the bioactive compounds from aqueous Olea europaea extract
as the capping and reducing agent, followed by various sophisti-
cated spectroscopic and microscopic characterization techniques
for confirmation. The sequential synergistic influence of micro-
wave and ultrasound in nanoparticles synthesis is not studied ear-
lier to the best of our knowledge. The influence of microwave
irradiation speeds up the reaction rate by rapid heating, and the
ultrasound maintains the particle size through uniform dispersion
[24]. The later part of the work focuses on the synthesized silver
nanoparticles’ biological applications, namely, antioxidant and
antimicrobial (MIC, MBC, and Well diffusion) assays. Lastly, we
have demonstrated the plausible interaction mechanism for the
in-vitro antimicrobial assay using in-silico computational tools.
Additionally, we discussed the drug-likeliness and toxicity
properties of the bioactive compounds. Thus, the present research
could effectively contribute to developing new drugs and delivery
routes in biological applications. Fig. 1 shows the detailed work-
flow of the research work.
2. Materials and methods
2.1. Chemicals and glassware
All chemicals used in this research work were of analytical
grade obtained from E. Merck, Mumbai, India unless specified. All
the microbiological media and allied chemicals were purchased
from HiMedia Laboratories, Mumbai, India. The chemicals were
used as received without further purification. The following chem-
icals were used viz., hexane (95% pure), toluene (99.9% pure),
acetone (99.8% pure), acetonitrile (99.9% pure), ethanol
(99.5% pure), magnesium ribbon (99.5% pure), hydrochloric
acid, sulphuric acid, nitric acid, sodium hydroxide (97% pure), fer-
V. Ragunathan and C. K
Fig. 1. Workflow of the research work.
ric chloride, gelatin, sodium chloride (99% pure), lead acetate
(95% pure), chloroform (99% pure), glacial acetic acid, benzene
(99.8% pure), ammonia solution, a-naphthol (99% pure), copper
sulphate (98% pure), sodium potassium tartrate, potassium
iodide (99% pure), iodine (99.9% pure), mercuric chloride
(99.5% pure), silver nitrate (99.7% pure, Fisher Scientific, USA),
L-ascorbic acid (99% pure), DPPH (90% pure), methanol
(99.5% pure), Mueller-Hinton agar, nutrient broth, agar-agar,
ampicillin sodium salt (90% pure), gentamycin sulphate (90%
pure), and silver (I) oxide (99% pure). Routine microbial cultures
S. aureus (MTCC 96) and E. coli (MTCC 2939) were procured from
Avanz Bio. Ltd., Chennai, for the antimicrobial assays. Borosilicate
glassware was used throughout the research work. Double-
distilled water (Milli-Q purified water) was used throughout the
experiment.
2.2. Collection and extraction of bioactive compounds Olea europaea
fruits
Olea europaea fruits were procured from local markets in Chen-
nai, India, washed with running tap water followed by double-
distilled water and soaked for 15 min to remove debris and salts.
The fruits were then pitted to remove the endocarp, and the meso-
carps were shade dried for three days, finely blended using an elec-
trical blender, and stored in a clean air-tight container at room
temperature. Next, the conventional Soxhlet extraction was per-
formed to prospect the value-added bioactive compounds from
3
Journal of Molecular Liquids 347 (2022) 117954
fruits. First, 30 g of powdered fruits were weighed (Shimadzu
BL220H electronic balance, Japan) and filled in the Whatman cellu-
lose extraction thimble with 250 mL of solvents filled in the round
bottom flask. The solvents used for extraction were chosen based
on the increase in polarity, viz.,
hexane < toluene < acetone < acetonitrile < ethanol < aqueous.
The extracts were immediately concentrated using R-210 Buchi
Rotavapor (Buchi, Switzerland) to remove excess solvents. The
concentrated extracts were then stored at 20 °C until further
use to prevent the loss of viability. The flow chart of extraction is
shown in Figure S1.
2.3. Preliminary qualitative phytochemical analysis of Olea europaea
fruit extracts
The qualitative test for the presence of various phytochemicals
viz., flavonoids, alkaloids, cardiac glycosides, coumarin glycosides,
tannins, phlobatannins, terpenoids, cholesterol, saponins, carbohy-
drates, and proteins in six different extracts was performed accord-
ing to the standard protocols [25–26]. In addition, the analyses
were performed in duplicates to maintain the concordance of
results.
2.4. Characterization of Olea europaea fruit extracts
The fruit before and after extraction was analysed using the
VEGA3-SBH scanning electron microscope (TESCAN, Czech Repub-
V. Ragunathan and C. K
lic) to study the morphological changes. The functional groups in
fruit extract were identified using JASCO FT/IR-4700 type Spec-
trometer (JASCO, Japan) in the mid-infrared region (4000 to
400 cm1) using the attenuated total reflectance (ATR) mode at
resolution 4 cm1. The bioactive compounds were detected using
Perkin Elmer Clarus 680 chromatography (Perkin Elmer, USA).
2.5. Optimization of O-AgNps synthesis using Box-Behnken (BB) design
of response surface methodology (RSM)
The optimization was done using the three-level (1,0+ 1) BB
design for the microwave-ultrasound-assisted O-AgNps synthesis
using the second-order polynomial equation [27]. The response
was studied by fitting the data onto the second-order quadratic
polynomial equation using the multiple regression method and is
given as follows [28].
Y ¼ b0 þ b1 X 1 þ b2 X 2 þ b3 X 3 þ b4 X 4 þ b12 X 1 X 2 þ b13 X 1 X 3
þ b14 X 1 X 4 þ b23 X 2 X 3 þ b24 X 2 X 4 þ b34 X 3 X 4 þ b11 X 21
þ b22 X 22 þ b33 X 23 þ b44 X 241
Equation (1) denotes the second-order quadratic polynomial
equation, where Y denotes the response recorded in the absor-
bance of O-AgNps at kmax. The equation gives the mathematical
relationship of the response-dependent variable (Y) to the inde-
pendent variables X1, X2, and X3. Here, b0 is a constant; b1, b2, b3,
and b4 denote the linear regression coefficients; b12, b13, b14, b23,
b24, and b34 denote the interactive coefficients; b11, b22, b33, and
b44 denote the quadratic regression coefficients; X1, X2, and X3
denote the independent variables.
Minitab 20.2.0 (Pennsylvania, USA) software trial version was
used for the statistical design of experiments and multiple regres-
sion analysis. Four independent variables were chosen based on
the literature [29–30] and preliminary laboratory experimentation,
precursor concentration, plant extract concentration, microwave
irradiation, and exposure time. These variables were assessed
using the three-level BB design of RSM. The independent variables
with their respective lower (-1), middle (0), and higher (+1) levels.
A total of 27 trials at randomized run orders were generated in a
single replicate. The significance of the test experiments was stud-
ied using the one-way or two-way analysis of variance (ANOVA) at
P < 0.05 and 95% statistical confidence interval below, which
rejecting the null hypothesis.
2.6. Microwave-ultrasound-assisted eco-friendly synthesis of O-AgNps
A 100 mM stock solution of silver nitrate was prepared by dis-
solving 16.98 g of silver nitrate precursor in 1000 mL of double-
distilled water in a 1 L Erlenmeyer flask. Appropriate dilutions
were made from the stock as per requirements and used through-
out the experiment. The nanoparticles were synthesized using the
precursor and aqueous fruit extract in 250 mL Erlenmeyer flasks
under dark and neutral pH conditions. Microwave heating was
irradiated directly into the flasks using a microwave oven (Sam-
sung, South Korea) with a proper condensation system. The O-
AgNps formed were homogenized to attain uniform size distribu-
tion using a 50-Watt digital ultrasonic cleaner (Labman Scientific
Instruments Pvt. Ltd., India) at 40 ± 3 kHz for 20 min at 30 °C. Next,
the O-AgNps were centrifuged at 8,000 rpm for 15 min (REMI R-24
centrifuge, India), the supernatant was discarded, the precipitate of
O-AgNps was washed thrice with absolute alcohol, and finally
washed twice with double-distilled water. The O-AgNps were then
dried in a hot air oven at 60 °C for 24 h and were stored in an air-
tight container.
Journal of Molecular Liquids 347 (2022) 117954
2.7. Characterization of O-AgNps
The preliminary characterization of O-AgNps was done using
UV-Visible spectrophotometer (Shimadzu UV-1800, Japan) in the
spectral range 800 to 300 nm at 1 nm resolution to observe the
kmax in the range 420 to 460 nm. The O-AgNps powder and dried
aqueous plant extracts were characterized using an ATR-FTIR spec-
trometer (Bruker, USA) in the attenuated total reflectance mode.
The O-AgNps were characterized using Bruker advance III
500 MHz solution-state 1H NMR spectrometer (Bruker, USA) to
study the chemical interactions and functional groups. The hydro-
dynamic particle size and zeta potential of O-AgNps were investi-
gated using the advanced Nanotrac Wave II (Microtrac MRB, USA)
particle size analyser. The size of O-AgNps was determined using
the JEM-2100 plus HR-TEM (JEOL, Japan). The Selected area elec-
tron diffraction (SAED) pattern and EDAX were also generated.
The surface topography and size of O-AgNps were characterized
using Park XE7 atomic force microscope (Park systems, South
Korea) using Park’s true non-contact mode.
2.8. DPPH antioxidant activity of O-AgNps
ð1Þ
The DPPH antioxidant assay was performed according to Blois
[31]. The O-AgNps were prepared in different concentrations
(200 to 1000 mg/mL), and L-ascorbic acid (200 to 1000 mg/mL)
was used as a comparison. To 3 mL of sample in a test tube,
1 mL of freshly prepared 0.1 mM DPPH solution was added and
incubated in the dark for 30 min at room temperature. The control
was prepared by adding 1 mL of 0.1 mM DPPH solution to 3 mL of
double-distilled water. The absorbance after dark incubation was
measured at 517 nm using a UV–Visible spectrophotometer (Lab-
man Scientific Instruments Pvt. Ltd., India). The DPPH free radical
scavenging potential percentage was calculated using equation
(2) below [32].
 
AB
%free radical scav enging potential ¼  100 ð2Þ
A
Where A and B denote the absorbance of the control and O-
AgNps samples, respectively.
The experiment was performed in duplicates to maintain the
concordance of results and was reported as mean ± standard devi-
ation. Microsoft Excel 2016, USA was used to determine the half-
maximum inhibitory concentration (IC50) value, graphical repre-
sentations, and data analysis using regression statistics at
P < 0.05 significance.
2.9. Antimicrobial activity of O-AgNps
The antimicrobial activity consisted of three tests: agar-well
diffusion, minimum inhibitory concentration (MIC), and minimum
bactericidal concentration (MBC) assays. All glassware and media
were autoclaved at 121 °C at 15 psi for 20 min before every assay.
The assays were performed in duplicates to maintain the concor-
dance of results and represented in terms of means ± standard
deviation at P < 0.05 significance. Before the assays, the O-AgNps
were filter sterilized using a 0.45 mm pore size Whatman cellulose
acetate filter to remove other contaminants and large-sized
particles.
2.9.1. Agar-well diffusion assay of O-AgNps
The agar-well diffusion antimicrobial assay was performed
against nosocomial pathogenic microorganisms viz., S. aureus
(gram-positive), and E. coli (gram-negative) according to Perez
et al. [33]. The O-AgNps were prepared using autoclaved double-
distilled water in 2, 4, 6, and 8 mg/mL concentrations. Broad-
4
V. Ragunathan and C. K
spectrum antibiotics ampicillin sodium salt and gentamycin sul-
phate at 10 mg/mL concentration were used as positive controls
along with crude Olea europaea (10 mg/mL) extract and silver (I)
oxide (Ag2O) for comparison. Mueller-Hinton agar was poured into
sterilized Petri dishes (100 mm  15 mm), and 100 mL of overnight
cultured microorganisms (
106 cells/mL) were inoculated and
spread using a sterile cotton swab to cover the entire Petri dish.
Then, four wells of 8 mm diameter each were punctured using a
sterile cork borer at equal intervals, and 50 mL of O-AgNps and con-
trols were pipetted onto each well. The plates were then sealed
using a stretch tape, incubated at 37 ± 1 °C for 18 to 22 h, and
the zones of inhibition were measured using a metric ruler.
2.9.2. Minimum inhibitory concentration and minimum bactericidal
concentration assays of O-AgNps
The MIC broth dilution assay was performed according to the
European Committee for Antimicrobial Susceptibility Testing
(EUCAST, 2003) [34] with slight modifications. First, the nanoparticles
were diluted using the four-fold serial dilution technique from
2500 mg/mL to 9.76 mg/mL for up to 5 dilutions. Then, overnight cul-
tured microorganisms (
106 cells/mL) were pipetted into all the
above mixtures. Overnight microbes in nutrient broth were a positive
control; negative control was the nutrient broth without microbes.
The test tubes were sealed using a stretch tape and incubated at
37 ± 1 °C for 18 to 22 h, the results were observed, and the visual
observance of turbidity determined the MIC. The percentage inhibi-
tion was calculated using equation (3) by recording the absorbance
of the complexes after incubation using a UV–Visible spectropho-
tometer (Labman Scientific Instruments Pvt. Ltd., India) at 600 nm.
 
AB
%inhibition ¼  100 ð3Þ
A
Where A and B denote the absorbance of positive control and
test MICs at various concentrations of O-AgNps, respectively.
The MBC assay was performed according to Parvekar et al. [35].
2.10. In-silico molecular docking analysis using AutoDock Vina
The software AutoDock Vina [36] was used for molecular dock-
ing. Two vital bacterial enzymes, deoxyribonucleic acid (DNA) gyr-
ase from S. aureus (PDB Id: 3U2D) and E. coli (PDB Id: 1KZN) [37–
38], were retrieved from the protein data bank (PDB). These two
proteins were selected based on the literature references [39–
40]. The enzymes’ grid box and active site were determined using
MetaPocket 2.0 webserver and compared with the literature refer-
ences for confirmation. Molecular docking was performed for 100
Genetic Algorithm runs with exhaustiveness set to 8. The docking
procedure was validated by removing and re-docking the actual
co-crystallized inhibitors of PDB Id: 3U2D and PDB Id: 1KZN into
their respective ligand-binding sites with the above set grid
parameters. The re-docked complexes were then superimposed
onto the actual co-crystallized structure using LigPlot + v.2.2 and
PyMOL 2.3, and the root-mean-square deviations (RMSD) concern-
ing the reference structures were studied. The extent of inhibition
from docking was studied using the free binding energy (DG) of
docked complexes. The docked complexes were then visualized
using Biovia Discovery studio 2020 and PyMOL 2.3 to study the
molecular interactions. The inhibition constant (Ki value) was cal-
culated using the formula given in equation (4).
 
DG
Ki ¼ exp ð4Þ
RT
Where DG denotes the estimated free binding energy (calories/-
mol); R denotes gas constant (1.986 calories/K mol); T denotes
temperature (298 Kelvin).
5
Journal of Molecular Liquids 347 (2022) 117954
The drug-likeliness and toxicity properties of ligands were pre-
dicted using admetSAR 1 and 2 [41] and SwissADME [42]
webservers.
3. Results and discussion
The extraction of bioactive compounds from Olea europaea fruit
extract was done using the Soxhlet extraction apparatus. The fruit
morphology before and after extraction using SEM, characteriza-
tion of the extracts using ATR-FTIR, and GC–MS were also per-
formed. The presence of various phytochemicals in Olea europaea
extracts was detected qualitatively to assess the feasibility of using
it in the synthesis of O-AgNps. Further, the antioxidant and antimi-
crobial activity of synthesized O-AgNps were studied.
3.1. Characterization of Olea europaea extracts
3.1.1. SEM analysis of Olea europaea fruit
The morphological changes in fruit biomass before and after
Soxhlet extraction were shown in Figure S2 (a) & (b). It is evident
from Figure S2 that the surface of fruit was intact before extraction,
whereas, after extraction, the surface erosion, broken cell walls,
and tissues were observed that indicates effective leaching of valu-
able bioactive compounds. A similar morphological observation
was reported by Pereira et al. [43].
3.1.2. Preliminary qualitative phytochemical analysis
The qualitative phytochemical analysis tests revealed the pres-
ence and the absence of various phytochemicals like flavonoids,
alkaloids, cardiac glycosides, coumarin glycosides, tannins, phlo-
batannins, terpenoids, cholesterol, saponins, carbohydrates, and
proteins in different extracts reported were in Table S1. The aque-
ous and ethanol extract possessed the highest number of phyto-
chemical abundances, including flavonoids, cardiac glycosides,
tannins, terpenoids, carbohydrates, and proteins, than any other
extract. Moreover, the aqueous extract of the fruit showed the
highest yield (78.43 ± 3.67%) compared to other solvent extracts
(Data not shown), which denotes the greater abundance of polar
phytocompounds. It showed the presence of flavonoids (Shinoda
test and Alkaline reagent test), cardiac glycosides (Keller Kiliani’s
test), condensed tannins (ferric chloride test), terpenoids
(chloroform-sulphuric acid test), carbohydrates (Molisch’s test),
and proteins (Xanthoproteic test). The phytochemical abundance
hierarchy in all the extracts was
aqueous > ethanol > acetonitrile > toluene > hexane > acetone.
From Table S1, it is clear that the polar solvents aqueous and etha-
nol extracted a higher number of phytochemicals compared to
mid-and non-polar solvents. Flavonoid polyphenols play a vital
role in reducing metal ions to nanoparticles through capping and
stabilization [44]. In general, Olea europaea is a rich source of
unique polyphenolic bioactive compounds like oleuropein, lignans,
verbascoside, tyrosol, and hydroxytyrosol [45]. As flavonoid
polyphenols play a vital role in reducing metal ions to nanoparti-
cles through capping and stabilization [44], it was decided to use
the aqueous extract of Olea europaea for synthesizing O-AgNps in
this study.
3.1.3. Characterization of extracts using ATR-FTIR
It is essential to characterize the Olea europaea to determine
functional groups as they play a significant role in reducing silver
nitrate to silver nanoparticles [46]. The ATR-FTIR spectra of aque-
ous Olea europaea fruit extract are shown in Fig. 2. The functional
groups are the indicators of various bioactive metabolites present
in phytochemicals. The following functional groups were detected
in the concentrated liquid aqueous Olea europaea extract: a med-
V. Ragunathan and C. K Journal of Molecular Liquids 347 (2022) 117954

105 2008 library, and the top best hit for each compound was recorded.
Aqueous Olea europaea extract The following compounds, namely, glycerol (RT = 12.407 mins;
area = 47.709%) triglyceride and erucic acid (RT = 20.896 mins;
100 area = 45.243%), which is a monounsaturated omega-9 fatty acid,

2886.92
were found to be the major constituents. n-hexadecanoic acid

2790.49
2980.45
(RT = 19.890 mins; area = 2.953%) which is a commonly found sat-
Transmittance %

urated fatty acid; 2-methyl-6-methylene-octa-1,7-dien-3-ol

95
(RT = 27.653 mins; area = 2.726%); oleic acid (RT = 24.012 mins;

825.384
1645.95
area = 1.369%) which is a mono-unsaturated omega-9 fatty acid
were detected as minor constituents in the aqueous extract [47–

722.211
688.214
90 48]. Thus, these fatty acids were detected as the major bioactive

614.217
compounds in the aqueous Olea europaea fruit extract using GC–
MS analysis. The details of the compounds identified and their
85 molecular structures are shown in Table S2 & Figure S3.

3.2. Optimization of parameters for O-AgNps synthesis using Box-

80
4000 3500 3000 2500 2000
-1
Wavenumber (cm )
Fig. 2. ATR-FTIR spectrum of aqueous Olea europaea fruit extract.
ium peak at 2980.45 cm1 and 2886.92 cm1 correspond to CAH
functional group, a vibration at 2790.49 cm1 denotes the presence
of ACHO functional group, a medium peak at 1645.95 cm1
denotes the presence of C@N functional group, a medium stretch-
ing at 825.384 cm1 denotes the presence of C@C functional group
in the extract, a medium peak at 722.211 cm1 denotes the pres-
ence of CAH, C@O, or a benzene derivative, a peak at
688.214 cm1, and 614.217 cm1 corresponds to the presence of
CABr functional group. These functional groups attribute to the
presence of various classes of phytochemicals present in the
extract.
3.1.4. Characterization of aqueous Olea europaea fruit extract using
GC–MS
The GC–MS analysis of aqueous Olea europaea fruit extract
revealed five bioactive compounds at various retention times
(Fig. 3). The spectra and retention time (RT) fingerprints were com-
pared with the National Institute of Standards and Technology-
Behnken (BB) design of experiments
1500 1000
Box-Behnken design of experiments was adopted to optimize
the parameters affecting nanoparticle synthesis. The various coded
levels of independent variables designed using the BB design of
RSM were shown in Table 1.
The BB design containing 27 randomized trials with experimen-
tal and predicted absorbance values was studied. The absorbance
as a response variable was preferred, according to Ibrahim et al.
[49]. The pH throughout the experiment was maintained at 7
based on the previous experimental studies [50]. All the response
absorbance values were obtained in the range 420 to 460 nm
caused due to the localized surface plasmon resonance (LSPR).
Depending on the contrast between the four independent vari-
ables, the experimental absorbance values varied from 0.129 to
0.822 (Table 2). The highest experimental absorbance of 0.822
was in the third trial of BB design with the following conditions,
precursor concentration 30 mM; plant extract concentration
10 mL; microwave irradiation 450 W; exposure time 1 min. The
corresponding predicted absorbance was 0.816. The consolidated
LSPR of all the 27 trials were shown in Fig. 4.
3.3. Analysis of variance (ANOVA)
The ANOVA was studied to examine the variance and signifi-
cance (P < 0.05) of the generated RSM model using T- and F-tests

Fig. 3. GC–MS chromatogram of aqueous Olea europaea fruit extract.

6
V. Ragunathan and C. K Journal of Molecular Liquids 347 (2022) 117954

Table 1
Coded levels of independent variables designed using BB design.

S. No Independent variable Symbol Coded levels

1 0 +1
1 Precursor concentration (mM) X1 10 30 50
2 Plant extract concentration (mL) X2 1 5.5 10
3 Microwave irradiation (W) X3 300 450 600
4 Exposure time (min) X4 1 3 5

Table 2
Experimental and predicted responses of O-AgNps using BB design of RSM.

Std Order Run Order X1 X2 X3 X4 Experimental value Predicted value

21 1 30 1 450 1 0.274 0.166
16 2 30 10 600 3 0.457 0.394
13 3 30 1 300 3 0.188 0.133
9 4 10 5.5 450 1 0.618 0.754
22 5 30 10 450 1 0.822* 0.816
19 6 10 5.5 600 3 0.610 0.469
17 7 10 5.5 300 3 0.536 0.520
11 8 10 5.5 450 5 0.537 0.500
14 9 30 10 300 3 0.669 0.588
26 10 30 5.5 450 3 0.323 0.352
10 11 50 5.5 450 1 0.422 0.341
23 12 30 1 450 5 0.347 0.390
8 13 30 5.5 600 5 0.129 0.233
15 14 30 1 600 3 0.194 0.157
4 15 50 10 450 3 0.286 0.299
1 16 10 1 450 3 0.129 0.194
7 17 30 5.5 300 5 0.579 0.576
20 18 50 5.5 600 3 0.143 0.195
3 19 10 10 450 3 0.817 0.806
25 20 30 5.5 450 3 0.341 0.352
5 21 30 5.5 300 1 0.425 0.399
12 22 50 5.5 450 5 0.687 0.433
6 23 30 5.5 600 1 0.491 0.572
24 24 30 10 450 5 0.284 0.429
18 25 50 5.5 300 3 0.137 0.314
2 26 50 1 450 3 0.132 0.221
27 27 30 5.5 450 3 0.394 0.352
*
Best run with the highest absorbance

(Table 3). The F-value of the quadratic regression model was found
to be 3.56 and was found to be significant (P < 0.05). The F-value of
Lack-of-fit was calculated by dividing the mean square of lack-of-
fit by the mean square of pure error, for the model was 16.55
(P > 0.05) and indicated that the model was slightly insignificant;
thus, the predicted model correlated and well fitted with the
experimental data [51]. The F-value of the model 3.56 was more
significant than the lack-of-fit 0.022544 at 95% significance that
denotes the model adequacy. The model’s coefficient of determina-
tion (R2) was found to be 0.8058, and the adjusted coefficient of
determination (Adj-R2) was 0.5793 with a standard error of
0.1378. Thus, the R2 and Adj-R2 were in moderate agreement with
each other. The precursor and plant extract concentrations were
the most significant on the O-AgNps synthesis, whereas microwave
irradiation and exposure time were insignificant. The regressions
equation was generated by fitting the experimental data onto the
second-order quadratic polynomial equation shown in equation
(5).

Y ¼ 0:526  0:0081 X 1 þ 0:1780X 2 þ 0:00241X 3 þ 0:033X 4

þ 0:000106X 1  X 1  0:00072X 2  X 2  0:000001X 3  X 3
þ 0:0281X 4  X 4  0:001483X 1  X 2  0:000006X 1  X 3
þ 0:00216X 1  X 4  0:000081X 2  X 3  0:01697X 2  X 4
 0:000430X 3  X 4 ð5Þ
Where Y denotes the dependent Response variable.
Totally six contour plots and their corresponding three-
dimensional surface plots were generated to explain the effects
of various levels of independent variables on O-AgNps synthesis.
Fig. 5 (a) depicts the effect of precursor concentration and plant
extract concentration on absorbance. The highest absorbance > 0.8
Fig. 4. UV–Visible spectra of synthesized O-AgNps using BB design of RSM. was attained at 10 to 15 mM precursor concentration and 8.5 to
7
V. Ragunathan and C. K Journal of Molecular Liquids 347 (2022) 117954

Table 3
Analysis of variance of experiments.

Source DF Adj SS Adj MS F-Value P-Value

Model 14 0.94691 0.067636 3.56 0.017*
Linear 4 0.57182 0.142955 7.52 0.003*
X1 1 0.17280 0.172800 9.09 0.011*
X2 1 0.35742 0.357420 18.80 0.001*
X3 1 0.02168 0.021675 1.14 0.307
X4 1 0.01993 0.019927 1.05 0.326
Square 4 0.10094 0.025234 1.33 0.315
X1 * X1 1 0.00958 0.009577 0.50 0.491
X2 * X2 1 0.00112 0.001121 0.06 0.812
X3 * X3 1 0.00205 0.002054 0.11 0.748
X4 * X4 1 0.06750 0.067500 3.55 0.084
2-Way Interaction 6 0.27415 0.045692 2.40 0.092
X1 * X2 1 0.07129 0.071289 3.75 0.077
X1 * X3 1 0.00116 0.001156 0.06 0.809
X1 * X4 1 0.02993 0.029929 1.57 0.234
X2 * X3 1 0.01188 0.011881 0.62 0.445
X2 * X4 1 0.09333 0.093330 4.91 0.047
X3 * X4 1 0.06656 0.066564 3.50 0.086
Error 12 0.22817 0.019014
Lack-of-Fit 10 0.22544 0.022544 16.55 0.058
Pure Error 2 0.00272 0.001362
Total 26 1.17508
*
denotes significance < 0.05

10 mL plant extract concentration; the absorbance then decreased
with increased precursor concentration and could be attributed to
the fact that the plant extracts were utterly utilized in the reduc-
tion of silver nitrate to nanoparticles; therefore, no further reduc-
tion occurred upon increasing the precursor concentration. Fig. 5
(b) depicts the effect of precursor concentration and microwave
irradiation on absorbance. The highest absorbance of 0.5 to 0.6
was attained within 15 mM precursor concentration and 300 to
500 W microwave irradiation. Further, the absorbance decreased
above 30 mM precursor concentration and above 500 W micro-
wave irradiation, respectively. The high temperature acted as a
limiting factor in the formation of O-AgNps. The effect of precursor
concentration and the exposure time was depicted in Fig. 5 (c),
where the highest absorbance of 0.8 was attained within 15 mM
precursor concentration and a short exposure time of 1 min. A
decrease in the absorbance was observed with an increase in the
precursor concentration and exposure time. The higher the precur-
sor concentration and exposure time were not favourable due to
the depleting plant extract concentration. The effect of plant
extract concentration and microwave irradiation on absorbance
was shown in Fig. 5 (d), where the highest absorbance of 0.5 to
0.6 was observed with plant concentration above 8 mL and micro-
wave irradiation 300 to 450 W. The effect of plant extract concen-
tration and exposure time shown in Fig. 5 (e) denotes the highest
absorbance > 0.8 was obtained with plant extract concentration
10 mL and exposure time 1 min. The effect of microwave irradia-
tion and exposure time on absorbance shown in Fig. 5 (f) denotes
the highest absorbance of 0.5 to 0.6 was obtained with microwave
irradiation 300 W and exposure time 5 min and 450 to 600 W and
exposure time 1 min. The O-AgNps yield was more significant at
high temperatures and a shorter exposure time interval, thus pre-
venting the aggregation of O-AgNps. Thus, from the contour plots,
we can conclude that the maximum absorbance was attained at
10 mM precursor concentration, 10 mL plant extract concentration,
microwave irradiation 600 W, and exposure time 1 min.
Further, the optimized solution for the synthesized O-AgNps
was obtained: 10 mM precursor concentration, 10 mL plant extract
concentration, microwave irradiation 600 W, and exposure time
1 min. Finally, the O-AgNps were synthesized using the predicted
optimized conditions to validate the experiment, and absorbance
of 0.830 was obtained at kmax 438 nm (Fig. 6).
8
3.4. Microwave-ultrasound-assisted green synthesis of O-AgNps
The O-AgNps were experimentally synthesized using the opti-
mized conditions to observe a colour change from light yellow to
a dark reddish-brown. The colour change was instantaneous
because of the influence of external microwave heating. The reduc-
tion of precursor silver nitrate ions (Ag2+) to O-AgNps (Ag0) could
be attributed to flavonoid polyphenols and fatty acids and showed
a characteristic absorption kmax between 420 and 460 nm of the
UV–Visible region upon striking of light. This absorption peak
and scattering were attributed to the vibration and excitation of
free electrons due to the localized surface plasmon resonance
effect of silver nanoparticles [52]. The effect of rapid and uniform
microwave heating has enhanced nucleation growth and nanopar-
ticle formation to a greater extent than the conventional green syn-
thesis. The particles tend to aggregate upon increasing the
temperature due to microwave irradiation [53] was overcome by
the influence of ultrasound cavitation that disaggregates them. Just
after microwave irradiation, as the temperature declined, the O-
AgNps were subjected to ultrasound vibrations by forming cavita-
tion bubbles to ensure uniform homogeneous dispersion of the
synthesized O-AgNps [54]. Thus, the non-aggregated particles give
rise to smaller and highly dispersed particles sizes essential for bio-
logical applications. The nanoparticles formed after sequential syn-
theses were characterized after drying.
3.5. Characterization of O-AgNps
3.5.1. ATR-FTIR spectrum of O-AgNps
The ATR-FTIR spectroscopy revealed the functional groups
responsible for the reduction of Ag2+ to Ag0. Interestingly, while
comparing the spectrum of both the Olea europaea fruit extract
and O-AgNps, several functional groups were found shifted, and
some of them have disappeared in O-AgNps and were as follows
(Fig. 7). The medium peaks from 3500 ± 1 cm1 to
3000 ± 1 cm1 attributed to aliphatic NAH and CAH stretching
detected in the fruit extract have entirely disappeared in the
OAAgNps. Also, medium peaks from 2782.04 ± 1 cm1 to 2384.4
4 ± 1 cm1 in the fruit extract responsible for CAH and SAH func-
tional groups were missing in O-AgNps. Medium peaks 2081.06 ±
V. Ragunathan and C. K
Fig. 5. Contour plots of synthesized O-AgNps using BB design of RSM.
1 cm1 and 1989.92 ± 1 cm1 corresponding to C@C@C functional
group detected in fruit extract were absent in O-AgNps. The finger-
print region peaks from 1750 ± 1 cm1 to 530 ± 1 cm1 present in
fruit extract representing the stretching of NAH, C@O, CAH, OAH,
CAO, CAN, CABr, and CAI groups were shifted in the OAAgNps
spectrum. Thus, these functional groups might have played a vital
role in the reduction of Ag2+ to Ag0. Similar experimental findings
of ATR-FTIR spectroscopy were reported by Chand et al. [55].
3.5.2. 1H NMR spectrum of O-AgNps
The 1H NMR spectrum of O-AgNps revealed various functional
groups that might have been responsible for the reduction of
Ag2+ to Ag0 (Fig. 8). Since the present study dealt with using the
crude aqueous extract of Olea europaea, it is impossible to predict
the exact structure of compounds responsible for reduction [56];
9
Journal of Molecular Liquids 347 (2022) 117954
however, the functional groups responsible could be detected
using 1H NMR spectroscopy. Tetramethylsilane was used as the
internal standard and was attributed to 0 ppm of the chemical
shift. As a result, several functional groups corresponding to the
chemical shifts were identified tabulated (Table S3). The functional
groups, namely, CAH, C@O, C@C, OAH, CABr, were found common
in both ATR-FTIR and 1H NMR that attributed to various phyto-
chemicals like polyphenolic flavonoids, tannins, terpenoids, sapo-
nins, glycosides, carbohydrates, and proteins, thus proving the
presence of strongly adsorbed capping and reducing biomolecules
on the surface of in-situ silver nanoparticles [57].
3.5.3. DLS analysis and zeta potential of O-AgNps
DLS analysis of O-AgNps revealed that the particles possessed a
hydrodynamic diameter of 318 ± 149 nm with a polydispersity
V. Ragunathan and C. K Journal of Molecular Liquids 347 (2022) 117954

index of 0.205, viscosity 0.981 cP at 20 °C, and conductivity 107 m/

S/V/cm (Fig. 9). The particles were uniformly shaped and homoge-
neously distributed. The experiment was performed thrice to
maintain the concordance of results, and the best readings were
reported. The DLS is dependent on the Brownian motion of parti-
cles in a suspension measured at 180° heterodyne-backscatter
arrangement. DLS measures the particle size along with the hydro-
dynamic diameter that comprises the charges, van der Waals
forces, capillary, and electrostatic forces [58] around the O-
AgNps. The zeta potential of O-AgNps was 56.8 mV, denoted a
repulsive force between the particles, and was non-
agglomerated. The negative charge could be attributed to the
capped-reducing agents such as fatty acids, polyphenols, and car-
bohydrates. The zeta potential of O-AgNps was found to be the best
when compared to several previous experimental findings
reported [59–60] with a higher negative zeta potential. The O-
AgNps were highly dispersed and non-agglomerated due to shal-
Fig. 6. Synthesized O-AgNps for the optimized conditions: (a) Olea europaea
extract, (b) silver nitrate, and (c) O-AgNps.
low negative zeta potential.

Fig. 7. ATR-FTIR spectra of (a) O-AgNps and (b) aqueous Olea europaea fruit extract.

10
V. Ragunathan and C. K
Fig. 8. 1H NMR spectrum of O-AgNps showing various functional groups.
Fig. 9. Hydrodynamic particle size distribution of O-AgNps characterized using DLS.
3.5.4. HR-TEM-EDAX analysis of O-AgNps
The particle size, shape, internal structure, and crystallinity of
O-AgNps were studied using HR-TEM-EDAX showed in Fig. 10 (a)
to (c). HR-TEM-EDAX analysis revealed the O-AgNps were spheri-
cal with an ultra-low average particle size of 10.47 ± 9.19 nm
(n = 64) (P < 0.001) and a low polydispersity index of 0.191. The
O-AgNps was found to be significantly calculated by the non-
linear Gaussian curve fit distribution analysis (Fig. 10 (d)) using
Origin 2021 learning edition (OriginLab Corporation, USA) soft-
ware. The size of particles was tiny, thus facilitating enhanced opti-
cal and electronic properties essential for biological applications.
The low polydispersity index denotes that the nanoparticles are
homogeneously distributed with uniform shape, size, and molecu-
lar weight. The R2 and Adj-R2 values of Gaussian particle size dis-
tribution were found to be 0.9829 and 0.9756, respectively, and
were in good agreement. The d-spacing of O-AgNps was calculated
using ImageJ software [61] and found to be 0.25 nm. Elemental
analysis using EDAX confirmed the presence of silver 20.93% (Ag)
at 3 keV, oxygen 3.18% (O), chloride 2.91% (Cl) and trace quantities
11
Journal of Molecular Liquids 347 (2022) 117954
of sodium 1.23% (Na) and sulphur 0.72% (S). Apart from these ele-
ments, carbon 47.22% (C) and copper 23.80% (Cu) were also
detected at high concentrations that arose due to the use of
carbon-coated copper grid mesh during TEM analysis (Fig. 10
(e)). The crystallinity of O-AgNps was studied using the SAED pat-
tern that produced a scattered diffraction pattern of polycrystalline
nature (Fig. 11). Joseph and Mathew attained a silver nanoparticle
size of 20.82 ± 1.8 nm characterized by TEM using Alpinia galanga
as a reducing agent through microwave irradiation [62] which is
twice the present work. Ashraf et al. reported that microwave-
assisted silver nanoparticles synthesis using Melia azedarach pos-
sessed a particle size of 12 to 46 nm. The present research reported
10 to 15 nm; this could be achieved due to the combined micro-
wave heating and ultrasound vibrations [63]. The size of the
nanoparticles reported in the present research work using Olea
europaea as a reducing agent is much smaller than the previously
reported similar experimental findings [14,64–65]. For instance,
Anjana et al. reported a particle size of 19 nm using the influence
of microwave alone, and the present study was able to attain a
much smaller particle size due to the sequential influence of
microwave and ultrasound [66]. Darmanin et al. reported a 45 to
130 nm particle size using various polyols as reducing agents with
the influence of microwave, which is much higher than this study
[67]; thus, the microwave-ultrasound-assisted technique has a
more substantial influence on particle size than the using micro-
wave alone.
3.5.5. AFM analysis of O-AgNps
The three-dimensional surface topography, size, and height of
O-AgNps were studied using the true non-contact mode of AFM
in the XY plane. The analysis was performed at different dimen-
sions viz., 10 mm  10 mm, 3 mm  3 mm, and 1 mm  1 mm to
get the best image of O-AgNps (Fig. 12 (a)). The non-contact mode
of AFM was very efficient and produced accurate results without
damaging the nanoparticles and cantilever. The three-
dimensional forward surface topography of O-AgNps denoted
round and elliptical shapes with particle heights ranging from 1
V. Ragunathan and C. K
to 25 nm along the red-coloured horizontal line with an average
height of
10 nm (Fig. 12 (b)).
3.6. Antioxidant activity of O-AgNps
The free radical scavenging potentials of O-AgNps and L-
ascorbic acid were studied using the DPPH assay that contains
stable free radicals [68]. The antioxidant ability of O-AgNps
Fig. 10. Particle size and EDAX spectrum of O-AgNps characterized using HR-TEM-EDAX.
12
Journal of Molecular Liquids 347 (2022) 117954
depends on reducing DPPH by hydrogen donor–acceptor interac-
tions between antioxidants and nitrogen atoms in DPPH [69].
The antioxidant activity of O-AgNps using Olea europaea fruit
extract is not previously reported. The scavenging potential of O-
AgNps increased linearly from 51.94 ± 0.001% to 88.90 ± 0.002%
(P < 0.05) with an increase in concentration from 200 to 1000 mg/
mL (Fig. 13). The O-AgNps showed a significant difference in con-
centrations and their respective antioxidant potentials, thus
V. Ragunathan and C. K
Fig. 11. SAED pattern showing polycrystalline nature of O-AgNps.
accepting the alternate hypothesis. Upon increasing the concentra-
tion of O-AgNps above 1000 mg/L (P > 0.05), no further significant
difference in antioxidant potential was observed (Data not shown).
The IC50 of O-AgNps was 155.08 mg/mL and greater than the poten-
tial of L-ascorbic acid with IC50 = 84.47 mg/mL. The antioxidant
activity of crude Olea europaea polyphenol extract was IC50 = 56.
5 mg/mL [70], nearly one-third lower than the IC50 of O-AgNps in
the present study. Almatroodi et al. reported the antioxidant
potential of Olea europaea fruit extract very similar to the O-
AgNps in the current research work; thus, no difference between
the crude and nano synthesized material [71]. Thus, the O-AgNps
showed moderate DPPH antioxidant activity than positive control
L-ascorbic acid and crude aqueous Olea europaea extract reported
in the literature.
3.7. Antimicrobial activity of O-AgNps
3.7.1. Agar-well diffusion assay
The antimicrobial susceptibility of O-AgNps against gram-
positive and gram-negative nosocomial pathogens S. aureus and
E. coli were assessed using the well diffusion method. The greater
the zone diameter size, the more the organisms are susceptible
to nanoparticles and vice-versa. The zone diameter was found to
increase as an increase in the concentration of O-AgNps
(P < 0.05). A well-established zone diameter of 19.25 ± 0.35 mm
was observed at the lowest tested concentration of 2 mg/mL, fol-
lowed by 22.5 ± 0.7 mm at 4 mg/mL, 24 mm at 6 mg/mL, and 24.
9 ± 0.14 mm at 8 mg/mL was observed for S. aureus (Fig. 14 (a)).
In addition, the organism was susceptible to broad-spectrum
antibiotic controls ampicillin sodium salt (10 mg/mL) and gen-
13
Journal of Molecular Liquids 347 (2022) 117954
tamycin sulphate (10 mg/mL) at 45 mm and 18 mm, respectively
(Fig. 14 (b)). S. aureus was not susceptible to plant crude aqueous
Olea europaea extract and chemically synthesized silver oxide.
E. coli also were also susceptible to O-AgNps at low concentra-
tions and showed zones of diameters 14.2 ± 0.28 mm at 2 mg/mL,
18.5 ± 0.7 mm at 4 mg/mL, 23.35 ± 0.49 mm at 6 mg/mL, and 24.
5 ± 0.7 mm at 8 mg/mL (Fig. 15 (a)). The control antibiotic ampi-
cillin sodium salt did not show any sign of inhibition; in other
words, E. coli was found resistant to ampicillin (10 mg/mL), whereas
gentamycin sulphate (10 mg/mL) showed a 10 mm inhibition zone
(Fig. 15 (b)). E. coli was susceptible to plant crude aqueous Olea
europaea extract and chemically synthesized silver oxide. From
the agar-well diffusion assay, it was clear that O-AgNps showed
excellent antimicrobial properties, and the results were compara-
ble with antibiotic control drugs. Qais et al. and Vu et al. reported
similar zones of inhibition diameter concerning the present study
[72–73].
3.7.2. MIC and MBC antimicrobial assay
A four-fold serial dilution technique was followed to find the
minimum concentration (MIC) of O-AgNps needed to inhibit the
growth and activity of microorganisms. This assay was performed
using the lowest concentration that inhibited the microorganisms
in the agar-well diffusion zone of inhibition assay (2 mg/mL). The
appearance of cloudy and turbid solution after incubation denoted
that the microorganisms were still alive and active. The appear-
ance of a clear and non-turbid solution denoted that the growth
of microorganisms was inhibited; however, the microorganisms
might not have been killed and would remain alive in the solution
[74]. The MIC of S. aureus and E. coli were 156.25 mg/mL, denoted by
V. Ragunathan and C. K
Fig. 12. Three-dimensional topography of O-AgNps characterized using AFM.
a non-turbid clear solution. The percentage inhibition of S. aureus
in the MIC assay varied from 76.66 ± 1.5% to 91.13 ± 0.09%
(P > 0.05) for every four-fold increase in the concentration of O-
AgNps from 9.76 to 2500 mg/mL (Table 4). The percentage inhibi-
tion of E. coli in the MIC assay varied from 28.14 ± 0.71% to 91.62
± 0.23% (P > 0.05) for every four-fold increase in the concentration
of O-AgNps from 9.76 to 2500 mg/mL (Table 5). The minimum bac-
tericidal concentration is the minimum concentration at which the
entire microorganisms are killed. Few colonies were observed after
inoculating 156.25 mg/mL of O-AgNps from the MIC assay of S. aur-
eus and E. coli onto the MHA plate. No colony was observed at
14
Journal of Molecular Liquids 347 (2022) 117954
625 mg/mL of O-AgNps against S. aureus and E. coli, respectively.
Thus, the MBC of O-AgNps against S. aureus and E. coli was
625 mg/mL, respectively. S. aureus was susceptible to O-AgNps
and antibiotics; however, E. coli were susceptible at slightly higher
concentrations than S. aureus. The MIC and MBC of O-AgNps were
very promising with tremendous potential compared to Buszewski
et al. [75]. Supporting the antimicrobial potential of O-AgNps in the
present study, Parvekar et al. also reported the same MIC and MBC
values of 625 mg/mL against the bacteria using commercially
purchased chemically synthesized silver nanoparticles (particle
size = 5 nm) [35]. The zones of inhibition, MIC, and MBC of
V. Ragunathan and C. K
Fig. 13. DPPH antioxidant activity of O-AgNps and L-ascorbic acid.
O-AgNps in the present study were satisfactory compared to the
results reported using Waltheria americana capped nanoparticles
[76].
3.8. In-silico molecular docking studies
The amino acids in the active sites of S. aureus and E. coli DNA
gyrase predicted using MetaPocket 2.0 are shown in Table S4 &
S5. The DNA gyrase (PDB Id: 3U2D) is an essential enzyme in S. aur-
eus contains Gyr A and B subunits and belongs to the family of
topoisomerase that plays a vital role in DNA synthesis and its inhi-
bition causes cell death that has become an attractive target in the
recent times [77]. Molecular docking was performed using Auto-
Dock Vina software using the bioactive compounds obtained from
GC–MS analysis of the aqueous extract as ligands against the DNA
gyrase of S. aureus. The ligands were ranked based on their increas-
ing order of binding energy (lowest to highest) in Table S6. The
lower the binding energy of ligands, the greater is the inhibition
potential. Out of the six bioactive compounds docked, oleic acid
ranked as the best compound to show inhibition against the
enzyme with a binding energy of 5.4 kcal/mol with an inhibition
Fig. 14. Agar-well diffusion antimicrobial assay (a) O-AgNps and (b) control denoting the zones of inhibition against S. aureus.
15
Journal of Molecular Liquids 347 (2022) 117954
constant (Ki) value of 109 mM interacting with VAL-21, GLY-24,
LEU-25, VAL-28, TYR-35, GLU-50, ILE-51, ASN-54, ILE-86, ILE-102,
LEU-103, THR-104, SER-128, SER-129, VAL-130, ALA-133, and
ILE-175 amino acid residues with one conventional hydrogen bond
within 3 Å. The conventional hydrogen bond was formed between
the oxygen atom of THR-104 and a hydrogen atom of the ligand.
Apart from the conventional hydrogen bond, other non-covalent
interactions like van der Waals, alkyl, and p-alkyl bonds were also
formed between the ligands and the amino acids in the enzyme’s
active site. The compound 2-methyl-6-methylene-1,7-octadien-3-
ol ranked second with 5.2 kcal/mol (Ki = 153 mM) interacting with
ILE-51, ASN-54, SER-55, GLU-58, VAL-79, ASP-81, GLY-85, ILE-86,
THR-173, and ILE-175 with amino acids in the active site. Conven-
tional hydrogen bonds, van der Waals, and alkyl bond interactions
were responsible for inhibiting the enzyme, thus inhibiting DNA
synthesis. Three hydrogen bonds ASP-81 and THR-173, were
formed between the enzyme and ligand within a distance of 3 Å.
The greater the number of intermolecular hydrogen bonds denotes
the greater the inhibition potential of ligands. The interaction of
erucic acid with the enzyme resulted in the binding energy of
4.8 kcal/mol (Ki = 300 mM) interacting with ILE-51, ASN-54,
SER-55, GLU-58, ASP-81, ARG-84, GLY-85, ILE-86, PRO-87, ILE-
102, LEU-103, SER-129, THR-173, and ILE-175 residues in the
enzyme. Two conventional intermolecular hydrogen bonds were
formed between the ligand and SER-55, ASP-81 residue of enzyme.
The compound n-hexadecanoic acid ranked fourth with 4.8 kcal/-
mol (Ki = 300 mM) interacting with ILE-51, ASN-54, SER-55, GLU-
58, VAL-79, ASP-81, GLY-85, ILE-86, PRO-87, ILE-102, THR-173,
VAL-174, and ILE-175 amino acids. One intermolecular conven-
tional hydrogen bond was formed between the oxygen acceptor
in THR-173 and the hydrogen bond donor in the ligand. Glycerol
with a binding energy 4.1 kcal/mol (Ki = 980 mM) interacted with
ASN-54, SER-55, GLU-58, ASP-81, GLY-83, ARG-84, GLY-85, ILE-86,
GLY-172, and THR-173 residues in the active site with seven con-
ventional hydrogen bonds at a distance within 3 Å. Glycerol
formed conventional hydrogen bonds with ASN-54, GLU-58,
ARG-84, GLY-85, and THR-173 amino acids. Thus, the compounds
of Olea europaea inhibited the S. aureus DNA gyrase enzyme, effec-
tively forming several interactions in the active site by forming
intermolecular conventional hydrogen bonds, alkyl, and p-alkyl
V. Ragunathan and C. K Journal of Molecular Liquids 347 (2022) 117954

Fig. 15. Agar-well diffusion antimicrobial assay (a) O-AgNps and (b) control denoting the zones of inhibition against E. coli.

interactions. A collective of bioactive ligands docked into S. aureus
DNA gyrase was shown in Fig. 16. The bioactive compounds docked
in the present study against S. aureus (PDB Id: 3U2D) were similar
and comparable with the binding energies and amino acid interac-
tions reported by Vijayakrishnan et al. [78]. The 2-dimensional
interaction profile diagram of bioactive compounds with the amino
acids of S. aureus DNA gyrase is shown in Figure S4.
The E. coli DNA gyrase (PDB Id: 1KZN) is involved in the negative
supercoiling of DNA during replication, thus facilitating replication
and transcription. Inhibiting the enzyme would ultimately lead to
bacterial cell death. Molecular docking revealed that n-
hexadecanoic acid with a binding energy of 5.4 kcal/mol
(109 mM) interacted with VAL-43, ASN-46, ALA-47, GLU-50, VAL-
71, ASP-73, ILE-78, ILE-90, VAL-93, LEU-94, HIS-95, ALA-96, GLY-
119, VAL-120, SER-121, and THR-165 amino acids in the active site
cleft by forming intermolecular hydrogen, van der Waals, and alkyl
interactions. n-hexadecanoic acid formed between seven hydrogen
bonds with ILE-90, VAL-93, ALA-96, VAL-120, and SER-121 amino
acids at a distance within 3 Å in the active site forming intermolec-
ular hydrogen bonds, van der Waals, and alkyl interactions
(Table S7). Oleic acid interacted with VAL-43, ASN-46, ALA-47,
GLU-50, VAL-71, ASP-73, ILE-78, ILE-90, HIS-95, ALA-96, GLY-119,
VAL-120, SER-121, THR-165, and VAL-167 residues of the enzyme
with a binding energy of 5.3 kcal/mol (129 mM) and two conven-
tional intermolecular hydrogen bonds VAL-120 and SER-121
within 3 Å. The compound 2-methyl-6-methylene-1,7-octadien-3
-ol showed binding energy of 5.3 kcal/mol (129 mM) interacting
with VAL-43, ASN-46, ALA-47, GLU-50, VAL-71, ASP-73, GLY-77,
ILE-78, VAL-120, THR-165, and VAL-167 residues by forming van
der Waals and alkyl bonds in the active site and one conventional
hydrogen bond with ASP-73 within 3 Å. Erucic acid showed bind-
ing energy of 5.1 kcal/mol (180 mM) interacting with VAL-43,
ASN-46, GLU-50, VAL-71, ASP-73, ILE-78, ILE-90, ALA-96, GLY-
119, VAL-120, SER-121, THR-165, and VAL-167 residues in the
active site of the enzyme forming conventional intermolecular
hydrogen bonds, alkyl, and van der Waals interactions. Four con-
ventional hydrogen bonds were formed with ASN-46, VAL-120,
and SER-121 residues in the enzyme’s active site. Glycerol pos-

Table 4
Percentage inhibition of S. aureus in MIC assay.

S. No Concentration (mg/mL) Absorbance % Inhibition

1 9.76 0.187 78.00
2 39.06 0.116 86.35
3 156.25 0.100 88.23
4 625.00 0.080 90.58
5 2500.00 0.074 91.29

Table 5
Percentage inhibition of E. coli in MIC assay.

S. No Concentration (mg/mL) Absorbance % Inhibition

1 9.76 0.644 26.82
2 39.06 0.348 60.45
3 156.25 0.142 83.86
4 625.00 0.078 91.13
5 2500.00 0.076 91.36 Fig. 16. Bioactive compounds bound to the active site of S. aureus DNA gyrase (PDB
Id: 3U2D).

16
V. Ragunathan and C. K
sessed binding energy of 3.9 kcal/mol (1.37 mM) interacting with
VAL-89, ILE-90, VAL-93, HIS-95, ALA-96, VAL-118, GLY-119, VAL-
120, and VAL-122 amino acid residues by forming van der Waals
and conventional hydrogen bonds. Conventional intermolecular
hydrogen bonds were formed between the ligand and ILE-90,
VAL-93, HIS-95, ALA-96, VAL-118, VAL-120, and SER-121 amino
acids in the active site of the enzyme. Thus, the compounds of Olea
europaea inhibited the E. coli DNA gyrase enzyme, effectively form-
ing several interactions in the active site by forming intermolecular
conventional hydrogen bonds, alkyl, and p-alkyl interactions. A
collective of bioactive ligands docked into E. coli DNA gyrase was
shown in Fig. 17. Boyapati et al. reported similar amino acid inter-
actions compared to the present study; however, our re-docking
and validation were better compared to them [79]. The 2-
dimensional interaction profile diagram of bioactive compounds
with the atoms of amino acids of E. coli DNA gyrase is shown in
Figure S5.
Thus, the bioactive compounds inhibited the vital enzymes of
nosocomial pathogens S. aureus and E. coli, essentially interacting
with their active site and inhibiting them and resulting in cell
death. Thus, a possible interaction mechanism for the in-vitro
antimicrobial potential of O-AgNps has been predicted using the
in-silico molecular docking. Furthermore, the elucidated interac-
tion chemistry of the docked complexes between the ligands and
enzymes could have resulted in a conformational change, thus,
causing the inhibition and eventually microbial cell death.
3.8.1. Re-docking and validation
The re-docked complexes (shown in blue in Figure S6) were
superimposed onto their respective native co-crystallized-
enzyme (shown in red in Figure S5) from the protein data bank
using PyMOL software 2.3, and root mean square deviation (RMSD)
Fig. 17. Bioactive compounds bound to the active site of E. coli DNA gyrase (PDB Id: 1KZN).
17
Journal of Molecular Liquids 347 (2022) 117954
were calculated and found to be 0.103 Å for S. aureus 3U2D (Fig-
ure S6 (a)) enzyme and 0.087 Å for E. coli 1KZN (Figure S6 (b))
enzyme respectively. A superimposed complex with a low RMSD
value typically < 2 Å is considered the best and acceptable. There-
fore, the enzymes’ RMSDs were very low (3U2D: 0.103 Å; 1KZN:
0.087 Å), indicating that the set docking parameters, grid size,
and protocols were valid.
3.8.2. Predicted drug-likeliness and toxicity properties of ligands
The predicted drug-likeliness properties of the bioactive ligands
were predicted and reported in Table S8. All the five bioactive com-
pounds satisfied Lipinski’s rule of oral drug-likeliness to enable
them to be used as an oral drug [80]. None of the compounds sat-
isfied the Ghose filter [81] except the saturated fatty acid n-
hexadecanoic acid; the rest of the compounds violated at least
one property. Glycerol and 2-Methyl-6-methylene-1,7-octadien-3
-ol satisfied Veber filter [82] based on the properties that influence
a drug’s oral bioavailability, rest of the compounds violated at least
one property. Erucic acid and oleic acid did not satisfy the Egan fil-
ter [83]. These drug-likeliness filters are essential criteria for a
molecule to be used as a drug, and it is based on its physical, chem-
ical, and pharmacological properties. All the bioactive compounds
of Olea europaea possessed acceptable drug-likeliness properties;
thus, they can be taken for further in-vivo studies after lead opti-
mization to discover novel plant-based drug candidates against
pathogens.
The toxicity properties of bioactive compounds detected using
GC–MS were reported in Table S9. The compounds did not show
any signs of AMES toxicity, carcinogenicity, and hepatotoxicity
properties and are safe to be used for further applications. The
compounds were also reported to be biodegradable.
V. Ragunathan and C. K
The novelty in the present research is that the combined effects
of microwave and ultrasound irradiations have tremendously
influenced the particle size compared to the physical, chemical,
and photochemical methods reported till now. Previous similar
green syntheses were focussed on using different parts of plants
as reducing agents; however, the reaction rate was prolonged.
The nanoparticles synthesized in this study were rapid, cost-
effective, and reliable. As of our knowledge, no previous optimiza-
tion is carried out using Olea europaea fruit as capping and reduc-
ing agents. The polyphenols and fatty acids were responsible for
the reduction of precursor to silver nanoparticles. The synthesized
O-AgNps were characterized and found to possess excellent
antioxidant and antimicrobial properties.
Several authors have studied various routes of nanoparticles
synthesis, predominantly focussing on particle size and its biolog-
ical activities. Quintero-Quiroz et al. reported a particle size similar
to the present research work using the chemical reduction method,
their MIC and MBC against S. aureus and E. coli were lower than
the present research work [84]; however, the nanoparticles were
toxic to Vero and NiH3T3 cells. On the other hand, the O-AgNps
in the present research work did not show any toxic or carcino-
genic properties. Mehr et al. synthesized silver nanoparticles using
sodium borohydride as a reducing agent and observed a particle
size of 47 nm, four times greater than the average particle size
reported in the current study [85]. Apart from this, several studies
have reported using chemicals like trisodium citrate, glucose,
hydrazine, paraffin, dextrose, ethylene glycol, ascorbic acid, etc.,
as stabilizing agents to obtain a particle size of 20 to 650 nm
[86–88] and have shown excellent biological activities. The present
study had a smaller particle size than the nanoparticles synthe-
sized using physical methods such as electric arc discharge [89],
UV [90], X-ray [91], and triton X-100 [92]. Singaravelan and Alwar
synthesized the silver nanoparticles of size 10 to 50 nm using the
electrochemical method and possessed exceptional antibacterial
properties [93], similar to the present study. However, the chief
drawback in the physical, chemical and electrochemical synthesis
methods produces harmful by-products and is costlier than the
others [94]. Many studies have reported that the traditional green
synthesis method using several bacteria, fungi, algae, and plants as
reducing agents has several drawbacks like efficacy, reaction time,
and its mechanism [95–96]. The nanoparticles synthesized in the
present study have also proven to be excellent antioxidants and
antibiotics. The traditional green synthesis methods result in a par-
ticle size ranging from 30 to 50 nm, far greater than the
microwave-ultrasound technique using olives as reducing and cap-
ping agents; thus, this study helps researchers working in drug dis-
covery and nanobiotechnology in developing novel low molecular
weight drug carriers [97].
Hence, the highly small-sized O-AgNps
10 nm were synthe-
sized using the sequential two-step microwave-ultrasound tech-
nique and possessed excellent antioxidant and antimicrobial
properties. This reported technology is cost-effective, rapid, reli-
able, and effective than the previously reported similar works.
4. Conclusions
Thus, the novel sequential microwave-ultrasound-assisted
green silver nanoparticles showed promising results for using Olea
europaea as reducing and capping agents for biological applica-
tions. At first, the phytochemicals from Olea europaea fruits were
extracted using the conventional Soxhlet apparatus using various
solvents and were characterized using preliminary phytochemical
analyses, ATR-FTIR, and GC–MS for the identification of functional
groups and bioactive compounds. Then, silver nanoparticles were
synthesized using the aqueous extract of Olea europaea with the
18
Journal of Molecular Liquids 347 (2022) 117954
influence of microwave and ultrasound irradiations using the
Box-Behnken design of RSM. The following were the optimized
conditions, precursor concentration 10 mM, plant extract concen-
tration 10 mL, microwave irradiation 600 W, and exposure time
1 min, and an ultra-low particle size of
10 nm was attained with
a uniform particle dispersion polydispersity index of 0.191. The O-
AgNps were assessed for the free radical scavenging potential by
in-vitro DPPH assay and obtained a good IC50 of 155.08 mg/mL.
The antimicrobial agar-well diffusion assay showed impressive
results with a maximum zone diameter of 24.9 ± 0.14 mm at
8 mg/mL for S. aureus and 24.5 ± 0.7 mm at 8 mg/mL for E. coli.
The in-vitro MICs and MBCs of O-AgNps were 156.25 mg/mL and
625 mg/mL for both S. aureus and E. coli, which were noteworthy.
In-silico molecular docking studies were a supporting tool to eluci-
date the possible chemical interactions between the microbial
enzymes and the bioactive compounds. Future work aims to study
the molecular dynamics simulations of the docked complexes, in-
vitro cytotoxicity of synthesized O-AgNps and will be used as drug
carriers for targeted in-vivo applications. Thus, the present work
finds application in developing ultra-low-sized nano-drug carriers
for biological applications, as the proposed method is rapid, cost-
effective, and eco-friendly.
CRediT authorship contribution statement
Venkataraghavan Ragunathan: Investigation, Methodology.
Chithra Kumaran: Supervision, Validation, Conceptualization,
Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgement
The authors extend their gratitude to Ms. V. Yamini and Ms. D. Sar-
iga, Avanz Bio Pvt. Ltd, East Tambaram, Chennai, for their assis-
tance in antimicrobial assays.
Funding statement
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molliq.2021.117954.
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