J Mol Liq Venkat & Chithra

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

of silver nanoparticles has considerably increased in the present
Abbreviations: O-AgNps, Olea europaea capped silver nanoparticles; UV, Ultravi- generation for various applications, including medical, food, phar-
olet; ATR-FTIR, Attenuated total reflectance – Fourier transform infrared spec-
maceutical, environmental, and industrial [1]. Nanoparticles range
troscopy; 1H NMR, Proton nuclear magnetic resonance; DLS, Dynamic light
scattering; HR-TEM-EDAX, high-resolution transmission electron microscopy- between 1 and 100 nm in diameter in at least any dimensions with
energy dispersive X-ray spectroscopy; AFM, Atomic force microscopy; BET, enhanced properties than the bulk materials. Their remarkable
Brunauer-Emmett-Teller; DPPH, 2,2-diphenyl-1-picrylhydrazyl; MIC, minimum thermal, optical, electrical, catalytical, and conductivity properties
inhibitory concentration; MBC, Minimum bactericidal concentration; IC50, half-
influence a lot to be used in every sector [2]. There is always a keen
maximal inhibitory concentration; S. aureus, Staphylococcus aureus; E. coli, Escher-
ichia coli; DNA, Deoxyribonucleic acid; BB, Box-Behnken design; RSM, Response
on obtaining smaller particle sizes as the size influences their
surface methodology; MTCC, Microbial Type Culture Collection and Gene Bank; physicochemical properties and applications, which has been an
ANOVA, Analysis of variance; SAED, Selected area electron diffraction; EUCAST, incentive for developing novel technologies. Several methods for
European Committee for Antimicrobial Susceptibility Testing; GC–MS, Gas synthesizing nanoparticles, including the three conventional meth-
chromatography-mass spectrometry; RT, Retention time; min/mins, minute(s);
ods, chemical, physical, photochemical, electrochemical, and bio-
R2, Coefficient of determination; Adj-R2, Adjusted coefficient of determination; DF,
Degrees of freedom; Adj SS, Adjusted sum of squares; Adj MS, Adjusted mean logical [3], are practiced globally. Furthermore, the green or eco-
squares; P–Value, Probability value; F-Value, Fisher’s value Ag2+ Silver ions; Ag0, friendly synthesis of nanoparticles is gaining pace in the present
Silver nanoparticles; PDB, Protein data bank. scenario since these are simple in synthesis and do not produce
⇑ Corresponding author.
any toxic or harmful by-products during the process [4].
E-mail address: [email protected] (C. K).
https://doi.org/10.1016/j.molliq.2021.117954
0167-7322/Ó 2021 Elsevier B.V. All rights reserved.
V. Ragunathan and C. K Journal of Molecular Liquids 347 (2022) 117954
Moreover, green synthesis has several advantages in cost, sim- The use of Box-Behnken design (BB) of Response Surface
plicity, effectiveness, viability, and stability [5]. The reducing Methodology (RSM) in the optimization of synthetic process condi-
agents are often various parts of plants and microorganisms like tions helps in attaining the best results with statistical efforts [20].
bacteria and fungi used through redox reactions between the BB design of experiments is a three-level effective factorial design
donor and acceptor atoms. It has been well known that silver that gives many experimental trials and helps understand the
nanoparticles possess exceptional biological properties against interactions among independent variables to predict the depen-
microorganisms and tumor cell lines, making them more attractive dent variable using multiple polynomial regression statistics.
towards biotechnological and biomedical applications worldwide Several sophisticated characterization instruments are used to
[6]. The underlying mechanism for these properties is that the sil- confirm the synthesis and study the essential properties of
ver nanoparticles are capped with various capping and reducing nanoparticles. The most commonly used characterization includes
agents that are capable of penetrating the cell membrane integrity, UV–Visible spectroscopy, FTIR spectroscopy, XRD, SEM, TEM,
causing leakage, generating free radicals causing oxidative stress, EDAX, DLS, Zeta potential, NMR, BET, and AFM. Apart from these
DNA and protein rupture, inhibiting vital enzymes, interfering in characterization techniques, several other sophisticated instru-
the electron transport chain, and so on. [7]. However, even though mentations like UV-Diffused reflectance spectroscopy, vibrating
the green synthesized silver nanoparticles are widely used, it suf- sample magnetometry, nanoparticle tracking analysis, and X-ray
fers from few drawbacks; one such drawback is that the time taken photoelectron spectroscopic are also used depending on the appli-
for the synthesis in the ambient condition is contradictory to the cation of nanoparticles [21].
other methods such as chemical and physical. To overcome this, In-silico computational works have been constantly used as a
the increase in reaction temperature would favor the rate at which shred of supportive evidence to elucidate the chemical interactions
the nanoparticles are formed; this is due to rapid nucleation and and bonding between the target and the ligand. They are accom-
crystal growth [8–9]. Thus, here comes the idea of using an alter- plished using several high-accuracy software like AutoDock Vina,
native energy source of heat by incorporating microwave irradia- AutoDock 4.2, GOLD, Glide, rDock, etc. [22] and arrive at a possible
tion that causes uniform heating by penetrating swiftly; this is mechanism using the in-built algorithms. However, the software is
attributed to the influence of microwaves’ electrical and magnetic supportive and is reinforced with in-vitro and in-silico experimen-
properties, resulting in narrow particle sizes [10]. tal studies to conclude. Molecular dynamics simulations using
Furthermore, during the microwave-assisted synthesis process, high-performance computers aids in further validation by assess-
there is a possibility for the aggregation of nanoparticles [11] ing the stability of the docked complexes using various algorithms
which could be overcome by using ultrasound to disperse the and force fields [23].
aggregated particles. The significant advantage of using The main objectives of this research are to explore the Soxhlet
microwave-ultrasound irradiations over other conventional routes extraction of Olea europaea fruits and characterize the presence
is energy efficiency up to 90% than the other methods [12], facile of various phytochemicals using the conventional procedure for
approach, faster reaction kinetics, smaller particle size, amplified phytochemical analyses, ATR-FTIR, GC–MS, and SEM. Secondly,
physicochemical properties, and greater efficiency product yield we statistically optimized the synthesis of silver nanoparticles
[13]. Many recent studies have also reported the reproducibility using the bioactive compounds from aqueous Olea europaea extract
and reliability of nanomaterials synthesized using microwave irra- as the capping and reducing agent, followed by various sophisti-
diations than nanomaterials synthesized using other physical, cated spectroscopic and microscopic characterization techniques
chemical, and biological routes [14–15]. In addition, combining for confirmation. The sequential synergistic influence of micro-
bioactive compounds as reducing agents and microwave- wave and ultrasound in nanoparticles synthesis is not studied ear-
ultrasound irradiations would be an add-on benefit without releas- lier to the best of our knowledge. The influence of microwave
ing any harmful by-products. Here comes the role of selecting the irradiation speeds up the reaction rate by rapid heating, and the
appropriate plant source, a reservoir of plant secondary metabo- ultrasound maintains the particle size through uniform dispersion
lites for reducing bulk precursors to nanoparticles. [24]. The later part of the work focuses on the synthesized silver
Olea europaea, commonly known as olive, which belongs to nanoparticles’ biological applications, namely, antioxidant and
the Oleaceae family, is a popular fruit commonly cultivated in antimicrobial (MIC, MBC, and Well diffusion) assays. Lastly, we
European countries and consumed as a Mediterranean diet. Olive have demonstrated the plausible interaction mechanism for the
fruits are a rich source of bioactive compounds, fatty acids, in-vitro antimicrobial assay using in-silico computational tools.
esters, etc., that are believed to lower the risk of various cardio- Additionally, we discussed the drug-likeliness and toxicity
vascular diseases, also act as effective antimicrobials, anti-tumor properties of the bioactive compounds. Thus, the present research
agents, and antioxidants [16]. There have been several studies could effectively contribute to developing new drugs and delivery
that reported several bioactive compounds in olive leaf extract routes in biological applications. Fig. 1 shows the detailed work-
[17]. The olive fruits are rich in bioactive constituents such as flow of the research work.
polyphenolic flavonoids, phenolic acids, terpenoids, tannins,
saponins, glycosides, etc. Polyphenols like oleuropein, dimethyl-
oleuropein, ligstroside, nuzhenide, tyrosol, quercetin, rutin, caf- 2. Materials and methods
feic acid, luteolin, gallic acid, and fatty acids like oleic acid, lino-
leic acid, myristic acid, hexadecanoic, stearic acid [18–19] are 2.1. Chemicals and glassware
the major components in olives responsible for reducing precur-
sor bulk salt to nanoparticles. The presence of these active com- All chemicals used in this research work were of analytical
pounds facilitates the formation of nanoparticles. Till now, there grade obtained from E. Merck, Mumbai, India unless specified. All
has been a focus on olive oil and olive fruit pulp. However, the the microbiological media and allied chemicals were purchased
synthesis of silver nanoparticles from Olea europaea fruit extract from HiMedia Laboratories, Mumbai, India. The chemicals were
using microwave and ultrasound is not reported elsewhere. used as received without further purification. The following chem-
Thus, we decided to use the sequential effect of microwave irra- icals were used viz., hexane (95% pure), toluene (99.9% pure),
diation and ultrasound vibrations using Olea europaea as a acetone (99.8% pure), acetonitrile (99.9% pure), ethanol
reducing and capping agent towards the practical synthesis of (99.5% pure), magnesium ribbon (99.5% pure), hydrochloric
silver nanoparticles for biological applications. acid, sulphuric acid, nitric acid, sodium hydroxide (97% pure), fer-
2
Fig. 1. Workflow of the research work.
ric chloride, gelatin, sodium chloride (99% pure), lead acetate fruits. First, 30 g of powdered fruits were weighed (Shimadzu
(95% pure), chloroform (99% pure), glacial acetic acid, benzene BL220H electronic balance, Japan) and filled in the Whatman cellu-
(99.8% pure), ammonia solution, a-naphthol (99% pure), copper lose extraction thimble with 250 mL of solvents filled in the round
sulphate (98% pure), sodium potassium tartrate, potassium bottom flask. The solvents used for extraction were chosen based
iodide (99% pure), iodine (99.9% pure), mercuric chloride on the increase in polarity, viz.,
(99.5% pure), silver nitrate (99.7% pure, Fisher Scientific, USA), hexane < toluene < acetone < acetonitrile < ethanol < aqueous.
L-ascorbic acid (99% pure), DPPH (90% pure), methanol The extracts were immediately concentrated using R-210 Buchi
(99.5% pure), Mueller-Hinton agar, nutrient broth, agar-agar, Rotavapor (Buchi, Switzerland) to remove excess solvents. The
ampicillin sodium salt (90% pure), gentamycin sulphate (90% concentrated extracts were then stored at 20 °C until further
pure), and silver (I) oxide (99% pure). Routine microbial cultures use to prevent the loss of viability. The flow chart of extraction is
S. aureus (MTCC 96) and E. coli (MTCC 2939) were procured from shown in Figure S1.
Avanz Bio. Ltd., Chennai, for the antimicrobial assays. Borosilicate
glassware was used throughout the research work. Double-
2.3. Preliminary qualitative phytochemical analysis of Olea europaea
distilled water (Milli-Q purified water) was used throughout the
fruit extracts
experiment.
The qualitative test for the presence of various phytochemicals
viz., flavonoids, alkaloids, cardiac glycosides, coumarin glycosides,
2.2. Collection and extraction of bioactive compounds Olea europaea
tannins, phlobatannins, terpenoids, cholesterol, saponins, carbohy-
fruits
drates, and proteins in six different extracts was performed accord-
ing to the standard protocols [25–26]. In addition, the analyses
Olea europaea fruits were procured from local markets in Chen-
were performed in duplicates to maintain the concordance of
nai, India, washed with running tap water followed by double-
results.
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- 2.4. Characterization of Olea europaea fruit extracts
trical blender, and stored in a clean air-tight container at room
temperature. Next, the conventional Soxhlet extraction was per- The fruit before and after extraction was analysed using the
formed to prospect the value-added bioactive compounds from VEGA3-SBH scanning electron microscope (TESCAN, Czech Repub-
3
lic) to study the morphological changes. The functional groups in 2.7. Characterization of O-AgNps
fruit extract were identified using JASCO FT/IR-4700 type Spec-
trometer (JASCO, Japan) in the mid-infrared region (4000 to The preliminary characterization of O-AgNps was done using
400 cm1) using the attenuated total reflectance (ATR) mode at UV-Visible spectrophotometer (Shimadzu UV-1800, Japan) in the
resolution 4 cm1. The bioactive compounds were detected using spectral range 800 to 300 nm at 1 nm resolution to observe the
Perkin Elmer Clarus 680 chromatography (Perkin Elmer, USA). 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.
2.5. Optimization of O-AgNps synthesis using Box-Behnken (BB) design The O-AgNps were characterized using Bruker advance III
of response surface methodology (RSM) 500 MHz solution-state 1H NMR spectrometer (Bruker, USA) to
study the chemical interactions and functional groups. The hydro-
The optimization was done using the three-level (1,0+ 1) BB dynamic particle size and zeta potential of O-AgNps were investi-
design for the microwave-ultrasound-assisted O-AgNps synthesis gated using the advanced Nanotrac Wave II (Microtrac MRB, USA)
using the second-order polynomial equation [27]. The response particle size analyser. The size of O-AgNps was determined using
was studied by fitting the data onto the second-order quadratic the JEM-2100 plus HR-TEM (JEOL, Japan). The Selected area elec-
polynomial equation using the multiple regression method and is tron diffraction (SAED) pattern and EDAX were also generated.
given as follows [28]. The surface topography and size of O-AgNps were characterized
using Park XE7 atomic force microscope (Park systems, South
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 Korea) using Park’s true non-contact mode.
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
2.8. DPPH antioxidant activity of O-AgNps
þ b22 X 22 þ b33 X 23 þ b44 X 241 ð1Þ
Equation (1) denotes the second-order quadratic polynomial The DPPH antioxidant assay was performed according to Blois
equation, where Y denotes the response recorded in the absor- [31]. The O-AgNps were prepared in different concentrations
bance of O-AgNps at kmax. The equation gives the mathematical (200 to 1000 mg/mL), and L-ascorbic acid (200 to 1000 mg/mL)
relationship of the response-dependent variable (Y) to the inde- was used as a comparison. To 3 mL of sample in a test tube,
pendent variables X1, X2, and X3. Here, b0 is a constant; b1, b2, b3, 1 mL of freshly prepared 0.1 mM DPPH solution was added and
and b4 denote the linear regression coefficients; b12, b13, b14, b23, incubated in the dark for 30 min at room temperature. The control
b24, and b34 denote the interactive coefficients; b11, b22, b33, and was prepared by adding 1 mL of 0.1 mM DPPH solution to 3 mL of
b44 denote the quadratic regression coefficients; X1, X2, and X3 double-distilled water. The absorbance after dark incubation was
denote the independent variables. measured at 517 nm using a UV–Visible spectrophotometer (Lab-
Minitab 20.2.0 (Pennsylvania, USA) software trial version was man Scientific Instruments Pvt. Ltd., India). The DPPH free radical
used for the statistical design of experiments and multiple regres- scavenging potential percentage was calculated using equation
sion analysis. Four independent variables were chosen based on (2) below [32].
the literature [29–30] and preliminary laboratory experimentation,
AB
precursor concentration, plant extract concentration, microwave %free radical scav enging potential ¼ 100 ð2Þ
irradiation, and exposure time. These variables were assessed A
using the three-level BB design of RSM. The independent variables Where A and B denote the absorbance of the control and O-
with their respective lower (-1), middle (0), and higher (+1) levels. AgNps samples, respectively.
A total of 27 trials at randomized run orders were generated in a The experiment was performed in duplicates to maintain the
single replicate. The significance of the test experiments was stud- concordance of results and was reported as mean ± standard devi-
ied using the one-way or two-way analysis of variance (ANOVA) at ation. Microsoft Excel 2016, USA was used to determine the half-
P < 0.05 and 95% statistical confidence interval below, which maximum inhibitory concentration (IC50) value, graphical repre-
rejecting the null hypothesis. sentations, and data analysis using regression statistics at
P < 0.05 significance.
2.6. Microwave-ultrasound-assisted eco-friendly synthesis of O-AgNps
2.9. Antimicrobial activity 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- The antimicrobial activity consisted of three tests: agar-well
distilled water in a 1 L Erlenmeyer flask. Appropriate dilutions diffusion, minimum inhibitory concentration (MIC), and minimum
were made from the stock as per requirements and used through- bactericidal concentration (MBC) assays. All glassware and media
out the experiment. The nanoparticles were synthesized using the were autoclaved at 121 °C at 15 psi for 20 min before every assay.
precursor and aqueous fruit extract in 250 mL Erlenmeyer flasks The assays were performed in duplicates to maintain the concor-
under dark and neutral pH conditions. Microwave heating was dance of results and represented in terms of means ± standard
irradiated directly into the flasks using a microwave oven (Sam- deviation at P < 0.05 significance. Before the assays, the O-AgNps
sung, South Korea) with a proper condensation system. The O- were filter sterilized using a 0.45 mm pore size Whatman cellulose
AgNps formed were homogenized to attain uniform size distribu- acetate filter to remove other contaminants and large-sized
tion using a 50-Watt digital ultrasonic cleaner (Labman Scientific particles.
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 2.9.1. Agar-well diffusion assay of O-AgNps
centrifuge, India), the supernatant was discarded, the precipitate of The agar-well diffusion antimicrobial assay was performed
O-AgNps was washed thrice with absolute alcohol, and finally against nosocomial pathogenic microorganisms viz., S. aureus
washed twice with double-distilled water. The O-AgNps were then (gram-positive), and E. coli (gram-negative) according to Perez
dried in a hot air oven at 60 °C for 24 h and were stored in an air- et al. [33]. The O-AgNps were prepared using autoclaved double-
tight container. distilled water in 2, 4, 6, and 8 mg/mL concentrations. Broad-
4
spectrum antibiotics ampicillin sodium salt and gentamycin sul- The drug-likeliness and toxicity properties of ligands were pre-
phate at 10 mg/mL concentration were used as positive controls dicted using admetSAR 1 and 2 [41] and SwissADME [42]
along with crude Olea europaea (10 mg/mL) extract and silver (I) webservers.
oxide (Ag2O) for comparison. Mueller-Hinton agar was poured into
sterilized Petri dishes (100 mm 15 mm), and 100 mL of overnight 3. Results and discussion
cultured microorganisms (106 cells/mL) were inoculated and
spread using a sterile cotton swab to cover the entire Petri dish. The extraction of bioactive compounds from Olea europaea fruit
Then, four wells of 8 mm diameter each were punctured using a extract was done using the Soxhlet extraction apparatus. The fruit
sterile cork borer at equal intervals, and 50 mL of O-AgNps and con- morphology before and after extraction using SEM, characteriza-
trols were pipetted onto each well. The plates were then sealed tion of the extracts using ATR-FTIR, and GC–MS were also per-
using a stretch tape, incubated at 37 ± 1 °C for 18 to 22 h, and formed. The presence of various phytochemicals in Olea europaea
the zones of inhibition were measured using a metric ruler. extracts was detected qualitatively to assess the feasibility of using
it in the synthesis of O-AgNps. Further, the antioxidant and antimi-
2.9.2. Minimum inhibitory concentration and minimum bactericidal crobial activity of synthesized O-AgNps were studied.
concentration assays of O-AgNps
The MIC broth dilution assay was performed according to the 3.1. Characterization of Olea europaea extracts
European Committee for Antimicrobial Susceptibility Testing
(EUCAST, 2003) [34] with slight modifications. First, the nanoparticles 3.1.1. SEM analysis of Olea europaea fruit
were diluted using the four-fold serial dilution technique from The morphological changes in fruit biomass before and after
2500 mg/mL to 9.76 mg/mL for up to 5 dilutions. Then, overnight cul- Soxhlet extraction were shown in Figure S2 (a) & (b). It is evident
tured microorganisms (106 cells/mL) were pipetted into all the from Figure S2 that the surface of fruit was intact before extraction,
above mixtures. Overnight microbes in nutrient broth were a positive whereas, after extraction, the surface erosion, broken cell walls,
control; negative control was the nutrient broth without microbes. and tissues were observed that indicates effective leaching of valu-
The test tubes were sealed using a stretch tape and incubated at able bioactive compounds. A similar morphological observation
37 ± 1 °C for 18 to 22 h, the results were observed, and the visual was reported by Pereira et al. [43].
observance of turbidity determined the MIC. The percentage inhibi-
tion was calculated using equation (3) by recording the absorbance
3.1.2. Preliminary qualitative phytochemical analysis
of the complexes after incubation using a UV–Visible spectropho-
The qualitative phytochemical analysis tests revealed the pres-
tometer (Labman Scientific Instruments Pvt. Ltd., India) at 600 nm.
ence and the absence of various phytochemicals like flavonoids,

AB alkaloids, cardiac glycosides, coumarin glycosides, tannins, phlo-
%inhibition ¼ 100 ð3Þ batannins, terpenoids, cholesterol, saponins, carbohydrates, and
A
proteins in different extracts reported were in Table S1. The aque-
Where A and B denote the absorbance of positive control and ous and ethanol extract possessed the highest number of phyto-
test MICs at various concentrations of O-AgNps, respectively. chemical abundances, including flavonoids, cardiac glycosides,
The MBC assay was performed according to Parvekar et al. [35]. tannins, terpenoids, carbohydrates, and proteins, than any other
extract. Moreover, the aqueous extract of the fruit showed the
2.10. In-silico molecular docking analysis using AutoDock Vina highest yield (78.43 ± 3.67%) compared to other solvent extracts
(Data not shown), which denotes the greater abundance of polar
The software AutoDock Vina [36] was used for molecular dock- phytocompounds. It showed the presence of flavonoids (Shinoda
ing. Two vital bacterial enzymes, deoxyribonucleic acid (DNA) gyr- test and Alkaline reagent test), cardiac glycosides (Keller Kiliani’s
ase from S. aureus (PDB Id: 3U2D) and E. coli (PDB Id: 1KZN) [37– test), condensed tannins (ferric chloride test), terpenoids
38], were retrieved from the protein data bank (PDB). These two (chloroform-sulphuric acid test), carbohydrates (Molisch’s test),
proteins were selected based on the literature references [39– and proteins (Xanthoproteic test). The phytochemical abundance
40]. The enzymes’ grid box and active site were determined using hierarchy in all the extracts was
MetaPocket 2.0 webserver and compared with the literature refer- aqueous > ethanol > acetonitrile > toluene > hexane > acetone.
ences for confirmation. Molecular docking was performed for 100 From Table S1, it is clear that the polar solvents aqueous and etha-
Genetic Algorithm runs with exhaustiveness set to 8. The docking nol extracted a higher number of phytochemicals compared to
procedure was validated by removing and re-docking the actual mid-and non-polar solvents. Flavonoid polyphenols play a vital
co-crystallized inhibitors of PDB Id: 3U2D and PDB Id: 1KZN into role in reducing metal ions to nanoparticles through capping and
their respective ligand-binding sites with the above set grid stabilization [44]. In general, Olea europaea is a rich source of
parameters. The re-docked complexes were then superimposed unique polyphenolic bioactive compounds like oleuropein, lignans,
onto the actual co-crystallized structure using LigPlot + v.2.2 and verbascoside, tyrosol, and hydroxytyrosol [45]. As flavonoid
PyMOL 2.3, and the root-mean-square deviations (RMSD) concern- polyphenols play a vital role in reducing metal ions to nanoparti-
ing the reference structures were studied. The extent of inhibition cles through capping and stabilization [44], it was decided to use
from docking was studied using the free binding energy (DG) of the aqueous extract of Olea europaea for synthesizing O-AgNps in
docked complexes. The docked complexes were then visualized this study.
using Biovia Discovery studio 2020 and PyMOL 2.3 to study the
molecular interactions. The inhibition constant (Ki value) was cal- 3.1.3. Characterization of extracts using ATR-FTIR
culated using the formula given in equation (4). It is essential to characterize the Olea europaea to determine
functional groups as they play a significant role in reducing silver
DG
Ki ¼ exp ð4Þ nitrate to silver nanoparticles [46]. The ATR-FTIR spectra of aque-
RT
ous Olea europaea fruit extract are shown in Fig. 2. The functional
Where DG denotes the estimated free binding energy (calories/- groups are the indicators of various bioactive metabolites present
mol); R denotes gas constant (1.986 calories/K mol); T denotes in phytochemicals. The following functional groups were detected
temperature (298 Kelvin). in the concentrated liquid aqueous Olea europaea extract: a med-
5
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
Behnken (BB) design of experiments
4000 3500 3000 2500 2000 1500 1000
-1
Box-Behnken design of experiments was adopted to optimize
Wavenumber (cm ) the parameters affecting nanoparticle synthesis. The various coded
levels of independent variables designed using the BB design of
Fig. 2. ATR-FTIR spectrum of aqueous Olea europaea fruit extract.
RSM were shown in Table 1.
The BB design containing 27 randomized trials with experimen-
ium peak at 2980.45 cm1 and 2886.92 cm1 correspond to CAH tal and predicted absorbance values was studied. The absorbance
functional group, a vibration at 2790.49 cm1 denotes the presence as a response variable was preferred, according to Ibrahim et al.
of ACHO functional group, a medium peak at 1645.95 cm1 [49]. The pH throughout the experiment was maintained at 7
denotes the presence of C@N functional group, a medium stretch- based on the previous experimental studies [50]. All the response
ing at 825.384 cm1 denotes the presence of C@C functional group absorbance values were obtained in the range 420 to 460 nm
in the extract, a medium peak at 722.211 cm1 denotes the pres- caused due to the localized surface plasmon resonance (LSPR).
ence of CAH, C@O, or a benzene derivative, a peak at Depending on the contrast between the four independent vari-
688.214 cm1, and 614.217 cm1 corresponds to the presence of ables, the experimental absorbance values varied from 0.129 to
CABr functional group. These functional groups attribute to the 0.822 (Table 2). The highest experimental absorbance of 0.822
presence of various classes of phytochemicals present in the was in the third trial of BB design with the following conditions,
extract. 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
3.1.4. Characterization of aqueous Olea europaea fruit extract using LSPR of all the 27 trials were shown in Fig. 4.
GC–MS
The GC–MS analysis of aqueous Olea europaea fruit extract 3.3. Analysis of variance (ANOVA)
revealed five bioactive compounds at various retention times
(Fig. 3). The spectra and retention time (RT) fingerprints were com- The ANOVA was studied to examine the variance and signifi-
pared with the National Institute of Standards and Technology- 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
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 experimental data [51]. The F-value of the model 3.56 was more
to be 3.56 and was found to be significant (P < 0.05). The F-value of significant than the lack-of-fit 0.022544 at 95% significance that
Lack-of-fit was calculated by dividing the mean square of lack-of- denotes the model adequacy. The model’s coefficient of determina-
fit by the mean square of pure error, for the model was 16.55 tion (R2) was found to be 0.8058, and the adjusted coefficient of
(P > 0.05) and indicated that the model was slightly insignificant; determination (Adj-R2) was 0.5793 with a standard error of
thus, the predicted model correlated and well fitted with the 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
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 3.4. Microwave-ultrasound-assisted green synthesis of O-AgNps
with increased precursor concentration and could be attributed to
the fact that the plant extracts were utterly utilized in the reduc- The O-AgNps were experimentally synthesized using the opti-
tion of silver nitrate to nanoparticles; therefore, no further reduc- mized conditions to observe a colour change from light yellow to
tion occurred upon increasing the precursor concentration. Fig. 5 a dark reddish-brown. The colour change was instantaneous
(b) depicts the effect of precursor concentration and microwave because of the influence of external microwave heating. The reduc-
irradiation on absorbance. The highest absorbance of 0.5 to 0.6 tion of precursor silver nitrate ions (Ag2+) to O-AgNps (Ag0) could
was attained within 15 mM precursor concentration and 300 to be attributed to flavonoid polyphenols and fatty acids and showed
500 W microwave irradiation. Further, the absorbance decreased a characteristic absorption kmax between 420 and 460 nm of the
above 30 mM precursor concentration and above 500 W micro- UV–Visible region upon striking of light. This absorption peak
wave irradiation, respectively. The high temperature acted as a and scattering were attributed to the vibration and excitation of
limiting factor in the formation of O-AgNps. The effect of precursor free electrons due to the localized surface plasmon resonance
concentration and the exposure time was depicted in Fig. 5 (c), effect of silver nanoparticles [52]. The effect of rapid and uniform
where the highest absorbance of 0.8 was attained within 15 mM microwave heating has enhanced nucleation growth and nanopar-
precursor concentration and a short exposure time of 1 min. A ticle formation to a greater extent than the conventional green syn-
decrease in the absorbance was observed with an increase in the thesis. The particles tend to aggregate upon increasing the
precursor concentration and exposure time. The higher the precur- temperature due to microwave irradiation [53] was overcome by
sor concentration and exposure time were not favourable due to the influence of ultrasound cavitation that disaggregates them. Just
the depleting plant extract concentration. The effect of plant after microwave irradiation, as the temperature declined, the O-
extract concentration and microwave irradiation on absorbance AgNps were subjected to ultrasound vibrations by forming cavita-
was shown in Fig. 5 (d), where the highest absorbance of 0.5 to tion bubbles to ensure uniform homogeneous dispersion of the
0.6 was observed with plant concentration above 8 mL and micro- synthesized O-AgNps [54]. Thus, the non-aggregated particles give
wave irradiation 300 to 450 W. The effect of plant extract concen- rise to smaller and highly dispersed particles sizes essential for bio-
tration and exposure time shown in Fig. 5 (e) denotes the highest logical applications. The nanoparticles formed after sequential syn-
absorbance > 0.8 was obtained with plant extract concentration theses were characterized after drying.
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 3.5. Characterization of O-AgNps
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 3.5.1. ATR-FTIR spectrum of O-AgNps
high temperatures and a shorter exposure time interval, thus pre- The ATR-FTIR spectroscopy revealed the functional groups
venting the aggregation of O-AgNps. Thus, from the contour plots, responsible for the reduction of Ag2+ to Ag0. Interestingly, while
we can conclude that the maximum absorbance was attained at comparing the spectrum of both the Olea europaea fruit extract
10 mM precursor concentration, 10 mL plant extract concentration, and O-AgNps, several functional groups were found shifted, and
microwave irradiation 600 W, and exposure time 1 min. some of them have disappeared in O-AgNps and were as follows
Further, the optimized solution for the synthesized O-AgNps (Fig. 7). The medium peaks from 3500 ± 1 cm1 to
was obtained: 10 mM precursor concentration, 10 mL plant extract 3000 ± 1 cm1 attributed to aliphatic NAH and CAH stretching
concentration, microwave irradiation 600 W, and exposure time detected in the fruit extract have entirely disappeared in the
1 min. Finally, the O-AgNps were synthesized using the predicted OAAgNps. Also, medium peaks from 2782.04 ± 1 cm1 to 2384.4
optimized conditions to validate the experiment, and absorbance 4 ± 1 cm1 in the fruit extract responsible for CAH and SAH func-
of 0.830 was obtained at kmax 438 nm (Fig. 6). tional groups were missing in O-AgNps. Medium peaks 2081.06 ±
8
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 however, the functional groups responsible could be detected
group detected in fruit extract were absent in O-AgNps. The finger- using 1H NMR spectroscopy. Tetramethylsilane was used as the
print region peaks from 1750 ± 1 cm1 to 530 ± 1 cm1 present in internal standard and was attributed to 0 ppm of the chemical
fruit extract representing the stretching of NAH, C@O, CAH, OAH, shift. As a result, several functional groups corresponding to the
CAO, CAN, CABr, and CAI groups were shifted in the OAAgNps chemical shifts were identified tabulated (Table S3). The functional
spectrum. Thus, these functional groups might have played a vital groups, namely, CAH, C@O, C@C, OAH, CABr, were found common
role in the reduction of Ag2+ to Ag0. Similar experimental findings in both ATR-FTIR and 1H NMR that attributed to various phyto-
of ATR-FTIR spectroscopy were reported by Chand et al. [55]. chemicals like polyphenolic flavonoids, tannins, terpenoids, sapo-
nins, glycosides, carbohydrates, and proteins, thus proving the
presence of strongly adsorbed capping and reducing biomolecules
3.5.2. 1H NMR spectrum of O-AgNps on the surface of in-situ silver nanoparticles [57].
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 3.5.3. DLS analysis and zeta potential of O-AgNps
crude aqueous extract of Olea europaea, it is impossible to predict DLS analysis of O-AgNps revealed that the particles possessed a
the exact structure of compounds responsible for reduction [56]; hydrodynamic diameter of 318 ± 149 nm with a polydispersity
9
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
Fig. 8. 1H NMR spectrum of O-AgNps showing various functional groups.
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
Fig. 9. Hydrodynamic particle size distribution of O-AgNps characterized using DLS.
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
3.5.4. HR-TEM-EDAX analysis of O-AgNps
much smaller particle size due to the sequential influence of
The particle size, shape, internal structure, and crystallinity of
microwave and ultrasound [66]. Darmanin et al. reported a 45 to
O-AgNps were studied using HR-TEM-EDAX showed in Fig. 10 (a)
130 nm particle size using various polyols as reducing agents with
to (c). HR-TEM-EDAX analysis revealed the O-AgNps were spheri-
the influence of microwave, which is much higher than this study
cal with an ultra-low average particle size of 10.47 ± 9.19 nm
[67]; thus, the microwave-ultrasound-assisted technique has a
(n = 64) (P < 0.001) and a low polydispersity index of 0.191. The
more substantial influence on particle size than the using micro-
O-AgNps was found to be significantly calculated by the non-
wave alone.
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- 3.5.5. AFM analysis of O-AgNps
cal and electronic properties essential for biological applications. The three-dimensional surface topography, size, and height of
The low polydispersity index denotes that the nanoparticles are O-AgNps were studied using the true non-contact mode of AFM
homogeneously distributed with uniform shape, size, and molecu- in the XY plane. The analysis was performed at different dimen-
lar weight. The R2 and Adj-R2 values of Gaussian particle size dis- sions viz., 10 mm 10 mm, 3 mm 3 mm, and 1 mm 1 mm to
tribution were found to be 0.9829 and 0.9756, respectively, and get the best image of O-AgNps (Fig. 12 (a)). The non-contact mode
were in good agreement. The d-spacing of O-AgNps was calculated of AFM was very efficient and produced accurate results without
using ImageJ software [61] and found to be 0.25 nm. Elemental damaging the nanoparticles and cantilever. The three-
analysis using EDAX confirmed the presence of silver 20.93% (Ag) dimensional forward surface topography of O-AgNps denoted
at 3 keV, oxygen 3.18% (O), chloride 2.91% (Cl) and trace quantities round and elliptical shapes with particle heights ranging from 1
11
to 25 nm along the red-coloured horizontal line with an average depends on reducing DPPH by hydrogen donor–acceptor interac-
height of 10 nm (Fig. 12 (b)). tions between antioxidants and nitrogen atoms in DPPH [69].
The antioxidant activity of O-AgNps using Olea europaea fruit
3.6. Antioxidant activity of O-AgNps extract is not previously reported. The scavenging potential of O-
AgNps increased linearly from 51.94 ± 0.001% to 88.90 ± 0.002%
The free radical scavenging potentials of O-AgNps and L- (P < 0.05) with an increase in concentration from 200 to 1000 mg/
ascorbic acid were studied using the DPPH assay that contains mL (Fig. 13). The O-AgNps showed a significant difference in con-
stable free radicals [68]. The antioxidant ability of O-AgNps centrations and their respective antioxidant potentials, thus
Fig. 10. Particle size and EDAX spectrum of O-AgNps characterized using HR-TEM-EDAX.
12
Fig. 11. SAED pattern showing polycrystalline nature of O-AgNps.
accepting the alternate hypothesis. Upon increasing the concentra- tamycin sulphate (10 mg/mL) at 45 mm and 18 mm, respectively
tion of O-AgNps above 1000 mg/L (P > 0.05), no further significant (Fig. 14 (b)). S. aureus was not susceptible to plant crude aqueous
difference in antioxidant potential was observed (Data not shown). Olea europaea extract and chemically synthesized silver oxide.
The IC50 of O-AgNps was 155.08 mg/mL and greater than the poten- E. coli also were also susceptible to O-AgNps at low concentra-
tial of L-ascorbic acid with IC50 = 84.47 mg/mL. The antioxidant tions and showed zones of diameters 14.2 ± 0.28 mm at 2 mg/mL,
activity of crude Olea europaea polyphenol extract was IC50 = 56. 18.5 ± 0.7 mm at 4 mg/mL, 23.35 ± 0.49 mm at 6 mg/mL, and 24.
5 mg/mL [70], nearly one-third lower than the IC50 of O-AgNps in 5 ± 0.7 mm at 8 mg/mL (Fig. 15 (a)). The control antibiotic ampi-
the present study. Almatroodi et al. reported the antioxidant cillin sodium salt did not show any sign of inhibition; in other
potential of Olea europaea fruit extract very similar to the O- words, E. coli was found resistant to ampicillin (10 mg/mL), whereas
AgNps in the current research work; thus, no difference between gentamycin sulphate (10 mg/mL) showed a 10 mm inhibition zone
the crude and nano synthesized material [71]. Thus, the O-AgNps (Fig. 15 (b)). E. coli was susceptible to plant crude aqueous Olea
showed moderate DPPH antioxidant activity than positive control europaea extract and chemically synthesized silver oxide. From
L-ascorbic acid and crude aqueous Olea europaea extract reported the agar-well diffusion assay, it was clear that O-AgNps showed
in the literature. 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
3.7. Antimicrobial activity of O-AgNps [72–73].
3.7.1. Agar-well diffusion assay

The antimicrobial susceptibility of O-AgNps against gram- 3.7.2. MIC and MBC antimicrobial assay
positive and gram-negative nosocomial pathogens S. aureus and A four-fold serial dilution technique was followed to find the
E. coli were assessed using the well diffusion method. The greater minimum concentration (MIC) of O-AgNps needed to inhibit the
the zone diameter size, the more the organisms are susceptible growth and activity of microorganisms. This assay was performed
to nanoparticles and vice-versa. The zone diameter was found to using the lowest concentration that inhibited the microorganisms
increase as an increase in the concentration of O-AgNps in the agar-well diffusion zone of inhibition assay (2 mg/mL). The
(P < 0.05). A well-established zone diameter of 19.25 ± 0.35 mm appearance of cloudy and turbid solution after incubation denoted
was observed at the lowest tested concentration of 2 mg/mL, fol- that the microorganisms were still alive and active. The appear-
lowed by 22.5 ± 0.7 mm at 4 mg/mL, 24 mm at 6 mg/mL, and 24. ance of a clear and non-turbid solution denoted that the growth
9 ± 0.14 mm at 8 mg/mL was observed for S. aureus (Fig. 14 (a)). of microorganisms was inhibited; however, the microorganisms
In addition, the organism was susceptible to broad-spectrum might not have been killed and would remain alive in the solution
antibiotic controls ampicillin sodium salt (10 mg/mL) and gen- [74]. The MIC of S. aureus and E. coli were 156.25 mg/mL, denoted by
13
Fig. 12. Three-dimensional topography of O-AgNps characterized using AFM.
a non-turbid clear solution. The percentage inhibition of S. aureus 625 mg/mL of O-AgNps against S. aureus and E. coli, respectively.
in the MIC assay varied from 76.66 ± 1.5% to 91.13 ± 0.09% Thus, the MBC of O-AgNps against S. aureus and E. coli was
(P > 0.05) for every four-fold increase in the concentration of O- 625 mg/mL, respectively. S. aureus was susceptible to O-AgNps
AgNps from 9.76 to 2500 mg/mL (Table 4). The percentage inhibi- and antibiotics; however, E. coli were susceptible at slightly higher
tion of E. coli in the MIC assay varied from 28.14 ± 0.71% to 91.62 concentrations than S. aureus. The MIC and MBC of O-AgNps were
± 0.23% (P > 0.05) for every four-fold increase in the concentration very promising with tremendous potential compared to Buszewski
of O-AgNps from 9.76 to 2500 mg/mL (Table 5). The minimum bac- et al. [75]. Supporting the antimicrobial potential of O-AgNps in the
tericidal concentration is the minimum concentration at which the present study, Parvekar et al. also reported the same MIC and MBC
entire microorganisms are killed. Few colonies were observed after values of 625 mg/mL against the bacteria using commercially
inoculating 156.25 mg/mL of O-AgNps from the MIC assay of S. aur- purchased chemically synthesized silver nanoparticles (particle
eus and E. coli onto the MHA plate. No colony was observed at size = 5 nm) [35]. The zones of inhibition, MIC, and MBC of
14
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
Fig. 13. DPPH antioxidant activity of O-AgNps and L-ascorbic acid. 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
O-AgNps in the present study were satisfactory compared to the
erucic acid with the enzyme resulted in the binding energy of
results reported using Waltheria americana capped nanoparticles
4.8 kcal/mol (Ki = 300 mM) interacting with ILE-51, ASN-54,
[76].
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
3.8. In-silico molecular docking studies enzyme. Two conventional intermolecular hydrogen bonds were
formed between the ligand and SER-55, ASP-81 residue of enzyme.
The amino acids in the active sites of S. aureus and E. coli DNA The compound n-hexadecanoic acid ranked fourth with 4.8 kcal/-
gyrase predicted using MetaPocket 2.0 are shown in Table S4 & mol (Ki = 300 mM) interacting with ILE-51, ASN-54, SER-55, GLU-
S5. The DNA gyrase (PDB Id: 3U2D) is an essential enzyme in S. aur- 58, VAL-79, ASP-81, GLY-85, ILE-86, PRO-87, ILE-102, THR-173,
eus contains Gyr A and B subunits and belongs to the family of VAL-174, and ILE-175 amino acids. One intermolecular conven-
topoisomerase that plays a vital role in DNA synthesis and its inhi- tional hydrogen bond was formed between the oxygen acceptor
bition causes cell death that has become an attractive target in the in THR-173 and the hydrogen bond donor in the ligand. Glycerol
recent times [77]. Molecular docking was performed using Auto- with a binding energy 4.1 kcal/mol (Ki = 980 mM) interacted with
Dock Vina software using the bioactive compounds obtained from ASN-54, SER-55, GLU-58, ASP-81, GLY-83, ARG-84, GLY-85, ILE-86,
GC–MS analysis of the aqueous extract as ligands against the DNA GLY-172, and THR-173 residues in the active site with seven con-
gyrase of S. aureus. The ligands were ranked based on their increas- ventional hydrogen bonds at a distance within 3 Å. Glycerol
ing order of binding energy (lowest to highest) in Table S6. The formed conventional hydrogen bonds with ASN-54, GLU-58,
lower the binding energy of ligands, the greater is the inhibition ARG-84, GLY-85, and THR-173 amino acids. Thus, the compounds
potential. Out of the six bioactive compounds docked, oleic acid of Olea europaea inhibited the S. aureus DNA gyrase enzyme, effec-
ranked as the best compound to show inhibition against the tively forming several interactions in the active site by forming
enzyme with a binding energy of 5.4 kcal/mol with an inhibition intermolecular conventional hydrogen bonds, alkyl, and p-alkyl
Fig. 14. Agar-well diffusion antimicrobial assay (a) O-AgNps and (b) control denoting the zones of inhibition against S. aureus.
15
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 GLU-50, VAL-71, ASP-73, ILE-78, ILE-90, HIS-95, ALA-96, GLY-119,
DNA gyrase was shown in Fig. 16. The bioactive compounds docked VAL-120, SER-121, THR-165, and VAL-167 residues of the enzyme
in the present study against S. aureus (PDB Id: 3U2D) were similar with a binding energy of 5.3 kcal/mol (129 mM) and two conven-
and comparable with the binding energies and amino acid interac- tional intermolecular hydrogen bonds VAL-120 and SER-121
tions reported by Vijayakrishnan et al. [78]. The 2-dimensional within 3 Å. The compound 2-methyl-6-methylene-1,7-octadien-3
interaction profile diagram of bioactive compounds with the amino -ol showed binding energy of 5.3 kcal/mol (129 mM) interacting
acids of S. aureus DNA gyrase is shown in Figure S4. with VAL-43, ASN-46, ALA-47, GLU-50, VAL-71, ASP-73, GLY-77,
The E. coli DNA gyrase (PDB Id: 1KZN) is involved in the negative ILE-78, VAL-120, THR-165, and VAL-167 residues by forming van
supercoiling of DNA during replication, thus facilitating replication der Waals and alkyl bonds in the active site and one conventional
and transcription. Inhibiting the enzyme would ultimately lead to hydrogen bond with ASP-73 within 3 Å. Erucic acid showed bind-
bacterial cell death. Molecular docking revealed that n- ing energy of 5.1 kcal/mol (180 mM) interacting with VAL-43,
hexadecanoic acid with a binding energy of 5.4 kcal/mol ASN-46, GLU-50, VAL-71, ASP-73, ILE-78, ILE-90, ALA-96, GLY-
(109 mM) interacted with VAL-43, ASN-46, ALA-47, GLU-50, VAL- 119, VAL-120, SER-121, THR-165, and VAL-167 residues in the
71, ASP-73, ILE-78, ILE-90, VAL-93, LEU-94, HIS-95, ALA-96, GLY- active site of the enzyme forming conventional intermolecular
119, VAL-120, SER-121, and THR-165 amino acids in the active site hydrogen bonds, alkyl, and van der Waals interactions. Four con-
cleft by forming intermolecular hydrogen, van der Waals, and alkyl ventional hydrogen bonds were formed with ASN-46, VAL-120,
interactions. n-hexadecanoic acid formed between seven hydrogen and SER-121 residues in the enzyme’s active site. Glycerol pos-
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,
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
sessed binding energy of 3.9 kcal/mol (1.37 mM) interacting with were calculated and found to be 0.103 Å for S. aureus 3U2D (Fig-
VAL-89, ILE-90, VAL-93, HIS-95, ALA-96, VAL-118, GLY-119, VAL- ure S6 (a)) enzyme and 0.087 Å for E. coli 1KZN (Figure S6 (b))
120, and VAL-122 amino acid residues by forming van der Waals enzyme respectively. A superimposed complex with a low RMSD
and conventional hydrogen bonds. Conventional intermolecular value typically < 2 Å is considered the best and acceptable. There-
hydrogen bonds were formed between the ligand and ILE-90, fore, the enzymes’ RMSDs were very low (3U2D: 0.103 Å; 1KZN:
VAL-93, HIS-95, ALA-96, VAL-118, VAL-120, and SER-121 amino 0.087 Å), indicating that the set docking parameters, grid size,
acids in the active site of the enzyme. Thus, the compounds of Olea and protocols were valid.
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 3.8.2. Predicted drug-likeliness and toxicity properties of ligands
collective of bioactive ligands docked into E. coli DNA gyrase was The predicted drug-likeliness properties of the bioactive ligands
shown in Fig. 17. Boyapati et al. reported similar amino acid inter- were predicted and reported in Table S8. All the five bioactive com-
actions compared to the present study; however, our re-docking pounds satisfied Lipinski’s rule of oral drug-likeliness to enable
and validation were better compared to them [79]. The 2- them to be used as an oral drug [80]. None of the compounds sat-
dimensional interaction profile diagram of bioactive compounds isfied the Ghose filter [81] except the saturated fatty acid n-
with the atoms of amino acids of E. coli DNA gyrase is shown in hexadecanoic acid; the rest of the compounds violated at least
Figure S5. one property. Glycerol and 2-Methyl-6-methylene-1,7-octadien-3
Thus, the bioactive compounds inhibited the vital enzymes of -ol satisfied Veber filter [82] based on the properties that influence
nosocomial pathogens S. aureus and E. coli, essentially interacting a drug’s oral bioavailability, rest of the compounds violated at least
with their active site and inhibiting them and resulting in cell one property. Erucic acid and oleic acid did not satisfy the Egan fil-
death. Thus, a possible interaction mechanism for the in-vitro ter [83]. These drug-likeliness filters are essential criteria for a
antimicrobial potential of O-AgNps has been predicted using the molecule to be used as a drug, and it is based on its physical, chem-
in-silico molecular docking. Furthermore, the elucidated interac- ical, and pharmacological properties. All the bioactive compounds
tion chemistry of the docked complexes between the ligands and of Olea europaea possessed acceptable drug-likeliness properties;
enzymes could have resulted in a conformational change, thus, thus, they can be taken for further in-vivo studies after lead opti-
causing the inhibition and eventually microbial cell death. mization to discover novel plant-based drug candidates against
pathogens.
3.8.1. Re-docking and validation The toxicity properties of bioactive compounds detected using
The re-docked complexes (shown in blue in Figure S6) were GC–MS were reported in Table S9. The compounds did not show
superimposed onto their respective native co-crystallized- any signs of AMES toxicity, carcinogenicity, and hepatotoxicity
enzyme (shown in red in Figure S5) from the protein data bank properties and are safe to be used for further applications. The
using PyMOL software 2.3, and root mean square deviation (RMSD) compounds were also reported to be biodegradable.
Fig. 17. Bioactive compounds bound to the active site of E. coli DNA gyrase (PDB Id: 1KZN).
17
The novelty in the present research is that the combined effects influence of microwave and ultrasound irradiations using the
of microwave and ultrasound irradiations have tremendously Box-Behnken design of RSM. The following were the optimized
influenced the particle size compared to the physical, chemical, conditions, precursor concentration 10 mM, plant extract concen-
and photochemical methods reported till now. Previous similar tration 10 mL, microwave irradiation 600 W, and exposure time
green syntheses were focussed on using different parts of plants 1 min, and an ultra-low particle size of 10 nm was attained with
as reducing agents; however, the reaction rate was prolonged. a uniform particle dispersion polydispersity index of 0.191. The O-
The nanoparticles synthesized in this study were rapid, cost- AgNps were assessed for the free radical scavenging potential by
effective, and reliable. As of our knowledge, no previous optimiza- in-vitro DPPH assay and obtained a good IC50 of 155.08 mg/mL.
tion is carried out using Olea europaea fruit as capping and reduc- The antimicrobial agar-well diffusion assay showed impressive
ing agents. The polyphenols and fatty acids were responsible for results with a maximum zone diameter of 24.9 ± 0.14 mm at
the reduction of precursor to silver nanoparticles. The synthesized 8 mg/mL for S. aureus and 24.5 ± 0.7 mm at 8 mg/mL for E. coli.
O-AgNps were characterized and found to possess excellent The in-vitro MICs and MBCs of O-AgNps were 156.25 mg/mL and
antioxidant and antimicrobial properties. 625 mg/mL for both S. aureus and E. coli, which were noteworthy.
Several authors have studied various routes of nanoparticles In-silico molecular docking studies were a supporting tool to eluci-
synthesis, predominantly focussing on particle size and its biolog- date the possible chemical interactions between the microbial
ical activities. Quintero-Quiroz et al. reported a particle size similar enzymes and the bioactive compounds. Future work aims to study
to the present research work using the chemical reduction method, the molecular dynamics simulations of the docked complexes, in-
their MIC and MBC against S. aureus and E. coli were lower than vitro cytotoxicity of synthesized O-AgNps and will be used as drug
the present research work [84]; however, the nanoparticles were carriers for targeted in-vivo applications. Thus, the present work
toxic to Vero and NiH3T3 cells. On the other hand, the O-AgNps finds application in developing ultra-low-sized nano-drug carriers
in the present research work did not show any toxic or carcino- for biological applications, as the proposed method is rapid, cost-
genic properties. Mehr et al. synthesized silver nanoparticles using effective, and eco-friendly.
sodium borohydride as a reducing agent and observed a particle
size of 47 nm, four times greater than the average particle size CRediT authorship contribution statement
reported in the current study [85]. Apart from this, several studies
have reported using chemicals like trisodium citrate, glucose, Venkataraghavan Ragunathan: Investigation, Methodology.
hydrazine, paraffin, dextrose, ethylene glycol, ascorbic acid, etc., Chithra Kumaran: Supervision, Validation, Conceptualization,
as stabilizing agents to obtain a particle size of 20 to 650 nm Writing – review & editing.
[86–88] and have shown excellent biological activities. The present
study had a smaller particle size than the nanoparticles synthe- Declaration of Competing Interest
sized using physical methods such as electric arc discharge [89],
UV [90], X-ray [91], and triton X-100 [92]. Singaravelan and Alwar The authors declare that they have no known competing finan-
synthesized the silver nanoparticles of size 10 to 50 nm using the cial interests or personal relationships that could have appeared
electrochemical method and possessed exceptional antibacterial to influence the work reported in this paper.
properties [93], similar to the present study. However, the chief
drawback in the physical, chemical and electrochemical synthesis Acknowledgement
methods produces harmful by-products and is costlier than the
others [94]. Many studies have reported that the traditional green The authors extend their gratitude to Ms. V. Yamini and Ms. D. Sar-
synthesis method using several bacteria, fungi, algae, and plants as iga, Avanz Bio Pvt. Ltd, East Tambaram, Chennai, for their assis-
reducing agents has several drawbacks like efficacy, reaction time, tance in antimicrobial assays.
and its mechanism [95–96]. The nanoparticles synthesized in the
present study have also proven to be excellent antioxidants and
Funding statement
antibiotics. The traditional green synthesis methods result in a par-
ticle size ranging from 30 to 50 nm, far greater than the
This research did not receive any specific grant from funding
microwave-ultrasound technique using olives as reducing and cap-
agencies in the public, commercial, or not-for-profit sectors.
ping agents; thus, this study helps researchers working in drug dis-
covery and nanobiotechnology in developing novel low molecular
weight drug carriers [97]. Appendix A. Supplementary material
Hence, the highly small-sized O-AgNps 10 nm were synthe-
sized using the sequential two-step microwave-ultrasound tech- Supplementary data to this article can be found online at
nique and possessed excellent antioxidant and antimicrobial https://doi.org/10.1016/j.molliq.2021.117954.
properties. This reported technology is cost-effective, rapid, reli-
able, and effective than the previously reported similar works. References
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Journal of Molecular Liquids 347 (2022) 117954

Contents lists available at ScienceDirect

Journal of Molecular Liquids

Sequential microwave-ultrasound-assisted silver nanoparticles

Due to their excellent chemical and physical properties, the use

Fig. 1. Workflow of the research work.

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

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

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

S. No Independent variable Symbol Coded levels

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

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

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

Fig. 5. Contour plots of synthesized O-AgNps using BB design of RSM.

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

Fig. 8. 1H NMR spectrum of O-AgNps showing various functional groups.

Fig. 11. SAED pattern showing polycrystalline nature of O-AgNps.

3.7.1. Agar-well diffusion assay

Fig. 12. Three-dimensional topography of O-AgNps characterized using AFM.

constant (Ki) value of 109 mM interacting with VAL-21, GLY-24,

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

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

[1] S. Meenakshi, S. Devi, K. Pandian, K. Chitra, P. Tharmaraj, Aniline-mediated

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