Analysis of structural and biomimetic characteristics of the green-synthesized Fe3O4 nanozyme from the fruit peel extract of Punica granatum

Abstract

A vast array of industries routinely uses enzymes to reduce environmental toxicity and to improve the quality of product. Apart from their conventional role in textile and food industries to remove hydrogen peroxide, enzymes like catalases are being used for novel applications as in the development of biosensors. However, being biological in nature, these enzymes are prone to degradation; they are vulnerable to pH and temperature changes that affect their effectiveness. This inconsistent behavior of natural enzymes at high temperatures and pH conditions makes them unsuitable for commercial use. Artificial enzymes are synthesized to compensate the drawbacks of natural enzymes. Metal oxide nanoparticles are prominent among these artificial enzymes. In the current study, iron oxide nanozymes (Fe3O4 NZs) were synthesized from the fruit peel extract of pomegranate using microwave-assisted extraction. Scanning Electron Microscopy (SEM) confirmed the cubical structure of the synthesized Fe3O4 NZs. High-resolution transmission electron microscopy/selected area electron diffraction (HR-TEM/SAED) revealed an average particle size of 17.8 ± 6.5 nm. The nanoparticle was further characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS). Further, the enzyme-mimicking activities of the Fe3O4 NZs were tested using peroxidase, catalase, and superoxide dismutase (SOD)-mimicking assays, revealing that the green-synthesized Fe3O4 NZs are good mimics of natural enzymes. This is the first report on the green synthesis, characterization and multi-enzyme-mimicking activity study of metal oxide nanozymes synthesized from the extract of pomegranate fruit peel.

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Chemical Papers
https://doi.org/10.1007/s11696-022-02130-2
ORIGINAL PAPER
Analysis ofstructural andbiomimetic characteristics
ofthegreen‑synthesized Fe3O4 nanozyme fromthefruit peel extract
ofPunica granatum
DeepaMundekkad1 · AVinothaAlex1
Received: 20 November 2021 / Accepted: 11 February 2022
© Institute of Chemistry, Slovak Academy of Sciences 2022
Abstract
A vast array of industries routinely uses enzymes to reduce environmental toxicity and to improve the quality of product.
Apart from their conventional role in textile and food industries to remove hydrogen peroxide, enzymes like catalases are
being used for novel applications as in the development of biosensors. However, being biological in nature, these enzymes
are prone to degradation; they are vulnerable to pH and temperature changes that affect their effectiveness. This inconsistent
behavior of natural enzymes at high temperatures and pH conditions makes them unsuitable for commercial use. Artificial
enzymes are synthesized to compensate the drawbacks of natural enzymes. Metal oxide nanoparticles are prominent among
these artificial enzymes. In the current study, iron oxide nanozymes (Fe3O4NZs) were synthesized from the fruit peel
extractof pomegranate using microwave-assisted extraction. Scanning Electron Microscopy(SEM) confirmed the cubical
structure of the synthesized Fe3O4NZs. High-resolution transmission electron microscopy/selected area electron diffrac-
tion (HR-TEM/SAED) revealed an average particle size of 17.8 ± 6.5nm. The nanoparticle was further characterized by
X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS).
Further, the enzyme-mimicking activities of the Fe3O4NZs were tested using peroxidase, catalase, and superoxide dismutase
(SOD)-mimicking assays, revealing thatthe green-synthesized Fe3O4NZs are good mimics of natural enzymes. This is the
first report on the green synthesis, characterization and multi-enzyme-mimicking activity study of metal oxide nanozymes
synthesized from the extract of pomegranate fruit peel.
Keywords Enzyme-mimicking nanozyme· Green synthesis· Fe3O4nanozymes· Catalase· Peroxidase· SOD
Abbreviations
Fe3O4NZs Iron oxide nanozymes
XRD X-ray diffraction spectroscopy
FT-IR Fourier transform infrared spectroscopy
DLS Dynamic light scattering
XPS X-ray photoelectron spectroscopy
HR-TEM High-resolution transmission electron
microscopy
SAED Selected-area electron diffraction
SEM Scanning electron microscope
EDAX Energy-dispersive analysis of X-rays
SOD Superoxide dismutase
OPD O-Phenylenediamine
TMB 3,3',5,5'-Tetramethylbenzidine
HRP Horseradish peroxidase
MAE Microwave-assisted extraction
LC–MS Liquid chromatography–mass spectrometry
TA Terephthalic acid
DMF Dimethylformamide
HE Hydroethidine
GAE Gallic acid equivalent
AAE Ascorbic acid equivalent
Introduction
Enzymes help to reduce the overall consumption of energy,
water and other resources that are non-renewable. Various
industries such as food, agriculture, pharmaceutical, paper,
sugar, and textile employ enzymes in a vast array of applica-
tions that increases efficiency and shorten production time.
* Deepa Mundekkad
deepamundekkad@gmail.com; m.deepa@vit.ac.in
1 Centre forNanobiotechnology, Vellore Institute
ofTechnology, VIT (PO), Tamil Nadu, Vellore,
India632014

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Food industry employs enzymes such as cellulase, xylanase,
and lactase to enhance taste, extraction and clarification of
food components (Kant Bhatia etal. 2021). In agriculture,
enzymes are used to bring back altered soil matrix, enrich
soil with nutrients and organic matter. Textile and leather
industry heavily depends on enzymes such as lipase (Chan-
dra etal. 2020), cutinase (Sooksai etal. 2019), and glu-
taminase (Duarte etal. 2020) for processing and finishing
products. Peroxidase and superoxide dismutase (SOD) are
biologically relevant enzymes that are employed in cosmetic
(Gomes etal. 2020) and diary (Fetilă etal. 2012) industries.
Pharmaceuticals is another industry which depends exten-
sively on the use of enzymes such as chitinase (Singh etal.
2021) and streptokinase (Yilmazel Ucar 2019). However,
the broad-spectrum applications of natural enzymes such as
catalase and SOD are constrained by the strict physiologi-
cal conditions needed for their optimal activity (Robic etal.
2017).As a consequence, artificial enzymes with significant
catalytic activity, along with excellent thermal and chemical
stability, were developed (Wang etal. 2019).Among these
artificial enzymes, metal- or metal oxide-based nanoparticles
are gaining more acceptance. Many of these metal-based
nanoparticles are capable of mimicking natural enzymes
(Cao etal. 2017; Zhou etal. 2017) and are aptly named
nanozymes (Wei and Wang 2013). Low cost of production,
controlled synthesis, highly tunable catalytic activity, high
stability even at harsh temperatureand pH conditions, etc.
are features of nanozymes that make them superior to natural
enzymes. A variety of nanozymes have been developed over
the last few years that has the efficiency to replace natural
enzymes (Vernekar etal. 2014; Jia etal. 2016; Zhang etal.
2016, 2017; Zeng etal. 2016; Jiang etal. 2017; Naganuma
2017; Singh etal. 2017; Vazquez-Gonzalez etal. 2017;
Wang etal. 2017; Yan etal. 2017; Yang etal. 2017).
Plant-based green synthesis is emerging as the most
environment-friendly method for nanoparticle synthesis.
Green synthesis of nanoparticles relies on the presence of
bioactive compounds present in the plants and is much more
reliable, fast, efficient, and less toxic than chemical synthe-
sis.Researchers have reported the synthesis of a variety of
metal oxide nanoparticles by green synthesis (Deepak etal.
2019). Utilizing the food waste resources for nanoparticle
synthesis is a sustainable and effective mode of managing
plant biomass. Though they are poor in nutritional value,
the polyphenol-rich biomolecules present in food waste
serve as reducing agents in the synthesis of metallic nano-
particles (Abdel-Shafy and Mansour 2018).A wide variety
of nanoparticles have been synthesized from various food
wastes.Juglans regia green husk extract was used for the
green synthesis of iron oxide nanoparticles (Izadiyan etal.
2020).Silver nanoparticles were prepared from banana
peels, grape stalk,etc.using silver nitrate as the metal pre-
cursor (Ibrahim 2015; Bastos-Arrieta etal. 2018).Titanium
dioxide nanoparticles were synthesized from the leaves
ofCarica papaya (Kaur etal. 2019). Silver and manganese
oxide nanoparticles were synthesized from the peel extracts
of beetroot (Yaow etal. 2019).Watermelon waste was used
for the synthesis of gold nanoparticles (Chums-ard etal.
2019).
Iron oxide nanoparticles (Fe3O4NPs) are one of the
most studied among the metal oxide nanoparticles because
of the significant variable oxidation state, magnetic prop-
erty, tunable size, and crystalline structure of iron oxide.
The magneto-structural properties, high Curie temperature,
and the ability to generate more heat makes the Fe3O4NPs a
highly favorable model for hyperthermia (Koli etal. 2019).
The magnetic Fe3O4NPs, coated by natural rubber latex,
were used as MRI contrast agents and proved to be a power-
ful tool for non-invasive clinical diagnosis (Arsalani etal.
2019).Besides, itwas found that coating other nanoparti-
cles with the magnetically active Fe3O4NPs makes them
suitable for the photocatalytic degradation of many harmful
industrial dyes (Beketova etal. 2020). Fe3O4nanoparticles
are also the first-ever nanoparticles to be reported as having
intrinsic enzyme-mimicking activity (Gao etal. 2007), and
hence, termed as nanozymes (NZs) (Stasyuk etal. 2020).
Ever since their surprising discovery, the multifunctional
enzyme-mimetic property of metal oxide nanoparticles was
exploited in a variety of applications as in the biomedical
field (Gao etal. 2020), to combat biofilm formation (Herget
etal. 2020), in tumor theranostics (Meng etal. 2020), etc.
However, most of these metal nanoparticles with enzyme-
mimicking activities were chemically synthesized and, con-
sequently, are associated with complex reactions and other
limitations (Yan and Gao 2020). To remedy this, green syn-
thesis was successfully employed (Deepak etal. 2019). The
current work exploits the potential of the polyphenols-rich
fruit peel extract of Punica granatum (pomegranate) in syn-
thesizing Fe3O4 NZs which has intrinsic enzyme-mimick-
ing activity. Though many studies describe the synthesis of
enzyme-mimicking Fe3O4 NZs, most of them are by chemi-
cal means (Yan and Gao 2020). This is the first such report
where a multi-enzyme-mimicking nanozyme was synthe-
sized from the fruit peel extracts of pomegranate.
Experimental
Chemicals andinstruments
All chemicals and solvents used were analytical grades;
Ferric chloride (FeCl3), hydrogen peroxide (H2O2),
3,3',5,5'-tetramethylbenzidine (TMB) andO-phenylen-
ediamine (OPD) were procured from Sigma Chemi-
cals, USA. All other chemicals were fromHiMedia. All
enzyme-related assays were carried out using the standard

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phosphate-buffered saline (PBS, pH 7.4) unless otherwise
specified. De-ionized water from Milli-Q (Pall-Cascada)
was used throughout the experiments.A microwave device
(Whirlpool,Magicook20S) was used for microwave-assisted
extraction (MAE). A probesonicatorwith a heating system
(Sonics VCX 320, US) was used for thesonication. The
mean hydrodynamic size and zeta potential were analyzed
by 90Plus Particle Size Analyzer (Brookhaven Instruments
Corporation). The powdered nanoparticle was subjected to
X-ray diffraction (XRD) analysis, and the data were recorded
on a Powder X-Ray diffractometer (Bruker D8 Advanced,
Germany). The elemental composition and chemical stateof
the Fe3O4NZswere studied using X-ray Photoelectron Spec-
troscopy(XPS—K-Alpha surface analysis, Thermo Fisher
Scientific, U.K.). FT-IR data were recorded usingIRAffin-
ity—1 (Shimadzu). Morphology and element distribution of
the Fe3O4NZswere studied by SEM/EDAX (FEI-Quanta
FEG 200F).High-resolution analytical TEM (Jeol/JEM
2100) was used to understand the physical and crystalline
nature of the nanoparticles.
Extract preparation
Fresh pomegranate fruits procured from the local market
were rinsed with water and thoroughly cleaned with ethanol
to remove residual dirt. The fruit peels were separated and
dried in a hot air oven at 60°C for 48h. The dried peels were
coarsely ground and the resultant powder was extracted with
100% ethanol using microwave (Veggi etal. 2012) to yield
the microwave-assisted extract (MAE) that was collected
by centrifugation. The extract was aliquoted and stored at
– 20°C till further use. The chemical composition of the
extract was determined by LC–MS analysis.
Green synthesis of Fe3O4NZs
The green synthesis of Fe3O4NZs was carried out by slowly
mixing 0.1M FeCl3and MAE in a 1:2 (v/v) ratio (Mishra
etal. 2016).The solution was stirred continuously for
2h.and sonicated for 20min. The resulting, black-colored
precipitate was washed with methanol until all traces of
plant organic molecules were removed and then dried in
a vacuum oven over 3 to 4days to obtain the nanoparticle
powder. The dried powder was subjected to various charac-
terization techniques to understand the identity and chemical
entity of the newly synthesized nanoparticle.
Characterization of Fe3O4 NZs
NZs were characterized by various techniques to understand
the morphology and the structural/chemical attributes. Mor-
phology of the nanoparticles was determined by SEM and
EDAX, which also determined the purity of the Fe3O4 NZs.
Dynamic light scattering (DLS) was employed to understand
the particle size distributionwhereas the surface chargewas
analyzed usingZeta Potential. Structural characterization
was carried out by HR-TEM where the exact size of the
particles was determined. SAED measured the d-spacing
values from which the lattice planes were determined. XRD
analysis was also carried out to understand and confirm the
lattice plane from which the spinel structure was deduced.
The phase formation was also predicted at this level in addi-
tion to the size of the nanoparticles. The average crystallite
sizewas calculated following the Debye–Scherrer equation:
where D represents the average crystallite size (nm), K, the
Scherrer constant value (0.9), λ, the wavelength of the X-ray
source (0.15406nm),β, the Full Width at Half Maximum in
radians(FWHM) and θ, the Bragg diffraction angle (Nna-
dozie and Ajibade 2020). Chemical characterization was
done using XPS where the possibility of the presence of
different phases of the Fe3O4 NZs was explored. The pres-
ence of various functional groups on the surface of the syn-
thesized Fe3O4NZswill explain the possible mechanism of
Fe3O4NZs’ formation. FT-IR spectroscopy was employed
to study the various functional groups that were involved in
the synthesis of the Fe3O4NZs.
Enzyme‑mimicking activity ofthe Fe3O4NZs
It is understood that metal oxide nanoparticles, especially
Fe3O4 NZs, can mimic natural enzymes (Gao etal. 2007).
The enzyme-like activities of Fe3O4 NZs were investigated
by monitoring the absorbance variation of the sample reac-
tion solution at suitable wavelengths in the presence of vari-
ous concentrations of Fe3O4 NZs. The resemblance of the
Fe3O4 NZs to peroxidase, catalase and SOD was studied and
the enzyme-mimicking activities were reported.
Peroxidase‑mimicking activity
The Fe3O4NZs can catalyze the oxidation of TMBand
OPDin the presence of H2O2 (Cao etal. 2017).In a typi-
cal TMB/OPDoxidation reaction catalyzed by Fe3O4NZs,
the reaction mixture contained a solution of 12.7 M
H2O2, 0.8 mM TMB/2 mM OPD and 23.8 × 10–11 M
Fe3O4NZs/2.38 × 10–11MHRP.Control experiments with-
out HRP or Fe3O4NZs were also carried out.The steady-
state kinetic assays were done separately with a reaction
mixture containing Fe3O4NZs as the enzyme mimic in the
presence ofvarying concentrations ofTMB/OPDand H2O2.
The initial velocity (v) of the catalytic reaction was calcu-
lated as
D=(K𝜆)∕(𝛽Cos𝜃),

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where e is the molar extinction coefficient of TMB at 653nm
(3.9 × 104 M−1 cm−1)(Karaseva etal. 2002; Ye etal. 2016)
and l is the length of the cuvette (in cm). Plotting 1/v vs. 1/
[S], a straight line will be obtained; y-intercept corresponds
to 1/Vmax and slope represents Km/Vmax (Khyade etal. 2019).
From this double reciprocal plot (or Lineweaver–Burk plot),
the kinetic parameters such as the maximal reaction velocity
attained by the system (Vmax) and Michaelis constant (Km)
are determined (Seibert and Tracy 2014; Wei etal. 2020).
Catalase‑mimicking activity
The catalytic elimination of H2O2 by the Fe3O4NZs was
studied by fluorescent method to evaluate the catalase-mim-
icking activity of the nanoparticles. H2O2 will decompose to
hydroxyl radical in normal cases. The hydroxyl radicals thus
produced can react with terephthalic acid (TA) to produce
the highly fluorescent 2-hydroxyterephthalic acid (excita-
tion—320nm and emission—425nm) (Wei etal. 2020).
However, in the presence of catalase (or any catalase-mim-
icking substance), instead of producing hydroxyl radicals,
the H2O2 decomposes to water and oxygen molecules (Yao
etal. 2018; Rasheed etal. 2020). Consequently, the fluo-
rescent molecule, 2-hydroxyterephthalic acid is not gener-
ated and the resulting fluorescent signal is lesser than that
of the sample without catalase (or any catalase-mimicking
substance). The assay involves adding 10mM H2O2 (in
25mM phosphate buffer, pH 7.4) to various concentrations
of Fe3O4NZs/catalase with very rapid mixing. The sam-
ple mixture was incubated for 6h at RT. TA (prepared in
0.5mM dimethyl formamide—DMF) was added to the reac-
tion mixture and the fluorescence intensity of the solution
was measured.
SOD‑mimicking activity
The SOD-mimicking activity of the Fe3O4NZs was exam-
ined by two of the most popular assays—the pyrogallol
autoxidation and the xanthine–xanthine oxidase method.
Pyrogallol autoxidation
Pyrogallol autoxidation, proposed originally by Marklund,
produces superoxide anion radicals (Marklund and
Marklund 1974). The natural enzyme SOD, which can
scavenge these radicals, can prevent the autoxidation of
pyrogallol. Here, superoxide anion (•O2 −) radical pro-
duced in the process serves as the chain propagating
v
=
Slope(initial)
𝜀×l,
species, but SOD or SOD-mimics inhibits pyrogallol
autoxidation. Owing to the SOD-like activity, various
amounts of Fe3O4NZs can also bring about the inhibi-
tion of autoxidation of pyrogallol. The assay conditions
followed for the inhibition studies are: Tris–HCl buffer
(50mM; pH 8.2) containing 1 M EDTA, pyrogallol
(0.2mM final concentration) and various concentrations
of Fe3O4NZs. The rate of autoxidation of pyrogallol was
monitored by the increase in the absorbance of 0.2mM
pyrogallol in Tris–EDTA buffer over a period. Inhibition
of autoxidation of pyrogallol following the addition of
Fe3O4NZs was observed in the same manner. The super-
oxide scavenging ability is calculated as
where ∆Ac is the change in absorbance of the control at
320nm, ∆As is the change in absorbance of the sample at
320nm and T is the time in minutes (Li 2012).
Xanthine–xanthine oxidase method
Xanthine–xanthine oxidase method was also carried out
to check the SOD-mimicking activity of Fe3O4NZs (Yao
etal. 2018). In this assay, the superoxide anion radicals
generated by reacting xanthine and xanthine oxidase was
scavenged by the Fe3O4 NZs. The superoxide-specific
probe (hydroethidine—HE) is added to the reaction sys-
tem. HE reacts with the superoxide radicals generated to
produce the fluorescent product ethidium (Li etal. 2020a)
that emits a strong fluorescence around 610nm. In short,
0.6mM xanthine and 0.05 U/mL xanthine oxidase was
prepared in 0.1M phosphate buffer (pH 7.4) to produce
superoxide anion radicals. The reaction was carried out for
40min in RT. The Fe3O4NZs were added to the mixture
and incubated for another 40min. This was followed by
the addition of 0.5mg/ml HE. After vortexing, the solution
was left undisturbed for another 40min and the resultant
fluorescence was measured (excitation—470nm and emis-
sion—610nm). The superoxide scavenging potential of
Fe3O4NZs is measured in terms of the fluorescent inten-
sity of the ethidium produced in the reaction mixture and
represented as
%
scavenging =
ΔAc
T−
ΔAs
T
ΔAc
T
×
100,
Superoxide scavenging
%=
F0−F
F0
×
100,

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where F0 is the fluorescent intensity of ethidium in the
absence of Fe3O4NZs/SOD, and F is the fluorescent inten-
sity of ethidium in the presence of Fe3O4NZs/SOD.
Statistical analysis
All assaysand tests were performed in triplicates. Data were
expressed as mean ± standard deviation and plotted using
Origin (8.5) software. Turkey’s Multiple Comparison Test
was conducted; the test conditions were compared to control
and the significance level was tested at p < 0.05 (N = 3).
Results anddiscussion
Synthesis of Fe3O4NZs
The green synthesis of Fe3O4NZs was carried out by fol-
lowing the green route, and therefore, the use of highly toxic
chemical entities like sodium borohydride could be avoided.
The LC–MS profile of the MAE (ethanol) extract of pome-
granate fruit peelrevealed a variety of compounds (Fig.1,
Table1) strengthening the hypothesis that this extract could
act as a strong reducing agent for nanoparticle synthesis. In
addition, unlike chemical synthesis, it leaves behind zero
wastage in the environment after the process (Deepak etal.
2019).
Morphological characterization of Fe3O4NZs
The morphology ofthe Fe3O4NZswas studied using SEM
(Fig.2a) and EDAX(Fig.2b).The Fe3O4NZswerepredom-
inantlycubical and slightlyaggregated as shown inFig.2a.
The elemental composition tested by EDAX confirmed the
presence ofcarbon (25 wt.%), oxygen (25.83 wt.%), and
Fe (49.17 wt.%)in thesynthesized Fe3O4NZs (Fig.2b).
The strong signals at 0.7keV, 6.39keV and 7.09keVwere
observed for Fe. Briefly, the EDAX spectrum validated that
the synthesized nanoparticle is indeed iron oxide and not any
other metal oxide as it containedonly carbon, oxygen and
iron (Wei etal. 2012; Tufa etal. 2020). This analysis helped
to rule out the presence of any contaminating element other
than organic carbon.
The HR-TEM images of prepared Fe3O4NZs repre-
sented in Fig.3a, c, demonstrated the cubical structure of
the green-synthesized Fe3O4NZs with noticeable agglom-
eration.The agglomeration may be due to the clumping of
particlespromoted by the magnetostatic interaction among
the Fe3O4 NZs (Yew etal. 2016).Besides, the appearance
of the tinyspherical particles around the cubical struc-
tureispossibly from the peel extract residues.The HR-
TEM particle size distribution histogram (N = 150) prepared
using Image J software revealed an average particle size of
17.8 ± 6.5nm (Fig.3b).The particle size observed in HR-
TEM is smallerthan the MeanHydrodynamic Diameter
Fig. 1 LC-MS profile of the ethanol extract of pomegranate fruit peel

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(MHD)value (112.69 ± 3.5nm)obtained from DLS. The
higher value from DLS may be due tothepresence ofa
solvation layeraround the Fe3O4NZs.In the case of HR-
TEM, the evaporation of solvents during sample prepara-
tion resulted in a smaller particle size. The d-spacing values
0.297nm and 0.25nm, measured from the SAED pattern
(Fig.3d), correspond to (220) and (311) lattice planes of
Fe3O4NZs, respectively (Saiphaneendra etal. 2017). This
result substantiates the data obtained from XRD analysis
(Fig.4).
The size distributionand the surface chargeof the
prepared Fe3O4NZs wereinvestigated using DLSand
Zeta Potential, respectively (Fig.S1). TheMHDwas
found to be112.69 nm with a polydispersity index
(PDI)of0.328which explains that thebio-synthesized
magnetite NZs possess narrow particle size distribu-
tion (Wen etal. 2008; Demirezen etal. 2019).Further,
the negative zeta potential value −42.92mVsuggests
the stronganionic charge on thesurface of the prepared
magnetite NZs. These preliminary results proved that
the polyphenols, flavonoids and other biomolecules from
pomegranate fruit peel extractserved as anextraordinary
stabilizingagent for Fe3O4 NZs’ synthesis by generating
strong repulsive force among the particles (Sathishkumar
etal. 2018; Khatami etal. 2019).
Table 1 Some of the
components identified by LC–
MS in the fruit peel extract of
pomegranate
Sl. no Name of the compound Retention time Mass m/z
1 11-Hydroxyiridodial glucoside
pentaacetate 5.298 556.2028 579.191
2 Hydroxytolbutamide 5.556 286.1001 309.088
3 O-Desmethylquinidine 5.647 486.19 487.202
4 Doxycycline 5.972 444.1512 467.1401
5 Picropodophyllotoxin acetate 6.348 456.1509 479.1403
6 Irigenin,7benzyl ether 6.353 450.138 451.1459
7 4-hydroxy pelargonic acid 6.382 174.122 197.1117
8 Tolbutamide 6.847 270.105 293.093
9 Cucurbitacin I 13.87 514.3004 537.2898
10 6,11hexadecadienal 17.528 236.2096 259.1989
11 Campestanol 17.609 402.3773 425.3665
12 Bilirubin 18.142 584.2543 607.2391
13 Khivorin 18.384 586.2659 609.2553
14 Rutin 20.193 610.1418 633.1333
15 Novobiocin 20.208 612.2276 635.2167
Fig. 2 a SEM and b EDAX of the green-synthesized Fe3O4NZs

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Structural characterization of Fe3O4NZs
The XRD pattern of the synthesized Fe3O4 NZs showed
characteristic peaks at 2θ with correspondinglatticeplanes
(Fig.4). The 2θ values of 30.4° (220), 35.7° (311), 45.88°
(400) and 63.08° (440) confirmed the inverse cubic spinel
structure for the bio-synthesized Fe3O4 NZs (JCPDS file
no.19–0629) (Karade etal. 2017).The extra, prominent
peak at 63.08° signified the loss of the amorphous nature
of the Fe3O4 NZs. This is indicative of the excellent stabi-
lizing effect of biomolecules in the extract favoring Fe3O4
NZs’ synthesis (Nnadozie and Ajibade 2020). In addi-
tion, the absence of peaks at 2θ = 24° and 33.1° indicated
the absence of hematite phase formation (Saiphaneendra
etal. 2017). The lattice constant (a) was estimated to be
8.3Å which is analogous to the actual value for magnetite
NZs (a = 8.379Å, JCPDS File No. 88–0315) (Dutta etal.
2018).Further, the average crystallite size of 17.75nm,
calculated for the intense peak at 2θ = 35.7°, was ingood
agreementwiththeTEM particle size result (Nnadozie and
Ajibade 2020).
Chemical characterization of Fe3O4NZs
XPS was performed to distinguish the different phases of
iron oxide NZs and represented as XPS survey (Fig.5a),
Fig. 3 a HR-TEM micrograph, bparticle size distribution histogram, c high-resolution image and d SAED pattern of the green-synthesized
Fe3O4NZs

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high-resolution Fe 2p (Fig.5b), O1s (Fig.5c), and C1s
(Fig.5d) scans. The Fe2p scan revealed peaks at binding
energy 724.6eV(Fe 2p1/2) and 711.4eV (Fe 2p3/2) and
specifically verified magnetite phase formation (Saif etal.
2019).The absence of satellite peak corresponding to
Fe3+inγ-Fe2O3 between 115 and 722eV, in particular, indi-
cated the formation of Fe3O4NZs (Tufa etal. 2020).Further,
the spectral analysis of core O1s and C1s demonstrated the
presence of various functional groups (like carboxyl group).
These core molecules provided stable binding sites for nano-
particle formation. In the high-resolution O1s spectrum, the
peak at 533.5eV (that was analogous to the adsorbed mois-
ture and oxygen content in Fe3O4NZs) was represented by
O = C–O and C–OH groups (Smith etal. 2016). Acetal and
hemiacetal components of protein also appeared around this
peak (Ma etal. 2018). It may also be due to the presence of
O–H groups in the sample. The C1s spectrum revealed bind-
ing energies at 284.6eV corresponding to the carbon–carbon
(C–C) and the carbon–hydrogen (C–H) bonds. The small
Fig. 4 XRD of the green-synthesized Fe3O4 NZs
Fig. 5 a XPS survey b Fe2p scan,c O1s scanand d C1s scan of the green-synthesized Fe3O4 NZs

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binding energy peak at 289.1eV could be attributed to the
carboxylate (C = C–OH) and amide carbon (N–C = O) of
the combining molecules. The XPS results obtained for the
green-synthesized Fe3O4NZs are well corroborating with
prior reports (Cheng etal. 2005; Lu etal. 2010).
Thesurface functional groups of pomegranate peel
extract andsynthesized Fe3O4NZswere verified using
FT-IR spectroscopy (Fig.6). The FT-IR spectrum revealed
apeakat 3320 cm−1correspondingto the stretching vibra-
tions ofthe O–H and N–H bonds in polyphenols and amide
groups, respectively. The band at 2924 cm−1representsthe
C–H stretching bond of methyl groups inthe carboxylic acid
(Fernandes etal. 2019; Yusefi etal. 2020).The band shift
observed from 1424 to 1428 may be due to the interaction
of Fe in the nanoparticles (Sari and Yulizar 2017). Further,
the peaks at 1667 cm−1and 1428 cm−1belong to the in-ring
stretching of the C = C amine and the aromatic C = C vibra-
tion, respectively. The band at 1428 cm−1alsodenotesthe
C = C– aromatic bond and the bending vibration of the N–H
bond. There are reports where the FT-IR spectrum demon-
strated peaks at 1682 and 1420 cm−1 for iron oxide nanopar-
ticles formed due to the asymmetric and symmetric stretch-
ing vibration of the –COO– group (Lesiak etal. 2019). The
occurrence of stretching C–O acid groups is confirmed
by the bands at 1325 and 1224 cm−1. Further, the band at
1052 cm−1corresponds to the C–O (amides), N–H (amines)
and it may also belong to δ (N–H) of amino acids, and ν
(C = O) of flavonoids.Finally, the peak at 870 cm−1could
be related to the ethanol or vibration of the aromatic ring
(Yusefi etal. 2020).The obtained FT-IR peaks validate the
occurrence of polyphenol compounds in the peel extract
which is strengthened by the results of LC–MS. The simi-
lar functional groups present in the Fe3O4NZs’ spectrum
attested tothe contribution of peel extract in Fe3O4NZs’ for-
mation as the peaks in the region between 400 and 600 cm−1
correspond to Fe3O4 (Yew etal. 2016).
Possible mechanism of Fe3O4NZs’ formation
Thepolyphenolic compounds present in the fruit peel
extract of Punica granatum served as an excellent reducing
and stabilizing agent for Fe3O4NZs’ synthesis.Thebio-
extractwas slowly added to the FeCl3solution, resulting
in a dark-black precipitate indicating the formation of the
Fe3O4nanoparticles. It has been reported that the reac-
tion of Fe3+ions with hydroxyl groups of polyphenols
produced ferric hydroxide which is then partially reduced
by the biomolecules to form the Fe3O4 NZs (Sathishku-
mar etal. 2018).Finally, the synthesized magnetite NZs
were methodically characterized using many techniques
which approved the active participation of peel extract
in stable Fe3O4NZs’ production. The Total Phenolic
Content (TPC)and Ferric Reducing Antioxidant Power
(FRAP)assays revealed22.6 ± 0.1mg GAE/g powder
and52 ± 1.074mgAAE/g powder, respectively. Theradi-
cal scavenging activity checked by 2,2-diphenyl-1-picryl-
hydrazyl (DPPH)and 2,2'-azino-bis(3-ethylbenzothiazo-
line-6-sulfonic acid (ABTS)assaysrevealed 54.44 ± 1.9%
and 26.26 ± 3.051%ofactivity respectively,indicatingthe
superior reducing potential of peel extract (Fig. S2). In
addition, the multiple peaks displayed in LC–MS cor-
responding to phenolic compounds, polyhydroxy phe-
nols, flavonoids, alkaloids, and other biomoleculesfur-
therdemonstrated the involvement of polyphenols in the
peel extract in the synthesis of Fe3O4NZ. Moreover, zeta
potential value – 42.92mV denoted the successful poly-
phenol capping on magnetite NZs. The above concept was
also supported by FT-IR results. The identical functional
groups noticed in the FT-IR spectra of peel extract and
the Fe3O4NZsconfirmedthat the O–H,C(= O) OHand
other groups facilitatedthe formation of Fe3O4 NZs (Ravi-
kumar etal. 2019).
Fig. 6 FT-IR of a extract and b the green-synthesized Fe3O4 NZs

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Enzyme‑mimicking activity ofthe Fe3O4 NZs
HRP‑mimicking activity
Fe3O4NZs are among the prominent entities that can
mimic natural enzymes. The newly synthesized Fe3O4NZs
werealso found to have enzyme-mimicking activities
where the particles imitated the behavior of the natural
enzymes horseradish peroxidase(HRP), catalase and
superoxide dismutase (SOD). In the current study, the
reaction conditions that are typical for the oxidation of the
two common substrates of HRP—3,3',5,5'-tetramethylb-
enzidine (TMB) and O-phenylenediamine (OPD)—were
replicated with varying concentrations of Fe3O4NZs and it
was found that theyfollowed a patterncomparable toHRP
in its reaction with TMBand OPD.All the samples con-
taining different concentrations of Fe3O4NZs were able to
catalyze the oxidation of TMBand OPD. Control reaction
carried out without Fe3O4NZs showed relatively less color
change over time emphasizing the role of Fe3O4NZs in the
oxidation process. The changes in absorbance observed at
650nm(for TMB oxidation) and 420nm(for OPD oxida-
tion)were recorded. The oxidation pattern of TMB in the
presence of the Fe3O4NZs was like that of HRP. The vary-
ing concentrations of Fe3O4NZs catalyzed the oxidation
of TMB to produce the TMB cation free radical which is a
single-electron oxidation product. This product is respon-
sible for the blue color which is negligible in the control
reaction. The time-dependent absorption spectra observed
(Fig.7a) showed that the Fe3O4NZs were able to catalyze
the oxidation reaction of TMB by H2O2 suggesting the
HRP-like activity. Previous studies on Ru NZs produced
visible color changes in support of TMB oxidation (Cao
etal. 2017). Oxidation of OPD by HRPresults in the gen-
eration ofa2,3-diaminophenazine (DAP) whichformsa
brownish-yellow solution. Typically, the HRP-mediated
oxidation of OPD can also be emulated by nanoparticle
activity (Cao etal. 2017).Similar tothe enzymatic activity
observed for TMB, upon addition of NP, the oxidation of
OPD happened instantly and the yellow-colored DAP was
formed. The time-dependent absorbance spectra observed
when NP was added to the reaction mixture containing
OPD and H2O2 are represented in Fig.7b. In the case of
natural enzymes,Kmis an index for the affinity between
the enzyme and its substrate; a lowerKm value symbolizes
the higher affinity of the enzyme towards the specific sub-
strate. Aseries of experimentswithTMB/OPDas the sub-
strate in one case and H2O2in another case were carried
out to study thekinetic parameters of theenzyme-mimick-
ing Fe3O4NZs.Lineweaver–Burk plots of 1/υ and 1/[S]
were constructed and fitted to the Michaelis–Menten equa-
tion to finally derive the Michaelis constant (Km) and the
maximum velocity (Vmax). The Lineweaver–Burk plots of
the experiments where TMB/OPD and H2O2served as the
substrates showed that Fe3O4NZs had alowerKmvalue
when TMB was used as the substrate (2.25mM)as com-
pared to the Kmvalue of HRP (20.92mM)(Fig. S3—A,
Fig. 7 The HRP-like activity of Fe3O4 NZs during oxidation of TMB/
OPD. a Time-dependent absorbance changes upon oxidation of TMB
to oxTMB in the presence of Fe3O4 NZs and HRP b Time-dependent
absorbance spectra generated upon the oxidation of OPD to DAP in
the presence of Fe3O4 NZs and HRP

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B, C, and D, respectively).This showed that when TMB
was used as the substrate, the Fe3O4NZs had more affin-
ity towards the substrate than HRP (Table2). The pro-
pensity of having a higheraffinityof Fe3O4NZs forthe
substratesthanHRPwas also observed in the first-ever
report of the enzyme-mimicking activity of Fe3O4NZs
(Gao etal. 2007)andin subsequent studies (Cao etal.
2017; Tian etal. 2017).This may be because of the smaller
size and greater surface-to-volume ratio of Fe3O4NZs that
are available for interaction with substrates (Jamkhande
etal. 2019).In addition,an HRP molecule has only one
iron ion, in contrast to theones that are present on the
surfaceof the Fe3O4NZs.Thekey tothecatalysisof
the Fe3O4NZs may be thepresence of ferrous and ferric
ionson the surfaceofthe nanoparticles (Gao etal. 2007).
Higher quantities of TMB/H2O2 will be required to obtain
maximum activity for Fe3O4NZs when compared to HRP
(Cao etal. 2017; Yaow etal. 2019).
Catalase‑mimicking activity ofthe Fe3O4 NZs
Further, to explore the catalase-mimicking activity of the
synthesized Fe3O4NZs, terephthalic acid (TA) was reacted
with H2O2 to produce highly fluorescent 2-hydroxytere-
phthalic acid (excitation wavelength—425nm) (Yao etal.
2018). Later, various concentrations of Fe3O4NZs and natu-
ral catalase (10μg/ml) were added to the reaction mixture
separately and the % inhibition of H2O2 was determined in
both cases. In the presence of Fe3O4NZs, terephthalic acid
failed to react with H2O2 and the fluorescent product was not
formed. There was a concentration-dependent decrease in
fluorescent intensity (Fig.8a). This decrease in fluorescent
intensity specifically implies the efficient inhibition of H2O2
by Fe3O4NZs (65.56%) which acted as a catalase mimic.
Natural catalase exhibited 73.99% inhibition of H2O2 under
same conditions (Fig.8b).
SOD‑mimicking activity ofthe Fe3O4NZs
Superoxide radicals are one of the most destructive species
among the reactive oxygen species ever. The idea that the
Fe3O4NZs can contribute to the scavenging of superoxide
radicals is, without doubt, an interesting outcome. Conse-
quently, to verify this, the SOD-mimicking activity of the
Fe3O4NZs was also measured. Two methods—the inhibi-
tion of pyrogallol autoxidation and the more common X-XO
system—were used to assess the SOD-mimicking activity of
the Fe3O4NZs. Pyrogallol is a highly unstable compound
that is instantly oxidized to purpurogallin with an active
participation of superoxide (O2• −) radicals (Marklund and
Marklund 1974). In the absence of SOD or any SOD-mim-
ics, the pyrogallol autoxidation is evidenced by an increase
in absorbance, reflecting the rapid production of purpurogal-
lin. In the current study also, a steady increase in absorb-
ance (that indicated the autoxidation of pyrogallol) was
observed (Fig.9a). Conversely, the removal of superoxide
radical by SOD or SOD-mimics brings about the inhibition
of pyrogallol autoxidation. The autoxidation of pyrogallol
was inhibited by Fe3O4NZs indicated by the decrease in
absorbance as represented in Fig.9a. The percent elimina-
tion of superoxide radicals by the Fe3O4NZs as compared
to the natural enzyme SOD during pyrogallol autoxidation
is represented in Fig.9b. This result indicated the efficient
SOD-mimicking capacity of the Fe3O4NZs, strengthening
the role of the Fe3O4NZs as a ubiquitous superoxide radical
scavenger.
Table 2 Comparison of kinetic
parameters ofdifferent catalysts Sl. no Catalyst Substrate Km(mM) Vmax (Ms−1) Reference
1Fe3O4NZs TMB 0.098 3.44 × 10 –8 (Gao etal. 2007)
2Fe3O4NZs H2O2154 9.78 × 10–8 (Gao etal. 2007)
3 HRP TMB 4.34 × 10–1 10.00 × 10−8 (Gao etal. 2007)
4 HRP H2O20.370 × 1018.71 × 10–8 (Gao etal. 2007)
5 Pt NP TMB 0.16 4.72 × 10–8 (Li etal. 2020b)
6 Pt NP H2O20.58 11.6 × 10–8 (Li etal. 2020b)
7 AgPtNZs OPD 0.129 8.971 × 10 –5 (Gharib etal. 2019)
8 AgPtNZs H2O276.05 1.2849 × 10–4 (Gharib etal. 2019)
9Fe3O4NZs TMB 2.25 3.64 × 10 –13 This work
10 Fe3O4NZs H2O26.59 × 10–2 6.60 × 10–13 This work
11 HRP TMB 20.92 3.97 × 10–8 This work
12 HRP H2O21.4 × 1022.09 × 10–12 This work
13 Fe3O4NZs OPD 3.46 × 10 –1 7.69 × 10–13 This work
14 Fe3O4NZs H2O24.162 1.97 × 109This work
15 HRP OPD 1.997 0.0342 This work
16 HRP H2O24.875 × 1045.9897 × 10–11 This work

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Fig. 8 Catalase-mimicking activity a and percent inhibition of H2O2 b by catalase and Fe3O4NZs. Turkey’s Multiple Comparison Test was con-
ducted; the test conditions were compared to control, and significance level was tested at p < 0.05, R2 = 1
Fig. 9 SOD-mimicking activity of the Fe3O4 NZs by pyrogallol
autoxidation method. a The absorbance changes observed during
autoxidation of pyrogallol in the presence/absence of Fe3O4 NZs/
SOD. b The percent elimination of superoxide radicals by the
Fe3O4 NZs as compared to the natural enzyme SOD during pyro-
gallol autoxidation. Turkey’s Multiple Comparison Test was con-
ducted; the test conditions were compared to control and significance
level was tested at p < 0.05, R2 = 0.9988)

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The reaction between X-XO generated the superoxide
radicals in the reaction mixture that was detected by the
fluorescence intensity of the superoxide-specific probe,
HE (Yao etal. 2018). It was observed that the addition
of the Fe3O4NZs substantially decreased the intensity of
the resultant fluorescent ethidium as the superoxide anion
radical generated in the reaction mixture was scavenged by
the Fe3O4 NZs (Fig.10A). The percent elimination of the
superoxide radicals was determined, and it was found that
the Fe3O4NZs eliminated the harmful ROS as steadily as
the natural enzyme, SOD (Fig.10B).
The versatility of nanozymes in terms of size and shape
guarantees a strong binding to its substrate. This is espe-
cially important for imparting the biomimetic effect which
is complementary to the shape and charge distribution on the
target substrate. Nanoparticles which display high affinity
and catalytic activity towards specific target molecules can
be designed in such a way as to mimic the catalytic active
site on natural enzymes. The high surface to area ratio and
smaller diameter enhances the catalytic efficiency (Shang
etal. 2020). The surface ligation as well as shape of metal
oxide nanoparticles has a great influence in optimizing the
catalytic activity. A histidine modified iron oxide nanopar-
ticle was apparently shown to have tenfold increase in its
affinity towards H2O2 as substrate (Fan etal. 2017).
Conclusion
To conclude, Fe3O4NZsweresynthesized from pomegran-
ate peels by following a green synthesis route. The engi-
neered nanomaterials were characterized using various tech-
niques, which in turn, established the identity and nature
of the iron oxide nanoparticles as Fe3O4. Furthermore, the
Fe3O4NZs could catalyze the oxidation of the peroxidase-
substrate TMB and OPD in the presence of H2O2,to produce
colored products. This might be due to the capacity of the
Fe3O4NZs to accelerate the electron transfer between the
substrates and H2O2. The catalase- and SOD-like activity of
the nanozymes was also explored. In the next phase of the
study, experiments are envisaged to assess the effect of the
Fe3O4NZs in mammalian cell lines.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s11696- 022- 02130-2.
Acknowledgements The authors are thankful to Dr. Amitava Mukher-
jee, Professor, Higher Academic Grade & Director, CNBT, VIT for
his mentorship, support, and valuable suggestions and to Dr. N Chan-
dra Sekaran, Professor, Higher Academic Grade, CNBT, VIT for the
facilities provided for the study. Characterization of the nanoparti-
cles was carried out using sophisticated analytical instruments avail-
able at prestigious institutes across the country. FT-IR was done at
Fig. 10 SOD-mimicking activity of the Fe3O4 NZs by Xanthine–
Xanthine oxidase method. a The fluorescent spectra of HE, in the
absence/presence of Fe3O4 NZs in the X-XO reaction system. b
The percent elimination of superoxide radicals in the presence of
Fe3O4NZs and natural SOD. Turkey’s Multiple Comparison Test was
conducted; the test conditions were compared to control and signifi-
cance level was tested at p < 0.05, R2 = 0.9999

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CNBT, VIT; XRD at SAIF, Chandigarh; TEM SAED at STIC, Kochi;
LCMS at SAIF, IIT Mumbai; SEM-EDAX and VSM at SAIF, IITM,
Madras; XPS at SASTRA University. All the help rendered are duly
acknowledged.
Author contributions DM conceived, designed, and performed the
experiments including data analyses, interpretation, and manuscript
writing. VA contributed to manuscript writing and data interpretation.
Funding This work was supported by grant from the Department of
Science and Technology (DST), Government of India, in the form of
project fund (SR/WOS-A/LS-371/2017).
Availability of data and materials The datasets used and/or analyzed
during the current study are available from the corresponding author
on reasonable request.
Declarations
Competing interests The authors declare that they have no competing
interests.
Ethics statement This article does not contain any studies with human
participants or animals performed by any of the authors.
Consent for publication Not applicable.
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