Article

Fabrication of Multifunctional Green-Synthesized Copper

Oxide Nanoparticles Using Rumex vesicarius L. Leaves for
Enhanced Photocatalytic and Biomedical Applications
Seham S. Alterary 1,2, *, Ali Aldalbahi 1 , Raneem Aldawish 1 , Manal A. Awad 2, * , Hind Ali Alshehri 3 ,
Zainah Ali Alqahtani 4 , Reem Hamad Alshathri 5 , Noura S. Aldosari 3 , Leen Abdullah Aldwihi 5 ,
Shorouq Mohsen Alsaggaf 3 , Khulood Ibrahim Bin Shuqiran 5 , Raghad B. Alammari 5 ,
Bushra Ibrahim Alabdullah 6 , Hissah Abdullah Aljaser 3 and Shaykha Alzahly 2

1 Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia;
aaldalbahi@ksu.edu.sa (A.A.); raneemdd@gmail.com (R.A.)
2 King Abdullah Institute of Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia;
shaykha.alzahly@hotmail.com
3 Department of Botany and Microbiology, College of Science, King Saud University,
Riyadh 11459, Saudi Arabia; hindali.o19@gmail.com (H.A.A.); nsaldosari@ksu.edu.sa (N.S.A.);
shorouqme@gmail.com (S.M.A.); hessaabdullah2030@gmail.com (H.A.A.)
4 Department of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia;
zainah.aalqahtani@gmail.com
5 Department of Clinical Pharmacy, College of Pharmcy, King Saud University, Riyadh 11451, Saudi Arabia;
ralshathri@yahoo.com (R.H.A.); 436202414@student.ksu.edu.sa (L.A.A.); khloudsn@gmail.com (K.I.B.S.);
raghad.alammari9@gmail.com (R.B.A.)
6 Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia;
bushra.ib.alabdullah@gmail.com
* Correspondence: salterary@ksu.edu.sa (S.S.A.); mawad@ksu.edu.sa (M.A.A.)
Citation: Alterary, S.S.; Aldalbahi, A.;
Aldawish, R.; Awad, M.A.; Ali Abstract: Recently, the use of plant extracts has emerged as an innovative approach for the pro-
Alshehri, H.; Ali Alqahtani, Z.;
duction of various nanoparticles. Enhancing green methods for synthesizing copper oxide (CuO)
Alshathri, R.H.; Aldosari, N.S.;
nanoparticles (NPs) is a key focus in the field of nanotechnology. This study presents a novel and
Aldwihi, L.A.; Mohsen Alsaggaf, S.;
eco-friendly synthesis of CuO NPs using Rumex vesicarius L. leaf extracts, offering a cost-effective
et al. Fabrication of Multifunctional
and efficient method. The synthesized CuO NPs were evaluated for their cytotoxic effects against
Green-Synthesized Copper Oxide
Nanoparticles Using Rumex vesicarius L.
human cervical carcinoma (HeLa) cells, as well as their photocatalytic and antimicrobial activities.
Leaves for Enhanced Photocatalytic The morphology, size, and structural properties of the CuO NPs were characterized using various
and Biomedical Applications. Catalysts analytical techniques. X-ray diffraction (XRD) analysis confirmed the pure crystalline structure of the
2024, 14, 800. https://doi.org/ CuO NPs with a size of 19 nm, while transmission electron microscopy (TEM) showed particle sizes
10.3390/catal14110800 ranging from 5 to 200 nm. The photocatalytic performance of the CuO NPs was assessed through
Academic Editor: Natalia
the photodegradation of crystal violet (CV) and methylene blue (MB) dyes under UV light. The
Martsinovich NPs exhibited excellent decolorization efficiency, effectively degrading dyes in aqueous solutions
under irradiation. Furthermore, the green-synthesized CuO NPs displayed strong antibacterial and
Received: 28 September 2024
antifungal activities against a variety of human pathogens. They also demonstrated significant
Revised: 31 October 2024
dose-dependent cytotoxicity against the HeLa cancer cell line, with an IC50 value of 8 ± 0.54 µg/mL.
Accepted: 4 November 2024
Published: 8 November 2024
Keywords: Rumex vesicarius L.; CuO nanoparticles; green synthesis; characterization; photoluminescence;
EDX; cytotoxic properties; photocatalytic and antimicrobial activities

Copyright: © 2024 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article 1. Introduction
distributed under the terms and
Nanotechnology is an emerging field that integrates various disciplines of science,
conditions of the Creative Commons
physics, chemistry, biology, materials science, and medicine. It primarily focuses on the
Attribution (CC BY) license (https://
nanonization of metals and their oxides to synthesize particles of varying dimensions
creativecommons.org/licenses/by/
chemical compositions, and dispersities [1,2]. Many researchers all over the world have
4.0/).

Catalysts 2024, 14, 800. https://doi.org/10.3390/catal14110800 https://www.mdpi.com/journal/catalysts

Catalysts 2024, 14, 800 2 of 18

begun to study and exploit the versatile applications offered by nanotechnology. Much
effort has been put towards the synthesis and characterization of metal and metal oxide
nanomaterials to generate novel materials that demonstrate electronic and optical properties
which are different from those observed on the bulk scale. Nanomaterials have recently
garnered immense attention for their potential biomedical applications including medicine,
agriculture, food, cosmetics, paints, catalysis, and textiles [3,4].
Recently, metal oxide nanoparticles have been widely used in research, with re-
searchers exploring their ability to alter the physical, optical, and electronic properties of
compounds [5,6]. Among various types of transition metal nanoparticles, copper oxide
nanoparticles (CuO NPs) are considered to be one of the most important metal oxide
nanomaterials [7]. CuO is a native p-type semiconductor with a band gap in the range
of 1.2–2.6 eV, which facilitates the absorbance of radiation in the visible region [8]. CuO
NPs are viable candidates in several fields and have shown considerable utilization for
their role in nanofluids, sensors, antimicrobial applications, catalysis, superconductors,
energy storage systems, and as anticancer agents [9]. For example, the decomposition of
synthetic dyes and organic effluents in wastewater using nanoparticles has been widely
explored by researchers in the field of catalysis. Among many photocatalysts, CuO is an
ideal photocatalytic system for the remediation of environmental contaminants because it is
inexpensive, nontoxic, efficient, photostable, and abundant, and because it generates high
photocatalytic performance under a solar spectrum [10]. In recent years, CuO NPs have
been at the forefront owing to their antimicrobial and biocidal properties and are widely
used for biomedical applications. CuO NPs have demonstrated significant microbial prop-
erties against microorganisms such as Bacillus, Staphylococcus aureus, Escherichia coli, and
Pseudomonas bacteria [11,12], and a prominent fungicidal influence on Penicillium spp. [13].
CuO NPs have also been used for their antioxidant status and cytotoxic activity, for example
in the antitumor activity that has been demonstrated preclinically in various cancer types,
including hepatocarcinoma, lung carcinoma (A549), nasopharynx cancer, breast cancer,
cervical carcinoma (HeLa), and pancreatic cancer [14–16].
The mode of synthesis plays a key role in the field of nanotechnology. There have
been several techniques reported for the fabrication of CuO NPs, which are categorized
as physical, biological, and chemical methods [17]. Recently, biosynthesis has emerged
as an important mode in the preparation of metal oxide nanoparticles by excluding the
use of toxic chemicals produced by chemical reactions and avoiding the use of organic
solvents. Current trends in research indicate that plant-derived metal nanoparticles are
safe, reliable, and eco-friendly compared with the physical or chemical systems. Plant-
mediated nanoparticle synthesis is also inexpensive and therefore economically viable for
large-scale production [18]. Several studies on the green synthesis of CuO NPs using plant
extracts have been published, including Musa acuminata [19], Aglaia elaeagnoidea flower [20],
Aloe vera [21], Saraca indica leaves [22], Piper betle [23], and Carica papaya [24].
Rumex vesicarius L. (R. vesicarius), commonly known as bladder dock (Arabic: Humeidh),
is an annual plant belonging to the family Polygonaceae. The word Rumex takes its
origin from the Latin word for dart, suggesting the shape of the leaves [25]. Previous
literature reports numerous ethnobotanical and ethnopharmacological studies referring to
the occurrence and traditional uses of Rumex species [26]. R. vesicarius is widely utilized as a
medicinal and culinary herb. It is widely found throughout Saudi Arabia [27]. R. vesicarius
was identified as a plant that showed a significant level of antiangiogenic activity [28], and
it is used in traditional medicine as an antiflatulent, tonic, digestion enhancer, laxative,
antiemetic, analgesic, and an antiangiogenic agent, and for the treatment of bronchitis,
spleen disorders, asthma, and some hepatic diseases prevalent in Egypt, India, and Saudi
Arabia [29].
Catalysts 2024, 14, 800 3 of 18

With this premise, the present study focused on synthesizing CuO nanoparticles using
green chemistry, with Rumex vesicarius extract serving a stabilizing and shaping role in the
process. The synthesized CuO NPs were characterized with advanced techniques, including
X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray
spectroscopy (EDX), scanning electron microscopy (SEM), and photoluminescence (PL)
spectroscopy. The major objectives of the present study are to investigate the diverse
applications of the prepared CuO NPs. These include evaluating their photocatalytic
efficiency in degrading pollutant dyes, specifically crystal violet (CV) and methylene blue
(MB), assessing their antimicrobial activity against both Gram-negative and Gram-positive
bacteria as well as three fungal strains, and investigating their cytotoxic effects on the
HeLa human cervical cancer cell line. To our knowledge, this is the first report on the
phytomediated synthesis of CuO NPs using an aqueous R. vesicarius extract [30].

2. Results and Discussion

2.1. Analysis of the Optical Properties of Synthesized CuO NPs
The optical absorption properties of the synthesized CuO NPs were determined using
UV–Vis and photoluminescence (PL) spectroscopies. The UV–Vis spectra of the resulting
CuO NPs exhibited absorption peaks in the range of 200–600 nm. The absorption spectra
of the synthesized CuO NPs showed two peaks (Figure 1a), which is in line with previous
reports [30,31]. The sharp absorption peak at 279 nm in Figure 1a corresponds to the
surface plasmon resonance (SPR) of CuO NPs, which is attributed to the oscillation of
surface conduction electrons excited by the incident electromagnetic radiation [32]. The
weak and broad peak observed at 377 nm was attributed to the SPR band of some metallic
Cu colloids from nonoxidized Cu NPs [33]. This is within the framework of Mie’s theory
that postulates that the SPR frequency or wavelength depends on the sizes and shapes of
the nanoparticles; spherical nanoparticles have a single SPR band [34,35].
The photoluminescence (PL) spectrum of synthesized CuO NPs is shown in Figure 1b,
where the excitation wavelength was 279 nm. The PL spectra of the synthesized CuO NPs
displayed a sharp emission peak in the blue region at 450 nm, which is attributed to singly
ionized Cu vacancies [36]. Muthuvel et al. reported that in green-synthesized CuO NPs
oxygen vacancies and defects will bind to a photoinduced electron to easily form excitons,
leading to a decrease in the PL intensity. Overall, the enhanced PL intensity and the highly
crystalline nature of the green-synthesized CuO NPs are desirable properties for use in
catalysis [37].
The band gap of the green-synthesized CuO NPs was determined from the absorption
data using the Tauc plot method, as written in Equation (1):

n
(αhν) = A hν − E g (1)

where α is the absorption coefficient, h is Planck’s constant, ν is the frequency, A is a constant

related to the material and the matrix element of the transition, Eg is the band gap, and
n is a coefficient that depends on the nature of the transition, where n = 1/2 for a direct
transition [38]. For indirect band gap materials, the energy gap is determined by plotting
(Ahν)1/2 versus hν and finding the intercept on the hν axis. The best linear relationship is
obtained by plotting against photon energy (hν), indicating that the absorption edge is due
to a direct allowed transition in the CuO NPs. From the Tauc plot, the indirect band gaps
were determined to be approximately 3.52 and 2.93 eV, as shown in Figure 1c. These results
are in moderate consensus with the data reported by Agam et al. and Talluri et al. [39,40].
Catalysts 2024, 14, 800 4 of 18

(a) (b)

(c)
Figure
Figure1.1.(a)(a)
UV–Vis spectrum;
UV–Vis (b) PL
spectrum; (b) spectrum; (c) Tauc
PL spectrum; plot toplot
(c) Tauc determine the energy
to determine gap of green-
the energy gap of
synthesized CuO NPs.
green-synthesized CuO NPs.

2.2. The photoluminescence

Dynamic (PL) spectrum
Light Scattering (DLS) of synthesized CuO NPs is shown in Figure
Measurements
1b, where the excitation wavelength was
DLS is an advanced tool used to analyze 279 nm.theThesize
PL and
spectra of the synthesized
distribution CuO
of synthesized
NPs displayed a sharp emission peak in the blue region at 450 nm, which is
nanoparticles, and it is one of the most commonly used techniques to determine the size attributed to
singly ionized Cu vacancies
of nanoparticles. [36]. Muthuvel
The Brownian motion ofetnanoparticles
al. reported that in green-synthesized
disperses CuO
the light at varying
NPs oxygen vacancies and defects will bind to a photoinduced electron to
intensities. By analyzing these dispersed light intensities, DLS can be used to determineeasily form
excitons, leading
the average size to
of athe
decrease in the PL
nanoparticles in intensity. Overall,
solution. The the enhancedranged
size distribution PL intensity and
from about
the highly crystalline nature of the green-synthesized CuO NPs are desirable
90 to 200 nm (Figure 2), and the polydispersity index indicated narrowly sized particles properties
for useagglomeration
with in catalysis [37].
and diversity in size.
The band gap of the green-synthesized CuO NPs was determined from the
absorption data using the Tauc plot method, as written in Equation (1):
Catalysts 2024,
Catalysts 14,14,x xFOR
2024, FORPEER
PEERREVIEW
REVIEW 5 of 519of 19

Catalysts 2024, 14, 800 5 of 18

90 to
90 to 200
200 nm (Figure
(Figure 2),
2),and
andthe
thepolydispersity index
polydispersity indicated
index narrowly
indicated sized
narrowly particles
sized particles
with agglomeration
with agglomeration and
anddiversity
diversityininsize.
size.

Figure 2. DLS technique for the measurement of synthesized CuO NPs.

Figure
Figure 2.
2. DLS
DLS technique
technique for
for the
the measurement
measurement of
of synthesized CuO NPs.
synthesized CuO NPs.
2.3. TEM and HR-TEM Analysis of Synthesized CuO NPs
2.3. TEM and HR-TEM
The particle Analysis
size and of Synthesized
morphology CuO NPs nanoparticles were determined
of the synthesized
usingThe
TEM. The TEM
particle micrographs
size and morphology(Figure 3a)synthesized
of the show that the synthesized were
nanoparticles CuO NPs are
determined
roughly
using TEM.
using spherical
TEM. The
The TEM and irregular
TEM micrographs in
micrographs (Figureshape,
(Figure 3a)with variable
3a) show
show that sizes
that the and some
the synthesized degree
CuO NPs
synthesized CuO NPsof are
are
agglomeration,
roughly spherical corresponding with the UV–Vis analysis. Further analysis using high-
roughly sphericaland andirregular in shape,
irregular in shape, withwith
variable sizes and
variable sizessome
and degree
some of agglom-
degree of
resolution
eration, TEM (HR-TEM)
corresponding withshowed
thewith the lattice
UV–Vis fringes of theanalysis
nanoparticles high-resolution
(Figure 3b),
agglomeration, corresponding the analysis. Further
UV–Vis analysis. Further using
analysis using high-
indicating
TEM the crystalline
(HR-TEM) nature of the synthesized nanoparticles. (Figure 3b), indicating the
resolution TEM showed
(HR-TEM) the showed
lattice fringes of thefringes
the lattice nanoparticles
of the nanoparticles (Figure 3b),
crystalline the
indicating nature of the synthesized
crystalline nature of the nanoparticles.
synthesized nanoparticles.

(a) (b)
Figure 3.
Figure 3. (a)
(a) TEM
TEM image
imageofofsynthesized
synthesizedCuO
CuONPs;
NPs;(b)(b)
HR-TEM image
HR-TEM of aofCuO
image NP.NP.
a CuO
(a) (b)
2.4. Structural Analysis of Synthesized CuO NPs
Figure 3. (a) TEM image of synthesized CuO NPs; (b) HR-TEM image of a CuO NP.
The synthesized CuO NPs were characterized using a variety of techniques. The XRD
pattern of the CuO NPs, prepared using copper acetate, is presented in Figure 4. XRD
measurements of the synthesized CuO NPs were obtained by coating glass substrates with
the NPs in the form of a powder. The spectrometer was a Bruker D8 ADVANCE X-ray
diffractometer operating at 40 kV and 40 mA with Cu K<alpha> radiation at a wavelength
of 1.5418 Å. The XRD analysis indicated major diffraction peaks at 2θ values of 34◦ , 38◦ ,
 2.4. Structural Analysis of Synthesized CuO NPs
The synthesized CuO NPs were characterized using a variety of techniques. The XRD
Catalysts 2024, 14, 800 pattern of the CuO NPs, prepared using copper acetate, is presented in Figure 4. 6XRD of 18
measurements of the synthesized CuO NPs were obtained by coating glass substrates with
the NPs in the form of a powder. The spectrometer was a Bruker D8 ADVANCE X-ray
diffractometer operating at 40 kV and 40 mA with Cu K<alpha> radiation at a wavelength
48◦ , 58◦ , 61◦ , 68◦ , and 74◦ . The peaks at 38◦ , 58◦ , and 74◦ correspond to the crystal planes
of 1.5418 Å. The XRD analysis indicated major diffraction peaks at 2θ values of 34°, 38°,
(111), (200), and (220), respectively, of the cubic phase of CuO NPs. The other peaks are
48°, 58°, 61°, 68°, and 74°. The peaks at 38°, 58°, and 74° correspond to the crystal planes
assumed to be the reflection lines of monoclinic CuO NPs. The pattern (COD 9012954) is
(111), (200), and (220), respectively, of the cubic phase of CuO NPs. The other peaks are
consistent with the planes [41,42]. The crystallite size (L) of synthesized CuO NPs was
assumed to be the reflection lines of monoclinic CuO NPs. The pattern (COD 9012954) is
found to be 19.8 nm, calculated from XRD data using Scherrer’s equation:
consistent with the planes [41,42]. The crystallite size (L) of synthesized CuO NPs was
found to be 19.8 nm, calculated from XRD data Kλusing Scherrer’s equation:
A = 𝐾𝜆
𝐴= β cos(θ )
𝛽 cos (𝜃)
where AAis is
where thethecrystallite size size
crystallite (in nanometers), K is theKScherrer
(in nanometers), is the constant, is the wavelength
Scherrer λconstant, λ is the
wavelength of the X-rays, β is the full width at half maximum (FWHM) ofpeak
of the X-rays, β is the full width at half maximum (FWHM) of the diffraction in radians,
the diffraction
and θin
peak is radians,
the Bragg andangle
θ is(the angle corresponding
the Bragg angle (the angletocorresponding
the peak position).
to the peak position).

Figure 4. XRD
Figure 4. XRD spectrum
spectrum analysis of CuO
analysis of CuO NPs.
NPs.

2.5. EDX Analysis of Synthesized CuO NPs

EDX andandelementary
elementarymapping
mappingwerewereused to determine
used to determine the purity and elemental
the purity and elemental com-
position of theofNPs.
composition the The
NPs.constituent elementselements
The constituent of the as of
prepared
the as CuO NPs were
prepared CuOdetermined
NPs were
by EDX analysis
determined by carried out at 8 keV
EDX analysis and out
carried are shown in Figure
at 8 keV and 5.areThe measurements
shown in Figurerevealed
5. The
the presence of copper (Cu) and oxygen (O) elements in the CuO NPs
measurements revealed the presence of copper (Cu) and oxygen (O) elements in the CuO and indicated that
the nanoparticles were nearly stoichiometric. There were no other elemental
NPs and indicated that the nanoparticles were nearly stoichiometric. There were no other impurities
found in the
elemental EDX spectra,
impurities foundconfirming the spectra,
in the EDX formation of pure CuO
confirming the NPs. The elemental
formation of pure CuO anal-
ysis
NPs.ofThe
theelemental
sample showedanalysisthat the sample
of the prepared samplethat
showed was copper
the oxide,
prepared whichwas
sample is in good
copper
agreement
oxide, whichwith thegood
is in XRDagreement
analysis. Inwith
the the
EDXXRDspectra, a peak
analysis. Inwas observed
the EDX at 8 akeV,
spectra, peakwhich
was
was attributed to the absorption of copper oxide nanocrystallites arising from
observed at 8 keV, which was attributed to the absorption of copper oxide nanocrystallites SPR [43]. The
optical absorption band for the NPs was in the range of 1 to 9 keV, which is typical for the
absorption of CuO NPs [44].
 Catalysts 2024, 14, x FOR PEER REVIEW 7 of 19

Catalysts 2024, 14, 800 7 of 18

arising from SPR [43]. The optical absorption band for the NPs was in the range of 1 to 9
keV, which is typical for the absorption of CuO NPs [44].

Figure 5. EDX analysis of synthesized CuO NPs.

Figure 5. EDX analysis of synthesized CuO NPs.
2.6. Cytotoxicity
2.6. Cytotoxicity Assessments
Assessments
The antiproliferative activity of the synthesized CuO NPs was evaluated by an in
The antiproliferative activity of the synthesized CuO NPs was evaluated by an in vitro
vitro assay with the HeLa cell line. The MTT assay was employed to assess the anticancer
assay
activitywith theCuO
of the HeLa NPscell
at line. Theconcentrations
different MTT assay was employed
(5, 10, toand
20, 30, 50, assess
100 the
µg anticancer
mL−1) activity
of the CuO
against NPscells.
the HeLa at different
The resultsconcentrations
of the MTT assay(5, 10, 20,that
indicated 30,the
50,synthesized
and 100 µg CuOmL−1 ) against
the
NPs HeLa cells. decreased
significantly The results of the of
the viability MTT assayadenocarcinoma
a cervical indicated thatcell the
linesynthesized
(HeLa) in CuO NPs
a dose-dependent
significantly mannerthe
decreased compared
viabilitytoofthe R. vesicarius
a cervical leaf extract. The
adenocarcinoma cellinhibitory
line (HeLa) in a dose-
concentration,
dependent IC50 (cytotoxicity
manner compared level
to of
the50%
R. dead/alive
vesicarius cells), was observed
leaf extract. to be >10 µgconcentration,
The inhibitory
mL−1 for the CuO NPs and >100 µg mL−1 for the R. vesicarius extract. At this concentration,
IC50 (cytotoxicity level of 50% dead/alive cells), was observed to be >10 µg mL−1 for the
the CuO NPs showed a remarkable inhibition of growth of the HeLa cells by 50%. These
CuO NPs and >100 µg mL−1 for the R. vesicarius extract. At this concentration, the CuO
results indicate that CuO NPs have a potent cytotoxic effect as compared to R. vesicarius
NPs showed
leaf extract a remarkable
against the HeLa cellinhibition
line (Figureof6). growth of the HeLa cells by 50%. These results
indicate
Catalysts 2024, 14, x FORthat have a potent cytotoxic effect as compared to R. vesicarius leaf extract
PEERCuO NPs
REVIEW 8 of 19

against the HeLa cell line (Figure 6).

100
90
80
Cell viability %

70
60
50
40
30
20
10
0
0 20 40 60 80 100 120
µg/mL

R. vesicarius extract CuO NPs

Figure 6. CytotoxicFigure
activity of CuO NPs and R. vesicarius extract in the HeLa cell line using MTT assay.
6. Cytotoxic activity of CuO NPs and R. vesicarius extract in the HeLa cell line using MTT
assay.
The cytotoxicity results show significant agreement with previous studies. However,
CuO NPs synthesized Thefrom R. vesicarius
cytotoxicity showed
results show more
significant profound
agreement cytotoxicity
with previous studies.compared
However,
CuO NPsin
to the findings reported synthesized from R.that
other studies vesicarius
usedshowed more profound cytotoxicity
green-synthesized CuO NPs. compared to
Vinardell
the findings reported in other studies that used green-synthesized CuO NPs. Vinardell
Rehana et al. reported the synthesis of CuO NPs using a green approach with different
Rehana et al. reported the synthesis of CuO NPs using a green approach with different
plant extracts and compared
plant their
extracts and cytotoxic
compared their efficacy against
cytotoxic efficacy the HeLa
against cellcell
the HeLa line
lineand
and other
other
cell lines [45,46].cell
Nagajyothi
lines [45,46].etNagajyothi
al. reportedet al.that CuOthat
reported NPsCuOefficiently reduced
NPs efficiently the pro-
reduced the
proliferation of HeLa cells via apoptosis [47]. Synthesized CuO NPs in the present study
showed higher cytotoxic potential against all cell lines in a dose-dependent manner with
IC50 >10 µg mL−1 than these reported by Vinardell and Nagajyothi. Previous studies also
reported that the nanoparticles showed no toxicity on normal human dermal fibroblast
cells [45,47]. Some studies reported that cancer cells treated with CuO NPs displayed an
Catalysts 2024, 14, 800 8 of 18

liferation of HeLa cells via apoptosis [47]. Synthesized CuO NPs in the present study
showed higher cytotoxic potential against all cell lines in a dose-dependent manner with
IC50 > 10 µg mL−1 than these reported by Vinardell and Nagajyothi. Previous studies also
reported that the nanoparticles showed no toxicity on normal human dermal fibroblast
cells [45,47]. Some studies reported that cancer cells treated with CuO NPs displayed an
altered morphology of the mitochondria, where they exhibited condensed clump structures.
Condensed nuclei and the clumped structure of the cancer cell mitochondria indicate
apoptosis, i.e., reactive oxygen species (ROS)-mediated DNA and mitochondrial damage
leading to apoptosis [15,48]. Others reports confirm that CuO NPs may cause disarray in
the cellular integrity of cancer cells by damaging their DNA and other vital molecules
required for successful survival and progression. The CuO NPs were found to induce
cytotoxicity in a human carcinoma cell line in a dose-dependent manner, which is likely
mediated through ROS generation and oxidative stress [49]. Overall, the findings suggest
that the release of copper ions contributes to CuO NP-induced vascular endothelial cell
death [50].

2.7. Photodegradation Results

The photocatalytic activity of the CuO NPs is demonstrated in Figure 7a,b. The syn-
thesized CuO NPs showed a DE for the CV dye of 92.126% after 36 h under UV irradiation
(Figure 7a) and 99.8435% for the MB dye after 12 h (Figure 7b). For the CV dye, after 4 h of
UV irradiation a weak indication of Cu+ ion generation was detected by a change in the
degradation percentage, and after 32 h a complete disappearance of color was observed.
For the MB dye, after 4 h of UV irradiation a strong indication of Cu+ ion generation
Catalysts 2024, 14, x FOR PEER REVIEW was de-
9 of 19
tected by a change in the degradation percentage, and after 12 h a complete disappearance
of color was observed. In the presence of CuO NPs, the primary absorption peak of the dyes
decreased
completegradually withofan
disappearance increase
color of UV exposure
was observed. time. This
In the presence of CuOdemonstrates the photo-
NPs, the primary
catalytic degradation
absorption peak of theof the
dyesCV and MBgradually
decreased dyes in thewithpresence of CuO
an increase of UVNPs. Previous
exposure time.reports
This demonstrates
showed the photocatalytic
that the crystallographic degradation
structure, of the CVand
morphology, andsize
MB ofdyes
theinparticles
the presence
affects the
of CuO NPs. Previous reports showed that the crystallographic structure, morphology,
photocatalytic activity of metallic nanoparticles [51]. Seerangaraj et al. [35] reported that
CuOandNPs
sizesynthesized
of the particles affects
using the photocatalytic
Ruellia tuberosa were activity of metallic
effective nanoparticles
as coating [51].cotton
agents over
Seerangaraj et al. [35] reported that CuO NPs synthesized using Ruellia
fabrics and in the photocatalytic degradation of CV dye under direct sunlight. Similarly,tuberosa were
effective as coating agents over cotton fabrics and in the photocatalytic degradation of CV
Roy et al. reported the photodegradation of methylene blue and Congo red after 72 h
dye under direct sunlight. Similarly, Roy et al. reported the photodegradation of
using CuO NPs synthesized with Impatiens balsamina leaf extract [52]. Navid Rabiee et al.
methylene blue and Congo red after 72 h using CuO NPs synthesized with Impatiens
also demonstrated
balsamina the[52].
leaf extract degradation
Navid Rabiee of MB dye
et al. bydemonstrated
also CuO NPs biosynthesized
the degradationfrom
of MBAchillea
millefolium leaf extract [53].
dye by CuO NPs biosynthesized from Achillea millefolium leaf extract [53].

(a)
(b)

Figure 7. Percentage decolorization of (a) CV and (b) MB dyes in the presence of synthesized CuO
Figure 7. Percentage decolorization of (a) CV and (b) MB dyes in the presence of synthesized
NPs.
CuO NPs.
According to Lahmar and Vivek [54,55], the possible photocatalytic degradation
mechanism for MB and CV dyes using CuO NPs is shown in Figure 8 and below in
Equations (2)–(11):
CuO + hv → (h+) + (e−) (2)
Catalysts 2024, 14, 800 9 of 18
Catalysts 2024, 14, x FOR PEER REVIEW 10 of 19

According to Lahmar and Vivek [54,55], the possible photocatalytic degradation

mechanism for MB and CV dyes using CuO NPs is shown in Figure 8 and below in
Dye + (O2•–, OOH− or OH•) → degradation products + CO2 + H2O (11)
Equations (2)–(11):
The photochemical reaction CuO
of UV+light + (e−NPs
(h+ )CuO
hv →with ) leads to the formation of holes (2)
(h+) and electrons (e−) (Equation (2)). UV light excites an electron from the valence band
(VB) to the conduction band (CB) when e− +the → O2 •−of the light is equal to or greater than
O2 energy (3)
the band gap of the CuO nanoparticles.•−The movement of the excited electron from the
H2 O + +O2 → OOH• + OH− (4)
VB to the CB produces a hole (h ) in the VB and an electron (e−) in the CB. The
photogenerated electron in the CB2OOHacts as• a→reducing
O2 + H2 O agent
2
in the reaction with an oxygen (5)
molecule (O2) to generate the superoxide anion (O2•–) (Equation (3)), hydroperoxide
−
radical (Equation (4)), and H 2 O2 + e →
hydrogen HO• + OH
peroxide −
(Equation (5)). In the VB, the (6)
photogenerated hole acts as an oxidizing agent to form hydroxyl radicals (OH•) from
water. In addition, the number O2 •− → HO
H2 O2of+ hydroxyl •
+ OH
radicals
− 2−
can +beOincreased by the degradation (7)
of hydrogen peroxide or hydroperoxide+radicals• (Equations (6) and (7)). The resulting
H O + h → OH + H+ (8)
ROS, including hydroxyl radicals2 (OH•−) (Equation (9)) and superoxide anions (O2•–)
(Equation (3)), generated by the oxidation
OH− + h+and → OHreduction
• processes are responsible for (9)
degrading the CV or MB dyes to water and carbon dioxide under UV light irradiation [56].
The efficient degradation of the organic Dye + h+dyes
→ Dye in +aqueous solution by the CuO NPs (10)
synthesized using •− R. vesicarius
−
leaf
•
extract suggests a wide range of additional
Dye applications,
photocatalytic + (O2 , OOH suchorasOH ) →remediation
water degradationand products + CO
pollution 2 + H2 O
control. ( 11)

Figure 8.
Figure 8. Schematic
Schematic diagram
diagram of
of the
the possible
possible mechanism
mechanism for
for photocatalytic
photocatalytic degradation
degradation of
of MB
MB and
and
CV dyes using CuO NPs under UV light irradiation.
CV dyes using CuO NPs under UV light irradiation.

A comparison
The of the reaction
photochemical photocatalytic
of UVactivity of copper
light with oxideleads
CuO NPs synthesized in this study
to the formation of
is made
holes (h+with
) and copper
electronsoxide
(e− )prepared
(Equationusing various
(2)). UV lightplant extracts,
excites as well
an electron fromasthe
with other
valence
metal(VB)
band oxides, such
to the as titanium
conduction bandand
(CB)zinc,
when synthesized
the energy through different
of the light is equalmethods and
to or greater
commercially available as shown in Table 1.
than the band gap of the CuO nanoparticles. The movement of the excited electron from
the VB to the CB produces a hole (h+ ) in the VB and an electron (e− ) in the CB. The
photogenerated electron in the CB acts as a reducing agent in the reaction with an oxygen
molecule (O2 ) to generate the superoxide anion (O2 •− ) (Equation (3)), hydroperoxide
radical (Equation (4)), and hydrogen peroxide (Equation (5)). In the VB, the photogenerated
hole acts as an oxidizing agent to form hydroxyl radicals (OH• ) from water. In addition,
the number of hydroxyl radicals can be increased by the degradation of hydrogen peroxide
or hydroperoxide radicals (Equations (6) and (7)). The resulting ROS, including hydroxyl
Catalysts 2024, 14, 800 10 of 18

radicals (OH•− ) (Equation (9)) and superoxide anions (O2 •− ) (Equation (3)), generated by
the oxidation and reduction processes are responsible for degrading the CV or MB dyes
to water and carbon dioxide under UV light irradiation [56]. The efficient degradation
of the organic dyes in aqueous solution by the CuO NPs synthesized using R. vesicarius
leaf extract suggests a wide range of additional photocatalytic applications, such as water
remediation and pollution control.
A comparison of the photocatalytic activity of copper oxide synthesized in this study is
made with copper oxide prepared using various plant extracts, as well as with other metal
oxides, such as titanium and zinc, synthesized through different methods and commercially
available as shown in Table 1.

Table 1. Performance of the synthesized CuO NPs compared with published data on CuO pre-
pared using plant extracts and other metal oxides (ZnO and TiO2 nanoparticles) under UV and
sunlight irradiation.

Decolorization Irradiation Source of Particles Size

of Dye (%) Time (min) Light Band Gap (eV) (nm)/Morphology Nano-Material Source of Plants

11/agglomerated Amaranthus dubius

84%/MB dye 150 Solar 2.23 CuO NPs
shape leaf [57]
84.23%/methyl Tribulus terrestris
120 Lamp 3.1 58.7/spherical shape CuO NPs
orange dye seed [58]
97.35%/ 36/irregular Arundinaria gigantea
80 UV 2.59 CuO NPs
AR88 dye morphology shape (giant cane) [59]
72%/methyl 27.99/less
240 Sunlight - CuO NPs Lemon peel [60]
orange dye agglomeration
96%/MB and
32/rectangular
99%/methyl 540 Sunlight 3.57 (330 nm) CuO NPs Aegle marmelos leaf [61]
shape
orange dyes
86%/RB21 dye 60 UV 2.04 25/spherical shape CuO NPs Tragacanth gum [62]

240 - 30–40/agglomerated CuO NPs Elaeagnus indica

76%/MB dye Sunlight NPs leaf [63]
91%/aniline 20–80/spherical Santa Maria
1000 Sunlight 3.56 CuO NPs
blue dye shapes feverfew [64]
82.31%/MB dye
15.88 nm/irregular
and 88.54%/ 150 UV - CuO NPs Allahabad Safeda [65]
CV dye surface shape

55.73/fakes and Trigonella

88.37%/MB dye 90 UV 2.97 irregular spherical ZnO NPs foenum-graecum aqueous
shape seed [66]
97%/rhodamine 160 UV 3.6 35/nanorod needle ZnO NPs Cymbopogon
B dye shape proximus [67]
75.8%/acid
red-88 (AR-88) 120 UV 2.79 30/irregular shape ZnO NPs Aloe vera latex [68]
dye

79%/MB dye 30 UV 3.38 (366 nm) 582.35 ± 52.40 nm/ ZnO NPs Stevia rebaudiana
flower-shape leaves [69]
88%/red-141 22.13/spherical
120 UV 3.36 ZnO NPs Chemical method [70]
azo dye shape
93.1%/CV dye,
90.6%/MB dye,
36–81/spherical
76.7%/methyl 240 UV 3.58 TiO2 NPs Ludwigia octovalvis [71]
shape
orange, 72.4%/
alizarin red
 Catalysts 2024, 14, 800 11 of 18

Table 1. Cont.

Decolorization Irradiation Source of Particles Size

Band Gap (eV) Nano-Material Source of Plants
of Dye (%) Time (min) Light (nm)/Morphology
15–28/spherical Nervilia aragoana
56%/MB 40 UV 3.47 TiO2 NPs
morphology leaf [72]
97%/MB,
99%/methyl 90 Sunlight 3.2 22/spherical shape TiO2 NPs Wrightia tinctoria [73]
orange
47% and 240 UV - TiO2 NPs
32.3/spherical shape Commercial P25 [74]
32%/MB
19.8/roughly
92% CV dye, 99% 36 h, and Rumex vesicarius L.
UV 3.52, 2.93 spherical and CuO NPs
MB dye 12 h [current study]
irregular in shape
Catalysts 2024, 14, x FOR PEER REVIEW 12 of 19

2.8. Evaluation of the Antimicrobial Activity of Synthesized CuO NPs

The antimicrobial activity of CuO NPs synthesized using R. vesicarius leaf extract was
various strains
assessed usingofa microbes. The Gram-positive
standard well-diffusion method bacterial strains, Gram-negative
with Mueller–Hinton agar media for Bacillus
cereus (B. strains
various cereus) ofand Staphylococcus
microbes. aureus (S.bacterial
The Gram-positive aureus), strains,
and the Gram-negative
Gram-negative bacterial
Bacillus
cereus Escherichia
species (B. cereus) andcoli Staphylococcus
(E. coli) and aureus (S. aureus),
Pseudomonas and the(P.
aeruginosa Gram-negative bacterial
aeruginosa) were used as
species Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa)
model bacterial species, while Helminthosporium, Alternaria alternata (A. alternata), were used as and
model bacterial
Fusarium oxysporum species, while Helminthosporium,
(F. oxysporum) were the model Alternaria alternata used
fungal species (A. alternata), and the
to investigate
Fusarium oxysporum (F. oxysporum) were the model fungal species used to investigate the
antimicrobial activity of the green-synthesized CuO NPs. The antimicrobial activities of
antimicrobial activity of the green-synthesized CuO NPs. The antimicrobial activities of the
thesynthesized
synthesized CuO NPs are shown in Figure 9.
CuO NPs are shown in Figure 9.

Figure 9. 9.
Figure Mean
Mean± ±
SESEofofinhibition
inhibition zone (mm)introduced
zone (mm) introducedbyby synthesized
synthesized CuOCuO
NPs NPs against
against various
various
strains of of
strains microbes.
microbes.

Theininvitro
The vitro antibacterial
antibacterialactivity of the
activity of CuO NPs against
the CuO E. coli, P.E.
NPs against aeruginosa,
coli, P. S. aureus,
aeruginosa, S.
and B. cereus were assessed by
aureus, and B. cereus were assessed −by determining their zones of inhibition. The synthesized
determining their zones of inhibition. The
CuO NPs at a concentration of 10 µg mL 1 showed the highest antibacterial performance
synthesized CuO NPs at a concentration of 10 µg mL−1 showed the highest antibacterial
against S. aureus, B. cereus, and P. aeruginosa, demonstrating inhibition zones of 20.0 ± 0.03,
performance against
16.3 ± 0.3, and 15.3 ± S. 0.3
aureus,
mm, B. cereus, andThe
respectively. P. aeruginosa, demonstrating
least antibacterial activity was inhibition
observedzones
of against
20.0 ± 0.03, 16.3 ± 0.3, and 15.3 ± 0.3 mm, respectively. The least antibacterial
E. coli with an inhibition zone of 5 ± 0.0 mm. No inhibitory ability of the R. vesicarius activity
was observed
extract against E.
was observed coli with
against an inhibition
any strains zone
of bacteria. of 5 results
These ± 0.0 mm.
clearlyNoshow
inhibitory ability of
that Gram-
thepositive
R. vesicarius extract
bacteria were wasmoreobserved against
sensitive to CuO NPsany than
strains of bacteria. These
Gram-negative results
bacteria. clearly
This is
in consensus
show with previous
that Gram-positive studieswere
bacteria that showed the inhibition
more sensitive to CuOzoneNPs of synthesized CuO
than Gram-negative
bacteria. This is in consensus with previous studies that showed the inhibition zone of
synthesized CuO NPs with Ailanthus altissima leaf extract against S. aureus and E. coli [75],
with Adhatoda vasica Nees extract against P. aeruginosa [76], and with Eryngium caucasicum
extract against B. cereus [77].
Catalysts 2024, 14, 800 12 of 18

NPs with Ailanthus altissima leaf extract against S. aureus and E. coli [75], with Adhatoda
vasica Nees extract against P. aeruginosa [76], and with Eryngium caucasicum extract against
B. cereus [77].
The findings of the antibacterial performance analysis revealed that the CuO NPs were
highly effective in inhibiting the growth of bacterial species. The plausible explanation
for this marked performance of antibacterial activity could be the direct interaction of
the NPs with the cell membranes of the bacteria. The CuO NPs generate ROS, and their
interaction with the bacterial cell membrane could facilitate the penetration of individual
NPs into the cell. The inhibition of bacterial growth is also possibly due to dysfunction
of the cell membrane caused by the NPs that results in a malfunction of the cell enzymes
affecting cellular integrity. The NPs could also destroy bacterial membranes with ROS
(superoxide and hydroxyl radicals) due to the lipid peroxidation causing oxidative damage
to the bacterial cell. Another probable explanation for the potent anti-microbial activity of
the CuO NPs is the presence of amines and carboxyl groups on the cell surface of bacterial
species which enhance the affinity of Cu2+ ions for the cells [1].
The antifungal activity of the CuO NPs prepared with R. vesicarius extract was eval-
uated against three types of fungi: Fusarium oxysporum, Aspergillus flavus, and Alternaria
alternata. The results demonstrated that the synthesized CuO NPs inhibited the growth
of F. oxysporum with an inhibition zone of 30 ± 0.0 mm, slightly higher than the aqueous
extract (28.5 mm). For A. flavus, the inhibition zone was also 30 ± 0.0 mm, but no inhibi-
tion was observed for the aqueous extract. Finally, the inhibition zone of A. alternata was
20.5 ± 0.03 mm and 6 mm for the CuO NPs and aqueous extract, respectively (Figure 9).
The results indicated that the CuO NPs were more efficacious than the aqueous extract
against all the fungal strains evaluated. Similar studies have been reported on the anti-
fungal activity of CuO NPs synthesized from Celastrus paniculatus Willd. leaf extract [78],
Pseudomonas fluorescens extract [79], and Brassica oleracea var. italica extract [7]. The possible
mechanisms of the activity of CuO NPs are based on induced changes in the structure and
function of the fungi. Furthermore, the NPs can affect the DNA, hindering replication and
protein synthesis, which eventually leads to death of the fungi [80].

3. Materials and Methods

3.1. Preparation of Rumex vesicarius L. Extract
Fresh leaves of Rumex vesicarius L. (known as Humeidh) were collected from the Al
Muzahimiyah area and sun dried for a week. The dried leaves of R. vesicarius were washed
with tap water several times before a final wash with deionized water, and again allowed
to dry. Next, 100 mL of boiled deionized water was added to 10 g of the dried and cleaned
R. vesicarius leaves. The mixture was gently stirred at a slow speed and allowed to soak
overnight at room temperature with a cover to prevent evaporation or contamination. The
liquid extract was then isolated by filtration with a grade 1 Whatman paper and stored at
4 ◦ C.

3.2. Preparation of Copper Oxide Nanoparticles

Two grams of copper acetate monohydrate (Sigma-Aldrich, St. Louis, MO, USA)
were dissolved in 50 mL of deionized water and stirred for 10 min at room temperature
to prepare a copper acetate solution. Then, 100 mL of R. vesicarius extract (prepared as
described earlier) was added to the copper acetate solution. The mixture was stirred for
30 min at room temperature, during which the initial light blue color changed to light
green. The stirring continued for an additional 3 h at room temperature, after which the
temperature was raised to 60 ◦ C. Next, a specific amount of ammonia solution was added
dropwise to the mixture with continuous magnetic stirring to adjust the pH to 11. This
led to the formation of a brown precipitate, indicating the preliminary formation of CuO
nanoparticles (CuO NPs). Once the addition was complete, the precipitate was isolated by
centrifugation at 10,000 rpm for 10 min to separate the liquid from the solid. The resulting
pellet was transferred to a covered ceramic vessel and dried in an oven at 80 ◦ C for 12 h.
Catalysts 2024, 14, 800 13 of 18

Finally, the dried material was calcined at 400 ◦ C for 4 h in a muffle furnace (CWF 1300,
Carbolite, Hope Valley, UK) to produce the CuO NPs [30].

3.3. Characterization of the Copper Oxide Nanoparticles

The synthesized CuO NPs were characterized using a variety of techniques. The
morphology of the copper oxide nanoparticles was characterized using TEM with a JEM-
2100 (JEOL, Peabody, MA, USA). The optical absorption spectrum of the CuO NPs was
recorded using a UV–Vis spectrometer (UV-1800, Shimadzu, Muttenz, Switzerland). The
photoluminescence (PL) spectra were measured for the CuO NPs dispersed in double-
distilled water using a Perkin–Elmer photoluminescence spectrophotometer equipped
with a xenon lamp as the excitation source. All experiments were conducted at room
temperature. Energy dispersive X-ray (EDX) spectroscopy was conducted (JSM-2100F,
JEOL, USA), and a SEM (JOEL JSM-7600F, USA) was used to identify and map the elements
in the CuO NPs samples. For the microscope measurements, an aqueous solution of CuO
NPs was sonicated, and a drop was placed on carbon-coated Cu grids and allowed to dry.

3.4. Anticancer Activities of the Copper Oxide Nanoparticles

Trypan blue dye was used as a metabolic indicator for assessing cell viability. A methyl
thiazol tetrazolium (MTT) assay was used to determine the cytotoxicity of the synthesized
CuO NPs for the HeLa cervical carcinoma cell line (obtained from American Type Culture
Collection, Manassas, VA, USA). HeLa cells were cultured at the optimal growth conditions
of 5% CO2 in air and 37 ◦ C and passaged regularly until use. The total number of cells
used in the experiments was determined with a 0.4% trypan blue exclusion test using a
cell counter. HeLa cells were seeded in a 96-well plate at a density of 2 × 105 cells/well in
100 µL of the optimized medium (Dulbecco’s Modified Eagles Medium (DMEM)). After
24 h, the cells were fed with fresh medium and treated with one of six concentrations of
CuO NPs (5, 10, 20, 30, 50, and 100 µg mL−1 ) and R. vesicarius leaf extract as a positive
control. The treated cells were incubated and allowed to grow further for 24 h. After the
incubation time, 20 µL of the MTT solution (Cell Titer 96® Aqueous One Solution Reagent,
Catalog Number G3581, Promega Corporation, Madison, WI, USA) was added to each well
and further incubated for 4 h. The absorbance was then read at 540 nm using an automatic
microplate reader (Molecular Devices–SPECTRA max–PLUS384, San Jose, CA, USA). Each
experiment was performed in four replicates.

3.5. Photodegradation of Dyes by Copper Oxide Nanoparticles

The photocatalytic activity of the synthesized CuO NPs was demonstrated in the
degradation of pollutant dyes, specifically crystal violet (CV) and methylene blue (MB)
dyes, under UV lamp irradiation. A 1 mg L−1 solution of the synthesized CuO NPs
(i.e., 0.03 mg CuO NPs) was added to 30 mL of either CV or MB dye solution (dye solutions
were 0.1 wt.% aqueous solutions) in 50 mL glass beakers. The synthesized CuO NP/dye
solutions were stirred and irradiated at a set distance from a UV lamp (365 nm wavelength:
0.7 AMPS). The range of wavelengths applied was from 190–950 nm. Optical absorption
spectra were collected after different light exposure durations using a UV–Vis spectropho-
tometer. The rate of degradation was monitored by recording the absorption intensity of
the dye at the maximum wavelength as it decreased. The degradation efficiency (DE) was
calculated according to the following equation:

A0 − A
 
(%DE) = × 100 (12)
A0

where (%DE) is the degradation efficiency, A0 is the initial absorption intensity, and A is
the absorption intensity after the photodegradation of the dyes.
Catalysts 2024, 14, 800 14 of 18

3.6. Antibacterial Activities of the Copper Oxide Nanoparticles

The CuO NPs were tested for antimicrobial activity against various bacterial and
fungal strains. Minimum inhibitory concentration (MIC) of the CuO NPs against each
bacterial pathogen was determined by the micro-dilution broth method. To match the
McFarland (turbidity) standard, all microbial dilutions were standardized, showing a
bacterial density of 1.5 × 108 CFU/mL. The antibacterial activity of the synthesized CuO
NPs was evaluated using the agar well-diffusion method. Four bacterial strains, Gram-
negative Bacillus cereus (B. cereus) and Staphylococcus aureus (S. aureus), and the Gram-
negative bacterial species Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa),
along with three fungal strains Helminthosporium, Alternaria alternata (A. alternata), and
Fusarium oxysporum (F. oxysporum) were tested. The bacteria were grown in blood agar at
37 ◦ C for 18 h, collected and suspended in 0.85% NaCl, and adjusted to 0.5 McFarland
(108 CFU/mL) turbidity. The fungal and bacterial suspensions were smeared on Muller–
Hinton agar (MHA) plates. 10 µg mL−1 of the synthesized CuO NPs and R. vesicarius leaf
extract was loaded in the agar, and the plates were incubated at 37 ◦ C for 18–24 h for bacteria
and at 28 ◦ C for 48–72 h for fungi. After incubation, clear zones of inhibition appeared
around the wells, and the diameter of each inhibition zone was measured and recorded.

3.7. Statistical Analysis

Statistical significance among groups was analyzed by one-way analysis of variance
(ANOVA) using SPSS version 17. Group comparisons were made using Tukey’s post hoc
test. Values of p ≤ 0.05 were considered statistically significant.

4. Conclusions
In summary, the key findings of this study highlight a simple, cost-effective, and
rapid method for the green synthesis of CuO nanoparticles (NPs) using copper acetate
and the aqueous extract of R. vesicarius at room temperature. Various standard techniques,
including UV–Vis, XRD, EDS, PL spectroscopy, as well as TEM and SEM microscopy,
confirmed the successful formation of CuO NPs. The synthesized nanoparticles were
irregular in shape and formed a network due to agglomeration, with particle sizes ranging
from 5 to 200 nm.
Cytotoxicity studies revealed the anti-proliferative effects of CuO NPs against the
HeLa cancer cell line, with an IC50 value of 8 µg/L, while the R. vesicarius extract itself
showed no cytotoxic effects, even at an IC50 of 50 µg/L. Additionally, the CuO NPs
exhibited significant antimicrobial activity, as demonstrated by inhibition zones against
various fungal and bacterial strains, indicating their therapeutic potential for treating
infectious diseases. Overall, the multifunctionality of the synthesized CuO NPs suggests
their promising potential for a wide range of future applications.

Author Contributions: Conceptualization, M.A.A., S.S.A. and A.A.; Data curation, A.A., S.S.A. and
M.A.A.; Formal analysis, M.A.A., S.S.A. and A.A.; Funding acquisition, S.S.A. and A.A.; Investigation,
M.A.A., A.A. and S.S.A.; Methodology, M.A.A., R.A., H.A.A. (Hind Ali Alshehri), Z.A.A., R.H.A.,
N.S.A., L.A.A., S.M.A., K.I.B.S., R.B.A., B.I.A., H.A.A. (Hissah Abdullah Aljaser) and S.A.; validation,
S.S.A., A.A. and M.A.A.; resources, A.A. and S.A.; writing—original draft preparation, M.A.A.,
R.A., H.A.A. (Hind Ali Alshehri), Z.A.A., R.H.A., N.S.A., L.A.A., S.M.A., K.I.B.S., R.B.A., B.I.A.,
H.A.A. (Hissah Abdullah Aljaser) and S.A.; writing—review and editing, M.A.A., A.A. and S.S.A.;
supervision, S.S.A., M.A.A. and A.A.; project administration, A.A. and S.S.A. All authors have read
and agreed to the published version of the manuscript.
Funding: The authors gratefully acknowledge the financial support from Researchers Supporting
Project number (RICSP-24-1), King Saud University, Riyadh, Saudi Arabia.
Data Availability Statement: The original contributions presented in the study are included in the
article, further inquiries can be directed to the corresponding authors.
Acknowledgments: The authors gratefully acknowledge the financial support from Researchers
Supporting Project number (RICSP-24-1), King Saud University, Riyadh, Saudi Arabia.
Catalysts 2024, 14, 800 15 of 18

Conflicts of Interest: The authors declare no conflicts of interest or state.

References
1. Bayda, S.; Adeel, M.; Tuccinardi, T.; Cordani, M.; Rizzolio, F. The history of nanoscience and nanotechnology: From chemical–
physical applications to nanomedicine. Molecules 2019, 25, 112. [CrossRef] [PubMed]
2. Sarma, I.D.; Bhowmick, D.; Bhilkar, P.; Sharma, R.; Chaudhary, R.G.; Sarma, I.D.; Chaudhary, R.G. Nanotechnology: Fundamental
Aspects and Biomedical and Technological Applications. Nanobiomater. Perspect. Med. Appl. Diagn. Treat. Dis. 2023, 145, 1–18.
3. Radulescu, D.M.; Surdu, V.A.; Ficai, A.; Ficai, D.; Grumezescu, A.M.; Andronescu, E. Green synthesis of metal and metal oxide
nanoparticles: A review of the principles and biomedical applications. Int. J. Mol. Sci. 2023, 24, 15397. [CrossRef]
4. Burlec, A.F.; Corciova, A.; Boev, M.; Batir-Marin, D.; Mircea, C.; Cioanca, O.; Danila, G.; Danila, M.; Bucur, A.F.; Hancianu, M.
Current overview of metal nanoparticles’ synthesis, characterization, and biomedical applications, with a focus on silver and
gold nanoparticles. Pharmaceuticals 2023, 16, 1410. [CrossRef]
5. Chavali, M.S.; Nikolova, M.P. Metal oxide nanoparticles and their applications in nanotechnology. SN Appl. Sci. 2019, 1, 607.
[CrossRef]
6. Ashour, M.; Mansour, A.T.; Abdelwahab, A.M.; Alprol, A.E. Metal oxide nanoparticles’ green synthesis by plants: Prospects in
phyto-and bioremediation and photocatalytic degradation of organic pollutants. Processes 2023, 11, 3356. [CrossRef]
7. Renuga, D.; Jeyasundari, J.; Shakthi, A.S.; Brightson Arul Jacob, Y. Synthesis and Characterization of Copper Oxide Nanoparticles
Using Brassica oleracea var. Italic Extract for Its Antifungal Application. Mater. Res. Express 2020, 7, 045007. [CrossRef]
8. Safeyah Al-Shehri, N.A.S.; Altuwirqi, R.; Bayahya, A.; Al-Shammari, F.; Wang, Z.; Hala, A.J. Green Synthesis of CuxO Nanoscale
MOS Capacitors Processed at Low Temperatures. Surf. Coat. Technol. 2017, 320, 246–251. [CrossRef]
9. Mali, S.C.; Dhaka, A.; Sharma, S.; Trivedi, R. Review on biogenic synthesis of copper nanoparticles and its potential applications.
Inorg. Chem. Commun. 2023, 149, 110448. [CrossRef]
10. Ganesan, K.; Jothi, V.K.; Natarajan, A.; Rajaram, A.; Ravichandran, S.; Ramalingam, S. Green Synthesis of Copper Oxide
Nanoparticles Decorated with Graphene Oxide for Anticancer Activity and Catalytic Applications. Arab. J. Chem. 2020, 13,
6802–6814. [CrossRef]
11. Rafique, M.; Tahir, M.B.; Irshad, M.; Nabi, G.; Gillani, S.S.A.; Iqbal, T.; Mubeen, M. Novel Citrus aurantifolia Leaves Based
Biosynthesis of Copper Oxide Nanoparticles for Environmental and Wastewater Purification as an Efficient Photocatalyst and
Antibacterial Agent. Optik 2020, 219, 165138. [CrossRef]
12. Vennila, S. Eco-Friendly Synthesis of Metal Oxide Nanoparticles Using Carissa Carandas fruit extract. World J. Pharm. Res. 2016,
5, 806–812.
13. Rajesh, K.M.; Ajitha, B.; Reddy YA, K.; Suneetha, Y.; Reddy, P.S. Assisted Green Synthesis of Copper Nanoparticles Using
Syzygium aromaticum Bud Extract: Physical, Optical and Antimicrobial Properties. Optik 2018, 154, 593–600. [CrossRef]
14. Benguigui, M.; Weitz, I.S.; Timaner, M.; Kan, T.; Shechter, D.; Perlman, O.; Sivan, S.; Raviv, Z.; Azhari, H.; Shaked, Y. Copper
Oxide Nanoparticles Inhibit Pancreatic Tumor Growth Primarily by Targeting Tumor Initiating Cells. Sci. Rep. 2019, 9, 12613.
[CrossRef]
15. Sankar, R.; Maheswari, R.; Karthik, S.; Shivashangari, K.S.; Ravikumar, V. Anticancer Activity of Ficus religiosa Engineered
Copper Oxide Nanoparticles. Mater. Sci. Eng. C Mater. Biol. Appl. 2014, 44, 234–239. [CrossRef]
16. Luque-Jacobo, C.M.; Cespedes-Loayza, A.L.; Echegaray-Ugarte, T.S.; Cruz-Loayza, J.L.; Cruz, I.; de Carvalho, J.C.; Goyzueta-
Mamani, L.D. Biogenic synthesis of copper nanoparticles: A systematic review of their features and main applications. Molecules
2023, 28, 4838. [CrossRef]
17. Wang, W.; Zhu, Y.; Cheng, G.; Huang, Y. Microwave-Assisted Synthesis of Cupric Oxide Nanosheets and Nanowhiskers. Mater.
Lett. 2006, 60, 609–612. [CrossRef]
18. Kannan, S.K.; Sundrarajan, M. Biosynthesis of Yttrium Oxide Nanoparticles Using Acalypha indica Leaf Extract. Bull. Mater. Sci.
2015, 38, 945–950. [CrossRef]
19. Muhammad, A.; Umar, A.; Birnin-Yauri, A.U.; Sanni, H.A.; Elinge, C.M.; Ige, A.R.; Ambursa, M.M. Green synthesis of copper
nanoparticles using Musa acuminata aqueous extract and their antibacterial activity. Asian J. Trop. Biotechnol. 2023, 20.
20. Manjari, G.; Saran, S.; Arun, T.; Vijaya Bhaskara Rao, A.V.B.; Devipriya, S.P. Catalytic and Recyclability Properties of Phytogenic
Copper Oxide Nanoparticles Derived from Aglaia elaeagnoidea Flower Extract. J. Saudi Chem. Soc. 2017, 21, 610–618. [CrossRef]
21. Kerour, A.; Boudjadar, S.; Bourzami, R.; Allouche, B. Eco-Friendly Synthesis of Cuprous Oxide (Cu2 O) Nanoparticles and
Improvement of Their Solar Photocatalytic Activities. J. Solid State Chem. 2018, 263, 79–83. [CrossRef]
22. Murthy, H.A.; Abebe, B.; Prakash, C.H.; Shantaveerayya, K. A review on green synthesis of Cu and CuO nanomaterials for
multifunctional applications. Mater. Sci. Res. India 2018, 15, 279–295. [CrossRef]
23. Praburaman, L.; Jang, J.S.; Muthusamy, G.; Arumugam, S.; Manoharan, K.; Cho, K.M.; Min, C.; Kamala-Kannan, S.; Byung-
Taek, O. Piper Betle-Mediated Synthesis, Characterization, Antibacterial and Rat Splenocyte Cytotoxic Effects of Copper Oxide
Nanoparticles. Artif. Cells Nanomed. Biotechnol. 2016, 44, 1400–1405. [CrossRef]
24. Sankar, R.; Manikandan, P.; Malarvizhi, V.; Fathima, T.; Shivashangari, K.S.; Ravikumar, V. Green Synthesis of Colloidal Copper
Oxide Nanoparticles using Carica Papaya and its Application in Photocatalytic Dye Degradation. Spectrochim. Acta A Mol. Biomol.
Spectrosc. 2014, 121, 746–750. [CrossRef]
Catalysts 2024, 14, 800 16 of 18

25. Saleh, N.A.M.; El-Hadidi, M.N.; Arafa, R.F.M. Flavonoids and Anthraquinones of Some Egyptian Rumex species (Polygonaceae).
Biochem. Syst. Ecol. 1993, 21, 301–303. [CrossRef]
26. Verma, K.K.; Gautam, R.K.; Choudhary, A.; Gupta, G.D.; Singla, S.; Goyal, S. A Review on Ethnobotany, Phytochemistry and
Pharmacology on Rumex hastatus. Res. J. Pharm. Technol. 2020, 13, 26. [CrossRef]
27. Harley, R.M. Flora of Eastern Saudi-Arabia–Mandaville, Jp Tls. Times 1991, 4598, 20.
28. Farooq, M.; Abutaha, N.; Mahboob, S.; Baabbad, A.; Almoutiri, N.D.; Wadaan, M.A.A. Investigating the Antiangiogenic Potential
of Rumex vesicarius (Humeidh), Anticancer Activity in Cancer Cell Lines and Assessment of Developmental Toxicity in Zebrafish
Embryos. Saudi J. Biol. Sci. 2020, 27, 611–622. [CrossRef] [PubMed]
29. Vasas, A.; Orbán-Gyapai, O.; Hohmann, J. The Genus Rumex: Review of Traditional Uses, Phytochemistry and Pharmacology. J.
Ethnopharmacol. 2015, 175, 198–228. [CrossRef]
30. Aldalbahi, A.; Aldawish, R.; Awad, M.A.G.; Aldosari, N.S.; Alshathri, R.H.; Aldwihi, L.A.; Alammari, R.; Shoqiran, K.I.B.
Synthesis of Copper Oxide Nanoparticles. U.S. Patent 10,995,010 B1, 4 May 2021.
31. Udayabhanu; Nethravathi, P.C.; Pavan Kumar, M.A.; Suresh, D.; Lingaraju, K.; Rajanaika, H.; Nagabhushana, H.; Sharma, S.C.
Tinospora cordifolia Mediated Facile Green Synthesis of Cupric Oxide Nanoparticles and Their Photocatalytic, Antioxidant and
Antibacterial Properties. Mater. Sci. Semicond. Process. 2015, 33, 81–88.
32. Saif, S.; Tahir, A.; Asim, T.; Chen, Y. Plant Mediated Green Synthesis of CuO Nanoparticles: Comparison of Toxicity of Engineered
and Plant Mediated CuO Nanoparticles Towards Daphnia magna. Nanomaterials 2016, 1, 205. [CrossRef] [PubMed]
33. Monte-Filho, S.S.; Andrade, S.I.E.; Lima, M.B.; Araujo, M.C.U. Synthesis of Highly Fluorescent Carbon Dots from Lemon and
Onion Juices for Determination of Riboflavin in Multivitamin/Mineral Supplements. J. Pharm. Anal. 2019, 9, 209–216. [CrossRef]
[PubMed]
34. Sukumar, S.; Rudrasenan, A.; Padmanabhan Nambiar, D. Green-Synthesized Rice-Shaped Copper Oxide Nanoparticles Using
Caesalpinia bonducella Seed Extract and Their Applications. ACS Omega 2020, 5, 1040–1051. [CrossRef] [PubMed]
35. Vasantharaj, S.; Sathiyavimal, S.; Saravanan, M.; Senthilkumar, P.; Gnanasekaran, K.; Shanmugavel, M.; Manikandan, E.;
Pugazhendhi, A. Synthesis of Ecofriendly Copper Oxide Nanoparticles for Fabrication over Textile Fabrics: Characterization of
Antibacterial Activity and Dye Degradation Potential. J. Photochem. Photobiol. B Biol. 2019, 191, 143–149. [CrossRef] [PubMed]
36. Salopek, B.; Krasic, D.; Filipovic, S. Measurement and Application of Zeta-Potential. Rud. Geol. Naft. Zb. 1992, 4, 147.
37. Muthuvel, A.; Jothibas, M.; Manoharan, C. Synthesis of Copper Oxide Nanoparticles by Chemical and Biogenic Methods:
Photocatalytic Degradation and In Vitro Antioxidant Activity. Nanotechnol. Environ. Eng. 2020, 5, 14. [CrossRef]
38. Dagher, S.; Haik, Y.; Ayesh, A.I.; Tit, N. Synthesis and Optical Properties of Colloidal CuO Nanoparticles. J. Lumin. 2014, 151,
149–154. [CrossRef]
39. Talluri, B.; Prasad, E.; Thomas, T. Ultra-Small (r < 2 nm), Stable (>1 Year) Copper Oxide Quantum Dots with Wide Band Gap.
Superlattices Microstruct. 2018, 113, 600–607. [CrossRef]
40. Agam, M.A.; Awal, N.N.; Hassan, S.A.; Yabagi, J.A.; Qabel, M. Energy Band Gap Investigation of Polystyrene Copper Oxide
Nanocomposites Bombarded with Laser. J. Adv. Res. Fluid Mech. Therm. Sci. 2020, 66, 125–135.
41. Nemade, K.R.; Waghuley, S.A. Optical and Gas Sensing Properties of CuO Nanoparticles Grown by Spray Pyrolysis of Cupric
Nitrate Solution. J. Mater. Sci. Eng. 2014, 2, 63–66. [CrossRef]
42. Chen, J.S.; Mao, S.; Xu, Z.; Ding, W. Various Antibacterial Mechanisms of Biosynthesized Copper Oxide Nanoparticles Against
Soilborne Ralstonia solanacearum. RSC Adv. 2019, 9, 3788–3799. [CrossRef] [PubMed]
43. Murthy, H.C.A.; Desalegn, T.; Kassa, M.; Abebe, B.; Assefa, T. Synthesis of Green Copper Nanoparticles Using Medicinal Plant
Hagenia abyssinica (Brace) JF. Gmel. Leaf Extr. Antimicrob. Prop. J. Nanomater. 2020, 12, 3924081. [CrossRef]
44. Mishra, L.; Dwivedi, V.K.; Dara, H.K.; Chakradhary, V.K.; Ithineni, S.; Prabhudessai, A.G.; Nehar, S. Core/Shell-Like Magnetic
Structure and Optical Properties in CuO Nanoparticles Synthesized by Green Route. ACS Sustain. Resour. Manag. 2024. [CrossRef]
45. Vinardell, M.P.; Mitjans, M. Antitumor Activities of Metal Oxide Nanoparticles. Nanomaterials 2015, 5, 1004–1021. [CrossRef]
46. Rehana, D.; Mahendiran, D.; Kumar, R.S.; Rahiman, A.K. Evaluation of Antioxidant and Anticancer Activity of Copper Oxide
Nanoparticles Synthesized Using Medicinally Important Plant Extracts. Biomed. Pharmacother. 2017, 89, 1067–1077. [CrossRef]
[PubMed]
47. Nagajyothi, P.C.; Muthuraman, P.; Sreekanth, T.V.M.; Kim, D.H.; Shim, J. Green Synthesis: In-Vitro Anticancer Activity of Copper
Oxide Nanoparticles against Human Cervical Carcinoma Cells. Arab. J. Chem. 2017, 10, 215–225. [CrossRef]
48. Elemike, E.E.; Onwudiwe, D.C.; Singh, M. Eco-Friendly Synthesis of Copper Oxide, Zinc Oxide and Copper Oxide–Zinc Oxide
Nanocomposites, and Their Anticancer Applications. J. Inorg. Organomet. Polym. Mater. 2020, 30, 400–409. [CrossRef]
49. Sharma, M.; Sharma, A.; Majumder, S. Synthesis, Microbial Susceptibility and Anti-Cancerous Properties of Copper Oxide
Nanoparticles-Review. NANO Express 2020, 1, 012003. [CrossRef]
50. Elavarasan, N.; Kokila, K.; Prakash, S.; Sujatha, V. Exploration of Bio-synthesized Copper Oxide Nanoparticles Using Pterolobium
hexapetalum Leaf Extract by Photocatalytic Activity. J. Clust. Sci. 2019, 30, 1157–1168. [CrossRef]
51. He, H.; Zou, Z.; Wang, B.; Xu, G.; Chen, C.; Qin, X.; Yu, C.; Zhang, J. Copper Oxide Nanoparticles Induce Oxidative DNA Damage
and Cell Death via Copper Ion-Mediated P38 MAPK Activation in Vascular Endothelial Cells. Int. J. Nanomed. 2020, 15, 3291–3302.
[CrossRef]
52. Roy, K.; Ghosh, C.K.; Sarkar, C.K. Degradation of Toxic Textile Dyes and Detection of Hazardous Hg2+ by Low-Cost Bioengineered
Copper Nanoparticles Synthesized Using Impatiens balsamina Leaf Extract. Mater. Res. Bull. 2017, 94, 257–262. [CrossRef]
Catalysts 2024, 14, 800 17 of 18

53. Rabiee, N.; Bagherzadeh, M.; Kiani, M.; Ghadiri, A.M.; Etessamifar, F.; Jaberizadeh, A.H.; Shakeri, A. Biosynthesis of Copper
Oxide Nanoparticles with Potential Biomedical Applications. Int. J. Nanomed. 2020, 15, 3983–3999. [CrossRef] [PubMed]
54. Balázs, N.; Mogyorósi, K.; Srankó, D.F.; Pallagi, A.; Alapi, T.; Oszkó, A.; Dombi, A.; Sipos, P. The Effect of Particle Shape on the
Activity of Nanocrystalline TiO2 Photocatalystsin Phenol Decomposition. Appl. Catal. B 2008, 84, 356–362. [CrossRef]
55. Lahmar, H.; Benamira, M.; Douafer, S.; Akika, F.Z.; Hamdi, M.; Avramova, I.; Trari, M. Photocatalytic Degradation of Crystal
Violet Dye on the Novel CuCr2 O4 /SnO2 Hetero-System under Sunlight. Opt. Int. J. Light Electron. Opt. 2020, 219, 165042.
[CrossRef]
56. Vivek, E.; Senthilkumar, N.; Pramothkumar, A.; Vimalan, M.; Potheher, I.V. Synthesis of Flower-Like Copper Oxide Microstructure
and Its Photocatalytic Property. Phys. B Condens. Matter 2019, 566, 96–102. [CrossRef]
57. Ansari, P.M.Y.; Muthukrishnan, R.M.; Khan, R.I.; Vedhi, C.; Sakthipandi, K.; Kader, S.A. Green synthesis of copper oxide
nanoparticles using Amaranthus dubius leaf extract for sensor and photocatalytic applications. Chem. Phys. Impact 2023, 7, 100374.
[CrossRef]
58. Meena, J.; Kumaraguru, N.; Sami Veerappa, N.; Shin, P.K.; Tatsugi, J.; Kumar, A.S.; Santhakumar, K. Copper oxide nanoparticles
fabricated by green chemistry using Tribulus terrestris seed natural extract-photocatalyst and green electrodes for energy storage
device. Sci. Rep. 2023, 13, 22499. [CrossRef]
59. Jayasimha, H.N.; Chandrappa, K.G.; Sanaulla, P.F.; Dileepkumar, V.G. Green synthesis of CuO nanoparticles: A promising
material for photocatalysis and electrochemical sensor. Sens. Int. 2024, 5, 100254. [CrossRef]
60. Sangeetha, A.; Abarna, B. Lemon peel assisted synthesis of copper oxide nanoparticles for photocatalytic degradation. Mater.
Today Proc. 2023. [CrossRef]
61. Ali, S.G.; Haseen, U.; Jalal, M.; Khan, R.A.; Alsalme, A.; Ahmad, H.; Khan, H.M. Green synthesis of copper oxide nanoparticles
from the leaves of Aegle marmelos and their antimicrobial activity and photocatalytic activities. Molecules 2023, 28, 7499. [CrossRef]
62. Taghavi Fardood, S.; Moradnia, F.; Heidarzadeh, S.; Naghipour, A. Green synthesis, characterization, photocatalytic and
antibacterial activities of copper oxide nanoparticles of copper oxide nanoparticles. Nanochem. Res. 2023, 8, 134–140.
63. Indhira, D.; Krishnamoorthy, M.; Ameen, F.; Bhat, S.A.; Arumugam, K.; Ramalingam, S.; Priyan, S.R.; Kumar, G.S. Biomimetic
facile synthesis of zinc oxide and copper oxide nanoparticles from Elaeagnus indica for enhanced photocatalytic activity. Environ.
Res. 2022, 212, 113323. [CrossRef]
64. Mishra, U.K.; Chandel, V.S.; Yadav, A.K.; Gautam, A.K.; Anand, A.D.; Varun, J.; Rai, A.K.; Singh, S.P. Synthesis, characterization,
and study of photocatalytic degradation of aniline blue dye using copper oxide nanoparticles prepared by Santa Maria feverfew
leaf extract. Nanotechnol. Environ. Eng. 2024, 9, 1–10. [CrossRef]
65. Relhan, A.; Guleria, S.; Bhasin, A.; Mirza, A.; Zhou, J.L. Biosynthesized copper oxide nanoparticles by Psidium guajava plants
with antibacterial, antidiabetic, antioxidant, and photocatalytic capacity. Biomass Convers. Biorefin. 2024, 1–18.–18. [CrossRef]
66. Alshehri, A.A.; Malik, M.A. Biogenic fabrication of ZnO nanoparticles using Trigonella foenum-graecum (Fenugreek) for
proficient photocatalytic degradation of methylene blue under UV irradiation. J. Mater. Sci. Mater. Electron. 2019, 30, 16156–16173.
[CrossRef]
67. Awad, M.A.; Hendi, A.A.; Ortashi, K.M.; Alnamlah, R.A.; Alangery, A.; Ali Alshaya, E.; Alshammari, S.G. Utilizing Cymbopogon
Proximus Grass Extract for Green Synthesis of Zinc Oxide Nanorod Needles in Dye Degradation Studies. Molecules 2024, 29, 355.
[CrossRef]
68. Avinash, B.; Ravikumar, C.R.; Basavaraju, N.; Abebe, B.; Kumar, T.N.; Manjula, S.N.; Murthy, H.A. Facile green synthesis of zinc
oxide nanoparticles: Its photocatalytic and electrochemical sensor for the determination of paracetamol and D-glucose. Environ.
Funct. Mater. 2023, 2, 133–141. [CrossRef]
69. Hoon Seo, K.; Markus, J.; Soshnikova, V.; Oh, K.H.; Anandapadmanaban, G.; Elizabeth Jimenez Perez, Z.; Mathiyalagan, R.; Kim,
Y.J.; Yang, D.C. Facile and green synthesis of zinc oxide particles by Stevia rebaudiana and its in vitro photocatalytic activity. Inorg.
Nano-Met. Chem. 2019, 49, 1–6. [CrossRef]
70. Singh, K.; Nancy Bhattu, M.; Singh, G.; Mubarak, N.M.; Singh, J. Light-absorption-driven photocatalysis and antimicrobial
potential of PVP-capped zinc oxide nanoparticles. Sci. Rep. 2023, 13, 13886. [CrossRef]
71. Ramasamy, K.; Dhavamani, S.; Natesan, G.; Sengodan, K.; Sengottayan, S.N.; Tiwari, M.; Vikram, S.S.; Perumal, V. A potential
role of green engineered TiO2 nanocatalyst towards enhanced photocatalytic and biomedical applications. Environ. Sci. Pollut.
Res. 2021, 28, 41207–41223. [CrossRef]
72. Rathi, V.H.; Jeice, A.R. Green fabrication of titanium dioxide nanoparticles and their applications in photocatalytic dye degradation
and microbial activities. Chem. Phys. Impact 2023, 6, 100197. [CrossRef]
73. Muthuvel, A.; Said, N.M.; Jothibas, M.; Gurushankar, K.; Mohana, V. Microwave-assisted green synthesis of nanoscaled titanium
oxide: Photocatalyst, antibacterial and antioxidant properties. J. Mater. Sci. Mater. Electron. 2021, 32, 23522–23539. [CrossRef]
74. Sangchay, W.; Sikong, L.; Kooptarnond, K. Comparison of photocatalytic reaction of commercial P25 and synthetic TiO2 -AgCl
nanoparticles. Procedia Eng. 2012, 32, 590–596. [CrossRef]
75. Akl, M.A.; Mohammad, W.A. Biosynthesis of Copper Oxide Nanoparticles Using Ailanthus altissima Leaf Extract and Antibacterial
Activity. Chem. Int. 2020, 6, 113–114.
76. Bhavyasree, P.G.; Xavier, T.S. Green Synthesis of Copper Oxide/Carbon Nanocomposites Using the Leaf Extract of Adhatoda
vasica Nees, Their Characterization and Antimicrobial Activity. Heliyon 2020, 6, e03323. [CrossRef]
Catalysts 2024, 14, 800 18 of 18

77. Hasheminya, S.M.; Dehghannya, J. Green synthesis and characterization of copper nanoparticles using Eryngium caucasicum
Trautv aqueous extracts and its antioxidant and antimicrobial properties. Part. Sci. Technol. 2020, 38, 1019–1026. [CrossRef]
78. Mali, S.C.; Dhaka, A.; Githala, C.K.; Trivedi, R. Green Synthesis of Copper Nanoparticles Using Celastrus paniculatus Willd. Leaf
Extract and Their Photocatalytic and Antifungal Properties. Biotechnol. Rep. 2020, 27, e00518. [CrossRef]
79. Gomaa, E.Z.; Housseiny, M.M.; Omran, A.A.A.K. Fungicidal Efficiency of Silver and Copper Nanoparticles Produced by
Pseudomonas fluorescens ATCC 17397 Against Four Aspergillus species: A Molecular Study. J. Clust. Sci. 2019, 30, 181–196.
[CrossRef]
80. Pariona, N.; Mtz-Enriquez, A.I.; Sánchez-Rangel, D.; Carrión, G.; Paraguay-Delgado, F.; Rosas-Saito, G. Green-Synthesized
Copper Nanoparticles as a Potential Antifungal against Plant Pathogens. RSC Adv. 2019, 9, 18835–18843. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.