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Scientific African 17 (2022) e01365
Contents lists available at ScienceDirect
Scientific African
journal homepage: www.elsevier.com/locate/sciaf
Cytotoxicity and antibacterial effects of silver doped zinc
oxide nanoparticles prepared using fruit extract of Capsicum
Chinense
Makiwa S. Mthana
a
, Mziwenkosi Nhlanhla Mthiyane
a , b
, Anthony C. Ekennia
c
,
Moganavelli Singh
d
, Damian C. Onwudiwe
e , f , ∗
a
Department of Animal Science, School of Agricultural Sciences, Faculty of Natural and Agricultural Sciences, North-West University,
(Mahikeng Campus), Private Bag X2046, Mmabatho, South Africa
b
Food Security and Safety Focus Area, Faculty of Natural and Agricultural Sciences, North-West University, (Mahikeng Campus),
Mmabatho 2735, South Africa
c
Department of Chemistry, Alex Ekwueme Federal University, Ndufu-Alike, Ebonyi, Nigeria
d
Department of Biochemistry, Nano-Gene and Drug Delivery Laboratory, University of KwaZulu-Natal, 16 Private Bag X54001, Durban
40 0 0, South Africa
e
Material Science Innovation and Modelling (MaSIM) Research Focus Area, Faculty of Natural and Agricultural Sciences, North-West
University, (Mahikeng Campus), Private Bag X2046, Mmabatho, South Africa
f
Department of Chemistry, School of Physical and Chemical Sciences, Faculty of Natural and Agricultural Sciences, North-West University,
(Mahikeng Campus), Private Bag X2046, Mmabatho, South Africa
a r t i c l e i n f o
Article history:
Received 4 March 2022
Revised 19 June 2022
Accepted 5 September 2022
Edited by Editor name: DR B Gyampoh
Keywo rds:
Silver-zinc oxide
Antibacterial assay
Anti-cancer
Green synthesis
Capsicum Chinense
a b s t r a c t
Phyto-mediated nanoparticles (NPs) have attracted great attention in recent times due
to their unique properties such as high biocompatibility, eco-friendliness, and cost-
effectiveness. In this study, silver- zinc oxide nanoparticles (Ag/ZnO-NPs) were prepared
using the fruit extract of Capsicum Chinense . The results obtained from both structural,
and optical characterization of the nanoparticles confirmed the purity of the composites
and their high crystallinity. The nanoparticles exhibited two absorption peaks at 376 and
258 nm with a bang gap energy of 3.53 eV, and the nanoparticulate size of the materials
exhibited antibacterial and cytotoxic activities. The Ag/ZnO-NPs exhibited better prolifer-
ative activity against HEK 293 cells than HeLa cells. The antibacterial study showed that
both the pristine and the composite nanoparticles (ZnO and Ag/ZnO-NPs) have high bac-
tericidal effect on all tested bacterial pathogens, with Ag/ZnO-NPs displaying better an-
tibacterial activity. Cytotoxicity studies showed a dose-dependent proliferative relationship
between Ag/ZnO-NPs and tested cancer cells (HEK 293 and HeLa). Antibacterial results
showed significant zones of bactericidal inhibition for all tested bacterial pathogens using
the biosynthesized ZnO and Ag/ZnO-NPs. These findings demonstrated two-fold applica-
tions of biosynthesized Ag/ZnO-NPs, as anticancer and antibacterial agents.
©2022 The Author(s). Published by Elsevier B.V. on behalf of African Institute of
Mathematical Sciences / Next Einstein Initiative.
This is an open access article under the CC BY-NC-ND license
( http://creativecommons.org/licenses/by-nc-nd/4.0/ )
∗Corresponding author at: Material Science Innovation and Modelling (MaSIM) Research Focus Area, Faculty of Natural and Agricultural Sciences, North-
West University, (Mahikeng Campus), Private Bag X2046, Mmabatho, South Africa.
E-mail address: Damian.Onwudiwe@nwu.ac.za (D.C. Onwudiwe) .
https://doi.org/10.1016/j.sciaf.2022.e01365
2468-2276/© 2022 The Author(s). Published by Elsevier B.V. on behalf of African Institute of Mathematical Sciences / Next Einstein Initiative. This is an
open access article under the CC BY-NC-ND license (
http://creativecommons.org/licenses/by-nc-nd/4.0/ )
M.S. Mthana, M.N. Mthiyane, A.C. Ekennia et al. Scientific African 17 (2022) e01365
Introduction
In the last two decades, high bacterial infections and abuse of antibiotics have triggered off drug-resistance microorgan-
isms that have resulted in high morbidity and mortality globally [ 1 , 2 ]. This consequently leads to a higher cost of health-
care. Similarly, cancer is a life-threatening disease, which is responsible for high mortality worldwide [ 3 , 4 ]. In 2018, cancer
claimed about 9.6 million lives and about 15% of the recorded death were women [5] . Furthermore, the burgeoning preva-
lence of chronic human cancer, antibiotic-resistant pathogenic infections, and the urgent need for enhanced efficiency in
producing food to sustainably feed the rapidly growing human population, have resulted in the engineering of advanced
nanotechnological systems with multitudinous applications in medicine, agriculture, and other fields.
In agriculture and medical fields, the advent of new bio-based nanomaterials offers alternative route to minimise or
eradicate the abuse and over-use of already limited natural resources as well as the impact and pressure on them [6–8] . In
particular, the engineering of hybrid (composite or coated) nano-systems with versatile modulatory properties and functions
offers revolutionary opportunities in various application fields. Their design involves combination of multiple nano-materials
into ordered assemblies with two or more unique nanoparticles put together in a functional nano-scale dimensional pat-
tern, whose biological effects are better than those that are obtainable from any simple mixture of separate components
[ 6 , 9 ]. In this regard, Ag/ZnO-NPs with enhanced mechanical and chemical properties (e.g., catalysis, optical, semi-conduction,
thermal, magnetism and electric responses) have been synthesized through chemical [10–14] and phytogenic [15–17] ap-
proaches. Research has previously demonstrated that nanoparticles synthesised using chemical approach are aften toxic and
very expensive [18] . Therefore, this has motivated researchers to develop advanced and reliable cost-effective and non-toxic
nanomaterials using extract of plant materials.
Phytogenic (plant-mediated) hybrid nanoparticles involving Ag are favoured for not only their environmental friendliness
and low cost of production [ 19 , 20 ] but also for their broad-spectrum antibacterial activity against some Gram-positive and
Gram-negative pathogens that are drug-resistant [21] . When compared with other notable metals, Ag nanoparticles have
been found to have substantial recognition as an antimicrobial agent [22–24] . Furthermore, the high surface energy of Ag-
NPs allowed it to gain interest in nanomaterial synthesis, which indorse surface chemistry [25] . So, when ZnO is combined
with Ag, it is expected that the antibacterial efficacy of the former would be pronounced [26] . Indeed, doping, or composit-
ing ZnO-NPs with other nanomaterial has been reported to increase their activity through induction of oxidative stress in
bacteria [27] , disruption of bacterial membrane, and bacterial death [28] .
In the current study, the aim was to synthesise, characterise green Ag/ZnO-NPs using C. chinense fruit extract as a me-
diating agent and evaluate the antibacterial and cytotoxicity potency. To the best of our knowledge, no studies have so far
investigated the antibacterial and cytotoxicity potency of ZnO composited with Ag-NPs obtained via this plant, C. chinense
fruit extract.
Materials and methods
Materials
Zinc acetate dihydrate [Zn(CH
3
CO
2
)
2
• 2H
2
O] and silver nitrate (AgNO
3
) were acquired from Merck (Pty) Ltd. Orange Ha-
banero pepper ( Capsicum Chinense Jacq) fruits were collected from Molelwane experimental farm of North-West University,
situated in Mahikeng Local Municipality, North-West Province, South Africa.
Capsicum Chinense fruit extract preparation
The fruits were washed thoroughly with double distilled water. About 50 g of fruits with100 mL of distilled water were
blended into pulp. The pulp was squeezed using a cloth sieve to obtain all the liquid available. The liquor was filtered twice
using filter paper with pore size of 11 μm to obtain a clear solution of the extract (pH = 4.6), which was kept in a sealed
container and reserved in a cooler until needed.
Nanoparticle synthesis
The ZnO-NPs were successfully synthesised using C. chinense fruit extract as a mediating agent in our previous study [29] .
Briefly, the ZnO-NPs were obtained from dissolving 4.4 g of Zn(CH
3
CO
2
)
2
• 2H
2
O into 20 mL of distilled water, then to this
solution was added 20 mL of the extract and stirred for 2 h at 85 °C. A similar method was also applied for the synthesis
of Ag/ZnO-NPs. However, in this case 4.4 g of Zn(CH
3
CO
2
)
2
• 2H
2
O was dissolved in 20 mL of distilled water, and a solution
of 0.5 g AgNO
3
in 10 mL AgNO
3 was added. Thereafter, 20 mL aqueous extract of C. chinense was added to the solution, and
the pH was adjusted to 7 by the addition of 1 M sodium hydroxide solution in a dropwise manner. The solution was heated
to 85 °C and the temperature was maintained for the duration of the reaction. After 2 h, the reaction was stopped and the
solution was centrifuged for 15 min at 5500 rpm. The paste obtained was washed with water/ethanol solution three times
to eradicate any contaminants. The product was then transferred into a crucible and calcinated at 350 °C for 2 h.
2
M.S. Mthana, M.N. Mthiyane, A.C. Ekennia et al. Scientific African 17 (2022) e01365
Characterisation of nanoparticles
The crystalline structure of the Ag/ZnO-NPs was analysed using X-ray diffraction (XRD) instrument (D8 Advance, BRUKER
AXS, Germany), with Cu-K αradiation (k = 1. 5 4 0 6 ˚
A). The average crystallite size of the Ag/ZnO-NPs was calculated using
the Debye-Scherrer equation (D = K λ/ βcos θ), where D is the particles size (nm), K is the Scherrer’s constant (0.90), λis
the X-ray wavelength (1.5406 ˚
A), βis the full width at half the maximum (FWHM) and θis the Braggs’s angle of reflection.
The surface morphology of the NPs was studied using scanning electron microscopy (SEM) (FE-SEM FEI 430 Nova NanoSEM
system) (JSM-7600F), while the average particle size and distribution of the NPs was determined using a TECNAI G2 (ACI)
Transmission electron microscopy (TEM) (FEI, Bellaterra Spain). The energy-dispersive X-ray (EDX) was employed to deter-
mine the elemental composition. For optical examination, the UV-visible analysis of the samples was conducted using the
Spectroquant® Prove 300 Spectrophotometer (Merck KGaA, D-64293 Darmstadt, Germany). The band gap energy of Ag/ZnO-
NPs was further estimated using Tauc’s plot equation [ αh ν= C(h ν–Eg)
m
], where αis the absorbance coefficient, h is the
Planck’s constant, νis the photon frequency, C is the proportionality constant, Eg is the optical band gap, and m is 1/2 for
direct band gap semiconductors.
In vitro cytotoxicity analysis
The cytotoxic assay was evaluated following similar procedure reported by Adeyemi et al. [30] . Two cell lines were used
which includes the immortal embryonic kidney (HEK 293) and human cervical carcinoma (HeLa) cell lines. The ATCC in
Manassas, Virginia, USA provided these cells. Cells were cultured in tissue flasks of a 25 cm
2
surface area by using Dulbecco’s
Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum, U/mL penicillin, and 100 μg/mL streptomycin100. The
3-(4,5-dimethylthiazol-2-yl)-2,6-diphenyltetrazolium bromide (MTT) assay was carried out using 100 μL of DMEM in a 96-
well plate containing 2.5 ×10
2
cells/well. Prior inoculation, the cells were incubated overnight at 37 °C. The cells were then
further incubated at 36 °C for 48 h with increasing concentrations of Ag/ZnO-NPs (10, 25, 50, and 100 μg/mL), followed by
the MTT assay. The standard used for comparison was 5-fluorouracil (5-FU). The medium in the assay was replaced by a
fresh medium that contains 10% MTT reagent and further incubated at 37 °C for 4 h. After incubation, 100 μL of dimethyl
sulphoxide (DMSO) was introduced to dissolve the insoluble formazan crystals, and the absorbance was analysed at 570 nm,
using DMSO as a blank. The assays were replicated three times to obtain the average absorbance.
Antibacterial analysis
Four clinical isolates consisting of Gram negative ( Escherichia coli and Klebsiella oxytoca ) and Gram positive (Bacillus sub-
tilis and Staphylococcus aureus) bacteria strains were obtained from Alex Ekwueme Federal University, Ndufu-Alike, Nigeria.
The Agar diffusion method was used to screen the in-vitro antibacterial potency of test samples. The petri plates were pre-
pared using Sterile Muller–Hinton agar (MHA) which were introduced on several petri plates and inocula of test cultures
(10
6 CFU/mL) were streaked on them using a sterilized cotton swab. The plates were subjected to drying for 15 min. Differ-
ent concentrations (5, 7.5 and 10 μg/mL) of the samples (nanoparticles) were prepared from the stock solution, using DMSO
and were sonicated. A cork borer was used to bore holes of 6.0 mm diameter on the plates and was later impregnated with
25 μL of the test sample solutions. The plates were incubated for 24 h at 37 °C before readings were taken. The control
drug used was a commercially available antibacterial drug (Streptomycin). To evaluate the antibacterial activity of each bac-
terial species by the samples, measurements of inhibitory zones (including disk diameters) on the agar surface were taken.
The values less than 6 mm were considered not active against bacteria strains. The experiment was conducted in triplicate.
The minimum inhibitory concentration (MIC) was obtained by serial dilution of the stock solution and screening against the
pathogens using the same procedure.
Data analysis
Data for antimicrobial activity was subjected to analysis of variance (ANOVA) procedures using GenStat Statistical Package
9.2, 9th edition. The mean separation was determined by Duncan’s Multiple Range Tests (DMRT). The data were deemed
significant where P -value was less or equal to 0.05 between means ( P ≤0.05).
Results and discussion
X-ray diffraction studies
Fig. 1 presents the XRD patterns of Ag/ZnO-NPs, which was synthesized using C. chinense extract. The diffractions peaks
identified at 2 θvalues of 31.70 ˚, 34.37 ˚, 36.23 ˚, 47.50 ˚, 56.60 ˚, 62.98 ˚, 67.93 ˚, 69.10 ˚and 76.98 ˚could be indexed to the (100),
(002), (101), (102), (110), (103), (112), (201) and (202) planes, respectively, of ZnO, with hexagonal wurtzite structure (JCPDS
card no. 36-1451), which has lattice constants of a = b = 3.242 ˚
A and c = 5.205 ˚
A [31] . Additional peaks were observed at
2 θvalues of 38.17 ˚, 44.37 ˚, 64.50 ˚, and 76.98 ˚, and could be indexed to the (111 ) , (200), (220), and (311) planes, respectively,
of Ag. They correspond to face centred cubic structure of Ag-NPs with the reported JCPDS card no. 04-0783. Furthermore,
the strong intensity of the diffraction peaks of the Ag/ZnO-NPs indicated that they were highly crystalline in nature [32] .
3
M.S. Mthana, M.N. Mthiyane, A.C. Ekennia et al. Scientific African 17 (2022) e01365
Fig. 1. XRD analysis of Ag/ZnO-NPs synthesised using C. chinense fruits extract.
Tabl e 1
Elemental composition of Ag, Zn, and O.
Elements Weight % Atomic %
Ag 7.98 2.7
Zinc 65.45 36.6
Oxygen 26.57 60.69
Tota l 100 100
The absence of any impurity peaks confirms that the Ag/ZnO-NPs was successfully formed using C. chinense fruit extract as
a mediating agent, with the average crystallite size of 23.88 nm. The observed crystallite size was, however, lower than the
one reported by Sorbium et al. [33] . This suggested that the reported crystallite size in the current study might have higher
surface chemistry due to lower crystallite size.
SEM, TEM, and EDX micrographs
The SEM, TEM, and EDX micrographs are presented in Fig. 2 . The lower magnification ( Fig. 2 a) and the higher magnifi-
cation of the SEM micrograph ( Fig. 2 b) show agglomerated spherical morphological images of Ag/ZnO-NPs. Agglomeration
might be ascribed to the result of the high temperature of calcination, which increased the surface reactivity [34] . The TEM
images of Ag/ZnO-NPs ( Fig. 2 c) confirmed the ascribed spherical shape (from SEM analysis) and an average particle size of
27.3 nm ( Fig. 2 d). Similar surface morphology was observed by Abou-Oualid et al. [35] , from Ag/ZnO-NPs synthesised using
sodium alginate, however with larger average particle size of 45 nm. The EDX spectrograph of Ag/ZnO ( Fig. 2 e) revealed the
presence of silver, zinc, and oxygen peaks only, which indicates that the Ag/ZnO-NPs were successfully formed. The elemen-
tal composition of each element in Ag/ZnO-NPs is shown in Table 1 . The weight percentage of Ag, Zn, and O was found to
be 7.9 8 %, 65.45 %, and 26.57 % with atomic weight percentages of 2.7 %, 36.6 %, and 60.69 %, respectively, giving credence
to the formation of the silver doped zinc oxide particles. Fig. 3 presents the EDX elemental mapping of the Ag/ZnO-NPs. The
elemental mapping showed that all component elements are uniformly distributed across the surface of the nanoparticles.
4
M.S. Mthana, M.N. Mthiyane, A.C. Ekennia et al. Scientific African 17 (2022) e01365
Fig. 2. SEM images at (a) low and (b) high magnification, (c) TEM image, (d) particles distribution, and (e) EDX spectrum of Ag/ZnO-NPs synthesized using
C. chinense fruits extract.
UV-visible spectroscopic results
The UV–visible spectroscopy was executed to assess the effect of Ag on the optical absorption of ZnO-NPs in the syn-
thesised Ag/ZnO-NPs. Two peaks of narrow and strong intensities were detected at 376 and 258 nm, respectively ( Fig. 4 a).
The first absorption peak is attributed to the electronic transition of ZnO, while the second peak resulted from the presence
of Ag in Ag/ZnO-NPs [36] . Absorption spectrum of Ag nanoparticles is associated with a distinctive band gap transition in
visible region around 420 to 300 nm, which is due to localised surface plasmon resonances (LSPR). In the current study,
5
M.S. Mthana, M.N. Mthiyane, A.C. Ekennia et al. Scientific African 17 (2022) e01365
Fig. 3. Elemental mapping of (a) Ag/ZnO, (b) O, (c) Zn, and (d) Ag elements on Ag/ZnO-NPs biosynthesised using C. chinense fruits extract.
Fig. 4. (a) UV-visible absorption spectra and (b) Tauc plot of Ag/ZnO-NPs biosynthesised using 20 mL aqueous extract of C. chinense fruits.
6
M.S. Mthana, M.N. Mthiyane, A.C. Ekennia et al. Scientific African 17 (2022) e01365
Tabl e 2
Cell viability of Ag/ZnO-NPs on HEK 293 and HeLa cancer cells.
Cell
lines
Samples
Sample concentrations (μg/mL) IC
50
(μg/mL)
10 25 50 10 0
HEK
293
5-FU 77.36 ±0.048 51.92 ±0.003 35.34 ±0.010 11.33 ±0.017 6.05
Ag/ZnO-NPs 80.81 ±0.048 53.72 ±0.061 31.89 ±0.051 22.47 ±0.023 30.05
HeLa 5-FU 78.40 ±0.03 58.89 ±0.02 50.47 ±0.02 37.99 ±0.02 17.48
Ag/ZnO-NPs 70.14 ±0.077 58.04 ±0.049 45.54 ±0.018 25.86 ±0.070 33.25
5-FU (Fluorouracil) is the standard.
Fig. 5. The percent cell viability of HEK 293 and HeLa Cells treated with increasing levels of Ag/ZnO-NPs.
this peak clearly demonstrates the compositing of Ag and ZnO [
37 , 38 ]. The estimated band gap energy of Ag/ZnO-NPs was
found to be 3.53 eV ( Fig. 4 b). The observed bang gap is in the same ranger (3.54 eV) with Ag/ZnO-NPs synthesised using
Urtica dioica plant extract [39] .
Cytotoxicity studies
Based on the cytotoxicity results obtained, the Ag/ZnO-NPs exhibited a concentration/dose-dependent proliferative activ-
ity against the growth of both HEK 293 and HeLa immortal cell lines ( Table 2 ). However, the standard used (5-Fluorouracil)
exhibited better cell viability on both HEK 293 and HeLa cell lines as confirmed by their IC
50
values of 6.05 and 17.48μg/mL,
respectively, when compared to Ag/ZnO-NPs. Nevertheless, Ag/ZnO-NPs demonstrated higher antitumor activity on HEK 293
cells compared to HeLa cells ( Fig. 5 ). The difference in the proliferative activity of Ag/ZnO-NPs against tested cell lines may
be attributed to different metabolism and multiplication rate of the cells [40] . The compositing of Ag into ZnO increased the
antitumor activity in both HEK 293 and HeLa cells compared to the results earlier reported on ZnO-NPs synthesised with C.
chinense fruit extract [29] . The high cytotoxic effect of C. chinense bio-mediated Ag/ZnO-NPs on tested cancer cells may be
due to high surface to volume ratio and small particle size, which led to increased surface chemistry of the nanoparticles
against cancer cells [ 25 , 41 ]. The oxidative state of Zn
2 +
, which could have contributed inducing the reactive extracellular
oxygen species (ROS) production that eventually killed cancer cells [42] . Ag on its own has widely been reported to have
high ability of increasing the concentration of free radicals which cause apoptosis and DNA degradation in cancer cells [43] .
Therefore, it is predictable that higher toxicity of the biosynthesized Ag/ZnO-NPs may be due to their high cellular uptake
and retention by the cancer cells [44] . The observed result in the current study indicates that C. chinense bio-mediated
Ag/ZnO-NPs could be considered as a promising anti-cancer drug.
Antibacterial studies
Antibacterial study was conducted to examine the bactericidal potentials of the nanoparticles and to evaluate the effect
of Ag on the antibacterial potentials of ZnO-NPs by comparing the pristine and composited nanoparticles. The inhibition
zones (mm) of increasing concentrations of pristine ZnO-NPs ( Table 3 ) and Ag/ZnO-NPs ( Table 4 ) against both Gram-negative
7
M.S. Mthana, M.N. Mthiyane, A.C. Ekennia et al. Scientific African 17 (2022) e01365
Tabl e 3
Inhibition zone of ZnO-NPs against Gram-negative and Gram-positive pathogenic bacteria.
Pathogens
ZnO-NPs Concentrations (μg/mL)
P -value
Control 5 7.5 10
Escherichia coli 21 ±0.3 8.0 ±0.7 12 ±03 17 ±0.7 < 0.001
Klebsiella oxytoca 24 ±0.0 0.0 ±0.0 11 ±0.7 20 ±0.07 < 0.001
Staphylococcus aureus 21 ±0.7 12 ±0.7 12 ±0.3 17 ±0.0 < 0.001
Bacillus cereus 21 ±0.7 8.0 ±0.3 11 ±0.0 13 ±0.7 < 0.001
Data are mean ±standard deviation.
Tabl e 4
Inhibition zone of Ag/ZnO-NPs against Gram-negative and Gram-positive pathogenic bacteria.
Pathogens
Ag/ZnO-NPs Concentrations (μg/mL)
P -value
Control 5 7.5 10
Escherichia coli 20 ±0.0 15 ±0.3 16 ±0.7 21 ±0.0 < 0.001
Klebsiella oxytoca 25 ±0.7 15 ±0.7 18 ±0.3 23 ±0.0 < 0.001
Staphylococcus aureus 20 ±0.3 9.0 ±0.3 14 ±0.7 19 ±0.7 < 0.001
Bacillus cereus 20 ±0.3 10 ±0.7 13 ±0.3 14 ±0.0 < 0.001
Data are mean ±standard deviation.
bacteria ( Escherichia coli and Klebsiella oxytoca ) and Gram-positive bacteria ( Staphylococcus aureus and Bacillus cereus ) are
presented as Tables 3 and 4 . The results show a dose dependant antibacterial activity as the inhibition zones increased
with increasing concentration levels of ZnO and Ag/ZnO-NPs for all bacteria strains. A highly significant bactericidal effect
( P < 0.05) was observed for all tested bacterial pathogens. These findings are in line with those reported by Bu ¸s il
˘
a et al.
and Rajaboopathi & Thambidurai [ 45 , 46 ].
The control drug showed better overall antibacterial activity, however, the results on the screening also showed that in
some instances, the antibacterial activity were comparable with those of the composite NPs for some pathogens at certain
concentrations. The Ag/ZnO-NPs exhibited better antibacterial properties compared to the pristine ZnO-NPs. These results
were expected since both metals are known to possess some antibacterial properties [47] , hence their combination pro-
duced an enhanced activity. In addition, Ag/ZnO-NPs are expected to be more permeating through the cellular walls of the
bacterial due to their smaller particle sizes and their uniform spherical morphology compared to the pristine [48] . Thus,
possess higher antibacterial activity than pristine ZnO-NPs. Similar results were reported in previous studies by other re-
searchers [49–51] , whereby introducing Ag to ZnO NPs showed better efficiency and faster kinetics in eradicating bacteria
with different cell wall type.
Microbes with Gram-negative cell walls were more susceptible to NPs than Gram-positive cells, in particular Bacillus
cereus . This could be attributed to the fact that Gram-positive bacteria strains are composed of thicker cell walls [47] than
the Gram-negative bacteria strains. Hence the test compounds are less permeable compared to those of Gram-negative
strains. The mechanism involved in anti-bacterial effect caused by NPs in general is not yet clearly understood. However,
the microscopic evidence has shown that when NPs comes to contact with bacterial cells, they mutilate the cell membrane
which causes bacterial cell death [52] . Furthermore, the other possible mechanism responsible for antibacterial properties
of Ag/ZnO-NPs may be the direct disturbance of the cell layer through the distribution of Ag and Zn ions and the oxidative
stress that was triggered by free radicals [25] . The minimum inhibitory concentrations (MIC) values of the test ZnO-NPs was
4, 5, 5 and 5 μg/mL against E. coli, K. oxytoca, S. aureus and B. cereus , respectively, while the MIC values Ag/ZnO-NPs was 3,
3, 5 and 5 μg/mL against E. coli, K. oxytoca, S.aureus and B. cereus , respectively.
Conclusion
This study presented facile green synthesis of Ag/ZnO-NPs using C. chinense fruit extract as a mediating agent. The struc-
tural and optical characterisation results revealed that the nanoparticles were pure and only composed of nanosized Ag
and ZnO particles, which confirmed the successful synthesis of Ag/ZnO-NPs. Ag/ZnO-NPs showed better proliferative activity
against HEK 293 cells than HeLa cells. Furthermore, both the pristine and the composite nanoparticles (ZnO and Ag/ZnO-
NPs) showed a high bactericidal effect in all tested bacterial pathogens, but the Ag/ZnO-NPs had better antibacterial activity.
The increasing prevalence of chronic human diseases including cancer, and the rising antibiotic-resistant pathogenic infec-
tions implies that the compositing of two biological active nanoparticles might be a worthwhile approach for more studies
aimed towards combating this menace. Thus, further research may be extended to focus on the effects of C. chinense fruit
extract mediated Ag/ZnO-NPs on other biological areas such as antioxidant and antiviral activities, and also in agricultural
fields.
8
M.S. Mthana, M.N. Mthiyane, A.C. Ekennia et al. Scientific African 17 (2022) e01365
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper
Acknowledgment
The authors gratefully acknowledge both the National Research Foundation (NRF), South Africa (Grand No. 141668 ) and
North-West University (NWU) for financial support. MSM further acknowledges NRF and NWU for the PhD bursary support.
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