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Green synthesis of zinc oxide nano particles using Allium cepa L. waste peel extracts
and its antioxidant and antibacterial activities

Md Faridul Islam, Shariful islam, Md Abdus Satter Miah, A.K. Obidul Huq, Anik Kumar
Saha, Zinia Jannat Mou, Md Mahmudul Hassan Mondol, Mohammad Nazrul Islam
Bhuiyan
PII: S2405-8440(24)01461-0
DOI: https://doi.org/10.1016/j.heliyon.2024.e25430
Reference: HLY 25430

To appear in: HELIYON

Received Date: 4 October 2023

Revised Date: 16 January 2024
Accepted Date: 26 January 2024

Please cite this article as: , Green synthesis of zinc oxide nano particles using Allium cepa L. waste peel
extracts and its antioxidant and antibacterial activities, HELIYON (2024), doi: https://doi.org/10.1016/
j.heliyon.2024.e25430.

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© 2024 Published by Elsevier Ltd.

 Green synthesis of zinc oxide nano particles using Allium cepa L.
waste peel extracts and its antioxidant and antibacterial activities

Md. Faridul Islam1, Shariful islam1, Md. Abdus Satter Miah1*, A. K. Obidul Huq2*, Anik Kumar Saha3,
Zinia Jannat Mou2, Md. Mahmudul Hassan Mondol4 and Mohammad Nazrul Islam Bhuiyan1

1
Institute of Food Science and Technology (IFST), Bangladesh Council of Scientific and

of
Industrial Research (BCSIR), Dr. Qudrat-I-Khuda Road, Dhanmondi, Dhaka-1205, Bangladesh.

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2
Department of Food Technology and Nutritional Science, Mawlana Bhashani Science and
Technology University, Santosh, Tangail-1902, Bangladesh.

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3
Department of Biochemistry and Molecular Biology, Bangabandhu Sheikh Mujibur Rahman
Agricultural University (BSMRAU), Salna, Gazipur-1706, Bangladesh.
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4
Department of Chemical Engineering, Khulna University of Engineering & Technology
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(KUET), Khulna-9203, Bangladesh.
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Corresponding authors details:

Dr. Md. Abdus Satter Miah, Institute of Food Science and Technology (IFST), Bangladesh
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Council of Scientific and Industrial Research (BCSIR), Dr. Qudrat-I-Khuda Road, Dhanmondi,
Dhaka-1205, Bangladesh. Email:[email protected]
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A. K. Obidul Huq, Email: [email protected]

Abstract

Synthesis of nanoparticles through the green approach using plant and vegetable extracts has
gained popularity since they are thought to be efficient and cost-effective materials. This study is
designed to synthesize zinc oxide nanoparticles (ZnO-NPs) from onion waste peel extract
(Allium cepa L.) via the green synthesis approach. The synthesized ZnO-NPs were characterized
by utilizing the UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), Energy
Dispersive X-ray (EDX), Field Emission Scanning Electron Microscopy (FE-SEM) and X-ray
Powder Diffraction (XRD)techniques. The nanoparticles formation was confirmed by the UV-

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Vis sharp absorption spectra at 318 and 322 nm. The synthesized ZnO-NPs size and shape was

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revealed by the XRD and SEM respectively. Smallest nanoparticle average crystallite size was
found 57.38 nm with hexagonal shape. The bioactive functional groups that are in charge of

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capping and stabilizing the ZnO-NPs was assured by the FTIR data. Further, prepared ZnO-NPs
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were used to assess their possible antioxidant and antibacterial properties. DPPH test for free
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radical scavenging showed potential antioxidant properties of the synthesized ZnO-NPs. The
antibacterial activity were studied against three clinical strains: P. aeruginosa, E. coli, and S.
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aureus with the maximum zone of inhibition 13.17 mm, 22.00 mm and 12.35 mm respectively at
100µg/mL subsequently minimum inhibitory concentration was found 50 µg/mL for P.
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aeruginosa, and S. aureus whereas 100 µg/mL for E. coli. Antioxidant and antibacterial activity
tests appear bio-resource based ZnO-NPs from Allium cepa L. extract have effects on free radical
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and growth of microorganisms.Therefore, it could be a promising candidates for agricultural and

food safety applications as an effective antimicrobial agent against pathogenic microorganisms
and also can address future biomedical applications after complete in vivo study.

Key words: Allium cepa L., antioxidant, antimicrobial, green synthesis, nanoparticle, zinc oxide.
1.Introduction

Nanotechnology has become a significant branch of research during the last few decades, mostly
employed for the evaluation and production of substances at the nanoscale, which typically falls
between 1 to 100 nm in at least one dimension [1]. The high surface-to-volume ratio of
nanoparticles enhanced the physical characteristics and also increased the reactivity of the
nanoparticles. Now-a-days, nanotechnology is referred to as a wonder of modern medicine,
since, this field has advanced to the point that new directions in nanoscience are possible,
especially in drug delivery, gene delivery, biosensing, and nanomedicine [2-4]. It is also playing

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a significant role in the field of agriculture, food processing industries, environmental sciences,

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and biotechnology [5-6].

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Metal and metal oxide (MO) NPs are regarded as the anticipated materials because of their
characteristics such as solubility, chemical stability, adhesiveness, and surface plasmon
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resonance [7-9]. Recently, ZnO, CuO, Ag2O, Fe2O3, CaO, NiO, and MgO NPs have received the
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most attention in literature as having antibacterial action [10-18]. Among those NPs, ZnO is
considered to be a prospective contender in the field of biomedicine, particularly for
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antibacterial, antioxidant, α-amylase inhibitory activity, biosensing, cell imaging, anti-

inflammatory, anti-cancer, and anti-diabetes applications [19-24]. So far, various physical and
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chemical techniques, including sol-gel, colloidal, thermal, sonochemical, hydrothermal,

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microwave irradiation, thermal breakdown, and rapid precipitation approach have been reported
to synthesize ZnO-NPs [25]. The risk of using hazardous chemicals in various NPs synthesis
process restricts them in applications in the domains of biotechnology, medicine, environment,
biology, and other industrial applications [26]. Synthesis based on bioresources could be one
way to overcome such restrictions since it is eco-friedly, less toxic and easy to adapt.
Additionally, using waste materials lowers the energy needs and synthesis costs when compared
to physical or chemical synthesis processes, highlighting the effectiveness of "green synthesis"
[27-28]. Recently, this approach has been applied for ZnO NPs synthesis to evaluate their
antioxidant and α-amylase inhibitory activity [23-24]. In addition, polyol present in the plant
component (like leaves, fruits, seeds, peels, pulps, roots, barks, and fruits) extracts as sources of
bioactive chemicals can stabilize the metallic NPs by acting as chelating and capping agents
during fast production of nanoparticles [29-30]. Preceded study showed that ZnO-NPs from leaf
extract of Melia azedarach and Cassia fistula exerted antimicrobial activity against two
medically significant strains namely Staphylococcus aureus (S. aureus) and Escherichia coli (E.
coli). The antibacterial activity of ZnO NPs produced from Ruta graveolens (L.) was observed
against four bacterial strains, specifically Escherichia coli (E. coli), Pseudomonas aeruginosa (P.
aeruginosa), Klebsiella aerogenes (K. aerogenes), and Staphylococcus aureus (S. aureus) [22].

However, Allium cepa L. (onion), being a cross-pollinated biennial important spice containing
several phytochemicals including phenolics, flavonoids, anthocyanins, and organic acids [31-32].
These polyol compounds make onion an outstanding option for the green synthesis of NPs. The

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ZnO-NPs using Allium cepa L. extract showed anti-inflammatory, anti-hyperlipidemic, and
cardiovascular protective effects [33-34]. In a recent investigation, Khamis et al., synthesized

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onion extracts mediated ZnO NPs at an average crystallite size 8.13 nm having hexagonal

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wurtzite phase and exhibited greated antibacterial activity than Cefotaxime [35]. Biogenic ZnO
NPs from Allium cepa extracts also showed potential antihyperlipidemic and cardiovascular
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protective effects by controlling biochemical parameters linked to lipid metabolism [36].
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Another study claimed that Allium cepa bulb based ZnO NPs have potential antimicrobial, in
vitro antioxidant and antidiabetic activity [37]. Most of the researchers uses the edible part of the
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onion to extract the reducing and capping agents. In Bangladesh, huge amounts of dry waste peel
of onion is generated everyday and dumped here and there. These are considered as a garbage
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and polluted the environment. But, it can be a potential source for the extraction of reducing
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agent since it contains several types of phytochemical such as polyphenols, flavonoids, tannins,
saponins, alkaloids and antioxidants etc. [35]. Therefore, to make dried onion waste peel usable,
development of an efficient extraction procedure for the synthesize of biogenic metal oxide
nanoparticles is indespensible. In addition, the biological method of producing zinc oxide
nanoparticles helps to create environmentally sustainable and biocompatible zinc oxide
nanoparticles while also reducing the amount of waste that is produced from onions.

In this research, Allium cepa L. (onion) waste peel extract were emploed as a reducing and
caping agent to synthesize ZnO-NPs. Different approach such as orbital shaking, microwave, and
hot plate heating was applied to optimize the extraction and synthesis method. The optimized
methods were then used to synthesize ZnO NPs and UV-Vis, FTIR, FE-SEM, and XRD analysis
were employed to characterize the synthesized ZnO NPs. The biogenic synthesized ZnO-NPs
were further employed to assess their potential antibacterial and antioxidant activities. The
antibacterial activity was investigated following the modified Kirby Bauer disc diffusion method
against three clinical strains: E. coli, P. aeruginosa, and S. aureus.

2. Materials and methods

2.1. Raw materials, chemicals and reagents

Dry onion peel waste (Allium cepa L.) were collected from a local market in Dhaka. Zinc acetate
(Zn(CH3CO2).2H2O), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), methanol, ethanol, and ascorbic

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acid were procured from Merck, Research-lab, India; Fine Chem Industries, India; Panreac, EU
and Merck, Germany, respectively.

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2.2. Onion peel extract preparation
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Dry waste peels of the onions (Allium cepa L.) were collected and washed under running water
repetedly to eliminate dust particles. The waste peel were then weighed and properly blended
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with the help of a food blender (Brand: PHILIPS, Model: Simply Silent, 600W). To get the
better results, it is important to extract the reducing agent efficiently. Numerous process
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variables such as the precursor to bio-source ratio, pH, temperature, reaction duration, kind and
concentration of the precursor solution are all the important factors in the green synthesis of zinc
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oxide nanoparticles. Therefore, optimisation of the factors involved in producing ZnO NPs is
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necessary to depict the relationship between different parameters. Considering all this, the extract
was prepared at three different concentrations under three distinct conditions. The sample
concentrations are: Condition A: (A1: 50g blended onion waste peel in 100 mL deionized water,
A2: 50 g blended onion waste peel in 150 mL deionized water, A3: 50 g blended onion waste
peel in 200 mL deionized water), Condition B: (B1: 50 g blended onion waste peel in 100 mL
deionized water, B2:50 g blended onion waste peel in 150 mL deionized water, B3: 50 g blended
onion waste peel in 200 mL deionized water), and Condition C: (C1: 50 g blended onion waste
peel in 100 mL deionized water, C2:50 g blended onion waste peel in 150 mL deionized water,
C3: 50 g blended onion waste peel in 200 mL deionized water). Microwave heat treatment with a
time interval of 2.0 min was applied on Condition A whereas mechanical shaking using an
orbital shaker (Brand: Grant-bio, Model: PSU-10i, England) at 200 rpm for 30 min was applied
on Condition B to prepare the extract solution respectively. Samples of condition C were kept for
microwave heat treatment with a time interval of 2 min followed by boiling for 15 min on a hot
plate to prepare the extract solution from condition C. To filter, Whatman No. 1 paper was used
and collected the supernatant. The light pink aqueous solutions were then stored at 4 °C to
synthesis ZnO NPs.

2.3. Synthesis of ZnO NPs

The synthesis of ZnO-NP were completed by following the modified method outlined by Degefa
A. et al. [38]. The 20.0 mL aqueous onion waste peel extract solution was transferred in a 250
mL beaker and positioned above a magnetic stirrer. (Phoenix, Model: RSM-0SH). The

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temperature and rotation per minute (rpm) was set 65 °C and 450 respectively. Then,100.0 mL of

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2 mM zinc acetate solution was was gradually added from a graduated burette. After the addition
of zinc acetate, the pH was adjusted to 8.0±0.10 and magnetically stirred until the reaction

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mixture took on a new hue from light pink to yellowish white. After that, it was set to cool to
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ambient temperature and stand for 4 h and then centrifuged (DSC, Model: 302SMD, Taiwan) at
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7000 rpm for 5 min. The supernatant disposed of and precipitate underwent two to three
washings with deionized water and subsequently with ethanol. The resulted residue was dried at
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130oC for 3 h to make it moisture free. The sample was then ground with mortar and pestle into
fine powders and calcined for 2 h at 500oC to get rid of any impurities. The powdered sample
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was transferred in a properly labeled airtight ziplock bag and stored at room temperature for
future analysis.
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2.4. Characterization

The physicochemical properties of ZnO-NPs produced from AlliumcepaL. extract have been
examined by employing a range of characterization techniques including FTIR, XRD, SEM,
EDX and UV-Vis spectrophotometer. The optical characteristics of the synthesized product were
analyzed by utilizing UV-Vis spectrophotometer (Model:Specord 205, Analytic Zena, Germany)
in the range of 280-450 nm. XRD was also used to examine the materials' crystallinity
(Model:Smart LAB, SE, Rigaku, Japan) equipped with Cu-k radiation and a wavelength of
1.5406 Å. The functional group responsible for the formation of the nano particle were
determined by using FTIR (Model:PerkinElmer, USA) spectroscopy between 600 -4000 cm-1.
The FESEM was used to record the NP's morphology (Model: JSM-7610F, JEOL, USA),
following their combination with acetone and drying on a glass slide to form a thin coating. The
elemental maping of the nanomaterial's were determined using SEM-EDX.

2.5. Antioxidant acivity

The antioxidant activity of biosynthesized ZnO NPs was determined using the DPPH method
described by D. Das et al. [39]. Two distinct procedures were used in the DPPH scavenging
assay; one experiment was time-dependent, while the other experiment was concentration-
dependent. In summary, 150 mg of produced nanoparticles were sonicated for 10 minutes at
room temperature in a glass tube containing 3.2 mL of a 100 μM DPPH solution. The absorbance

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was then measured at 517 nm at intervals of 0, 15, 30, and 45 minutes.

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To evaluate the DPPH scavenging activity at various doses, a solution containing 100 μM DPPH

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was produced in 99.9% methanol. The analysis was then carried out by combining 3.0 mL of
DPPH solution with 1 mL of sample solution at various doses (50 µg/mL, 100 µg/mL, 200
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µg/mL, 250 µg/mL, and 500 µg/mL). This combination was then allowed to incubate for 30
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minutes at ambient temperature in the dark. Ascorbic acid was used as the standard and methanol
as a blank. The optical density was then determined at 517 nm. To find the synthesized ZnO NPs'
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DPPH scavenging activity, the following equation (1) was applied:

% of DPPH inhibition = {(A0 – As)/A0} ×100 ………………………… (1)

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Here, control is A0 and sample is As to identify % of inhibition.

2.6. Antimicrobial acivity

2.6.1. Test Microorganisms

The research investigation examined the antibacterial activity of synthesised ZnO NPs against
three strains of S. aureus, E. coli, and P. aeruginosa that are resistant to antibiotics. First, tainted
food samples were used to extract the test bacteria. To create pure cultures, the isolated bacteria
were regularly subcultured on Nutrient Agar (NA, Hi-media, India) and then incubated at 37°C.
The isolated pure cultures of bacteria were kept at -20°C right away. The selected isolates were
then identified by performing the series of morphological (growth on suitable media),
microscopic, physiological, and biochemical tests. Using carbon sources and a chemical
sensitivity test, the BIOLOGTM identification system was used to determine the species level
[40].

2.6.2. Kirby Bauer Disc Diffusion Assay

Prior to the experiment, all of the glassware and associated items were autoclaved at 121 °C and
a pressure of 115 lb. The modified Kirby Bauer disc diffusion method followed by the Clinical
and Laboratory Institute (CLSI) guideline was utilized to assess the antibacterial susceptibility
test [41]. Amikacin 30µg disc was considered as a positive control whereas 10% DMSO soaked
filter paper disc was served as a negative control. ZnO-NPs (100 µg) were dissolved in 1.0 mL of

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10% DMSO. 100 µL of the prepared sample solution was applied on the antibiotic disc. The

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plates were subsequently kept at 4 °C for 3 h to allow nanoparticles to enter and diffuse across
the well, after which incubation at 37 °C was performed. Clear zones were discovered

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surrounding the wells after 24 h of incubation, and a slide calliper was used to measure these
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zones' diameters.
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2.6.3. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations

(MBC)
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The broth micro dilution method was employed to examine the MIC and MBC following the
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CLSI guideline [42]. Three concentrations doses of ZnO NPs: 50 μg, 100 μg and 200 μg in 10%
DMSO were investigated against the bacterial concentration of 5 × 105 CFU/mL. Bacterial
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suspension (50 μL) was added to each sample except the negative control (10% DMSO).
Amikacin 30 (30 μg) was considered as a positive control. Both positive control and ZnO-NPs
coated discs were incubated for 24 h at 37±2 oC. After incubation, MIC and MBC was
determined by visual inspection.

2.7. Statistical analysis

All the antioxidant and antibacterial experiments were carried out three times and the results are
presented as mean ± standard deviation. We used R Studio (version 4.3.0, 2019), OriginPro
(2022) and Microsoft Excel (Office 2016) to draw the graph. No test was performed to determine
the level of significant difference (p< 0.05).
3. Results and Discussion

3.1. UV-Vis Spectroscopic analysisand synthesis method optimization

The formation of ZnO-NP using secondary metabolites of onion waste peel were investigated by
UV-visible spectra in the range of 280 nm–450 nm. As shown in Fig.1(a), sample A1 exhibited
an absorption maximum at 322 nm, which corresponds to the ZnO-NPs whereas A2 and A3
didn’t exhibit any absorption maxima. A1 was synthesized from the most concentrated extract
and this indicated that the microwave heat treatment was more effective when the substrate to
solvent (w/v) ratio was maimtained 1:2 to prepare the extract solution. No absorption maxima

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were observed for condition B experiments shown in Fig.1(b) and this implied that ZnO-NPs

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were not formed when the extract was prepared only by orbital shaking. The reason might be due

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to the incomplete extraction of the reducing agent. So, orbital shaking is not an efficient
technologies to extract the capping and reducing agent from the dry onion waste peel to
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synthesize ZnO NPs. In case of condition C, the strongest peak was noted for sample C3 at 318
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nm whereas C1 and C2 did not show any significant absorption maxima presented in Fig.1(c).
Since the samples A1 and C3 only exhibited intense UV-visible absorption maxima among the
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nine samples and based on this data these two samples were chosen for further characterization.
Subsequently, evaluation of their antioxidant and antibacterial activities to make a comparative
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study in between the synthesized ZnO NPs utilizing extract solution prepared from two different
approaches.
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0.7
1(a) A1
0.6
0.5 A3
Absorbance

0.4 A2
0.3
0.2
0.1
0
280 300 320 340 360 380 400 420 440

Wavelength (nm)
 1
1(b) B1
0.8
B2
Absorbance

0.6 B3
0.4
0.2
0
280 330 380 430
Wavelength (nm)

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1(c)

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C3
0.2 )
C1

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Absorbance

0.15
C2
0.1
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0.05
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0
280 300 320 340 360 380 400 420 440
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Wavelength (nm)

Fig. 1: UV-Vis spectra of synthesized ZnO-NPs. 1(a): synthesized using extract from condition
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A, 1(b): synthesized using extract from condition B and 1(c): synthesized using extract from
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condition C

ZnO-NPs surface plasmon absorption properties are the cause of all the peaks [43]. These results
matched with the absorption pattern of conventional ZnO since oxide materials have shorter
wavelengths and broad band gaps [44-45]. The synthesized A1 and C3 samples estimated band
gap energy shown in the Tauc plot in Fig. 2(a) and 2(b) respectively.
 (a) (b)

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Fig. 2: Tauc plots for optical bandgap energy. For A1 sample (a) and C3 sample (b).

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Using the following equation (2), the band gap energy (Eg) is calculated by extrapolating the
linear part of (αhv)2 against energy plots [46].
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αhν = A(hν-Eg)n ---------------(2)
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where A is the constant, Eg is the optical band gap energy, hν is the photon energy, and α is the
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absorption coefficient. The value of n equals 1/2 in the case of permitted direct transitions, such
as those in ZnO. The energy band gaps are indicated by the intercepts of these graphs on the
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energy axis, as Eg = hν when (αhν)2 = 0. The computed band gap value was found 3.47 and 3.48
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eV respectively for our sample A1 and C3, and this value indicated that synthesized ZnO-NPs
absorb UV light. A band gap of 3.32 eV is estimated for ZnO NPs synthesized via green
synthesis [35]. So, it can be used for medicinal purposes like protection of skin from sun and/or
antibacterial ointments [47]. The broad spectrum of energy band gap of our synthesized ZnO-
NPs also indicates their possible use as metal oxide semiconductor-based systems such as
memory devices, sensors, UV-light emitting diodes, photocatalysts, solar cells, photodetectors
and piezo-electric transducers and is similar to the energy bandgap values that have been
previously reported for ZnO-NPs [48].

3.2. FE-SEM characterization of ZnO NPs

The morphological study of synthesized ZnO-NP were performed using FE-SEM scanned at
different magnification. The Fig. 3(A-H) showed the morphological study of the green
synthesized ZnO-NPs from samples A1 shown in Fig. 3 (A-D) and C3 presented in Fig. 3 (E-H).
At low magnification (50000x), the FE-SEM images of A1 and C3 showed notable
agglomeration, which point towards the distinct characteristics of ZnO-NPs fabrication mediated
by plant extracts. At higher magnifications (100000x and 150000x), the image revealed their
regularly clustered hexagonal-shaped nanoparticles for sample A1 that were heterogeneous in
nature. The size of the agglomerated particles for sample A1 ranged from 49 nm to below 100
nm as shown in Fig. 3(C). The agglomeration of smaller particles leads to the creation of larger
particles, which also gives irregular shapes to the structures [49]. The hexagonal morphology and
agglomeration with irregular distribution pattern of the synthesized ZnO-NPs were evident from

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previous studies [50-51].

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Fig.3: FE-SEM images of ZnO-NPs. (A-D) Forsample A1 and (E-H) for sample C3 captured at
various magnifications. The scale denotes 100 nm.
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The Fig. 3(G) shown the irregularly shaped nanoparticles with both round and hexagonal shapes,
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obtained from sample C3 using higher magnification levels (100,000x and 150,000x). These
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nanoparticles have sized ranged from 53 to 100 nm. The round shaped of ZnO-NPs has also been
observed in a research study conducted by Ihsan et al. [52]. The increased of the particle size and
distortion of the hexagonal structure to round shaped in sample C3 from sample A1 attributed to
the increased concentration of the extract in sample A1 than C3 and this effect of concentration
was consistent with the result reported in previous study [34]. ZnO-NPs formation was
confirmed by further XRD analysis.

3.3. Energy Dispersive X-ray Analysis (EDX)

The EDX pattern of sample A1 and sample C3 showed that the single peak of O was found
between 0 and 1 eV and three peaks for Zn were found at 1eV and between 8 and 10 eV shown
in Fig. 4(a) and 4(b) which confirmed the presence of zinc and oxygen in the oxide form. The
EDX spectra of synthesized ZnO-NPs also confirmed that Zn and O comprised 37.23 % and
25.62 % of the mass of sample A1 whereas 33.45 % and 29.32 % of the mass of sample C3.
Apart from Zn and O, the observed peak for C reflected the green feature of the nanoparticle
synthesis process, and the mass percentage of C accounts for the stabilizing and capping of the
green synthesized nanoparticles [53].

(a) (b)

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Fig.4: EDX spectrograph of bio-synthesized ZnO NPs. (a) For A1 and (b) C3 sample.

3.4. FT-IR analysis of ZnO-NPs

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The onion waste peel extract mediated synthesized ZnO NPs FTIR spectra were captured
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between wavenumber of 600-4000 cm-1 and presented in Fig. 5. Phytochemicals present in the
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onion waste peel extract could be involved in the capping and reduction of the Zn2+ ions. The
FT-IR analysis of sample A1 exhibited bands at 2302.99 cm-1, 1697.96 cm-1, 1649.08 cm-1,
1524.91 cm-1, 1426.08cm-1, 1045.16 cm-1, 747.03 cm-1, 664.40 cm-1, 634.40 cm-1 and 630.32 cm-
1
. The C=O double bond and C-O stretching with slight shift are indicated by a strong and
medium peak at 1649.08 cm-1 and 1697.96 cm-1, respectively [54]. A strong band at 1524.91 cm-
1
corresponds to the N-O stretching present in nitro compounds. Medium peak at 1426.08 cm-1 is
attributed to O-H bending of alcohols. The sharp band at 1045.16 cm-1, 747.03 cm-1, and 664.40
cm-1 were attributed to the stretching of S-O, C-Cl, and C-Br, respectively. Two sharp bands at
634.40 cm-1 and 630.32 cm-1 are observed due to the interaction of ZnO-NPs with other organic
moieties. The 500-900 cm-1 peaks are attributed to the metal-oxygen groups [55]. For sample C3,
bands found at 2322.73 cm-1, 1783.99 cm-1, 1750.52 cm-1, 1525.34 cm-1, 1038.46 cm-1, 822.61
cm-1 and 665.13 cm-1. The medium peak at 2322.73 cm-1 is attributed to C=C alkenes. Two
strong peaks at 1783.99 cm-1 and 1750.52 cm-1 correspond to C=O monomer. N-O stretching is
attributed by a strong band at 1525.34 cm-1 and this stretching is of nitro compounds. Intense
peak at 1038.46 cm-1 corresponds to S-O stretching of sulfoxide. ZnO intra-atomic weak
interactions are indicated by a strong peak at 822.61 cm-1 and 665.13 cm-1 which is allotted for
ZnO as reported by A. Jayachandran et al. [56].

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Fig. 5: FTIR pattern of synthesized ZnO NPs from two different conditions.
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The slight shift of FTIR spectra of ZnO-NPs could occurred for the different experimental
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conditions. The presence of C=C bonds, S-O stretching of sulfoxide, nitro compounds N-O
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stretching and C=O monomers reveals that these groups assist the reduction and capping of Zn2+
ions and the overall formation of ZnO-NPs and this is corroborated by previous studies [52,57].
The donor-acceptor mechanism was followed for the reduction of Zn2+ions in green synthesis.
The presence of hydroxyl or oxygen compounds in the onion waste peel extract give an electron
to an electrophile zinc complexes cause zinc ions to be reduced and OH groups to be oxidised
[58]. The S-O stretching represents the abundance of organo-sulfur compounds in Allium cepa L.
[59]. Despite multiple washings, the FTIR data showed the existence of metabolites, such as
flavonoids, alkaloids, carboxylic acid, and polyphenols, which chained to ZnO-NPs. These
substances, in particular flavonoids and phenolics, helped transform zinc ions into ZnO-NPs.
Apparently, the presence of carboxylic groups stabilizes ZnO-NPs by interacting with the Zn
surface [60]. Therefore, FTIR data revealed that the presence of different functional group
containing bioactive compouds in the onion waste peel extract made the synthesis procedure
effective and formation of NPs successful.

3.5. XRD analysis

The XRD pattern of A1 and C3 sample is depicted in Fig. 6(a) and Fig. 6(b). The details of the
crystal plane, nature, and size of the NPs provided by XRD [61]. For both sample A1 and C3, the
diffraction peaks at lattice planes (hkl) indexed as (100), (002), (101), (102), (110), (103), and
(112) have been used to identify the spherical to hexagonal phase of the produced ZnO-NPs. The
crystal’s peaks were found to match the peaks on the ICDD (International Centry for Diffraction

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Data) card no #01-079-0207 [57]. Line broadening in XRD peaks indicated the nanoscale range

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of the synthesized nano particles. Sharp peak also supported the crystalline nature of NPs [54].

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Intensity (a.u)

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10 20 30 40 50 60 70
2 theta (degree)
 (b)
Intensity (a.u)

10 20 30 40 50 60 70

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2 theta (degree)

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Fig. 6: X-ray diffraction spectra of synthesized ZnO NPs for A1 (a) and C3 (b).

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The Debye-Scherrer formula (equation 3) was employed to compute the diameter of ZnO

crystals size.
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𝑘𝜆
𝐷= Å---------------(3)
𝛽𝑐𝑜𝑠𝜃
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Here, D is the mean particle size of nonmaterials, k is the shape factor or Scherrer’s constant
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(0.9), λ is the wavelength of x-ray (1.5406Å), β is the full width at half maxima in radians and θ
is the Bragg’s diffraction angle in degrees. The more intense peaks corresponded to (101) planes
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and smallest crystal size was found for both samples. It revealed that most of the particles were
pointed in (101) direction. The others diifraction peak at higher 2θ value might be responsible for
the capping and stabilizing agent of onion waste peel extract species. The detail calulation of
particle size data is provided in the supporting information titled as Tab. S1. The average
crystallite size was found 72.60 and 57.38 nm, respectively, for samples A1 and C3. The result
was compatible with the findings of SEM. The particle size of nanomaterials plays crucial role in
their different activities especially in the antioxidant and antibacterial efficacy.

3.6. Antioxidant activity

As seen in Fig. 7(a) and 7(b), the antioxidant activity of the produced ZnO NPs was assessed
using the DPPH scavenging assay. For both samples shown in Fig. 7(a), the maximum intensity
at 517 nm gradually reduced with time in agreement with changes in color of the DPPH solution
in the presence of ZnO NPs. This finding correlated with the result observed by D. Das et al. in
2013 [39]. The drop in peak intensity proofed that ZnO nanoparticles capable to scavenge free
radicals. The DPPH scavenging activity of sample A1 and C3 were found 63.85% and 71.53% at
45 minrespectively. The DPPH free radical scavenging activity data at different concentration
has been provided in the supplementary documents S3.

(a)
(b)

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Fig. 7: ZnO NPs antioxidant activity at various time intervals (a) and at different concentrations
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(b).

The antioxidant activity of green synthesized ZnO-NPs was found higher when concentration
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was higher for both samples but lower than the standard. This results is accordant with the
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findings reported by Adamu AS S et al. [34]. Sample C3 showed increased free radical inhibition
activity than A1 at every concentration. Therefore, it was revealed that DPPH free radical
scevanging activity of ZnO NPs was dose dependent and NPs with smaller particle size exhibited
enhanced antioxidant activity at the same concentration.

3.7. Antimicrobial activity

The antimicrobial activities shown in Fig. 8 (A-F) of the synthesized complexes were studied in-
vitro against two gram-negative strains P. aeruginosa and E. coli and one gram-positive strain S.
aureus using disc diffusion method with reference antibiotic Amikacin 30 (30 µg disc). The
results exhibited that the synthesized ZnO-NPs possessed a noteworthy antibacterial properties
against all three tested microorganisms. It was shown that sample C3 showed significant zone of
inhibition than sample A1 at all concentration. This behavior might be for the small particle size
of sample C3 than A1. When the particle size decreased, surface area increased resultant in the
proper diffusion of NPs in the medium. This causes an elevated rate of surface oxygen species
formation to accelerate membrane rupture and ultimately cause pathogen death [62]. Another
explanation for ZnO NPs' antibacterial action could be that they come into direct contact with
pathogens' cell walls, breaking down their cell walls and releasing antimicrobial ions (Zn2+) as a
result. These free ions then bind with the carbohydrates and proteins of the pathogens, resulting
in the disintegration of every key bacterial activity [37]. In our study, 50, 100 and 200 µg/mL
concentrations of A1 and C3 samples were used to record the antibacterial activity after 24 h of
incubation. No labeled at the assay plate was for standard Amikacin 30. The average zone of

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inhibition calculation data of both samples at three different concentration is tabulated in the

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supplementary file S4 in detail. At 100 µg/mL concentration, gram positive bacteria S. aureus
showed average zone of inhibition diameter 12.00±1.52 mm and 13.50±0.76 mm for samples A1

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and C3 respectively. But, the scenario was quite different when dealing with gram negative
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bacteria E. Coli and P. aeruginosa. It is showed that sample A1 and C3 respectively cleared
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8.83±0.54 mm and 13.17±0.82 mm diameter zone against E. Coli whereas 13.00±1.00 mm and
22.00±0.58 mm diameter zone was inhibited when studied against P. aeruginosa. The lowest
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zone of inhibition diameter 8.83±0.54 mm was found for sample A1 against gram-negative E.
coli bacteria whereas highest zone of inhibition 22.00±0.58 mm was found for sample C3 against
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gram-negative P. aeruginosa bacteria at 100 µg/mL. Also, it was observed that for both sample
A1 and C3, the highest zone of inhibition was found against P. aeruginosa and S. aureus.
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Among the three studied starin, E. coli was less sensitive i.e., sample A1 and C3 did not inhibit
the significant zone when compared with the standard. However, between the studied sample, C3
exhibited highest zone of inhibition at all three concentrations, which indicated that the
antibacterial activity was directly related to the particle size of ZnO-NPs [63]. In line with the
earlier research, it was therefore demonstrated that as small as the the particle size, the higher the
antibacterial activity [62].
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Fig. 8: Antibacterial activity of ZnO-NPs against three bacterial strains at three different
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concentrations. (A-C) is for sample A1 and (D-F) is for sample C3.

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3.8. Minimum inhibitory and bactericidal concentration

The minimum bactericidal concentration (MBC) was the lowest amount of an antibacterial agent
required to completely eradicate a particular bacterium, while the minimum inhibitory
concentration (MIC) was considered the lowest amount of the materials (ZnO-NP) that
completely prevented bacterial growth. The data was displayed in Tab 1. It was observed that at
100 µg/mL, ZnO NPs were effective against three studied bacterial strains. The synthesized ZnO
NPs were found to be 100 µg/mL and 200 µg/mL of MIC and MBC, respectively, for all selected
strains. Therefore, ZnO-NPs synthesized with Allium cepa L. waste peel extact could be
suggested as a good antibacterial material.
Tab. 1: Antibacterial activity of assynthesizedZnO nanoparticles against test microorganisms.

Sample A1
Concentration Bacterial growth
(µg/mL) S. aureus E. coli P. aeruginosa
50 - + -
100 - - -
200 - - -
MIC 50 µg/mL 100 µg/mL 50 µg/mL
MBC 100 µg/mL 200 µg/mL 100 µg/mL
Sample C3
50 - - -

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100 - - -
200 - - -

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MIC 50 µg/mL 50 µg/mL 50 µg/mL
MBC 100 µg/mL 100 µg/mL 100 µg/mL

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Note:‘+’ indicates the presence of bacterial growth; ‘-’ indicates no bacterial growth. MIC =
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minimum inhibitory concentration and MBC = minimum bactericidal concentration
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3.9. Comparative study

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Numerous researchers have synthesized ZnO NPs via biogenic approach during the last two
decades and examined their antioxidant and antibacterial properties and have demonstrated the
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NPs' strong antibacterial activity. We have synthesized ZnO NPs via green system from two
different pathway and evaluated their structural and biological activity to explore its potentiality
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as an antioxidant and antibacterial materials. The elemental composition and biological activities
data summary of our studied materials was presented in Tab. 2 to make the comparision easier
between these two samples. Based on the analyzed data it could be concluded that C3 NPs were
superior than the A1 NPs considering the antioxidant and antibacterial activities.

Tab. 2: Elemental composition and biological activity data of the synthesized two ZnO NPs.

Characterization parameters ZnO NPs (A1) ZnO NPs (C3)

Absorption maxima 322 nm 318 nm
Estimated optical band gap
3.47 eV 3.48 eV
calculated from Tauc plot
Regularly clustered Regularly clustered
Morphology by FE-SEM hexagonal-shaped with 49 to hexagonal-shaped with 49 to
100 nm 100 nm
Average particle size calculated
72.60 nm 57.38 nm
from Scherrer formula
Antioxidant activity (at 100
61.53 % 65.39 %
µg/mL)
Antibacterial activity (MZI) . .
S. aureus 12.00±1.52 mm 13.50±0.76 mm
E. coli 8.83±0.54 mm 13.17±0.82 mm
P. aeruginosa 13.00±1.00 mm 22.00±0.58 mm
Antibacterial activity (MIC) . .
S. aureus 50 µg/mL 50 µg/mL
E. coli 100 µg/mL 50 µg/mL
P. aeruginosa 50 µg/mL 50 µg/mL
Antibacterial activity (MBC) . .

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S. aureus 100 µg/mL 100 µg/mL
E. coli 200 µg/mL 100 µg/mL

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P. aeruginosa 100 µg/mL 100 µg/mL

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* MZI = maximum zone of inhibition at 100 µg/mL, MIC = minimum inhibitory concentration,
and MBC = minimum bactericidal concentration.
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4. Conclusion
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ZnO-NP was synthesized successfully using aqueous Allium cepa L. waste peel extract through
the optimization of extraction and synthesis method. The procedure was economical and
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environmentally friendly since it used extract from onion waste peels. The characterization was
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conducted via different techniques like UV-vis, XRD, FTIR, and FE-SEM. The average crystal
size of the best-studied ZnO-NP was measured 57.38nm with hexagonal shape. In our study, we
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have shown the possible bioactivity of green synthesised ZnO NPs by studying the antioxidant
and antibacterial activity using standard method. When compared to the standard antibacterial
substance, the growth of inhibition in synthesized ZnO NPs was found exclusively greater. Also,
ZnO NPs and standard exhibited the similar antioxidant activity at higer concentration. The
larger surface to volume ratio of smaller particles is thought to be responsible for their improved
bioactivity. The bioresource-based ZnO NPs' exceptional bactericidal efficacy against gram
(+)ve and gram (-)ve bacterial strains may lead to new opportunities for its application in food
and medicine for the development of prospective antimicrobial drugs. However, in vivo research
is necessary to fully comprehend their biological activities and may be the focus of future
studies.
Credit Authorships

Md. Faridul Islam: Experiment design, writing-review and editing and coordination, Shariful
Islam: Data acquisition and interpretation, writing-original draft, review and editing, A. K.
Obidul Huq: Supervision, writing-review and editing, Md. Abdus Satter Miah: Review,
funding acquisition and supervision, Anik Kumar Saha: Experiment design, synthesis and data
acquisition, Zinia Jannat Mou: Formal analysis, Md. Mahmudul Hassan Mondol: Writing-
review and editing and Mohammad Nazrul Islam Bhuiyan: writing-review and editing

Declaration of competing interest

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The authors declare no known competing financial interests.

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Data availability

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Data will be made available on request. re
Acknowledgment
This study was supported by the Bangladesh Council of Scientific and Industrial Research
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(BCSIR).
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Declaration of interests

☒ 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.

☐ The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

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