Droepenu et al. Beni-Suef University Journal of Basic and Applied Sciences Beni-Suef University Journal of
           (2021) 10:1 
https://doi.org/10.1186/s43088-020-00091-7 Basic and Applied Sciences
RESEARCH Open Access
Biosynthesis, characterization, and
antibacterial activity of ZnO
nanoaggregates using aqueous extract
from Anacardium occidentale leaf:
comparative study of different precursors
Eric Kwabena Droepenu1,2* , Ebenezer Aquisman Asare1,2, Boon Siong Wee1*, Rafeah Binti Wahi1,
Frederick Ayertey4 and Michael Odoi Kyene3
Abstract
Background: Various parts of Anacardium occidentale plant possess curative qualities like antidiabetic, anti-
inflammatory, antibacterial, antifungal, and antioxidant. Aqueous extract of this plant leaf was used in
biosynthesizing zinc oxide (ZnO) nanoaggregates using two precursors of zinc salt (zinc acetate dihydrate
[Zn(CH3COO)2∙2H2O] and zinc chloride [ZnCl2]). The synthesized ZnO samples were used in a comparative study to
investigate the antibacterial activity against selected Gram-positive and Gram-negative microbes [Staphylococcus
aureus, Exiguobacterium aquaticum (Gram +ve) and Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii
(Gram −ve)]. The synthesized ZnO nanoaggregates from the two precursors were characterized using Fourier
transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), transmission electron microscopy
(TEM), and energy-dispersive x-ray spectroscopy (EDX) techniques.
Results: Micrographs of SEM and TEM confirmed nanoparticles agglomerated into aggregates. While spherical
nanoaggregates were identified in samples prepared from Zn(CH3COO)2∙2H2O, flake-like structures were identified
in samples synthesized from ZnCl2. Particle size determined by TEM was 107.03 ± 1.54 nm and 206.58 ± 1.86 nm for
zinc acetate dihydrate and zinc chloride precursors respectively. ZnO nanoaggregate synthesized using zinc acetate
as precursor gave higher antibacterial activity than its counterpart, zinc chloride with K. pneumonia recording the
highest inhibition zone of 2.08 ± 0.03 mm (67.53%) whereas S. aureus recorded the least inhibition zone of 1.06 ±
0.14 mm (34.75%) for ZnO nanoaggregate from zinc chloride precursor. Also, antibacterial activity increases with
increasing concentration of the extract in general. However, A. baumannii, E. aquaticum, and K. pneumoniae did not
follow the continuity trend with regards to the 250 ppm and 500 ppm concentrations.
(Continued on next page)
* Correspondence: kobladodzie01@yahoo.co; swboon@unimas.my
1Resource Chemistry Program, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
Full list of author information is available at the end of the article
© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
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Droepenu et al. Beni-Suef University Journal of Basic and Applied Sciences            (2021) 10:1 Page 2 of 10
(Continued from previous page)
Conclusion: Biosynthesis of ZnO nanoaggregates using aqueous extract of A. occidentale leaf from zinc acetate
dihydrate and zinc chloride as precursors was successful with the formation of nanospheres and nanoflakes. The
study suggested that A. occidentale sp. could be an alternative source for the production of ZnO nanoparticles and
are efficient antibacterial compounds against both Gram +ve and Gram −ve microbes with its promising effect
against infectious bacteria.
Keywords: Zinc oxide nanoaggregate, Zinc acetate dihydrate, Zinc chloride, Anacardium occidentale, Antibacterial
activity, Biosynthesis
1 Background Africa, as a cash crop. Different parts of this plant spe-
Nanotechnology is a growing field of study combining tech- cies possess curative qualities like antidiabetic, anti-
nology, bio-nanoscience, and material science together [1]. inflammatory, antibacterial, antifungal, and antioxidant.
Serious interests arose in the last few decades among scien- This was reported by different studies that investigated
tists into some unique properties: catalytic, magnetic, elec- into these activities and has proven its viability and ef-
tronic, optical, antibacterial, antimicrobial, anti- fectiveness [21–26].
inflammatory, and wound healing of nanoparticles [2–4]. Phytochemical constituents of this plant as investi-
When different precursors are used in the synthesis of ZnO gated by Ojezele and Agunbiade [27] reported the pres-
particles at different reaction conditions (reaction time, ence of tannin = 15.38 mg/g, total polyphenolics = 2.00
concentration, and temperature), literature shows that dif- mg/g, alkaloid = 39.90%, and oxalate = 8.13%. Also,
ferent morphologies, such as flower shape and needle Fadeyi et al. [28] identified fatty acid esters and a new
shape, are produced with different sizes [5–7]. The porosity androstane steroid derivative being reported for the first
of the synthesized nanoparticles enhances the surface area time. Bastos et al. [29] also in a study went further to
and chemical and photochemical stability [8, 9]. Research isolate anacardic acid, cardol, and cardanol compounds
has also shown that ZnO nanoparticles synthesized bio- from this plant to evaluate its inhibition against Trypa-
logically have shown strong antibacterial effect than those nosoma cruzi Sirtuins. From their report, isolation was
synthesized through chemical means [10, 11]. The type of successful with positive inhibition against pathogens
plant extract and its concentration used in a biosynthesis under study.
reaction have an effect on the morphology of ZnO nano- Nevertheless, literature survey reveals the synthesis of
particles. For instance, extract containing functional groups platinum nanoparticles from this plant species and its
such as alcohol, ketone, carboxylic acid, and amine creates catalytic and thermal applications but with very little in-
spherical-shaped ZnO nanoparticles, whereas extract with formation on zinc oxide nanoparticle synthesis [30].
–OH (hydroxyl) group gives quasi-spherical agglomerates Hence, the study sort to synthesize ZnO nanocrystals
[12, 13]. from aqueous extract of A. occidentale from two precur-
Natural products such as chitosan and extracts of sors and also investigate their antibacterial activity
fungi, bacteria, and different plant genus extracts are against selected Gram-positive and Gram-negative mi-
used as stabilizing and reducing agents which serve as crobes [Staphylococcus aureus, Exiguobacterium aquati-
alternative to chemical synthesis of nanoparticles [14]. cum (Gram +ve) and Escherichia coli, Klebsiella
The reason has been reported to be due to the fact that pneumoniae, Acinetobacter baumannii (Gram −ve)].
green synthesis of nanoparticles is environmentally fa- Aqueous extract was preferred for this study because the
vorable, simple, economical, and comparatively reprodu- extraction process is simple and free from contaminants.
cible. The concentration of metabolites in the plant
extracts, pH, as well as the temperature used in the plant
extract preparation has an effect on the morphology of 2 Methods
the nanoparticles to be formed [15–19]. ZnO nanoparti- 2.1 Collection and preparation of plant extracts
cles are known to be one of the multipurpose inorganic The plant species (A. occidentale) was formally identified
nanoparticles with effective antibacterial, antimicrobial, by the two individuals of the Centre for Plant Medicine
and antifungal activities [20] even at very low Research, Mampong-Akuapem, Ghana, who doubles as
concentrations. co-authors of this paper. A. occidentale fresh leaves were
Anacardium occidentale (cashew) belongs to the genus collected from a peasant farmer in Dormaa Ahenkro in
Anacardium and family Anacardiaceae, which is found the Brong Ahafo Region of Ghana, West Africa, in Sep-
worldwide. The plant is predominantly grown in Dor- tember 2019 upon a request. Since the plant species was
maa Ahenkro in the Brong Ahafo region of Ghana, West meant for study purposes and not for any commercial
reasons, permission was granted to harvest the leaves
Droepenu et al. Beni-Suef University Journal of Basic and Applied Sciences            (2021) 10:1 Page 3 of 10
without attaining any voucher specimen. The harvested 1230, Japan) in determining the morphological features
fresh leaves of the plant species were brought to the of the synthesized particles. The purity and functional
phytochemical lab of the Centre for Plant Medicine Re- groups present in the synthesized ZnO nanoparticle
search, Mampong-Akuapem, Ghana. The fresh plant were determined using energy dispersive x-ray spectros-
leaves were washed under running water and double- copy (EDX) (JEOL 6390LA) and Fourier transform infra-
distilled water. The method reported by Droepenu and red spectroscopy (FT-IR) (Thermo Scientific Nicolet
Asare [31] for aqueous plant extract preparation was iS10, USA). Further assessment into the optical proper-
adopted for this study. About 10.0 ± 0.1 g of the sample ties of the sample was carried out using ultraviolet vis-
(fresh leaves) was heated with 100.00 ml of distilled ible spectroscopy (UV-vis) (UV-1800 Series, SHIM
water at a temperature of 60 ± 2 °C for about 25 min. ADZU) techniques. Sample preparation prior to analysis
The filtrate after filtering using Whatman filter paper for the different characterization techniques was carried
was stored in a clean Schott bottle for further analysis. out using the method reported by Droepenu and Asare
[31, 34].
2.2 Phytochemical analysis
Preliminary qualitative phytochemical analysis was car- 2.5 Preparation of plant-mediated ZnO NPs test samples
ried out on the aqueous leaf extract of Anacardium occi- The antibacterial activity of A. occidentale-mediated zinc
dentale plant species. Fifty milliliter of the plant extract oxide nanoparticles at different concentrations (25, 50,
was screened for ten (10) selected phytochemical con- 100, 250, 500, and 1000 ppm) from the two precursors
stituents as per the methods reported by Trease and Ev- (zinc acetate dihydrate and zinc chloride) on the five se-
ans [32]. The phytoconstituents tested were saponins, lected pathogens (Staphylococcus aureus, Exiguobacter-
tannins/phenolic compounds, flavonoids, reducing ium aquaticum (Gram +ve) and Escherichia coli,
sugars, cyanogenic glycosides, alkaloids, triterpenes, phy- Klebsiella pneumoniae, Acinetobacter baumannii (Gram
tosterols, anthracinosides, and polyuronides. −ve)) was determined by adopting the disc diffusion
method reported by Umaru et al. [35].
2.3 Synthesis of ZnO nanoparticles
The method reported by Moazzen et al. [33] and cited by 2.6 Bacteria broth preparation
Droepenu et al. [34] was adopted with some modifications The procedure for bacteria broth preparation follows the
in this study. A weighed mass of 9.15 ± 0.1 g (0.05mol) one reported by Droepenu et al. [34] where a weighed
zinc acetate dihydrate [ZnCH3COO)2∙2H2O] and 6.81 ± mass of 2.60 ± 0.1 g of the dried broth was dissolved (in
0.1 g (0.05mol) anhydrous zinc chloride [ZnCl2] (Sigma- 200 mL deionized water) and autoclave at a temperature
Aldrich, India) was each dissolved in 50ml of deionized of 121 °C. Culture stains of the five Gram +ve and Gram
water in a 250ml Schott bottle and heated to a −ve bacteria were obtained from the Department of Bio-
temperature of 60 ± 2 °C with constant stirring using elec- chemistry Laboratory, University of Ghana-Legon, and
trical stirring hotplate (Favorit). Also, 2.80 ± 0.1 g (0.05 incubated with a shaker at a temperature of 37 °C [36]
mol) of potassium hydroxide (KOH) (VWR Amresco, for 16 h. The optical density (OD) of the bacterial broth
USA) was dissolved in 25ml of deionized water in a 100- after incubation was computed by UV Mini Spectropho-
ml Schott bottle under the same condition as the precur- tometer (1240 SHIMADZU) at wavelength 575 nm and
sors. The KOH solutions were slowly drained dropwise compared to the standard (0.6–0.9).
from a burette into each of the Zn(CH3COO)2 ∙ 2H2O
and ZnCl2 solutions at 60 ± 2 °C with vigorous stirring for 2.7 Inoculation of plate
an hour until white precipitate of zinc oxide was formed. Bacteria inoculation for this study follows the procedure
Fifty milliliter of the plant extract was drained dropwise reported by Droepenu et al. [34]. Approximately 1.0 ml
with a burette into each mixture under constant stirring at of the prepared bacterial broth was streaked over the en-
30 ± 2 °C for 3 h. The solution was centrifuged at 4000 tire agar plate surface in four different directions using
rpm for 15min with Hanil FLETA 5 Centrifuge machine; sterile cotton bud. A 10 μl volume of the ZnO nanopar-
the zinc oxide precipitate was thoroughly washed with de- ticle from the A. occidentale leaf extract from the two
ionized water and dried under hot air. The samples were precursors of concentrations 25, 50, 100, 250, 500, and
then preserved in air-tight container for characterization 1000 ppm was each pupated onto the prepared discs (6-
and antibacterial studies. mm diameter) and gently pressed onto the agar plate
and left for 10 min at room temperature. Commercial
2.4 Characterization of ZnO nanoparticles synthesized ZnO NPs (Sigma–Aldrich, USA) containing
Different characterization techniques were used includ- the same volume of methanol was taken as a control to
ing scanning electron microscopy (SEM) (SU3500, Hita- identify the activity of the solvent and ZnO nanoparticle.
chi) and transmission electron microscope (TEM) (JEOL In addition, 30 μg of tetracycline was used as positive
Droepenu et al. Beni-Suef University Journal of Basic and Applied Sciences            (2021) 10:1 Page 4 of 10
control. Each of the test samples was tested in triplicate Before analysis, the dried powdered ZnO sample was
for the bacterium used. The plate samples were then in- mixed with potassium bromide (KBr) in a ratio of 1:19.
cubated at temperature of 37 °C for 24 h, and the sensi- The sample was then placed in the metal hole, pressed
tivity of the pathogenic organisms to the ZnO extracts until the sample was compressed inside the hole before
from the two precursors were examined by the zone of being used for the analysis.
inhibition around every sample disc after incubation.
The inhibition zone data was computed in millimeters 3.3 SEM and TEM results
(mm) to show the presence of antibacterial activity for The SEM and TEM images of ZnO nanoaggregates from
all the samples compared to the positive control and an- aqueous extracts of A. occidentale using zinc chloride
alyzed using one-way analysis of variance (ANOVA) and zinc acetate dihydrate as precursor are illustrated in
with differences considered at P value < 0.05. Fig. 2a–d. Prior to analysis, the dried powdered solid
ZnO sample was coated on an aluminum plate with the
2.8 Drug entrapment efficiency of ZnO NPs help of adhesive membrane for SEM analysis. In the case
The entrapment efficiency of ZnO NPs was determined of TEM, the dried powdered ZnO sample was first di-
by an indirect method. Briefly, the precipitate of ZnO luted with absolute ethanol (95%) and sonicated with
NPs from the two precursors were each separated by ultrasonic cleaner (Elma, Germany) for 10 min. A 4 μl
centrifugation at 4000 rpm for 15min. Drug content in volume of the solution sample was loaded onto a For-
the supernatant was determined by measuring the ab- mvar film Copper grid (FF300-Cu) before being ob-
sorbance using UV–Vis spectrophotometer (UV-1800 served under the machine.
Series, Shimadzu, Japan) at 455 nm (λ max of extract).
The amount of drug entrapped was calculated by sub- 3.4 EDX results
tracting the amount of drug in the supernatant from the Elemental analysis on the two ZnO nanoaggregate sam-
total amount of drug added [37, 38]. The entrapment ef- ples was performed by dispersive x-ray and the graph il-
ficiency was calculated as follows: lustrated in Fig. 3 with percentage elemental
composition (inset) of the graph. The same procedure
Amunt of drug entrapped
Entrapment efficiency ð%Þ ¼  100% used for the TEM was also carried out for the EDX ana-
Total amount of drug entrapped
lysis, but in this case, the sonicated solution was loaded
onto an aluminum plate.
3 Result
3.1 Phytochemical analysis results 3.5 UV-vis results
The results of the qualitative phytochemical study car- The dry powdered ZnO sample was dispersed in a 95%
ried out on the aqueous leaf extract of Anacardium occi- absolute ethanol and sonicated for 10 min before the ab-
dentale plant species is illustrated in Table 1. sorbance recorded using UV-1800 Series (SHIMADZU)
in the range of 300–400 nm. The spectra for the two
3.2 FT-IR results samples are illustrated in Fig. 4 a and b.
The IR spectrum analysis of the two ZnO nanoaggre-
gates was determined at a spectral range of 4000–400 3.6 Antibacterial activity evaluation
cm−1 with resolution of 4 cm−1 as illustrated in Fig. 1. The mean diameter measured for the inhibition zone and
the comparison of the antibacterial activity of A. occiden-
Table 1 Phytochemical components of aqueous leaf extract of tale leaf extract-mediated biosynthesized ZnO nanoaggre-
Anacardium occidentale gates from the two precursors (zinc acetate dihydrate and
Constituent Results zinc chloride) for the selected microbes by disc diffusion
Saponins + method is illustrated in Table 2 and Fig. 5 a and b.
Tannins/phenolic compounds +
4 Discussion
Flavonoids +
4.1 Phytochemical analysis
Reducing sugars + The aqueous leaf extract of the plant under study re-
Cyanogenic glycosides vealed the presence of active constituents such as alka-
Alkaloids + loids, tannins/phenolic compounds, saponins, and
Triterpenes flavonoids. Similar study was carried out by Ojezele and
Phytosterols Agunbiade [27], where tannins, phenolic compounds, sa-
ponins, and alkaloids were identified in the aqueous ex-
Anthracinosides
tract of the same plant species. On the other hand,
Polyuronides Semecarpus anacardium L., another family of
Droepenu et al. Beni-Suef University Journal of Basic and Applied Sciences            (2021) 10:1 Page 5 of 10
Fig. 1 FT-IR spectra of synthesized ZnO nanoaggregates using Zn(CH3COO)2∙2H2O and ZnCl2 as precursors
Fig. 2 SEM images of ZnO NPs of a ZnAc and b ZnCl2, and TEM images of c ZnAc and d ZnCl2 from aqueous extract of A. occidentale
Droepenu et al. Beni-Suef University Journal of Basic and Applied Sciences            (2021) 10:1 Page 6 of 10
Fig. 3 EDX spectra of ZnO NPs from a ZnAc and b ZnCl2 precursor from aqueous extract of A. occidentale
Anacardium, recorded the presence of steroids and terpe- cm−1 for ZnO nanocrystals from zinc chloride precursor
noids in addition to what this study found [39]. These to 3451.66 cm−1 and 3505.69 cm−1 for ZnO nanocrystals
chemical constituents are known for their medicinal activ- from zinc acetate was identified to be the stretching vi-
ity [28]. brations of –OH groups. However, the peaks at 1632.15
cm−1 (C=O), 1400.75 cm−1 (C=C), and 1043.47 cm−1 (C–
4.2 FT-IR analysis N) for ZnO nanoaggregates from zinc chloride precursor
All IR spectrum peak analysis was done with reference shifted to 1632.09 cm−1, 1400.83 cm−1, and 1043.66 cm−1
to Socrates [40]. The spectra result shows a slight shift in the case of ZnO nanocrystals from zinc acetate dihy-
in the wavenumbers of the peaks which might be as a re- drate. The presence of these functional groups is a result
sult of the precursor used to synthesize the two samples of the constituents in the extract as indicated by litera-
from the same plant extract. ture. The corresponding wavenumber indicating ZnO
From the spectra result of this study, the shift in the stretching in the two samples is at absorption peak
absorption bands observed at 3451.62 cm−1 and 3493.71 bands of 467.73 cm−1 and 467.67 cm−1 for ZnO
0.414 0.820
a 0.8000.400 b
0.700
0.350
0.600
0.300 0.500
0.262 0.393
300.00 320.00 340.00 360.00 380.00 400.00 300.00 320.00 340.00 360.00 380.00 400.00
nm. nm.
[Measurement Properties] No. P/V Wavelength Abs. Description [Measurement Properties] No. P/V Wavelength Abs. Description
Wavelength Range (nm.): 300.00 to 400.00 1 360.90 0.401 Wavelength Range (nm.): 300.00 to 400.00 1 362.80 0.784
Scan Speed: Fast Scan Speed: Fast
Sampling Interval: 0.1 Sampling Interval: 0.1
Auto Sampling Interval: Enabled Auto Sampling Interval: Enabled
Scan Mode: Single Scan Mode: Single
[Instrument Properties] [Instrument Properties]
Instrument Type: UV-1800 Series Instrument Type: UV-1800 Series
Measuring Mode: Absorbance Measuring Mode: Absorbance
Slit Width: 1.0 nm Slit Width: 1.0 nm
Light Source Change Wavelength: 340.0 nm Light Source Change Wavelength: 340.0 nm
S/R Exchange: Normal S/R Exchange: Normal
[Attachment Properties] [Attachment Properties]
Attachment: None Attachment: None
[Operation] [Operation]
Threshold: 0.0010000 Threshold: 0.0010000
Points: 4 Points: 4
InterPolate: Disabled InterPolate: Disabled
Average: Disabled Average: Disabled
[Sample Preparation Properties] [Sample Preparation Properties]
Weight: Weight:
Volume: Volume:
Dilution: Dilution:
Path Length: Path Length:
Additional Information: Additional Information:
Fig. 4 UV-vis spectra of ZnO NPs from a ZnAc and b ZnCl2 precursor from aqueous extract of A. occidentale
Abs.
1
Abs.
1
Droepenu et al. Beni-Suef University Journal of Basic and Applied Sciences            (2021) 10:1 Page 7 of 10
Table 2 Mean inhibition zone results of A. occidentale leaf extract-mediated biosynthesized ZnO nanoaggregates from the two
precursors against selected microbes [S. aureus, E. aquaticum (Gram positive) and E. coli, K. pneumonia, A. baumanni (Gram negative)]
Inhibition
Concentration ZnO NPs E. coli (Gram S. aureus (Gram A. baumannii (Gram E. aquaticum (Gram K. pneumoniae (Gram
(ppm) type −ve) +ve) −ve) +ve) −ve)
Control 3.06 ± 0.01 mm 3.05 ± 0.06 mm 3.07 ± 0.02 mm 3.06 ± 0.01 mm 3.08 ± 0.03 mm
25 ZnAc 1.60 ± 0.04 mm 1.49 ± 0.03 mm 1.89 ± 0.08 mm 1.61 ± 0.11 mm 1.91 ± 0.10a mm
ZnCl2 1.12 ± 0.04 mm 1.06 ± 0.14 mm 1.27 ± 0.13 mm 1.25 ± 0.14 mm 1.30 ± 0.22
a mm
50 ZnAc 1.68 ± 0.03 mm 1.61 ± 0.11 mm 1.90 ± 0.02a mm 1.62 ± 0.07 mm 2.04 ± 0.15a mm
ZnCl2 1.13 ± 0.34 mm 1.23 ± 1.13 mm 1.29 ± 0.18
a mm 1.17 ± 0.05 mm 1.29 ± 0.12a mm
100 ZnAc 1.69 ± 0.04 mm 1.62 ± 0.11 mm 1.79 ± 0.09a mm 1.82 ± 0.16 mm 1.88 ± 0.02a mm
ZnCl2 1.23 ± 0.07 mm 1.27 ± 0.17 mm 1.24 ± 0.16
a mm 1.33 ± 0.21 mm 1.37 ± 0.16a mm
250 ZnAc 1.78 ± 0.06 mm 1.87 ± 0.10a mm 1.76 ± 0.05 mm 1.71 ± 0.31 mm 1.65 ± 0.17 mm
ZnCl 1.29 ± 0.19 mm 1.33 ± 0.15a2 mm 1.30 ± 0.17 mm 1.29 ± 0.19 mm 1.30 ± 0.12 mm
500 ZnAc 1.71 ± 0.16 mm 1.64 ± 0.11 mm 1.82 ± 0.09 mm 1.79 ± 0.07 mm 1.86 ± 0.12a mm
ZnCl2 1.37 ± 0.13 mm 1.35 ± 0.14 mm 1.35 ± 0.10 mm 1.37 ± 0.15 mm 1.38 ± 0.06
a mm
1000 ZnAc 1.94 ± 0.04 mm 1.90 ± 0.13 mm 2.01 ± 0.23ab mm 2.00 ± 0.10 mm 2.08 ± 0.03b mm
ZnCl2 1.39 ± 0.18
bmm 1.36 ± 0.08 mm 1.39 ± 0.12b mm 1.38 ± 0.09 mm 1.40 ± 0.10b mm
Values are mean ± SD for three determinations
aSignificantly (p < 0.05) higher compared at the same concentration in each row
bSignificantly (p < 0.05) higher compared at the same concentration in each column
nanoaggregates from zinc chloride and zinc acetate re- were recorded in a study by Droepenu et al. [34], Gopal
spectively. All the observed peaks were similar to previ- and Kamila [42], Rao and Guatam [43], Zheng et al.
ous findings reported by Dobrucka and Dugaszewska [44], and Tripathi et al. [45].
[41].
4.3 SEM and TEM analysis 4.4 EDX analysis
From the results, both samples show an aggregation of EDX results of aqueous extract of A. occidentale-medi-
particles (× 5000 magnification) with spherical and ated ZnO NPs synthesized using zinc acetate and zinc
flake-like structures for ZnO nanostructures synthesized chloride as precursors are shown in Fig. 3. The analysis
from zinc acetate and zinc chloride respectively. The confirmed the presence of oxide form of zinc nanoparti-
flake-like morphology was formed from an agglomer- cles with percent mass of Zn and O at 67.09% and
ation of nano-rods as in the TEM image (Fig. 2d). The 18.66% for sample synthesized from zinc acetate precur-
particle size determined for the nanospheres was ap- sor and 64.23% and 17.36% for samples synthesized from
proximately 107 nm whereas the length, width, and aver- zinc chloride respectively. The carbon content present
age mean size of the nanorods as per the TEM analysis might be due to the basic component of plant materials.
was 167 nm, 68 nm, and 206 nm. Similar observations The result obtained in this study could be said to be
Fig. 5 Comparative antibacterial evaluation of A. occidentale leaf extract-mediated biosynthesized ZnO nanoaggregates from a ZnAc and b ZnCl2
precursors for selected microbes
Droepenu et al. Beni-Suef University Journal of Basic and Applied Sciences            (2021) 10:1 Page 8 of 10
almost in conformity with the study by Mohammadi- perfectly with tannins (polyphenols) for effective precipi-
Aloucheh et al. [46]. tation of proteins (microbes). Moreover, the entrapment
efficiency determined for the two ZnO NPs using zinc
4.5 UV-vis analysis acetate dihydrate and zinc chloride as precursors (89.1%
The distinct absorption peak of the UV-vis analysis for and 87.4%) respectively also accounted for the variations
ZnO nanocrystals synthesized using zinc acetate dihy- in the activity. Ayepola and Ishola [55] in their study
drate and zinc chloride precursor for A. occidentale are with both aqueous and methanolic extracts of A. occi-
observed at 360.90 and 362.80 nm respectively. Accord- dentale leaf reported that methanolic extracts recorded a
ing to Gupta et al. [47], the adsorption edge shifts to higher antimicrobial activity against selected pathogens
lower wavelength as the particle size decreases. Wave- (B. subtilis, K. pneumonia, and E. coli) than the aqueous
length for the two samples in this study decreased from extract. The reason was that tannins are polyphenols
362 to 360 nm corresponding to the decrease in particle that can bind and precipitate proteins [56], thereby hav-
size from 206 to 107 nm. Literature also shows that ab- ing a broader range of antibacterial activity.
sorption depends on factors such as band gap, oxygen
deficiency, size and structure of the nanoparticles, sur- 5 Conclusion
face nature, and impurity centers [48]. These factors Biosynthesis of ZnO nanoaggregates using aqueous ex-
could affect the blue-shift of the absorption peak due to tract of A. occidentale leaf from zinc acetate dihydrate
the reduction of the particle size [49] as revealed by the and zinc chloride as precursors was successful, with
TEM analysis. Similar studies reported the absorption nanosphere and nanoflake morphologies and the con-
band for ZnO nanocrystals in the range of 355–380 nm firmation of elemental zinc from the EDX analysis for
[50–52]. the two samples. Particle size determined for the
nanoaggregates using zinc acetate dihydrate and zinc
4.6 Antibacterial activity analysis chloride precursors were 107 nm and 206 nm
Significant differences existed between the two ZnO respectively.
nanoaggregates and the microbes under study. From the The active ingredients present in the leaf plant from
results, ZnO nanoaggregates using zinc acetate dihydrate literature as well as the zinc nanoparticles have shown
as precursor were more effective than the one from zinc positive results in the antibacterial activity of the se-
chloride precursor (p > 0.05). Generally, zone of inhib- lected microbes. Based on the results of this study, the
ition increased with increase concentration of the two following conclusions were drawn:
extracts for all microbes as in Table 2 and Fig. 5 a and b.
Irregularity in this trend occurred mostly in the 250 ppm  Increasing the concentration of the extract in the
and 500 ppm for A. baumanni, E. aquaticum, and K. study increases the zone of inhibition generally for
pneumonia. At a concentration of 1000 ppm for K. pneu- all the pathogens under investigation. Irregularities
monia, ZnO nanocrystals from zinc acetate precursor in this trend occurred in A. baumanni, E.
showed maximum antibacterial activity of 67.53% (2.08 aquaticum, and K. pneumonia for the 250 ppm and
mm) as compared to that of zinc chloride at the same 500 ppm concentrations.
concentration (Fig. 5a) with an activity of 45.45% (1.40  ZnO nanoaggregates synthesized from zinc acetate
mm) with reference to the control used. Commercial dihydrate recorded higher antibacterial activity in all
synthesized ZnO NPs (Sigma–Aldrich, USA) containing the microbes used more than antibacterial activity of
the same volume of methanol was used as control to ZnO nanoaggregates from zinc chloride precursors.
identify the activity of the solvent and ZnO nanoparticle.  ZnO nanoaggregates using zinc acetate dihydrate as
On the other hand, the least antibacterial activity (zone precursor gave the highest antibacterial activity of
of inhibition) was recorded for S. aureus with 34.75% 67.53% (2.08 mm) in K. pneumonia at 1000 ppm
(1.06 mm) and 48.85% (1.49 mm) by extract using zinc concentration.
chloride and zinc acetate dihydrate precursors respect-  The least antibacterial activity occurred in S. aureus
ively at concentration of 25 ppm (Fig. 5b). The presence with 34.75% (1.06 mm) zone of inhibition at a
of high concentration of bioactive ingredients especially concentration of 25 ppm by extract used in
tannins in A. occidentale leaf from literature could prob- synthesizing ZnO nanoaggregates using zinc
ably account for the high antibacterial activities against chloride as precursor.
the microbes used in this study. Variations in the activity
of the two ZnO nanocrystals with the same active ingre- Abbreviations
dients in the extract could be associated with the particle ZnO: Zinc oxide; Zn(CH3COO)2∙2H2O: Zinc acetate dihydrate; ZnCl2: Zinc
chloride; Gram +ve: Gram positive; Gram −ve: Gram negative; FT-IR: Fourier
sizes of the two samples [53, 54] and the organic nature transform infrared spectroscopy; SEM: Scanning electron microscopy;
of the precursor, zinc acetate dihydrate which binds TEM: Transmission electron microscopy; EDX: Energy-dispersive x-ray
Droepenu et al. Beni-Suef University Journal of Basic and Applied Sciences            (2021) 10:1 Page 9 of 10
spectroscopy; UV-vis: Ultraviolet visible spectroscopy; OD: Optical density; 8. Kılıç B, Gür E, Tüzemen S (2012) Nanoporous ZnO photoelectrode for
KBr: Potassium bromide; KOH: Potassium hydroxide; ZnAc: Zinc acetate dyesensitized solar cell. J Nanomater DOI. https://doi.org/10.1155/2012/
dihydrate; NPs: Nanoparticles 474656
9. Li B, Wang Y (2011) Hierarchically assembled porous ZnO microstructures
Acknowledgements and applications in a gas sensor. Superlattice Microst 49:433–440
The authors acknowledge the contribution of colleagues from Faculty of 10. Vimala K, Sundarraj S, Paulpandi M, Vengatesan S, Kannan S (2013) Green
Resource Science and Technology (FRST) Geochemistry Laboratory and synthesized doxorubicin loaded zinc oxide nanoparticles regulate the Bax
Analytical Laboratory, Universiti Malaysia Sarawak; Department of and Bcl-2 expression in breast and colon carcinoma. Process Biochem 49:
Pharmaceutics, Phytochemistry Laboratory, Centre for Plant Medicine 160–172
Research, Mampong-Akuapem, Ghana; Chemistry Laboratory, Department of 11. Venkatachalam P, Jayaraj M, Manikandan R, Geetha N, Rene ER, Sharma NC
Biochemistry Laboratory, University of Ghana-Legon; and Mr. Obeng Wilson, et al (2016) Zinc oxide nanoparticles (ZnO NPs) alleviate heavy metal-
a peasant farmer in Dormaa Ahenkro in the Brong Ahafo region of Ghana. induced toxicity in Leucaena leucocephala seedlings: a physiochemical
analysis. Plant Physiol Biochem 110:59–69
Authors’ contributions 12. Elumalai K, Velmurugan S (2015) Green synthesis, characterization and
MOK and FA undertook formal identification and collection of the plant antibacterial activities of zinc oxide nanoparticles from the leaf extract of
species. EKD, AEA, BSW, and RBW designed the research. MOK, FA, EKD, BSW, Azadirachtaindica (L.). Applied Surface Science 345:329–336
RBW, and AEA conducted the review and editing. Centre for Plant Medicine 13. Matinise N, Fuku XG, Kaviyarasu K, Mayedwa N, Maaza M (2017) ZnO
Research, Mampong-Akuapem, Ghana, provided resources. EKD and AEA nanoparticles via Moringa oleifera green synthesis: physical properties and
wrote the paper. Finally, all authors have read and approved the manuscript mechanism of formation. Applied Surface Science 406:339–347
for publication. 14. Mohamad NAN, Arham NA, Jai J, Hadi A (2014) Plant extract as reducing
agent in synthesis of metallic nanoparticles: a review. Advanced Materials
Funding Research 832(2014):350–355
The authors received no specific funding for this work. 15. Dubey SP, Lahtinen M, Sarkka H, Sillanpaa M (2010) Bioprospective of Sorbus
aucuparia leaf extract in development of silver and gold nanocolloids.
Colloids Surf B 80(2010):26–33
Availability of data and materials 16. Christensen L, Vivekanandhan S, Misra M, Mohanty AK (2011) Biosynthesis of
The dataset used during the current study are available from the silver nanoparticles using Murraya Koenigii: an investigation on the effect of
corresponding author on reasonable request. broth concentration in reduction mechanism and particle size. Advanced
Materials Letters 2(2011):429–434
Ethics approval and consent to participate 17. Dwivedi AD, Gopal K (2010) Biosynthesis of silver and gold nanoparticles
Not applicable using Chenopodium album leaf extract. Colloids and Surf A 369(2010):27–33
18. Sathishkumar M, Sneha K, Yun Y-S (2010) Immobilization of silver
Consent for publication nanoparticles synthesized using Curcuma longa tuber powder and extract
Not applicable on cotton cloth for bactericidal activity. Bioresource Technology 101(2010):
7958–7965
Competing interests 19. Michiels JA, Kevers C, Pincemail J, Defraigne JO, Dommes J (2012) Extraction
The authors declare that they have no competing interests. conditions can greatly influence antioxidant capacity assays in plant food
matrices. Food Chem 130(2012):986–993
Author details 20. Gunalana S, Sivaraja R, Rajendranb V (2012) Green synthesized ZnO
1Resource Chemistry Program, Faculty of Resource Science and Technology, nanoparticles against bacterial and fungal pathogens. Program of Natural
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia. Science Material International 22(6):693–700
2Graduate School of Nuclear and Allied Sciences, University of Ghana, AE1, 21. Akinpelu DA (2001) Antimicrobial activity of Anarcardium occidentale bark.
Kwabenya, Accra, Ghana. 3Department of Pharmaceutics, Centre for Plant Fitoterapia 72:286–287
Medicine Research, Mampong-Akuapem, Ghana. 4Department of 22. Doss VA, Thangavel KP (2011) Antioxidant and antimicrobial activity using
Phytochemistry, Centre for Plant Medicine Research, Mampong-Akuapem, different extracts of Anacardium occidentale L. International Journal of
Ghana. Applied Biology and Pharmaceutical Technology 2:436–443
23. Souza NC, Oliveira JM, Morrone MS, Albanus RD, Amarante MSM, Camillo
Received: 6 March 2020 Accepted: 12 December 2020 CS, Langassner SMZ, Gelain DP, Moreira JCF, Dalmolin RJS, Pasquali MAB
(2017) Antioxidant and anti-inflammatory properties of Anacardium
occidentale leaf extract. Evidence-Based. Complementary and Alternative
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