Ultrasonics - Sonochemistry 55 (2019) 57–66

Contents lists available at ScienceDirect

Ultrasonics - Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

A facile ultrasonic-aided biosynthesis of ZnO nanoparticles using Vaccinium T

arctostaphylos L. leaf extract and its antidiabetic, antibacterial, and oxidative
activity evaluation
⁎
Abolfazl Bayramia, , Sanaz Alioghlia, Shima Rahim Pouranb, Aziz Habibi-Yangjehc,
⁎
Alireza Khataeed,e, S. Rameshb,
a
Department of Biology, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran
b
Centre of Advanced Manufacturing and Materials Processing, Department of Mechanical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur,
Malaysia
c
Department of Chemistry, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran
d
Research Laboratory of Advanced Water and Wastewater Treatment Processes, Department of Applied Chemistry, Faculty of Chemistry, University of Tabriz, 51666-
16471 Tabriz, Iran
e
Department of Materials Science and Nanotechnology Engineering, Faculty of Engineering, Near East University, 99138 Nicosia, North Cyprus, Mersin 10, Turkey

A R T I C LE I N FO A B S T R A C T

Keywords: The synthesis of nanoparticles often result in the generation of harmful chemical pollutants. As such, many
Bactericidal effect researchers have focused on developing green processes, which include the biosynthesis. In this research, ZnO
Bio-extract nanoparticles were prepared using the leaf extract of whortleberry (Vaccinium arctostaphylos L.) via a simple
Decolorization ultrasonic-assisted method. The morphology, crystal size and structure, surface, thermal, and optical properties
Diabetes mellitus
of the bio-mediated ZnO sample (ZnOext) were analyzed and compared with that produced without in-
Nanocatalyst
Ultrasound
corporating the extract (ZnOchem). The ZnO samples were evaluated for their antidiabetic, antibacterial, as well
as their sono- and photo-catalytic performances. Initially, the samples were intraperitoneal injected to alloxan-
diabetic rats to examine their treatment efficiency in terms of effects on fasting blood glucose, insulin, choles-
terol, high-density lipoprotein, and total triglyceride levels. The ZnOext showed significantly higher efficiency for
improving the health status of alloxan-diabetic rats in contrast with other tested treatments, vis. ZnOchem, in-
sulin, and only leaf extract. In addition, both the ZnO samples were assessed against gram-negative and gram-
positive bacteria and through sono- and photo-catalytic processes for removing rhodamine B, respectively. The
results of this study indicated that not only the ZnOext sample was pollution free, it also exhibited higher po-
tentials for treating diabetic rats, bacterial decontamination, and also oxidative removal of organic compounds
under the influences of ultrasound and UV irradiations when compared with ZnOchem sample.

1. Introduction resistance, and possess exceptional absorption filter [5]. Moreover, it

Today, nanotechnology is employed in almost every field of re-
search [1]. In parallel, nanomaterials have demonstrated the potential
to appoint a great impact on the quality of life of individuals with
health problems [2]. In this regard, there is a growing body of literature
on the application of semiconductor oxide nanoparticles in scientific
processes [3]. Among the semiconductor oxide nanoparticles, zinc
oxide has been one of the greatly studied metal oxides for medicinal
and environmental applications, since it has been certified as a safe
substance by food and drug administration (FDA), USA [4]. ZnO na-
noparticles are resistant to corrosion, have durable anti-oxidation
has exhibited satisfactory activity against harmful bacteria and cancer
cells coming from its ability in producing reactive oxygen species [6].
In recent years, plant-mediated synthesis has become a more pop-
ular method for ZnO production owing to be safe, simple, effective, and
environmentally benign [7]. Plants are natural and plentiful source of
secondary metabolites, which offer unique therapeutic properties to
improve human health condition. From therapeutic point of view, a
number of secondary metabolites such as flavonoids, steroids, tannins,
alkaloids, and terpenoids were reported as significant bioactive mole-
cules [8]. There are many literatures on the bio-synthesis of ZnO in the
presence of the extract obtained from different parts of plants such as

⁎
Corresponding authors.
E-mail addresses: a_bayrami@uma.ac.ir (A. Bayrami), ramesh79@um.edu.my (S. Ramesh).

https://doi.org/10.1016/j.ultsonch.2019.03.010
Received 28 January 2019; Received in revised form 1 March 2019; Accepted 9 March 2019
Available online 11 March 2019
1350-4177/ © 2019 Elsevier B.V. All rights reserved.
A. Bayrami, et al.
Scheme 1. Chemical structure of a number of compounds present in the leaf extract of V. arctostaphylos L.
Amaranthus caudatus [9], Silybum marianum L. seed [10], Momordica
charantia [11], Euphorbia prolifera leaf [12], and Ziziphus nummularia
leaf [13]. The rich content of plant extract not only performs as capping
agent to control the shape and size and inhibit the aggregation of the
ZnO nanoparticles but also it is involved in stabilizing the nanoparticles
while acting as reducing agent [7].
As reported in the literature, ultrasound irradiation has been em-
ployed as a promising approach for effective extraction and biosynth-
esis of ZnO nanoparticles [7]. The use of ultrasonic irradiation has been
effectively launched in the recent decades and is well-prevailing in
preparation of nanomaterials and therapeutic applications because it is
efficient, safe, and applicable. The interaction of the sound waves of
high intensity and frequency with biological systems gives rise to quick
diffusion of solute out to the solvent. The role of ultrasonication in an
effective extraction process has been attributed to several mechanisms
including the enhanced penetration and swelling, hydration process,
disruption of cells or capillary phenomenon [14]. On the other hand,
there has been a surge of interest in the ultrasound-assisted biosynthesis
of nanoparticles on account of rapid reaction, high efficiency, moderate
condition, and generating well-dispersed nanoparticles [15].
Diabetes mellitus is described as abnormal secretion or reception of
insulin, or post-receptor trials, which affect the metabolism including
the β cells of the pancreas, liver, and kidney. The common symptoms of
diabetes are overeating, thirst, and recurrent urination, which are not
typically taken seriously by the patient to think about remedy [16]. In
this regard, a shift in lifestyle toward a healthy diet, doing exercise, and
using hypoglycemic agents are required to control diabetes. For treat-
ment purpose, in addition to injectable insulin, some other oral medi-
cines are also prescribed. However, various complications including
lactic acidosis, diarrhea, low blood sugar, and liver damage are asso-
ciated with these chemical medications [17]. Zinc is considered as a
vital trace element in the body that is directly associated with the
glucose uptake physiology, partaking in the insulin production, storing,
secretion, performance, and transposition inside the cells [18], and
translocation of GLUT4 to cell surface for carrying glucose inside the
insulin-responsive cells [19]. It has been also reported that Zn shows
insulin-like properties, improves insulin mitogenic signaling, and sti-
mulates extracellular-signal-regulated kinases 1 and 2 [20,21].
ZnO has also demonstrated adequate activities against various pa-
thogenic bacteria. The bactericidal effect of ZnO against various pa-
thogens such as Escherichia coli [22], Campylobacter jejuni [23], Pseu-
domonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis [24] are
well-documented. Several mechanisms have been reported on ZnO ac-
tion when come in contact with bacteria cells. One frequently reported
reason is production of H2O2 and its consequent oxidative stress on
bacteria cells [23]. Another mechanism is anticipated with the direct
58
Ultrasonics - Sonochemistry 55 (2019) 57–66
contact of ZnO with cell membrane and its disruption, which inhibits
the bacterial growth and division [25]. One more mechanism is asso-
ciated with the increased solubility of ZnO because of the complexation
of the released Zn2+ ions [26].
Besides that, the multifunction ZnO is well-known for its good
photocatalytic activity. However, an energy larger than ZnO band gap
energy (Eg = 3.37) is required to produce electron-hole pairs in order to
start the oxidation reactions. Alternatively, this n-type semiconductor
can be modified through doping with other metals or metal oxides to
reduce the corresponding Eg and accordingly, the amount of irradiated
energy [5]. However, the effect of residual organic moieties from green
synthesis of ZnO nanoparticles on its photocatalytic activity is not fully
understood.
Caucasian whortleberry (Vaccinium arctostaphylos L.) is a perennial
plant, which commonly grow in Caucasus and western Asia (Iran and
Turkey). The fruits of this plant has shown beneficial effects on oxi-
dative stress, and hyperlipidemia [27], while the aqueous extract of its
leaves have high levels of phenolic compounds and flavonoids with
antidiabetic and anti-hypertensive activities [28]. Quercetin (inhibitory
effects on the activity of pancreatic α-amylase) [29], Rosmarinic acid,
caffeoylarbutin (phenolic compounds) [30], delphinidin, petunidin,
and malvidin (anthocyanidins) [31] are a number of identified com-
pounds in the leaf extract of V. arctostaphylos L. The chemical structure
of these compounds are given in Scheme 1. These compounds promote
the reduction of the metallic ions if used during the synthesis processes
[7].
In the present study, a facile and eco-benign approach was adapted
to produce ZnO nanoparticles. For this purpose, an aqueous extract of
Vaccinium arctostaphylos leaf was obtained via ultrasonic-assisted ex-
traction method and used to produce ZnO nanoparticles through a rapid
ultrasonic-assisted process, subsequently labeled as ZnOext. The sample
was characterized by XRD, TEM, SEM, EDX, FT-IR, BET, TGA, and
UV–vis DRS techniques and compared with the ZnO sample synthesized
without incorporating the extract, labeled as ZnOchem. The activity of
the samples were evaluated for their efficiency in treating alloxan-in-
duced diabetic rats, decomposition of gram-positive (Staphylococcus
aureus) and gram-negative (Escherichia coli) bacteria, and also deco-
lorization of a dye wastewater (Rhodamine B) through sono- and photo-
catalysis. In antidiabetic studies, the efficiency of ZnOext was compared
to other treatment alternatives, including ZnOchem, insulin, and only
whortleberry extract in relation to diabetic-control (not treated) and a
normal healthy group as control. In other studies, the comparisons in
efficacies were made between ZnOext and ZnOchem samples to explore
the best preparation method from environmental, effectiveness, and
economic points of view.
A. Bayrami, et al.
2. Materials and methods
2.1. Materials
In this work, the reagents of analytical grade were used without
additional purification. Alloxan monohydrate C4H2N2O4·H2O was ob-
tained from Sigma Aldrich. Zinc nitrate hexahydrate and sodium hy-
droxide were purchased from Loba Chemie Company whereas
Rhodamine B (RhB) was procured from Merck. The Vaccinium arctos-
taphylos L. leaves were collected from mountain areas located in Ardabil
province, Iran and validated by a botanist. The determination kits of
TG, TC, HDL, and FBS were obtained from Pars Azemoun Company,
Iran. Insulin level was established by commercial ELISA kit from
Mercodia, Sweden. Distilled water (DW) was used during the course of
preparation of samples and solutions.
2.2. Animal testing
Male Wistar rats of about 200 g and one month old, were bought
from animal house of the University of Tehran. The rats were kept in a
standard laboratory (23 ± 2 °C, 50 ± 5% humidity, under a 12 h
light-12 h dark cycles), and fed with ad libitum laboratory rodent diet
(vitamins 3%, lipids 12%. proteins 22%, carbohydrates 30%), and un-
restricted water access. National Institutes of Health (NIH) guidelines
were strictly followed to care and work with laboratory animals (NIH
Publications No. 8023, revised 1978), under the permission from the
Ardabil Animal Care and Use Committee (AACUC). The experiments
involving animal were carried out in the animal laboratory, Department
of Biology, Faculty of Basic Sciences, University of Mohaghegh Ardabili.
2.3. Extraction and preparation of ZnO samples
The whortleberry leaves were washed using double DW, dried in a
dust-free environment at room temperature and then crushed by a
grinding mill into a fine powder. About 100 g of the powder was mixed
with 200 mL double DW in a 1000 mL-flask. Initially, an ultrasonication
for 15 min was employed to assist the extraction. The resulted mixtures
was then heated at 60 °C while stirring on a magnetic heating stirrer
over a period of 1 h. The mixture was centrifuged (7000 rpm), filtered,
and kept in the refrigerator for subsequent applications.
To prepare ZnOchem sample, Zn (NO3)2·6H2O was dissolved in DW
(100 mL) and irradiated by ultrasonic waves for 15 min. The pH of the
mixture was then maintained at 10 using NaOH (5 M) and the sus-
pension was irradiated in a domestic microwave oven (Samsung,
2.45 GHz, 1000 W) for 10 min. The white sediment was then cen-
trifuged at 3000 rpm and washed with DW. Finally, the samples dried in
an oven at 333 K overnight.
In the case of ZnOext, 50 mL of extract was diluted with 50 mL of DW
in a flask. Then Zn (NO3)2·6H2O was added to the extract solution and
irradiated by ultrasound for 15 min. The initial pH of the mixture was
acidic (5.6), and became basic when NaOH (5 M) drop-wisely added
into the solution. The changes in the pH was evident from the change in
color (dark green to bright green). The same procedure as ZnOchem was
followed after reaching the pH to 10.
2.4. Characterization
The XRD measurements were carried out using Philips Xpert X-ray
diffractometer with Cu Kα radiation (λ = 0.15406 nm). The size, shape,
and morphology of the samples were observed using the images ob-
tained from SEM (model LEO1430VP) and TEM (model Zeiss-EM10C
180 kV). The same SEM was used to provide the EDX spectra. The FT-IR
analysis was carried out using a Perkin-Elmer Spectrum RXI device.
Nitrogen adsorption-desorption analysis was conducted to measure the
specific surface area and pore properties of the samples at −196 °C
using Belsorp device and Brunauer-Emmett-Teller (BET) and Barret-
59
Ultrasonics - Sonochemistry 55 (2019) 57–66
Joyner-Halenda (BJH) models. TGA curves of ZnO samples were col-
lected under air atmosphere using Linseis STA PT 1000 at the rate of
10 °C/min from 0 to 700 °C (resolution: 0.1 µg). UV–vis DRS data was
recorded using Scinco 4100 apparatus. The pH measurements were
carried out by Metrohm digital pH-meter (model 744).
2.5. Antidiabetic activities
The overnight-fasted Wister rats were imposed with diabetes by
single intraperitoneal injection of 70 mg alloxan per one kg body
weight. The hyperglycaemic animals (200–250 mg/dL) were picked for
antidiabetic work. The experiments were conducted for 6 groups of
five-rat: 1st group (control): typical healthy rats that received saline,
2nd group (Diabetic control): diabetic rats that received physiological
saline, 3rd group (insulin): diabetic rats that received 10 U/kg insulin,
4th group (ZnOchem): diabetic rats that received 10 mg/dL of ZnOchem
dissolved in saline, 5th group (EXT): diabetic rats that received plant
extract (150 mg/dL), and 6th group (ZnOext): Diabetic rats that received
ZnOext dissolved in saline (8 mg/dL). In all the groups, intraperitoneal
injection was daily performed over a period of 16 days. At the end,
Ketamine (80 mg/Kg)/Xylazine (15 mg/Kg) was used to anesthetize the
overnight-fasted rats. Then, blood samples were procured by heart
puncture, collected in sterile tubes, centrifuged (4000 rpm) for 15 min,
and clotting for 30 min. The resultant serum was used to estimate the
FBS, TG, HDL, and TC analysis using commercial testing kits. The in-
sulin level was determined by enzyme-linked immunosorbent assay
(ELISA) kits purchased from Mercodia AB, Sweden, using Microplate
reader, URIT Medical Electronic Co., Ltd, China. All results were sta-
tistically expressed as Mean ± SEM. The significance level of p < 0.05
was considered for all analyses
2.6. Antibacterial studies
The antibacterial activity of the synthesized ZnOchem and ZnOext
samples were evaluated against both gram-negative and gram-positive
bacteria (E. coli and S. aureus, respectively), in Muller-Hinton broth
using serial dilution technique ranging from 0 (control) and 0.05 to
0.8 mg/mL. At length, a number of Muller-Hinton broth tubes were
mixed by serial double dilution in the indicated range. Subsequently,
the culture media inoculated by the nightlong bacteria culture in Luria-
Bertani (LB) broth, and all tubes incubated for a day at 37 °C. Optical
density at 600 nm was used to verify the antibacterial performance of
the samples in triplicate trials. The percentage of the control was used
to express the viability of the bacteria in the nanoparticle-treated cul-
ture. The antibacterial effects of the ZnO samples were calculated and
described using minimum inhibitory concentration (MIC) and non-in-
hibitory concentration (NIC) [32]. The inhibited bacteria were reposi-
tioned to a LB agar in the MIC tubes and checked for their viability.
2.7. Catalytic experiments
In both catalytic processes, 0.1 g of either ZnOext or ZnOchem sample
was added into 250 mL of RhB solution (5 ppm) in a 500 mL Erlenmeyer
flask. During the experiments, the pH of the suspension was not altered
(pH0 ∼6.9). In the sonocatalytic process, an ultrasonic bath was used to
sonicate the flask (Sonic Spar 7500S; 20 kHz). During the sonication,
ice-water (2 °C) was added to the bath to prevent the temperature rise
due to ultrasonication. During the process, 7 mL of the supernatant was
sampled with a pipette, centrifuged at 3500 rpm for 5 min and catalyst
particles were removed by filtration through a 0.20 μm filter (nylon,
Whatman). Afterward, the concentration of residual OTC was measured
by a UV–vis spectrophotometer (UNICO-S2100SVIS) at λmax = 553 nm.
The degradation efficiency (%) was determined by DE% = [(A0 − At)/
A0] × 100, where, A0 and At are the initial and after t min RhB ab-
sorbance.
The photocatalytic activity of ZnO samples was studied via RhB
A. Bayrami, et al.
Fig. 1. XRD patterns of ZnO nanoparticles synthesized with and without ex-
tract.
decolorization at the same concentration of sonocatalytic experiment.
100 mg of each ZnO was added to two reaction chambers. The RhB-
catalyst mixture was initially stirred for 60 min before being exposed to
a LED-Source (50 W). About 2 mL of mixture was sampled every 30 min,
for 6 h, centrifuged to remove the catalyst, and tested for RhB content
using a UV–Vis spectrometer.
3. Results and discussion
3.1. Characteristics of the samples
Fig. 1 depicts the XRD patterns of the as-prepared ZnO nano-
particles. The diffraction peaks of both samples displayed the crystal-
line structure corresponding to wurtzite hexagonal (JCPDS No. 36-
3411). The sharpness of the peaks in both spectra is the sign of nano-
particles of high crystallinity wherein the dissimilarity in particle sizes
gave rise to different peak intensities. The crystallite sizes of the sam-
ples were calculated by Scherrer’s equation [3]. The determined
average crystallite sizes for the ZnOchem and ZnOext samples from the
(1 0 1) plane were 27.9 nm and 21 nm, and for (1 1 0) plane were 15 nm
and 12.4 nm, respectively. The results clearly indicated that the extract
of V. arctostaphylos leaves was effective in controlling the crystallite size
of ZnO.
SEM and TEM analyses were used to study the shape, and size of the
ZnO samples and the images are shown in Fig. 2. A distinct morphol-
ogies were observed for the two samples, i.e. the ZnOchem sample ex-
hibited spindle-shaped particles whereas particles of spherical character
were observed for the ZnOext sample. Although ultrasonically-produced
ZnO nanoparticles have been reported to form spindle-like shaped
morphology, in the present work, the extract could have altered the
particle shape to spherical [33]. In accordance with XRD results, the
ZnOext sample can be observed in smaller sizes than the ZnOchem
sample. This could be attributed to the bioactive compounds of the
extract, which act as capping agent that control the growth of the
crystallites [34].
EDX analysis was performed to determine the elements present in
the ZnOchem and ZnOext samples (Fig. 3). The EDX spectrum of the ZnO
sample that was prepared in water consisted of only two elements, Zn
and O. However, in addition to the peaks related to Zn and O, the
spectrum of the ZnOext sample encompassed the peaks associated with
carbon element which implied the existence of a strong link between
the biomolecules of the extract and ZnO nanoparticles despite re-
peatedly washing of the ZnOext sample.
The FT-IR spectra of the ZnO samples are shown in Fig. 4. The wide-
ranging absorption band at 3320 cm−1 is assigned to the OeH
stretching vibration of the adsorbed water molecules [35]. Moreover,
the absorption peak of ZneO bond located at 550 cm−1 discern the
60
Ultrasonics - Sonochemistry 55 (2019) 57–66
production of ZnO nanoparticles [36]. As for the ZnOext sample, addi-
tional peaks at 1610 and 1255 cm−1 were observed that correspond to
CeH stretching vibrations of CH2 group, and vibration peaks for C]C,
and C]O bonds, respectively [34]. The absorption band of the plant
extract showed a broad peak at 3100–3600 cm−1, which corresponds to
OeH and CeH stretching vibrations, and NeH stretch of amide group.
The sharp peak at 1630 cm−1 is associated with C]C stretch of alkane
group. The peaks at 1220 and 550 cm−1 strongly portrayed the oc-
currence of CeN stretch of amine groups and CeBr stretch of alkyl
Halide [37]. The occurrence of extract associated bonds in ZnOext ap-
proves the link between the two partitions.
TG analysis was conducted to assess the thermal stability of ZnO
samples and approximate the biomolecule content in the ZnOext sample
(Fig. 5a). The ZnOchem sample exhibited a steady weight-loss trend if
compared to the ZnOext sample. This loss could be associated mainly to
the evaporation of the adsorbed water molecules. The weight-loss
measured at 700 °C for this sample was about 10% if compared to 21%
for ZnOext. In contrast, the ZnOext sample initially experienced a rapid
weight decay from room temperature up to 250 °C as depicted by the
slope of the curve for this sample in Fig. 5(a) and this can be associated
to the evaporation of absorbed water [38]. The second region of weight-
loss started at 250 °C was observed for this sample and this is believed
to correspond to the combustion of biomolecules of extract, which was
connected onto the ZnOext surface.
UV–vis DR spectra were provided in the range of 250–850 nm to
investigate the electronic absorption features of the ZnO samples, as
depicted in Fig. 5b. A strong characteristic absorption peak at UV wa-
velengths of 365 nm was observed for the ZnOchem sample in the cor-
responding UV–Vis DR spectrum. Nonetheless, the absorption peak red-
shifted in the case of ZnOext sample, which could be assigned to the
bonding between the bioactive molecules of the extract and ZnO. The
enhanced photo-activity of ZnOext was further confirmed via computing
the band gap energies (Eg) of both ZnO samples when the linear area in
a plot of (Ahv)2 was extrapolated against (hv) [39]. This provided an
estimate on the Eg of ZnOchem and ZnOext that were respectively 3.15
and 2.6 and supported the results.
In order to investigate the effect of the extract on the surface
characteristics of ZnO, nitrogen absorption-desorption trials were con-
ducted and the resulted isotherms are presented in Fig. 5c. The gener-
ated curves show typical type II isotherms, which disclose the non-
porous characteristic of solids. Moreover, the active BET surface areas
of 11.06 m2g−1 and 26.3 m2g−1 were measured for ZnOchem and ZnOext
samples. As reported in XRD studies, larger surface area of smaller
ZnOext nanoparticles was expected. Although no remarkable change
was detected for pore diameters of the samples, the pore volume en-
hanced in ZnOext sample.
The leaf extract used in the present work is a plentiful source of
active elements, such as phenolics and anthocyanins [30,31]. A number
of mechanisms are suggested for the formation of the ZnO nanoparticles
in the literature. In a number of studies [40], the phenolic compounds
of extract are suggested as the reducing factors in the course of ZnO
preparation process. Based on this mechanism, the complexation of
Zn2+ ions and hydroxyl groups lead to a p-track conjugation effect [34].
3.2. Antidiabetic activities
The antidiabetic performance of the bio-fabricated ZnO was eval-
uated for treating alloxan-diabetic rats after 16 days by measuring the
FBS, insulin, HDL, TC, and TG levels in their blood serums. The results
were statistically compared to that of achieved by other treatments.
Alloxan is a hydrophilic diabetogen, which selectively destroys the β-
cells of pancreas and liver, leading to increased levels of blood glucose
upon injection to rats and imposes the diabetic condition [41]. The
variations in the blood glucose level in the control and diabetic rats are
shown in Fig. 6a and expressed as Mean ± SEM. The results clearly
shows that the FBS levels were significantly elevated after alloxan
A. Bayrami, et al.
Fig. 2. SEM images of (A) ZnOchem and (B) ZnOext. TEM images of (C) ZnOchem, (D) ZnOext and SEM image of (E) ZnOext after photocatalytic process.
injection (p < 0.05). On the 17th day, the recipient group of ZnOext
nanoparticles showed a significant reduction in FBS level compared to
the diabetic control group. This reduction was significantly lower when
compared with the decreases caused by extract and insulin treatments
(p < 0.05). Similar results were obtained for those treated by ZnOchem
sample compared to the diabetic control group. However, the difference
between both ZnO samples were not statistically significant.
Data for the effects on the insulin levels after alloxan and treatments
injections is shown in Fig. 6b. A significant drop in insulin level was
observed in the diabetic control group compared to that of healthy
control group (p < 0.05). However, the level of insulin was re-
markably enhanced in the ZnOext and insulin-treated groups in com-
parison to the diabetic control group (p < 0.05). Though, the differ-
ence in insulin levels induced in the ZnOext and insulin-treated groups
was not statistically significant. The main insulin-like action of Zn de-
pends on the glucose transport improvement, along with some other
intracellular effects [42]. Furthermore, Zn can offer insulin secreta-
gogue activities, since it does not inflict hypoglycemia in organisms
[43]. The influence of Zn on insulin signaling can be explained by
considering the insulin-like actions of Zn. One example of zinc-depen-
dent enzyme is insulin-regulated aminopeptidase (IRAP) which is ex-
pressed in adipose and muscle tissues as target tissue of insulin and can
preserve a required glucose transporter-4 level [44]. Normally, the Zn
content of healthy pancreas is particularly higher than the diabetic
61
Ultrasonics - Sonochemistry 55 (2019) 57–66
patients, which is mainly concentrated inside the thick core insulin-
secreting granules (ISG) and in the β cells [45]. On the other hand, the
interactive effect of extract with ZnO should also be considered wherein
both ZnO and extract showed lower efficiencies for enhancing insulin
levels when applied solely. It has been reported that whortleberry in-
duces antihyperglycemic effects, which could be associated with con-
structive secondary metabolites such as anthocyanins, myricetin, and
chlorogenic acid [46].
Hyperlipidemia is another complication in diabetic patient that
should be considered as a serious health threat. In this context, the
levels of a number of lipid profiles, vis. HDL, TC, and TG were analyzed
in the serums and assessed for the studied treatments (Fig. 6c, d, e). In
advance, the non-treated diabetic rats endured elevated levels of TG
along with extremely reduced HDLC levels. Notwithstanding, HDL le-
vels significantly enhanced in the serums of the rats treated by ZnOext
compared to the diabetic control group. On the other hand, ZnOext
helped for the TG level reduction. In both cases, the ZnOchem, extract,
and insulin efficiencies were respectively at lower levels. Likewise,
ZnOext displayed better effects in reducing TC level. Despite that, other
treatments did not show major alterations in the levels of serum TC
versus the diabetic control rats.
Dyslipidemia is an important risk factor for atherosclerotic com-
plications of diabetes. The refined lipid profile levels after treating with
ZnOext can be initially assigned to the key role of Zn in a number of
A. Bayrami, et al.
Fig. 3. The EDX spectra of ZnO nanoparticles.
Fig. 4. FT-IR spectra of whortleberry extract, and ZnO samples.
enzymatic reactions. Zn acts similar to α-blocker, as proven in the
short-term treatment of dyslipidemia [47]. The results were in good
agreement with related literature, which indicate the fundamental ac-
tion of zinc in metalloenzymes in charge for the lipid digestion and
absorption [48]. Apart from that, the water extract of V. arctostaphylos
was found effective in dealing with dyslipidemia as a fatal aspect for
atherosclerotic complications in diabetes. The organic moieties and
secondary metabolites in V. arctostaphylos are reportedly involved in
62
Ultrasonics - Sonochemistry 55 (2019) 57–66
enhancing the morphology and/or function of β-cells through avoid-
ance of their constant damage, rescue of moderately hurt β-cells, revival
of fresh β-cells, and insulin secretion stimulation [49]. On the other
hand, V. arctostaphylos is characterized with high potentials as anti-
hyperlipidemic mediator, which helps for pulling the lipids and lipo-
proteins levels down in the blood.
As provided from the present study, regardless of the preparation
method, both ZnO samples offered effective risk-reducing interventions
for treating alloxan-diabetic rats. In point of fact, the imperative role of
zinc in several cell functions need to be taken into account. Diabetes is
associated with the Zn insufficiency as it is a unit of insulin.
Administration of Zn to diabetic patients have generally shown positive
effects, especially among type 2 diabetic patients who have a Zn defi-
ciency [43]. This trace element is a significant nutrient, and cofactor of
several enzymes, which performs as a mediator for intracellular sig-
naling. Zn has found to be involved in effective utilization and meta-
bolism of glucose via its effect on the pathway of insulin signaling,
which in turn enhances the hepatic glycogenesis [50]. On the other
hand, the secondary metabolites of the V. arctostaphylos leaf extract
have shown healing properties to control diabetes mellitus [49]. From
the results, it can be concluded that the in cooperation of ZnO and
extract can induce a synergistic effect, which promote the antidiabetic
action of ZnOext nanoparticles.
3.3. Antibacterial activity
At the next step, a set of contrastive studies were conducted to assess
the prospective antibacterial activity of the ZnO nanoparticles synthe-
sized with and without whortleberry leaf extract. Fig. 7a, b display the
antibacterial results of both ZnO samples at the concentrations ranging
from 0.05 to 0.8 mg/mL against S. aureus and E. coli. Both samples
exhibited great performances for destruction of the studied bacteria, as
exposed from the survival rate results and pure culture images. More-
over, the data correspond to minimum inhibitory concentration (MIC)
and none inhibitory concentration (NIC) results of the samples revealed
the MIC of 0.4 mg/mL against S. aureus. Besides bacterial duplication
inhibition at ZnOext of 0.8 mg/mL, the bacteria was destroyed as well.
In the case of E. coli, both ZnO samples performed similarly wherein
MIC of 0.8 was obtained for either. This dominance can be primarily
elucidated by ZnO ability to produce reactive oxygen species such as
•
OH radicals wherein the cell membrane is damaged by these radicals
which ultimately kills bacteria. Moreover, the intracellular accumula-
tion of ZnO nanoparticles and released Zn2+ ions are reported as other
mechanisms involved in the ZnO antibacterial action [25,26].
At the same time, the anti-pathogenic role of the organic moieties
over ZnOext should not be neglected as they include heterocyclic and
phenolic compounds. The antibacterial results established that the bio-
synthesizing of ZnO NPs by leaf extract of V. arctostaphylos encompass
of inherently active antimicrobial biomolecules strongly prevented the
growth of the gram-positive bacteria and enhanced antimicrobial per-
formance of ZnO nanoparticles.
3.4. Oxidative performance
Rhodamine B (RhB) was chosen as the model organic compound to
assess the plausible effect of V. arctostaphylos leaf extract on the oxi-
dative activity of ZnO nanoparticles. In this regard, an LED source of
light was used to activate the ZnO nanoparticles. Fig. 8a shows the
optical absorption of the RhB aqueous solution before and during the
photocatalytic reaction of both ZnO samples. The degradation effi-
ciency (%) was gradually decreased as the reaction proceeded, in-
dicating the prominent degradation of RhB under the light irradiation.
Ultimately, RhB was totally removed by both samples after 270 min of
photocatalytic reaction. Although the photocatalytic DE% of ZnOchem
and ZnOext was not remarkably different at 270 min, it can be seen from
the figure that ZnOext showed higher efficiencies during the studied
A. Bayrami, et al.
Fig. 5. (a) TGA charts, (b) UV–vis DRS, and (c) Isotherms of nitrogen adsorption-desorption of ZnO samples.
reaction time especially between 60 and 180 min. This can be assigned
to the reduction in the band gap energy of ZnO when enriched by the
extract (Eg = 2.7), which brought about easier activation of the sample.
The SEM images of the ZnOext sample is also obtained after its appli-
cation in the photocatalytic system (Fig. 2E) and compared to that
obtained earlier. No obvious changes in the morphology and size of the
sample was observed, except the aggregation of the particles. This is
because the sonication process during the synthesis of the sample
helped for well-distribution of particles, while this factor was absent
during the photocatalytic activity.
At the next step, ultrasound irradiation took the place of light source
in order to evaluate the oxidative performance of both ZnO samples as
sonocatalyts. The activity of ZnOext and ZnOchem were comparable
wherein both of them effectively removed RhB within 240 and 270 min,
respectively (Fig. 8b). In this case, the sonocatalytic performance of
biologically produced ZnO was also higher than chemically produced
sample, primarily eventuated from the large number of the generated
holes under the influence of ultrasound irradiation and long lifespan of
charge carriers due to the electron-capturing by phenolic compounds of
extract.
From the oxidative experiments, ZnOext showed higher activities for
degradation of RhB under both photo and sono illumination. This can
be explained as follows: when ZnO nanoparticles are irradiated with
photons, the excitation of electrons from the valence band to the con-
duction band of ZnO is occurred that induces electron-hole pairs (e−/
h+). As above mentioned, the excitation of electrons and generation of
holes are easier in ZnOext owing to its smaller band gap energy. These
e−/h+ either recombine together or transfer to the photocatalyst sur-
face to begin the photocatalytic reactions. In the case of ZnOext, the
present phenolic compounds capture the generated electrons, leaving
the holes free for reactions. Subsequently, the holes can react with
63
Ultrasonics - Sonochemistry 55 (2019) 57–66
either adsorbed H2O or surface eOH groups to form hydroxyl radicals.
Consequently, more RhB molecules could be decomposed by ZnOext
sample compared to ZnOchem. It should be noted that the amount of the
linked biomolecules were very low, thus not impeding the photo-
catalytic activity of ZnO via absorbing the incident light.
4. Conclusion
Diabetes is a multifactorial disease, yet is not easily controlled and
treated. This study assessed the synergistic effects of Zn, as a key ele-
ment in various cellular bio-processes, and V. arctostaphylos leaf extract
with antidiabetic properties when present together for treating alloxan-
diabetic rats. The characterization results exposed that ZnO nano-
particles grew in smaller sizes when prepared with V. arctostaphylos leaf
extract. The company of whortleberry phytochemicals over ZnOext was
verified by EDX, FT-IR, TGA, and UV–vis DRS techniques. All the stu-
died treatments exhibited good antidiabetic activities for handling the
complications associated with diabetes. Nonetheless, ZnOext showed the
best performance for lowering the FBS, TC, and TG levels and pro-
moting the insulin and HDL levels in alloxan-diabetic rats in contrast to
other treatments. Additionally, the green synthesized sample showed
great efficiencies against microbial and organic pollutants. The domi-
nant action of ZnOext in contrast to ZnOchem and only extract clearly
indicate the synergy between them. The results obtained from this
study strongly proves that the application of V. arctostaphylos leaf ex-
tract during the synthesis prevents the chemical pollution and em-
powers the resulted ZnO towards effective antidiabetic, antibacterial,
and oxidative performances.
A. Bayrami, et al. Ultrasonics - Sonochemistry 55 (2019) 57–66

Fig. 6. (a) FBS, (b) Insulin, (c) HDL, (d) CT, and (e) TG levels in the studied groups during the experiments.

Fig. 7. Antibacterial activities of ZnO samples against (a) gram-positive, and (b) gram-negative bacteria.

64
A. Bayrami, et al.
Fig. 8. (a) photocatalytic and (b) sonocatalytic activities of ZnO samples for removing RhB (Experimental condition: pH = unchanged; [RhB]: 5 mg/L; LED lamp;
Catalyst: 0.1 g/L; and Ultrasound frequency = 20 kHz).
Acknowledgement
The support provided by the University of Mohaghegh Ardabili
(Iran) is greatly acknowledged.
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