Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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IN VIVO ACTIVITY OF GREEN ZINC OXIDE NANOPARTICLES
AGAINST Leishmania donovani USING ALBINO MALE RATS
Ahmed Hamad Saleh
Department of Medical Analysis,College of Al-Qalam ,Kirkuk, Iraq.
Keywords: green zinc oxide nanoparticles, oxidative stress, saffron
Corresponding Author:Ahmedeagle72@gmail.com
ABSTRACT
The present study was designed to show the potential role of zinc oxide nanoparticles against
the toxicity of L. donovani. The study were conducted on 15 adult male rats that arranged in
three groups (5 rates for each), The control group that received normal saline, The second is the
group with rats injected with L. donovani at dose rate of 1.2× 1 0 6 cell/ 0.2ml . intraperitoneally,
The third group is rats injected with L. donovani at a dose rate of 1.2× 1 0 6 cell/ 0.2ml and
treated with50ug/kg of ZnO-NPs for two weeks. The results show a high significant increased (P
< 0.05) in levels of Malondialdehyde (MDA) and a high significant decreased (P < 0.05) in
levels of GSH, catalase in the second group compared with the controls. The results of third
group showed non-significant changes (P < 0.05) in all parameters compared with controls when
using green zinc oxide nanoparticles. The results of the histopathological study of sections
prepared from animals of the second group indicated a thickening wall of bronchiole in most
regions with sever lymphocytes infiltration and damage in wall of alveoli. However , After
treatment, the sections that prepared from the third group show a semi-normal structure of
bronchiole, alveolar sac and alveoli. It has been was concluded that green zinc oxide
nanoparticles have the true been potential role against the toxicity of L. donovani in adult male
rats.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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.INTRODUCTION
Leishmaniasis is one amongst the foremost numerous and sophisticated diseases caused
by associate degree obligate intra cellular protozoan parasite happiness to
the mastigophoran [1]. Kala azar may be a major public pathological state within the
world, it's recognized as a vital public pathological state in Irak and also the most fatal kind,
classic that is, referred to as Kala-azar or black fever [2-3]. L. donovani attacks the RES and also
the symptoms area unit characterised by fever, hepato-splenomegaly, leukopenia, progressive
weakness and leanness, which may lead to death if left untreated [4-5]. Recently, associate
degree explosive strategic development has been habitually applied in
novel inexperienced synthesis protocols that produces nanoscale biocompatible
nanoparticles attributable to the chance of coming up with good bio-nanomaterials with specific
biological functionalities. In recent times, non-toxic, biomimetic, surroundings friendly
approaches are gaining additional importance thanks to their ability to create a good vary of
biocompatible nanomaterials paves the thanks to best fitted for medical specialty applications[6-
8]. Zinc oxide nanoparticles (ZnO-NP) encompasses a tremendous potential in biological
applications like biological sensing, biological labeling, sequence delivery, drug delivery and
nano-medicine [9-10] beside its bactericide, antifungal, acaricidal, pediculocidal, larvicidal and
anti-diabetic activities [11-12]. Therefore the aim of study is to indicate the potential role
of oxide nanoparticles against the toxicity of L. donovani.
MATERIALS AND METHODS
Source of Leishmania
The pure isolate of L. donovani was obtained and diagnosed at the Department of Biology –
College of Education for Pure Science – University of Samaraa .
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Plant extract
Saffron (Crocus sativus L.) flowers purchased from local markets were used. The aqueous
solution of the saffron was obtained by grinding dried flowers in the form of fine powder by an
electrolytic mill. 60 mg of the powder was added to 500 ml of distilled water and left at room
temperature for 24 hours and the solution was filtered using a Buchner funnel and Wattman No.1
filter paper, The filtration solution dried using an oven at a temperature of 45 C˚. after drying
distilled water was used to prepare the concentrations under study [13].
Green synthesis of zinc oxide nanoparticles
20 ml of the plant extract was heated at 50 °C for 10 min and 50ml of 91 mM of zinc acetate
solution (1 gm of zinc acetate was dissolved in 50 ml of distilled water) was added drop wise to
it under stirring. The reaction mixture became yellowish and cream colored precipitate of zinc
hydroxide was formed. The reaction mixture was left for 30 min for complete reduction to zinc
hydroxide. Then the precipitate was collected by centrifugation at 16000 rpm for 10 min at 4 °C.
The precipitate was vacuum dried at 30 °C and the sample (PZN30) was stored by using [14].
Experimental design
Fifteen adult male rats were used in this study and then divided as follows ( 5 rates for each
group):
1. control group: rats were received a normal diet only for two weeks and then killed.
2. Second group: rat injected intraperitoneal with L. donovani at dose 1.2× 1 0 6 cell/ 0.2ml
were, and then killed after infection.
3. Third group: rat injected intraperitoneal with L. donovani at dose 1.2× 1 0 6 cell/ 0.2ml
and treated with 50ug/kg ZnO-NPs for two weeks, and then killed.
4. Fourth group: rat treated with 50ug/kg ZnO-NPs for two weeks, and then killed.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
322
2.5. Prepare of blood solution
Blood samples are collected from rat heart, under anesthesia, blood was put in test tubes.
Then, tubes were centrifugation for 10 min to obtain serum that stored by deep freezing until
used.
2.5. Serological tests
MDA (malonedialdehyied) was measured according to colorimetric reaction with
thiobarbituric acid (TBA) using spectrophotometer [15]. Glutathione (GSH) level estimated by
mixed 2.3 ml buffer with 0.2ml of the sample and then added 0.5ml of DTNB (5,5-dithio-bis-
(2-nitrobenzoic acid). The mixture was analyzed by spectrophotometer [16].
Histopathological study
Lung biopsies were taken with 4mm punch and 2% xylocaine was used as an anesthetic. The
biopsies were fixed in 10% formalin, routinely processed and embedded in paraffin sections
which were stained with hematoxylin and eosin and examined under the microscope.
Statistical analysis
The Data were analyzed using a statistical Minitab program. A statistical difference between
the means of the experimental groups was analyzed using one way analysis of variance
(ANOVA) [17].
RESULTS
Oxidative stress (MDA) & antioxidant parameters (GSH and catalase)
The levels of MDA (2.67 ± 0.42), GSH (0.293 ± 0.019) and catalase (0.64 ± 0.03) in second
group show high significant changes (P < 0.05) compared with controls (1.53 ± 0.19; 0.423 ±
0.034 and 1.22 ± 0.08 respectively). The levels of MDA (1.63 ± 0.27), GSH (0.415 ± 0.028) and
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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catalase (1.19 ± 0.06) in third group show non-significant changes (P < 0.05) compared with
control rats as shown in Table (1).
Table (1): The levels of MDA, GSH and CAT in serum
Parameters
Groups
MDA (mmol/l) GSH (mol/l) Cata (mmol/l)
Control group 1.53 ± 0.19 0.423 ± 0.034 1.22 ± 0.08
Second group 2.67 ± 0.42 * 0.293 ± 0.019 * 0.64 ± 0.03 *
Third group 1.63 ± 0.27 0.415 ± 0.028 1.19 ± 0.06
Histopathological study
The sections that prepared with a control group that show normal in the normal structure of
bronchiole, alveolar sac and alveoli as shown in figure (1). The cross section that prepared from
second group that injected with L. donovani show thickening wall of bronchiole in most regions
with sever lymphocyte infiltration and damage in wall of the alveoli as shown in figure (2). The
sections that prepared from third group show a semi-normal structure of bronchiole, alveolar sac
and alveoli as shown in figure (3).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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BH
AV
AS
Figure (3): lung of third group show bronchiole (BH),
alveolar sac (AS) and alveoli (AV) H&E X400.
Figure (2): lung of second group show thickening wall of
bronchiole (TW), lymphocytes infiltration (IL) and alveoli
degeneration (DA) H&E X400.
IL
T
DA
Figure (1): lung of third group show normal structure of
bronchiole (BH), alveolar sac (AS) and alveoli (AV) H&E
X400.
BH
AV
AS
AV
AS
Figure (3): lung of fourth group show alveolar sac (AS) and
alveoli (AV) H&E X400.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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DISCUSSION
The results of ,present study show different significant changes in oxidative stress and
antioxidant enzymes, with different lesions in lung tissue of ,rat that injected with L. donovani.
The Same results were reported by Ali (2016) who referred that visceral Leishmaniasis that
caused by L. donovani lead to increase the levels of MDA (4.60±2.15) and decrease the levels of
catalase (0.75±0.58) compared to control group (1.35±0.31; 9.81±1.16 respectively) [18]. The
numerous increase of MDA level within the gift study powerfully reflects associate in
Nursing hyperbolic lipide peroxidation initiated by reaction of free radicals to unsaturated fatty
acids in biological membranes. lipide peroxidation is created by oxidative stress ensuing from
the over-production of ROS and element species (RNS) in, cutaneous Leishmaniasis [19]. Also,
Also, in an alternative study according a decrease in enzyme activity and also the increase MDA
levels in patient with Delhi boil are according. many inhibitor enzymes like SOD and CAT exist
that convert ROS into harmless merchandise [20]. About, , the treatment with nanoparticles,
compounds that found in plants extract, are bound to ions, and function as a complexing agent
[21]. There are a number ,of, biologically active constituents in Saffron (Crocus sativus)
,including more than 34 components that are terpenes, terpene alcohols and their esters among
which safranal is the main component. Non-volatile compounds, comprise crocins, crocetin,
picrocrocin and flavonoids (quercetin and kaempferol) [22]. Many studies on medicinal
properties of saffron have indicated that saffron, has a potent antioxidant activity which is
,mostly due to the presence of crocin as a unique carotenoid [23-24]. On the other hand, the role
of AgNPs within the treatment was incontestable through its propeities as inhibitor and anti
inflammatory. Whereas, David et al. according that Ag-NPs synthesized by American
elder possess exceptional medicinal drug properties [25]. El-Rafie and Hamed according that the
synthesized nanoparticles by Terminalia species have inhibitor activity thanks to the
capped phenolic resin and flavonoid compounds and might be used against hurtful effects of free
radicals and have powerfully medicinal drug activity [26].
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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