THE EFFICACY OF CHITOSAN NANOPARTICLE ALONE ......(Abubakar et al., 2007 and Kumar et al., 2013)....

www.wjpps.com Vol 8, Issue 10, 2019.

139

Mohamed et al. World Journal of Pharmacy and Pharmaceutical Sciences

THE EFFICACY OF CHITOSAN NANOPARTICLE ALONE VERSUS

CONJUGATED WITH Nigella sativa (EL BARAKA SEED OIL)

AGAINST Cryptosporidium parvum IN INFECTED

IMMUNOCOMPETENT AND IMMUNOSUPPRESSED MICE

Walaa A. Mohamed*1, Eglal A. Koura

1, Ibrahim Rabee

2, Olfat A. Hammam

2 and

Hanan M. Ismail1

¹Zoology Department, Faculty of Women for Arts, Science and Education- Ain Shams

University, Cairo, Egypt.

²Department of Parasitology, Theodore Bilharz Research Institute, Giza, Egypt.

ABSTRACT

Cryptosporidium is a protozoan parasites that affect the gastrointestinal

epithelium and other mucosal surfaces of their hosts, which include

humans and domestic and wild animals worldwide. Current diagnostic

methods include microscopic examination of stool for

Cryptosporidium species oocysts with acid-fast stains and modified

Ziehl-Neelsen. Nitazoxanide has limited activity in

immunocompromised individuals. The highest percentages of

reduction in the number of C. parvum oocysts were in the groups

receiving Baraka and chitosan nanoparticles. Histopathological

examination of the intestine, liver and kidney appeared complete

healing after treatment by Baraka and chitosan nanoparticles. Also

immunological studies against C. parvum as IgG and IgM showed improvement of immune

status of both treated groups after treatment by Baraka and chitosan nanoparticles. In this

study the findings indicated less toxicity in using Baraka and chitosan nanoparticles

compared to use alone.

KEYWORDS: Cryptosporidiosis, immunosuppressed, nitazoxanide, Baraka, chitosan,

Baraka and chitosan and immunocompetent.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 8, Issue 10, 139-161 Research Article ISSN 2278 – 4357

*Corresponding Author

Dr. Walaa A. Mohamed

Zoology Department,

Faculty of Women for Arts,

Science and Education- Ain

Shams University, Cairo,

Egypt.

Article Received on

07 August 2019,

Revised on 28 August 2019,

Accepted on 18 Sept. 2019

DOI: 10.20959/wjpps201910-14825


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INTRODUCTION

Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus

Cryptosporidium, is a common cause of diarrheal diseases and often fatal in

immunocompromised individuals (Mukerjee et al., 2015). Cryptosporidium is a great reason

of moderate to severe diarrhea in children younger than 2 years, particularly infants, and is

second only to rotavirus in this regard (Kotloff et al., 2013).

Cryptosporidium parvum is an important zoonotic parasitic disease worldwide, but the

molecular mechanisms of the host–parasite interaction are not fully understood (Wang et al.,

2019). There are several factors that may enhance the spreading of this parasite in human

population especially in young children (Khan et al., 2019). Cryptosporidium parvum is one

of the most species involved in human cryptosporidial infections (Ryan et al., 2014). In

humans, nearly 20 Cryptosporidium species were detected, among them the C. hominis and

C. parvum are the most reported species (Ryan and Xiao, 2014). The spreading of these

parasites occurs through faecal-oral route and also by consumption of contaminated water or

food and zoonotic or anthropogenic transmission (Xiao, 2010).

Current diagnostic methods include microscopic examination of stool for Cryptosporidium

species oocysts with acid-fast stains (modified Ziehl-Neelsen, Auramine-O, or Kinyoun’s),

direct fluorescent-antibody (DFA) tests, antigen-based enzyme immunoassays (EIAs),

Polymerase chain reaction (PCR), and lateral-flow immunochromatographic “strip” tests

(Johnston et al., 2003 and White, 2010).

This disease causes gastrointestinal distress, which can persist two weeks or more. However

in immunocompromised individuals, such as those with malnutrition, human

immunodeficiency virus (HIV), cancer, or organ transplants, this disease can be debilitating

and often fatal (O’Hara and Chen, 2011 and Checkley et al., 2015). Currently approved

therapeutics, nitazoxanide and paromomycin have limited activity in immunocompromised

individuals, creating an urgent need for the development of new anti-parasitic drugs

(Abubakar et al., 2007 and Kumar et al., 2013).

New antiprotozoal drugs with high effectiveness and low toxicity are urgently required.

Medicinal plants used in the treatment of these diseases can be an alternative resource of

novel antiprotozoal drugs (Freitas et al., 2006) Crude extracts (aqueous and alcoholic

extracts) and essential oil of Nigella sativa were proved to have many therapeutic effects. The


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N. sativa alcoholic extract was found to be as effective as metronidazole in the cure of

giardiasis (Bishara and Masoud, 1992).

The aim of the present work is to study the efficacy of chitosan nanoparticle alone versus

conjugated with Nigella sativa (El Baraka seed oil) used in the treatment of cryptosporidiosis

in the experimentally infected immunocompetent and immunocompromised mice.

MATERIALS AND METHODS

Experimental animals: 120 male Swiss albino mice, aged 6- 8 weeks, weighing 20–25 g,

was obtained from Schistosome Biological Supply Centre (SBSC), Theodor Bilharz Research

Institute (TBRI), Giza, Egypt. They will be housed in well ventilated cages with perforated

covers (cleaned every day), supplied with standard pellet food and water. Mice were

maintained throughout the study in an air conditioned room at 21°C and food content 24%

protein and water. The mice were allowed to adapt to the laboratory environment for one

week before the experiment. The experiment was carried out according to the Internationally

Valid Guidelines and an institution responsible for animal ethics.

Parasite: Isolation of Cryptosporidium oocysts from faeces of infected calves by Sediment

and flotation procedures (Waldman et al., 1986 and Zeibig, 1997).

Immune suppression and infection of the mice: Immune suppression of the mouse will be

performed by using dexamethasone orally at a dose of 0.25 mg/g/day for 14 successive days

prior to inoculation with Cryptosporidium oocysts (Abdou et al., 2013 and Rehg et al.,

1988). Each mouse will be infected by oral inoculation with the isolated Cryptosporidium

oocysts in a dose of about 10000 oocysts/ mouse (Gaafar, 2007).

Experimental design: Animals were divided into three main groups as follows:

Group I

Control group which will be subdivided into the following subgroups:

A- Non-infected groups (the negative-control group):

Subgroup A1: Non-infected immuno-competent.

Subgroup A2: Non-infected immuno-suppressed.


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Group II

Infected non treated and infected treated immunocompetent groups which will be subdivided

into the following subgroups:

B1- Infected non-treated groups (the positive-control group).

B2- Infected and treated with nitazoxanide (NTZ).

B3- Infected and treated with Nigella sativa (Baraka).

B4- Infected and treated with chitosan nanoparticle.

B5- Infected and treated with Baraka and chitosan nanoparticles

Group III

Infected non treated and infected treated immunosuppressed groups which will be subdivided

into the following subgroups:

C1- Infected non-treated groups (the positive-control group).

C2- Infectd and treated with nitazoxanide (NTZ).

C3- Infectd and treated with Nigella sativa (Baraka).

C4- Infectd and treated with chitosan nanoparticle.

C5- Infectd and treated with Baraka and chitosan nanoparticles.

Parasitological examination

1. Collection of faecal samples

Faecal samples were collected from each mouse. The samples were then put into clean, wide-

mouthed containers with tight-fitting covers and homogenized in PBS to evaluate C. parvum

oocysts shedding. The number of oocysts was counted and then calculated/gm faeces.

2. Microscopic examination of faecal sample

All faecal samples were subjected to the modified Ziehl-Neelsen staining technique as

described by (John and Petri, 2006).

Animal scarification: Scarification of animals was performed by rapid decapitation of all

mice. The part of small intestine and liver from each mouse was removed and subjected to

histopathological examination. Part of liver and kidney subjected to measurement toxicity of

nanoparticle combinations.

Histopathological examination: To clarify the histological features of different tissues,

segments of about 1cm long from the small intestine and liver of each animal were cut off


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and immediately fixed in 10% buffered formalin. All sections were microscopically studied

to evaluate the pathological changes that occurred due to cryptosporidiosis before and after

drug administration (under low power and high power).

Evaluation of toxic effects of nanoparticles: Determination of toxicity concentrations in

liver of all studied groups by measurement of Glutathione (GSH) and Lipid Peroxide

(Malondialdehyde) (MDA), using colorimetric method (Ohkawa et al., 1979 and Beutier et

al., 1963).

Statistical Analysis: Data were expressed as mean values ± SD by the statistical software

package SPSS (version 16.0). Continuous variables are presented, and frequencies with their

respective percentages are given for categorical variables. Comparisons between 2 groups

were done using the Student’s t-test. Percent inhibition compared to infected non treated

oocysts was determined using the following equation:

Infected non treated – Infected treated x 100 =

Infected non treated

The degree of significance (P-value) was obtained from corresponding tables.

The degree of significance was expressed as follows:

P>0.05 Non-significant.

P< 0.05 Significant.

P<0.01 Highly significant.

p< 0.001 Very highly significant.

RESULTS

Parasitological results of faecal examination

Mice began to shed oocysts with their faeces in day four post infection (PI). Maximum

shedding of Cryptosporidium parvum oocysts/gm in all immunocompetent mice groups were

observed on day 14 PI, with a mean of 7800±110.0 except B2 was observed 7720±142.9

while in all immunosuppressed mice groups, the mean number of oocysts/gm shed in the

stools on the same day were 8800±94.2809 except groups C1 and C4 were 9000±88.1 (Table

1 and 2).


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Table (1): Mean and standard deviation of parasitological examination of

immunocompetent groups (X±SD).

Time Infected

(B1)

Infected+

NTZ (B2)

Infected+

Baraka (B3)

Infected+

Chitosan (B4)

Infected+ Baraka

and chitosan (B5)

14days PI 7800±110.0 7720±142.9 7800±164.9 800±205.4 800±309.7

18days PI 7500±166.6 6150±81.64 6000±371.9 6200±218.5 0285±418.9

20days PI 6800±141.4 5050±100* 4500±200** 4850±106.8* 3750±89.44***

23days PI 6200±141.4 4000±70.71** 2750±68.41*** 2550±252.9*** 2100±100.7***

27days PI 6000±126.4 3250±75.82*** 2200±52.44*** 1750±161.2*** 1250±70.71***

30days PI 5700±139.1 2100±88.54*** 1750±89.44*** 1500±118.3*** 005±89.44***

Data are expressed as mean ± SD. p> 0.05*= significant,

p<0.01**= highly significant and p<0.001*** very highly significant.

Table (2): Mean and standard deviation of parasitological experiment of

immunosuppressed groups (X±SD).

Time Infected

(C1)

Infected+

NTZ (C2)

Infected+

Baraka (C3)

Infected+

chitosan (C4)

Infected+ Baraka

and chitosan (C5)

41days PI 9000±88.1 8800±94.2 8800±94.2 9000±88.19 8800±94.2

41days PI 8500±105.4 8230±67.49 7400±81.64 7500±164.9 6240±69.92*

02days PI 8200±192.3 7750±89.44 6750±89.44 7000±70.71 4750±100***

02days PI 7750±118.3 7000±137.8 5500±178.8* 5850±89.4* 3500±83.6***

02days PI 7500±114.0 6800±100 4750±137.8** 4250±89.4*** 2000±70.7***

22days PI 7000±70.71 6250±89.4 3000±77.4*** 3250±89.4*** 1500±89.4***


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Histopathological results

1. Small intestine

Immunocompetent groups Immunosuppressed groups


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Figure: Section of small intestine from immunocompetent groups A) normal control

group showing normal structure of the mucosa, lamina propria and normal small intestinal

crypt villous ratio (black arrows). B) Infected animal (+ve control) showing, blunting and

shortening of villous in mucosa and villous atrophy (red arrows), ulcerations (yellow arrow)

non-specific inflammatory infiltration of the lamina propria with lymphocytes (black arrow).

C) Infected animal and treated with nitazoxanide showed almost normal villous pattern,


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(yellow arrow), mild lymphocytic inflammatory response noticed in villi (red arrow). D)

Infected animal and treated with Baraka showed almost normal villous pattern, (black

arrow), mild lymphocytic inflammatory response noticed in villi (red arrow). E) Intestine of

infected animal and treated with chitosan showed almost normal villous pattern. F) Infected

animal and treated with Baraka and chitosan showed almost normal villous pattern. Section

of small intestine from immunosuppressed groups G) normal control group showing

normal structure of the mucosa, lamina propria and normal small intestinal crypt villous ratio

(black arrows). H) small intestine of infected animal (+ve control) showing, blunting and

shortening of villous in mucosa and villous atrophy (red arrows), ulcerations (yellow arrow)

non-specific inflammatory infiltration of the lamina propria with lymphocytes (black arrow).

I) Infected animal and treated with nitazoxanide showed almost normal villous pattern,

(yellow arrow), mild lymphocytic inflammatory response noticed in villi (red arrow). J)

Infected animal and treated with Baraka showed almost normal villous pattern, (black

arrow), mild lymphocytic inflammatory response noticed in villi (red arrow)

(H&Ex100,x200). K) Infected animal and treated with chitosan showed almost normal

villous pattern, L) Infected animal and treated with Baraka and chitosan showed almost

normal villous pattern (H&Ex200).


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2. Liver

Immunocompetent groups Immunosuppressed groups


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Figures: A) liver sections from immunocompetent groups, control group (-ve control)

showed preserved (intact) lobular hepatic architecture with thin plates of normal hepatocytes

(black arrow) and normal morphological appearance of hepatocytes. B) Infected animal (+ve

control) showing preserved (intact) lobular hepatic architecture with thin plates of

hepatocytes with moderate hydropic degeneration (black arrows), congested central vein


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(yellow arrow). C) Infected animal and treated with nitazoxanide showing preserved

(intact) lobular hepatic architecture with thin plates of almost normal hepatocytes (black

arrows), congested central vein (yellow arrow), small collection of interlobular

lymphocytes (red arrow), congested dilated sinusoids (green arrow). D) Infected animal and

treated with Baraka showing preserved (intact) lobular hepatic architecture with thin plates

of almost normal hepatocytes (black arrows), congested central vein (yellow arrow), mild

collection of interlobular lymphocytes (red arrow), congested dilated sinusoids (green

arrow). E) Infected animal and treated with Chitosan showing preserved (intact) lobular

hepatic architecture with thin plates of almost normal hepatocytes (black arrows), moderate

collection of interlobular lymphocytes (red arrow). F) Infected animal and treated with

Baraka and Chitosan showing preserved (intact) lobular hepatic architecture with thin plates

of almost normal hepatocytes (black arrows), small collection of interlobular lymphocytes

(red arrow) (H&E, x400). G) liver sections from immunosuppressed group, normal control

group (-ve control) showed preserved (intact) lobular hepatic architecture with thin plates of

normal hepatocytes (black arrow) and normal morphological appearance of hepatocytes,

central vein congestion (red arrow). H) Infected animal (+ve control) showing preserved

(intact) lobular hepatic architecture with thin plates of almost normal hepatocytes (black

arrows), large collection of interlobular lymphocytes (red arrow), congested central vein

(yellow arrow). I) Infected animal and treated with NTZ showing preserved (intact) lobular

hepatic architecture with thin plates of almost normal hepatocytes (black arrows), congested

central vein (yellow arrow), small collection of interlobular lymphocytes (red arrow),

congested dilated sinusoids (green arrow) (H&E,x400). J) Infected animal and treated with

Baraka showing preserved (intact) lobular hepatic architecture with thin plates of almost

normal hepatocytes (black arrows), congested central vein (yellow arrow), mild collection

of interlobular lymphocytes (red arrow). K) Infected animal and treated with Chitosan

showing preserved (intact) lobular hepatic architecture with thin plates of almost normal

hepatocytes (black arrows). L) Infected animal and treated with Baraka and Chitosan

showing preserved (intact) lobular hepatic architecture with thin plates of almost normal

hepatocytes (black arrows) (H&E, x400).

Evaluation of toxic effects of nanoparticles in liver

1. Measurement of glutathione (GSH): Glutathione designation was calculated in mmol/g

after evaluating the yellow product at 405 nm.


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1.1. In immunocompetent mice

Table (3) the level of GSH in normal control group of mice was (3.46±0.97), the infected

group showed decrease to about (1.68±0.205) mmol/g which give highly statistically

significant (p<0.001). The least significant (p<0.01) in the level of GSH was observed in

group of mice treated with chitosan nanoparticles was (1.94±0.21) mmol/g, compared with

all infected treated mice groups. Where the highest significant (p<0.001) in the level of GSH

was observed in group of mice treated with Baraka and chitosan nanoparticles the level was

(2.94±0.15) mmol/g. All groups are demonstrated in Figure (1).

Table 3: Mean and standard deviation of hepatic glutathione of immunocompetent

groups (X±SD).

Groups GSH Hepatic

Normal 3.46±0.97

Infected 1.68± 0.205

Infected+ NTZ 2.43±0.18

Infected+ Baraka 2.04±0.18

Infected+ chitosan 1.94±0.21

Infected+ Baraka and chitosan 2.94±0.15

Figure (1): Hepatic GHS mean values in infected and infected treated groups of

immunocompetent mice.


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1.2. In immunosuppressed mice

Table (4) the level of GSH in normal control group of mice was (4.33±0.179), the infected

group showed decrease to about (1.93±0.115) mmol/g, which give highly statistically

significant (p<0.001). the highest significant (p<0.001) in the level of GSH was observed in

group of mice treated with Baraka and chitosan nanoparticles the level was (3.98±0.059)

mmol/g.

Table 4: Mean and standard deviation of hepatic glutathione of immunosuppressed

groups (X±SD).

Groups Hepatic GSH

Normal 4.33±0.179

Infected 1.93±0.115

Infected+NTZ 3.35±0.161

Infected+Baraka 2.39±0.291

Infected+ chitosan 2.31±0.084


Figure (2): Hepatic GHS mean values in infected and infected treated groups of

immunosuppressed mice.

2. Measurement of Malondialdehyde (MDA): Malondialdehyde designation was

calculated in mmol/g after evaluating pink product at 534 nm wave-length.

2.1.In immunocompetent mice

The level of hepatic MDA in immunocompetent groups are demonstrated in Figure (3). The


153


level of MDA in infected control group was (35.81±1.445) mmol/g, as compared to this of

normal control the level was (24.35±1.019) mmol/g. MDA levels were found to be decreased

in all treated groups as compared to infected control group. The highest reduction in the

level of MDA was in group treated with Baraka and chitosan nanoparticles the level was

(27.34±1.242) mmol/g (Table 5 and figure 3).

Table (5): Mean and standard deviation of hepatic Malondialdehyde of

immunocompetent groups (X±SD).

Groups Hepatic MDA

Normal 24.35±1.019

Infected 35.81±1.445

Infected +NTZ 29±0.547


Infected+chitosan nanoparticles 33.9±0.387

Infecte+ Baraka and chitosan 27.34±1.242

Figure (3): Hepatic MDA mean values in infected and infected treated groups of

immunocompetent mice.

2.2. In immunosuppressed mice

The level of hepatic MDA in immunosuppressed groups are demonstrated in Figure (11). The

level of MDA in infected control group was (45±4.867) mmol/g, as compared to this of

normal control the level was (21.8±0.678) mmol/g. MDA levels were found to be decreased

in all treated groups as compared to infected control group. The highest reduction in the

level of MDA was in group treated with Baraka and chitosan nanoparticles the level was

(26.8±0.971) mmol/g.


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Table (6): Mean and standard deviation of hepatic Malondialdehyde of

immunosuppressed groups (X±SD).

Groups Hepatic MDA

Normal 21.8±0.678

Infected 45±4.867

Infected+NTZ 32.1±0.61


Infected+chitosan 41.7±1.65


Figure (4): Hepatic MDA mean values in infected and infected treated groups of

immunosuppressed mice.

DISCUSSION

Cryptosporidium parvum is one of the main of a zoonotic protozoan parasite that causes food

and waterborne gastrointestinal disease (Nash et al., 2018). C. parvum has a wide host range,

in which cattle and small ruminants are the principal reservoir hosts (Feng et al., 2018).

Contact with cattle has been implicated as a major risk factor in the epidemiology of human

cryptosporidiosis (Ryan et al., 2014).

The microscopic detection of stool parasites through staining methods have high rate of

parasites identification (Crannell et al., 2014). The molecular technique such as PCR for the

laboratory diagnosis of cryptosporidiosis exhibits an outstanding specificity and sensitivity in

the detection and identification of these parasites at specie level (El-Badry et al., 2010).


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Nanotechnology is widely used in different fields of science. In particular, nanoparticles

(NPs) have attracted significant attentions as anti-parasitic agents in recent years (Khan et

al., 2015). Chitosan (CS) is a natural polysaccharide resulting from deacetylation of chitin in

alkaline conditions and is composed of N-acetyl-d-glucosamine and d-glucosamine units. In

addition to its harmless nature, CS includes striking properties such as antibacterial,

antitumor and antifungal properties as well as abilities to heal wounds and stimulate the

immune system (Goy et al., 2009 and De Marchi et al., 2017). N. sativa has antioxidant and

neuroprotective effects in addition to many other therapeutic activities such as antitumor,

immunopotentiation, anti-inflammatory, antiasthmatic and antimicrobial properties (Agrawal

et al., 1979).

In this study the outputs of oocysts were still high at the end of experimental work in infected

immunocompetent group (B1) which was (5700±139.1402) . This result is in agreements

with (Abdou et al., 2013) where Swiss albino mice continued to shed oocysts until day 30

(PI) with C. parvum. In addition Lacroix et al. (2001) found that the duration of oocysts

shedding was about 3 weeks. Also Matsui et al. (2001) reported that the interval which

covers the natural shedding period of Cryptosporidium infection in mice is about 24 days. As

well as patients with acute infection of cryptosporidiosis, acute watery diarrhoea can be

persistent and last for up to 5 weeks (Borad and Ward, 2010).

In infected immunosuppressed mice (C1), high shedding of oocysts were observed on the end

of experiment which was (7000±70.7106) oocysts/g of faeces. In a retrospective study it was

shown that the level of oocysts excretion was higher in Dex-immunosuppressed animals

(Baishanbo et al., 2005 and Certad et al., 2007). This result is in agreement with

Benamrouz et al. (2012) who showed that in Dex severe combined immunodeficiency

(SCID) mice inoculated with low inocula, the parasite excretion increased, reaching a mean

of oocysts shedding of more than 10,000 oocysts/g of faeces at 45 days (PI).

Finally in the present study using natural product (Nigella sativa) conjugated with chitosan

nanoparticles against cryptosporidiosis gave strong reactivity in reduction of oocysts excreted

in both immunocompetent and immunosuppressed, but in contrast the use of nitazoxanide

gave non significant in immunosuppressed. on the other side using Nigella sativa alone or

chitosan nanoparticles alone gave the nearby reduction number that are significantly.


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Histopathological examination of small intestine in the present work in infected groups (B1

and C1) showed changes in the morphology of the intestinal mucosa as a result of infection

and appearing numerous number of Cryptosporidium oocysts adhere to epithelial cell and

intraepithelial this is in agreement with O’Hara and Chen, 2011. When percepted sections

of small intestine in group (B2) treated with NTZ showed development in the

histopathological changes, but few number of Cryptosporidium oocysts intraepithelial

appeared. In (C2) appeared constant severe villous atrophy. Epithelial cells presented a loss

of their normal surface with absence of mucin. Depleting of goblet cell and mononuclear

inflammatory cells infiltration in the lamina propria. Also, no losing in number of

Cryptosporidium noticed. This confirms that nitazoxanide is not efficient in

immunosuppressed cryptosporidiosis.

In B3, B4, C3 and C4 groups noticed improvement in the histopathological changes in the

form of healing of intestinal mucosa, reduction of number of Cryptosporidium oocysts

compared to B1 and C1 where a few number of Cryptosporidium oocysts intraepithelial

noticed. But groups B5 and C5 treated with Baraka and chitosan showed completely

improvement in the histopathological examination of intestine and no Cryptosporidium

oocysts intraepithelial noticed.

Histopathological liver results of groups B2, B3 and B4 (Infected treated with NTZ, Baraka

and chitosan respectively) and groups C2, C3 and C4 (Immunosuppressed) showed

preserved (intact) lobular hepatic architecture with thin plates of almost normal hepatocytes,

congested central vein, small collection of interlobular lymphocytes, but groups B5 and C5

(infected treated with Baraka and chitosan) showed preserved (intact) lobular hepatic

architecture with thin plates of almost normal hepatocytes and in group C5 small collection

of interlobular lymphocytes noticed this is in accord with Aboelwafa and Yousef (2015)

found that supplementation of hydrocortisone-treated rats with thymol reversed most of the

biochemical, histological, and ultrastructural alterations of the liver. So, thymol has strong

ameliorative effect against hydrocortisone-induced oxidative stress injury in hepatic tissues.

In the present study, liver could be also considered site of accumulation of the nanoparticles

in the mice, in this context, a toxicological study was performed on this organ. Glutathione

(GSH) designation for liver of immunocompetent groups showed the level of GSH in normal

control group A1 of mice was (3.46±0.97), the infected group showed decrease to about

(1.68±0.205) mmol/g which give highly statistically significant (p<0.001). The highest


157


significant (p<0.001) in the level of GSH was observed in group B5 treated with Baraka and

chitosan nanoparticles was (2.94±0.15) mmol/g, but the hepatic GSH in immunosuppressed

groups showed in normal control group A2 of mice was (4.33±0.179), the infected group

showed decrease to about (1.93±0.115) mmol/g which give highly statistically significant

(p<0.001). The highest significant (p<0.001) in the level of GSH was observed in group B5

was (3.98±0.059) mmol/g.

The level of hepatic Malondialdehyde (MDA) in immunocompetent groups was highly

significant (p<0.001) in infected untreated group was (35.81±1.445) mmol/g, as compared to

normal control was (24.35±1.019) mmol/g. Hepatic MDA levels were found to be decreased

in all treated groups as compared to infected control group. The highest reduction was in

group treated with Baraka and chitosan nanoparticles the level was (27.34±1.242) mmol/g. In

immunosuppressed groups the level of hepatic MDA was highly significant (p<0.001) in

infected untreated group the level was (45±4.867) mmol/g, as compared to normal control the

level was (21.8±0.678) mmol/g. Hepatic MDA levels were found to be decreased in all

treated groups as compared to infected control group. The highest reduction was in group

treated with Baraka and chitosan nanoparticles the level was (26.8±0.971) mmol/g and

(32.1±0.61 and 39.6±0.493) mmol/g. In addition, Okeola et al. (2011) revealed that N. sativa

seeds had a strong antioxidant property and might be a good phytotherapeutic agent against

Plasmodium infection in malaria, this is agreement with our results. In the case of anti-

leishmanial effects of N. sativa, Nilforoushzadeh et al. (2010) indicated that combination of

honey and N. sativa extract in patients with Cutaneous leishmaniasis (CL) receiving

glucantime was more effective in treating and improving clinical signs than honey alone.

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