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Research Article
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Influence of Nigella sativa Fixed Oil on Some Blood Parameters and Histopathology of Skin in Staphylococcal-Infected BALB/c Mice |
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Mariam A. Abu- Al-Basal
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ABSTRACT
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Nigella sativa has been used for a long time in Jordanian folk medicine to treat skin diseases like microbial infections and inflammation. Therefore, the present study was conducted to assess the healing efficacy of petroleum ether extract of Nigella sativa seeds (fixed oil) on staphylococcal-infected skin. Male BALB/c mice were infected with 100 μL of Staphylococcus aureus (ATCC 6538) suspension at a dose of 108 colony forming unit/mouse into shaved mild dorsal skin. Application of treatments for each group (100 μL sterile saline, 100 μL chloramphenicol (10 μg/mouse) and Nigella sativa fixed oil at a dose of 50, 100 or 150 μL/mouse) was performed at the site of infection twice a day for two consecutive days after 3 h of infection. At day 3 and 5 after infection, total White Blood Cells (WBCs) count; differential and absolute differential WBC counts and the number of viable bacteria present in the skin area were measured. At day 5 after infection, the animals were sacrificed and the histology of skin was examined. Results indicated that fixed oil of Nigella sativa seeds enhance healing of staphylococcal-infected skin by reducing total and absolute differential WBC counts, local infection and inflammation, bacterial expansion and tissue impairment. These effects provide scientific basis for the use of Nigella sativa in traditional medicine to treat skin infections and inflammations.
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Received: July 01, 2011;
Accepted: October 14, 2011;
Published: December 02, 2011
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INTRODUCTION
Nigella sativa L. (Family Ranunculaceae), commonly known as black seed,
is an annual herbaceous plant cultivated in different parts of the world, mainly
in countries bordering the Mediterranean Sea (Bourgou et
al., 2010). Seeds of Nigella sativa are frequently added to bread
and pickles as a flavouring agent. They have been used in Jordan and Palestine,
as a natural remedy, for health promotion and also to treat many diseases and
ailments. For example, fever, allergies, arthritis, hypercholesterolemia, lung
diseases, common cold, high blood pressure, immune disorders and skin diseases
including psoriasis, eczema, hair loss, microbial infections and inflammations
(Oran and Al-Eisawi, 1998; Lev and
Amar, 2002; Said et al., 2002; Abu-Irmaileh
and Afifi, 2003). Previous studies confirmed that the seed extract of Nigella
sativa possesses a remarkable effect in the treatment of many health problems.
These include immune stimulation (Salem, 2005), anti-inflammatory
(Ali and Blunden, 2003; El Gazzar
et al., 2006), anti-oxidant (Burits and Bucar,
2000), anti-microbial (Mashhadian and Rakhshandeh, 2005),
anti-tumor (Amara et al., 2008) and wound healing
stimulatory effects (Abu-Al-Basal, 2001).
Staphylococcus aureus is one of the most important human and veterinary
pathogens that cause infections ranging from benign to life threatening diseases
(Francois et al., 2010). The high incidence of
methicillin resistance in hospitals and the emergence of vancomycin-intermediate
Staphylococcus aureus complicated the prevention and treatment of serious
infections due to staphylococci (Griffith et al.,
2003). Skin and soft tissue abscesses and infected cysts are among the most
common infections caused by these organisms that may lead to serious local and
systemic complications (Brook, 2010). A number of experimental
models of staphylococcal skin infections have been previously described and
these showed that bacteria can readily invade the epidermis and dermis to produce
localized infection and cause a variety of pathologic changes in the skin, including
impetigo, furuncles, subcutaneous abscesses, scalded skin syndrome and necrotizing
fasciitis (Hahn et al., 2009). In fact, infections
due to multi-resistant Gram-positive organisms are increasing in frequency and
hence their early recognition, proper medical treatment and surgical management
are of primary importance and urgently required. Consequently, new treatment
strategies is crucial to deal with this emerging issue involving the use of
medicinal plants as a source for new therapeutic agents aimed at the management
and treatment of skin infections.
The oil and active ingredients of Nigella sativa seeds have been proved
to exert promising inhibitory effects against many strains of bacteria, including
those known to be highly resistance to drugs. (Hanafy and
Hatem, 1991; Salman et al., 2008). In a recent
study, organic solvents were used to extract fixed oil constituents from seeds
of Nigella sativa including petroleum ether. That had the most powerful
significant inhibitory effects both in vitro and in vivo against
clinical isolates from patients with skin wound infections (Abu-Al-Basal,
2009). As a matter of fact, in vivo studies on strains of pathogenic
microbes are scarce and fixed oil extracted by petroleum ether was never used
before at least with the seeds of Nigella sativa. Therefore, in a continuation
with efforts of previous study, the present research was followed for investigating
the effect of petroleum ether extract of Nigella sativa seeds on some
blood parameters and histopathology of skin in Staphylococcus aureus
infected BALB/c mice.
MATERIALS AND METHODS Plant selection: Seeds of Nigella sativa were purchased in March, 2011 from a herbal shop in Irbid, Jordan. The seeds were authenticated at the Herbarium of the Department of Biological Sciences, University of Jordan, Amman, Jordan. A voucher specimen (NO.NSJD-3-011) has been deposited in the Department of Biological Sciences, Faculty of Science, Al-al-Bayt University, Mafraq, Jordan for future reference.
Plant extract: Seeds of Nigella sativa were ground into slightly
coarse powder using electric blender. Fixed oil was prepared by soaking 100
g of the dried powder in 500 mL of petroleum ether (40-60°C) using a conical
flask plugged with cotton wool. The mixture was kept at room temperature for
48 h under continuous shaking (memmert shaker SV 1422, Germany) and filtered
through Whatman filter paper No. 2 under vacuum. The filtrate was evaporated
to dryness by Rota vapor (Ruchl R-114, Switzerland), where the rotary water
bath (Ruchl B-480) was adjusted to 55°C. The extract was kept overnight
under vacuum fume hood to obtain a constant dry weight and later stored in closed
vessel at 4°C in a refrigerator for further use.
Animals: Male BALB/c mice six week of age (18-20 g) were obtained from
the animal house of the Department of Biological Sciences, Yarmouk University,
Irbid, Jordan. Mice were kept under specific pathogenic-free conditions, housed,
fed and treated in accordance with the international guidelines principles of
laboratory animal use and care (Hedrich and Bullock, 2006).
They were maintained on standard pellet diet and water ad libitum for
two weeks to be acclimatized prior to the investigation.
Bacterial strain: Staphylococcus aureus (ATCC 6538) was kindly
obtained from Dar Al Dawa, Na our, Jordan. Bacterial culture was routinely grown
in nutrient broth at 37°C for 24 h. Active culture was harvested by centrifugation,
washed twice and resuspended in sterile saline. The bacterial suspension was
then diluted with sterile saline to 1x109 colony forming unit (CFU)
mL-1 according to method of Kuo et al.
(2005).
Infection of animals: Infection of BALB/c mice with 100 mL of Staphylococcus
aureus (ATCC 6538) suspension at a dose of 1 x 108 CFU/mouse
was conducted into shaved mid dorsal skin as described by Godin
et al. (2005).
Mice grouping and treatment: Infected mice were randomly divided into control and experimental groups of twelve mice each. Various doses of N. sativa fixed oil were prepared and preliminary tested for their tolerance in BALB/c mice to select the optimum dose intended for the treatment of experimental animals. Application of treatments for control or experimental groups was performed at the site of primary infection by cutaneous injection twice a day for two consecutive days using insulin syringes, after 3 h of infection. Group I: uninfected mice left without treatment, as normal control. Group II: infected mice treated with vehicle (100 μL sterile saline), as negative control. Group III: infected mice treated with 100 μL chloramphenicol antibiotic solution (10 μg/mouse dissolved in sterile saline), as positive control. Group IV: infected mice treated with 50 μL pure fixed oil. Group V: infected mice treated with 100 μL pure fixed oil. Group VI: infected mice treated with 150 μL pure fixed oil. Mice were housed and maintained on normal food and water ad libitum during the whole period of experiment.
Blood sampling and hematological methods: All blood samples were withdrawn
via cardiac puncture at day 3 and 5 after infection and analyzed for total,
differential and absolute differential White Blood Cell (WBC) counts by manual standard hematological
methods as described by Doeing et al. (2003)
and McGarry et al. (2010).
Bacterial counts: Counts of viable bacteria in mice infected skin at
day 3 and 5 after infection was performed as previously described by Kuo
et al. (2005) and Godin et al. (2005).
Behavioral responses of mice: Behavioral responses of mice were followed
as proposed by Kugelberg et al. (2005). The mice
were monitored at least twice a day for signs of fatigue, stress or aggressiveness
and the weight was recorded before and after each experiment.
Histology: Skin samples from each group were obtained at day 5 after
infection. Samples were dissected, fixed in 10% neutral formalin, dehydrated
in ascending grades of alcohol and imbedded in paraffin wax. Five-micrometer
sections were stained with hematoxylin and eosin for histological evaluation
and Gram's crystal violet solution for the identification of bacteria under
light microscope. Semi quantitative scoring system was carried out to characterize
the size and density of bacteria presence, the inflammatory response and infiltrating
cells in different layers of staphylococcal-infected skin (epidermis, dermis,
subcutis and muscular tissue) in BALB/c mice as described previously by Kugelberg
et al. (2005).
Statistical analysis: Results are expressed as Means± SEM (Standard Error of the Mean). Comparisons between groups were performed by using paired student's t-test on a statistical software package (SPSS). Differences were considered significant, if p value is less than 0.05. RESULTS
Blood parameters: Staphylococcal-infected mice treated with Nigella
sativa fixed oil exhibited a dose-dependent significant decrease (p<0.05)
in total and absolute differential White Blood Cell (WBC) counts compared to
those of control groups (Tables 1, 2). Remarkable
reduction in total WBC counts was observed at a dose of 150 μL of Nigella
sativa fixed oil/mouse (Group VI: 10.12±0.38x103 cells
μL-1), when compared to vehicle-treated control (Group II: 15.48±0.21x103
cells μL-1), at day 3 after infection (Table 1).
The same oil-treated group showed further significant decrease in total WBC
counts (Group VI: 09.20±0.31x103 cells μL-1)
which was very close to the level of normal control (Group I: 08.90±0.21x103
cells μL-1) but slightly higher than that of chloramphenicol-treated
mice (Group III: 08.20±0.21x103 cells μL-1),
at day 5 after infection (Table 2). Similar significant effect
was noticed at the same dose of oil in absolute differential WBC counts of treated
mice at day 3 and 5 after infection (Tables 1, 2).
This was highly illuminated by considerable decrease in absolute differential
counts of basophil to the level of normal control (Group I: 00.04±0.01x103
cells μL-1). Though, it was significantly much lower than those
of chloramphenicol-treated mice (Group III: 00.07±0.02x103
cells μL-1) and vehicle-treated mice (Group II: 00.09±0.03x103
cells μL-1), at day 5 after infection (Table 2).
In vivo anti-bacterial effect: The effect of Nigella sativa fixed
oil on Staphylococcus aureus (ATCC 6538) growth in the skin of mice at
day 3 and 5 after infection was shown in Fig. 1. Counts of
viable bacteria decreased considerably to a significant level (p<0.05) at
a dose-dependent manner in Nigella sativa fixed oil-treated groups. The
most significant anti-bacterial effect was observed at a dose of 150 μL
fixed oil/mouse (bars labeled 5), when compared to control groups.
Table 1: |
Influence of Nigella sativa fixed oil on some blood
parameters of staphylococcal-infected BALB/c mice at day 3 after cutaneous
infection |
 |
Data are expressed as Means±SEM for six mice in each
group. I: Uninfected normal control; II: Infected vehicle-treated group
(100 μL of sterile saline/mouse); III: infected chloramphenicol-treated
group (100 μL of antibiotic solution at a dose of 10 μg/mouse);
IV: Infected fixed oil-treated group (50 μL of Nigella sativa
petroleum ether extract/mouse); V: Infected fixed oil-treated group (100
μL of Nigella sativa petroleum ether extract /mouse); VI: Infected
fixed oil-treated group (150 μL of Nigella sativa petroleum
ether extract/mouse). aStatistically significant when compared
to uninfected normal control group (I) at p<0.05. b Statistically
significant when compared to infected vehicle-treated group (II) at p<0.05.
cStatistically significant when compared to infected chloramphenicol-treated
group (III) at p<0.05 |
Table 2: |
Influence of Nigella sativa fixed oil on some blood
parameters of staphylococcal-infected BALB/c mice at day 5 after cutaneous
infection |
 |
Data are expressed as Means±SEM for six mice in each
group. I: Uninfected normal control; II: Infected vehicle-treated group
(100 μL of sterile saline/mouse); III: infected chloramphenicol-treated
group (100 μL of antibiotic solution at a dose of 10 μg/mouse);
IV: Infected fixed oil-treated group (50 μL of Nigella sativa
petroleum ether extract/mouse); V: Infected fixed oil-treated group (100
μL of Nigella sativa petroleum ether extract /mouse); VI: Infected
fixed oil-treated group (150 μL of Nigella sativa petroleum
ether extract /mouse). aStatistically significant when compared
to uninfected normal control group (I) at p<0.05. bStatistically
significant when compared to infected vehicle-treated group (II) at p<0.05.
c Statistically significant when compared to infected chloramphenicol-treated
group (III) at p<0.05 |
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Fig. 1: |
Counts of viable Staphylococcus aureus in the skin
of BALB/c mice at day 3 and 5 after infection. Bars labeled 1: infected
vehicle-treated group (100 μL of sterile saline/mouse). Bars labeled
2: infected chloramphenicol-treated group (100 μL of antibiotic solution
at a dose of 10 μg/mouse). Bars labeled 3: infected fixed oil-treated
group (50 μL of Nigella sativa petroleum ether extract/mouse).
Bars labeled 4: infected fixed oil-treated group (100 μL of Nigella
sativa petroleum ether extract/mouse). Bars labeled 5: infected fixed
oil-treated group (150 μL of Nigella sativa petroleum ether
extract/mouse). Data are expressed as Means±SEM for six mice in each
group. a: Statistically significant when compared to infected
vehicle-treated group (bars labeled 1) at p<0.05. b: Statistically
significant when compared to infected chloramphenicol-treated group (bars
labeled 2) at p<0.05 |
This potent dose of fixed oil displayed significant reduction in bacterial
count reached to around 42x105 cfu g-1 of tissue, at day
3 after infection. In contrast, vehicle- and chloramphenicol-treated groups
had elevated bacterial counts, to around 86x105 and 56x105
cfu g-1 of tissue, respectively. Similar effect was observed at day
5 after infection, yet the bacterial numbers declined further to a significant
level in oil-treated group and reached approximately 4x105 cfu g-1
of tissue. On the contrary, mice treated with chloramphenicol had much higher
bacterial counts (11x105 cfu g-1 of tissue) which indicates
that Nigella sativa fixed oil is more effective in treating staphylococcal-infected
skin.
DISCUSSION
In the present study, the significant anti-bacterial properties of the fixed
oil (Fig. 1) could be related to potent bactericidal and/or
immune stimulating/modulating agents present in the oil. Chemical analysis of
petroleum ether extract of Nigella sativa seeds revealed the presence
of considerable amount of fatty acids in the oil. Linoleic acid (55.6%) and
oleic acid (23.4%) are the major components of unsaturated fatty acids that
constituting 82.5% of the total fatty acids identified in the oil (Nickavar
et al., 2003). Previous studies confirmed that lipids have an inhibitory
effect against pathogenic infections in skin and mucous membranes. For example,
long chain unsaturated fatty acids, medium chain saturated fatty acids and their
monoglycerides are the most active against pathogenic bacteria and viruses by
killing them rapidly in large amount.
Table 3: |
Microscopic evaluation of Nigella sativa fixed oil
effects on the size and density of the inflammatory response, infiltrating
cells and presence of bacteria in different layers of staphylococcal-infected
skin of BALB/c at day 5 after infection |
 |
Data are expressed as Means±SEM for six mice in each
group. I: Uninfected normal control; II: Infected vehicle-treated group
(100 μL of sterile saline/mouse); III: Infected chloramphenicol-treated
group (100 μL of antibiotic solution at a dose of 10 μg/mouse);
IV: Infected fixed oil-treated group (50 μL of Nigella sativa
petroleum ether extract/mouse); V: Infected fixed oil-treated group (100
μL of Nigella sativa petroleum ether extract /mouse); VI: Infected
fixed oil-treated group (150 μL of Nigella sativa petroleum
ether extract /mouse). ND: Not detected; +: Little/few; ++: Moderate; +++:
Severe/abundant |
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Fig. 2(a-f): |
Hematoxylin and eosin histological sections of Staphylococcal-infected
skin obtained from the controls and Nigella sativa treated BALB/c
mice at day 5 after infection. (a) Uninfected normal skin; (b) infected
vehicle-treated skin (100 μL of sterile saline/mouse); (c) infected
chloramphenicol-treated skin (100 μL of antibiotic solution at a dose
of 10 μg/mouse); (d) infected fixed oil-treated skin (50 μL of
Nigella sativa petroleum ether extract/mouse); (e) infected fixed
oil-treated skin (100 μL of Nigella sativa petroleum ether extract
/mouse) and (f) infected fixed oil-treated skin (150 μL of Nigella
sativa petroleum ether extract/mouse). Epidermis (e); dermis (d); hair
follicle (hf); striated muscle (m), the subcutis (sc), a layer of adipose
tissue; sebaceous gland (s); infiltration of inflammatory cells (white arrow
head). Scale bar: 50 μm (a-f) |
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Fig. 3(a-f): |
Hematoxylin and eosin histological sections of Staphylococcal-infected
skin obtained from the controls and Nigella sativa treated BALB/c
mice, revealing the persistence of inflammatory and immune cells at day
5 after infection. (a and b) Infected vehicle-treated skin (100 μL
of sterile saline/mouse); (c) infected chloramphenicol-treated skin (100
μL of antibiotic solution at a dose of 10 μg/mouse); (d) infected
fixed oil-treated skin (50 μL of Nigella sativa petroleum ether
extract/mouse); (e) infected fixed oil-treated skin (100 μL of Nigella
sativa petroleum ether extract /mouse); (f) infected fixed oil-treated
skin (150 μL of Nigella sativa petroleum ether extract /mouse).
Eosinophils (e); lymphocytes (l); active macrophages with variable appearance
(m); neutrophils (n). Persistence of large numbers of inflammatory cells
in considerably infected skin (black arrow head). Scale bar: 200 μm
(a-f) |
This anti-bacterial effect of Free Fatty Acids (FFAs) noticed to be increased
significantly by the addition of a double bond, and hence unsaturated fatty
acids, such as linoleic acids (C18:2) are more effective against Gram-positive
bacteria. A skin lipid was also found to destroy many species of microorganisms,
for instance the FFAs of stratum corneum in the epidermis, that known to participate
in skin immune defense by providing a physical barrier to bacterial invasion,
exhibiting substantial anti-bacterial effect against Staphylococcus aureus
(Thormar and Hilmarsson, 2007).
The significant changes in total and absolute differential WBC counts (Tables
1, 2) and marked histopathological observations (Table
3, Fig. 3) in fixed oil treated mice revealed improvement
in healing staphylococcal-infected skin, when compared to infected vehicle-treated
and normal untreated controls. However, the oil healing effect appeared to be
dose dependent and the clear potentiality was typically observed at a dose of
150 μL fixed oil/mouse, which proved to be more effective than the standard
drug, chloramphenicol in treating infection. This efficient dose of the oil
attenuates bacterium-induced inflammation by reducing the size and density of
inflammatory response, infiltrating cells and presence of bacteria in different
layers of infected skin at day 5 after infection (Table 3,
Fig. 3). Along with significant decrease in total and absolute
differential WBC counts that reached near to normal levels in the treated mice
(Table 2). Which indicate that the constituents of the oil
might have an impact on the host-bacterium interrelationship, and as a consequence
an effect on some mediators of inflammation. Such as oxidants, cytokines and
lytic enzymes secreted by neutrophils and macrophages and part of the inflammatory
response is the recruitment of these cells to site of infection (Salem,
2005). Reduced inflammation in the oil-treated mice seems also to be attributed
to the inhibition of eicosanoid generation in leukocytes as suggested by the
study of Houghton et al. (1995). On the contrary,
acute inflammatory response was high-lighted by significant increase in total
and absolute differential WBC counts (Table 2) along with
marked dense infiltrate of cells, mainly neutrophils, to tissues of infected
skin (Fig. 2) in the vehicle-treated mice at day 5 after infection,
when compared to normal untreated mice. This may be attributed to the increase
in the rate of neutrophil production at the bone marrow leading to neutrophil
egress into tissue layers of infected skin that appeared intensely later at
day 5 after infection, as a result of inflammation (Von
Vietinghoff and Ley, 2008). Neutrophils are the first defense cells against
invading bacteria and are rapidly recruited to the site of infection by chemotactic
factors. The major roles played by neutrophils in inflammatory and immune responses
are phagocytosis and killing of bacteria via the generation of reactive oxygen
intermediates and the release of lytic enzymes stored in granules (Molne
et al., 2000). Furthermore, marked histological findings in oil-treated
mice (Table 3, Fig. 3) indicated that rapid
recruitment of neutrophils and phagocytosis, in the initial stages of infection,
is critical for the clearance and inferior expansion of infection. That was
clearly demonstrated in vehicle-treated mice, in which the skin infection continues
despite the abundant occurrence of neutrophilic granulocytes at late stage of
inflammation (day 5 after infection) due to Staphylococcus aureus capability
in interfering chemotaxis and impairing function of neutrophils leading to disseminate
pathogen extensively in skin tissue layers and failure in the resolution of
inflammation (Iwatsuki et al., 2006; Hirsch
et al., 2008).
Late stage of inflammation is maintained by more complex interactions among
several cell types including lymphocytes and macrophages that dominate the inflammation
noticed in oil-treated mice at day 5 after infection (Fig. 3).
Tissue macrophages and newly recruited monocytes which differentiate into macrophages,
also function in phagocytosis and hence killing of bacteria. In addition, they
contribute to tissue repair and act as antigen presenting cells that are required
for the induction of specific immune responses (Young et
al., 2006). In response to inflammation, infected mice have increased
monocyte and lymphocyte counts and these are significantly decreased toward
the normal values after treatment with oil, as a result of increased recruitment
to infected skin. Similar effect observed in absolute differential counts of
eosinophil and basophil, however the standard drug showed significant increase
in basophil counts, revealing the occurrence of allergic skin reactions that
may be due to repeated treatments of antibiotic (Table 2,
Fig. 3) (Abbas and Lichtman, 2011).
These results clearly demonstrate that fixed oil constituents act synergistically
in reducing inflammation and stimulating immune response in staphylococcal-infected
mice. Previous findings displayed that Nigella sativa exerted a stimulatory
effect on macrophages through interleukin (IL)-3 which was secreted by T-lymphocytes
under the effect of fixed oil (Swamy and Tan, 2000).
It also increases the percentages of CD4-positive subsets of T-lymphocytes,
which activates macrophages and regulate their function (Szejda
et al., 1984). Production of IL-1α in a medium containing fixed
oil and macrophages confirmed that the former has a stimulatory effect on macrophage
either directly or through IL-3 (Haq et al., 1995).
In addition, oil exhibited stimulatory effect on peritoneal macrophages in streptozotocin-induced
diabetic hamsters by increasing phagocytic activity either directly or via stimulation
of lymphocytes (Fararh et al., 2004). In contrast,
treatment with Nigella sativa oil induced a 2-fold decrease in antibody
production in response to typhoid vaccination as compared with control rats
(Islam et al., 2004). Accordingly, Salem
(2005) suggested that crude extracts of Nigella sativa and its active
constituent, thymoquinone, may enhance T cell-mediated immune response but suppress
humoral immune response, and this required further scientific verification.
The oil healing effect was also marked by the absence of local signs of infection
and inflammation, such as necrotic regions and accumulation of edema in infected
treated skin (Fig. 2). That may be related to antioxidant
effect of the oil and contribution of active leukocytes in tissue repair at
the site of infection. The production of free radicals at or around the infected
site may delays the healing process through the destruction of lipids, proteins
and extracellular matrix elements (Burits and Bucar, 2000).
Constituents of the oil, having antioxidant property, are able to inhibit lipid
peroxidation and activate antioxidant enzymes, and thus protect tissue impairment
and improve healing of infected skin, as confirmed previously (El-Dakhakhny
et al., 2002; Hosseinzadeh et al., 2007;
Alkharfy et al., 2011).
CONCLUSION
The results of this study revealed that fixed oil of Nigella sativa
seeds might has potent bactericidal, anti-inflammatory, immune stimulating and/or
antioxidant agents that improve healing of staphylococcal-infected skin in BALB/c
mice, by inhibiting pathogenic growth and expansion, reducing inflammation and
prevent tissue impairment. These effects provide scientific basis for the use
of Nigella sativa in traditional medicine to treat skin infections and
inflammations.
ACKNOWLEDGMENTS The author acknowledge Dar Al Dawa, Na'our, Jordan, for kind contribution in supplying bacterial strain, Wasfi Al-bekearat for his support and technical assistance, Al-al-Bayt University, Department of Biological Sciences, Mafraq, Jordan, Ismail Zayed for the help in photographing histological sections, Yarmouk University, Department of Biological Sciences, Irbid, Jordan and Al-al-Bayt University, Mafraq, Jordan, for providing necessary facilities to conduct this work.
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