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Global Journal of Pharmacology 7 (1): 14-19, 2013
ISSN 1992-0075
© IDOSI Publications, 2013
DOI: 10.5829/idosi.gjp.2013.7.1.7383
Corresponding Author: Muhamed T. Osman, Centre of Pathology, Diagnostic and Research Laboratories,
Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sg. Buloh Campus,
47000 Sg Buloh, Selangor, Malaysia.
14
Nigella sativa Oil Has Significant Repairing Ability of Damaged Pancreatic
Tissue Occurs in Induced Type 1 Diabetes Mellitus
Afaf Jamal Ali Hmza, Effat Omar, Ariza Adnan and Muhamed T. Osman
Centre of Pathology, Diagnostic and Research Laboratories, Faculty of Medicine,
Universiti Teknologi MARA (UiTM), Sg. Buloh Campus, 47000 Sg Buloh, Selangor, Malaysia
Abstract: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that impairs production of insulin.
The disruption of insulin synthesis is caused by an autoimmune destruction of pancreatic islet cells. Nigella
sativa oil (NSO) was known as hypoglycemic agent in both types of diabetes but little known about its ability
of repairing the pancreatic damage occurred in T1DM. By intraperitoneal injection of a single dose of
streptozotocin (STZ) (65 mg/kg), T1DM was induced in overnight fasted 24 rats. They were equally divided into
four groups as following; (1) control group; (2) diabetic non treated group, (3) and (4) groups were treated with
different doses of NSO (0.2 and 0.4 ml/kg) respectively, for a period of 30 consecutive days. Blood glucose was
tested every morning through the experimental period. After completion the experimental protocol, blood
samples were collected and serum insulin was assayed using ELISA. The pancreatic tail was dissected and kept
in 10% formalin. The samples were processed using a tissue processor for histological study after H and E
staining. The control group showed normal cells in pancreatic islet of Langerhans. The diabetic group with no
treatment showed shrunken islets of Langerhans displaying degenerative and necrotic changes. Meanwhile,
the treatment with low dose NSO protected the majority of cells in the islet of Langerhans, however the high
dose NSO treatment showed a similar morphology as in normal control group (GA), so that resulted in
significant elevation of serum insulin level (p<0.005). The data suggests that NSO treatment has a therapeutic
effect against STZ induced T1DM rats.
Key words: Nigella sativa oil % Type1 diabetes mellitus % Blood glucose % Serum insulin % Pancreas
INTRODUCTION Streptozotocin (STZ) is the most commonly used
Type 1 Diabetes mellitus or insulin dependingtype 1 diabetes [6]. A single dose of 65mg/kg of STZ are
diabetes mellitus (T1DM) is the most severe form ofable to induced T1DM to experimental animals [6-7].
diabetes mellitus. According to WHO it accountsMore than 1000 different plants have been described
approximately 5-10% of all diabetic cases around thefor traditional treatment of diabetes [8]. Among these; the
world and usually appears during childhood [1]. T1DMNigella sativa (NS) which is a spice plant also knows as
is caused by an autoimmune destruction of pancreatic(black seed) has been used as an herbal medicine for more
$-cells, resulting in absolute deficiency in insulinthan 2000 years by different cultures to treat and prevent
production [2]. There are increase evidences that diabetes several diseases and illness. [9]. NS seeds have been
is combined by increase production of free radicals andreported to possess many biological activities including
reduction of antioxidant defense leading to developmentanti-inflammatory [10], anti-tumor [11], antihypertensive
of diabetes ant its complications [3]. Many mechanisms[12] and hypoglycemic properties [13, 14]. Moreover,
seem to be involved in the initiation of oxidative stressmany researchers have reported results supporting its
which has been reported in experimental diabetespotential value [15, 16]. NS is of great therapeutic benefit
animals as well as in T1DM [4]. Oxidative stress isin diabetic individuals and those with glucose intolerance,
involved in the origin of type 1 diabetes especiallyas it accentuates glucose-induced secretion of insulin,
through the destruction of pancreatic $ cells [5]. besides having a negative impact on glucose absorption
diabetogenic agent in the experimental animals to produce
Global J. Pharmacol., 7 (1): 14-19, 2013
15
from the intestinal mucosa [17]. NS was proved itintraperitoneal injection of a single dose of STZ (65 mg/kg
attenuates the damage to $-cells of the pancreas following body weight) to all animal groups except the normal
exposure to toxic elements such as cadmium [18].control group (25). STZ was dissolved in sodium citrate
Recently, it has been investigated that the effects of NSbuffer solution (pH 4.5) immediately before use. The rats
oil (NSO) on some physiological parameters inwith blood glucose above 13.9 mmol/L (250 mg/dL), which
streptozotocin (STZ)-induced diabetic rats. STZ inducedlasted for at least three days, were considered as type 1
diabetic rats showed a highly significant increase indiabetic rat [21-22, 25].
the levels of blood glucose compared to the controls.The experimental animal groups were divided into 4
Administration of NSO to diabetic rats resulted in agroups (6 rats each). The groups are: Group A (Normal
significant decrease in blood glucose levels after threecontrol group received 65mg/bw sodium citrate buffer),
weeks compared to untreated diabetic rats, indicated thatGroup B (Diabetic control group treated with STZ only 65
the oil of NS possess hypoglycaemic effect in STZ-mg/kg), Group C (Diabetic rats received i.p. NSO low dose
induced diabetic rats [19]. 0.2 ml/kg) and Group D (Diabetic rats received i.p. NSO
Many studies have been carried out to evaluate thehigh dose 0.4 ml/kg). Blood glucose was tested every
role of NSO for management of diabetes [20], but the exact morning (at 8 am) through the experimental period (30
antidiabetic mechanism is not yet established regardingdays). The blood glucose level was tested by using
type 1 diabetes mellitus, however, in this present studyglucometer purchased from (Roche, USA).
the effect of NSO on the reversal process of histological
damage caused by the disease process of IDDM wereLaboratory Tests: Blood samples were collected from
evaluated. overnight fasted rats, after completion of 30 days of
MATERIALS AND METHODS a chamber containing diethyl ether. Blood was collected
Experimental Animals: Twenty four Male Sprague-collection, the blood was transferred into fresh tube and
Dawley rats with an average weight of 150-250g and ancentrifuged at 3000 rpm for 10 minutes. The sera were
average age of 12-16 weeks were used throughout thestored at –80 °C until analysis. Serum was assayed for
experiments. The animals were obtained from Nano Lifeinsulin level by using enzyme-linked immunosorbent
Quest Company. Ethical clearance for performing theassay (ELISA) (USCNK, CHINA).
experiment on animals was approved by Animal Care and The animals were dissected after scarification and the
Use Committee (ACUC), Faculty of Medicine, Universitipancreas was taken for histology. This organ was placed
Teknologi MARA UiTM (ACUC-2/11). The rats werein 10% formalin for fixation. Paraffin was used to
acclimatized for a period of 21 days in the Laboratoryembedding the pancreas and then stained with
Animals Care Unit (LACU), Faculty of Medicine, Sg Bulohhematoxylin and eosin (H and E). The preparations were
Campus, Universiti Teknologi MARA (UiTM) Malaysia.evaluated by Olympus multiheaded microscope and
A standard environmental condition such as temperaturephotographed by camera (Optiphot 2; Nikon, Tokyo,
(20-22°C), relative humidity (45-55%) and 12 hrs. dark/lightJapan).
cycles was maintained. The animals were fed daily with
rodent pellet diet and tap water ad-libitum under strictStatistical Analysis: Data were analyzed by comparing
hygienic conditions. values for different treatment groups with the values for
Chemicals: The Streptozotocin (STZ) (2-deoxy-2-expressed as mean ± SD. The significant differences
({[methyl (nitroso) amino] carbonyl} amino)-$-D-among values were analyzed using by one way analysis
glucopyranose) used in the present study was purchasedof variance (ANOVA) carried out by SPSS 16 software
from Sigma, Germany. The Nigella sativa oil wasfollowed coupled with post-hoc least a p-value of < 0.05
produced by Kausar, Iran. It is a pure preparation, with no was considered as statistically significance.
additive. The oil was administered once a day by
intraperitoneal (i.p) injection at doses of either 0.2 ml/kg or RESULTS AND DISCUSSION
0.4 ml/kg for 30 days.
Induction of Type 1 Diabetes and Experimental Design:effect of NSO on daily blood glucose and serum levels by
T1DM was induced to overnight fasted animals byusing two different doses during 4 weeks of experimental.
experimental protocols. The animals were anesthetized in
from the heart by the cardiac puncture. Immediately after
the positive and negative control groups. Results are
Biochemical Results: In this study we had evaluated the
0
5
10
15
20
25
0 days 10 days 20 days 30 days
DAYS
Fasting blood Glucose (mmol/l
control
STZ
0.2ml/kg oil
0.4ml/kg oil
0
0.5
1
1.5
2
2.5
(A) control (B) STZ (C ) NSO
0.2ml/kg
0
0.5
1
1.5
2
2.5
(A) control (B) STZ (D) NSO 0.4
ml/kg
Global J. Pharmacol., 7 (1): 14-19, 2013
16
Fig. 1: Effects of NSO administration on Blood Glucose level. GA; control group, GB; untreated diabetic rats;
GC, diabetic rats treated with (0.2 ml/kg) NSO. GD, diabetic rats treated with (0.4 ml/kg) NSO. Data are expressed
as mean ±SD.
Fig. 2: Effects of NSO administration on level of Insulin production. GA; control group, GB; untreated diabetic rats; GC,
diabetic rats treated with (0.2 ml/kg) NSO. GD, diabetic rats treated with (0.4 ml/kg) NSO. Data are expressed as
mean ±SD.
After 10 days treatment with NSO 0.2 ml/kg, there was adifferent doses (0.2 - 0.4 ml/kg) cause a significant
slight, but non-significant decrease in blood glucoseincrease in serum insulin level after 30 days of treatment
levels compared with those in the untreated diabetic(p> 0.05) (Figure 2).
group (Group B). Administration of NSO (0.2ml/kg)
resulted in a significant lowering of elevated bloodHistological Results: In normal control rat group (GA) the
glucose levels after 30 days treatment comparedhistological sections showed normal pancreatic structure.
with those in the untreated diabetic group (p> 0.05).The islet of Langerhans appeared regular in shape
Moreover, treatment of diabetic rats with NSO (0.4 ml/kg) surrounded by thin capsule of connective tissue. The
resulted in a significant decrease in blood glucose levelsclusters of cells are embedded in the pancreatic exocrine
compared with the untreated diabetic group after 10 daystissue. The cells are polygonal cells on shape and have
treatment. After 30 days, blood glucose levels hadregular nuclei (Figure 3.a). However, in STZ diabetic rats
decreased to levels that did not differ significantly fromgroup with no treatment (GB), the findings on histologic
basal levels seen in the control group (p> 0.05) (Figure 1). sections of pancreatic tissues were degenerative, necrotic
The present study has clearly shown that NSO at twochanges and shrinkage in the islets of Langerhans.
Global J. Pharmacol., 7 (1): 14-19, 2013
17
(a) (b)
(c) (d)
Fig. 3:a. Control group, b. STZ Diabetic group, c. 0.2 ml/kg (NSO) treated, d. 0.4 ml/kg (NSO) treated group.
The islets were relatively small, atrophied and showed aDISCUSSION
reduction in the number of polygonal islet cells. The
nucleuses of necrotic cells are pynotic. The atrophied and The present study have shown that NSO produced
degenerated cells show hydropic degeneration and lossa significant, consistent and time-dependent decrease in
of granules within the cytoplasm. The cytoplasm is darkblood glucose levels and elevate insulin level production
and esonophilic (Figure 3.b). in STZ-T1DM induced diabetic rats compared with the
In (GC) diabetic animals treatment with 0.2 ml/kg NSO,untreated diabetic animals. The hypoglycemic effect
the histological sections showed that the islets wereobserved in the present study became more significant
relatively small in size and irregular in shape comparedafter daily administration of the oil for one month to the
with normal control (Figure 3.c). There were less hydropic diabetic animals. These results were consistent with other
degeneration, degranulation and necrosis in the islet cells. studies like Houcher et al., 2007, who showed that the use
More viable polygonal islet cells were observed compared of the commercial oil at a dose of 2.5 mL/ kg per day for 25
with in GB. Meanwhile, islets of Langerhans of (GD) rats days significantly reduced blood glucose [5]. Data from
treated with a high dose of NSO (0.4 ml/kg), showedother experimental study reported that blood glucose
similar appearance as in GA, there were more viable cells lowering effect of black seed oil was due to improved
present and less hydropic degeneration, degranulated andinsulin insensitivity in diabetic rats [23]. A study done by
necrosis compared with GB. The polygonal islet cellsother investigators reported that treatment with NSO
appeared with nuclei of various sizes (Figure 3.d). commenced 6 weeks after induction of diabetes at a dose
Global J. Pharmacol., 7 (1): 14-19, 2013
18
of 400 mg/kg body weight by gastric lavage reducedislet of Langerhans, however the high dose of NSO
blood glucose after the first, second, third and fourthtreatment showed a similar morphology as in normal
weeks of treatment. They indicated that thecontrol group, so that resulted in significant elevation
hypoglycaemic effect of NSO is due to, at least in part, aof serum insulin level. The data suggested that NSO
decrease in hepatic gluconeogenesis [24]. treatment has a therapeutic effect against induced
In the present study we proved that NSO hasT1DM rats.
significant increase on serum insulin levels, this result
may be due to the effect of thymoqionone, a majorACKNOWLEDGMENT
component of NSO, These results are consistent with
pervious study that have shown that daily gastricThis research was completely funded by international
administration of 80 mg/ kg thymoquinone, for 45 daysgrant from Libyan Embassy in Malaysia (RMI/INT
produces significant increase in insulin levels in STZ-4/2011). The authors would like to thank all staff of IMMB
diabetic rats [25]. From in vitro study, scientificinstitute and CPDRL Laboratories, Faculty of
investigations to the isolated pancreatic Islets ofMedicine, Universiti Teknologi MARA Malaysia for
Langerhans have proven that the N. sativa extract causestechnical help.
direct stimulation of insulin release [26].
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