Tazneem and Abdullah khan.

Int J Pharm 2017; 7(1): 109-115 ISSN 2249-1848

International Journal of Pharmacy

Journal Homepage: http://www.pharmascholars.com

Original Article CODEN: IJPNL6

EFFECT OF NIGELLA SATIVA IN EXPERIMENTALLY INDUCED

INFLAMMATORY BOWEL DISEASE IN RATS
Tazneem. B*1 , Abdullah khan2
1
Research Scholar, Mewar University, Rajasthan. India.
2
Research Supervisor, Mewar University, Rajasthan. India.

*Corresponding author e-mail: [email protected], [email protected].

Received on: 07-11-2016; Revised on: 29-11-2016; Accepted on: 26-12-2016

ABSTRACT

Inflammatory bowel disease (IBD) (ulcerative colitis and Crohn’s disease) is an idiopathic, chronic inflammatory
condition, which affects the gastrointestinal tract.The present study was carried out to evaluate the effect of
ethanolic extract of Nigella sativa in experimentally induced inflammatory bowel disease (IBD) in rats. Colitis was
induced by a single intra-colonic application of 20 mg 2,4,6-trinitrobenzene sulfonic acid (TNBS) dissolved in 35%
ethanol into the descending colon. Rats were divided into six groups. Animals were treated with vehicle (ethanol),
TNBS dissolved in 35% ethanol, Ethanolic extract of Nigella sativa seeds (ENS) 100, 200 and 400 mg/kg body
weight p.o. and sulfasalazine(SSZ) 360 mg/kg body weight p.o. for 14 days. After completion of 14 days of
treatment, animals were sacrificed and the following parameters were assessed morphological score, histopathology
and biochemical parameters like myeloperoxidase (MPO), malondialdehyde (MDA), reduced glutathione (GSH),
catalase (CAT), superoxide dismutase (SOD) activity and serum nitrate levels. Nigella sativa provided protection
against TNBS-induced colonic damage. There was significant protection with ENS 200 and 400 mg/kg body weight
compared to control (P <0.001). Morphological and histological score were significantly reduced in all the treated
groups (P<0.001). All parameters were altered in ulcerated rats, and improved in animals receiving ENS, an effect
that was comparable to that of the standard sulfasalazine, especially at the highest dose level. Results indicate
efficacy of ENS against TNBS induced experimental colitis in rats.

Key words: IBD, ENS, TNBS, MPO, MDA, Anti-oxidant enzyme.

INTRODUCTION initiates and perpetuates the inflammatory response

of the gut. The available treatment choices have
Inflammatory bowel disease (IBD) (ulcerative colitis major limits owing to associated side effects and
and Crohn’s disease) is an idiopathic, chronic toxicity. As a result, there is high prevalence of
inflammatory condition, which affects the complementary and alternative medicines for treating
gastrointestinal tract. The etiology of IBD remains IBD[1-3].
obscure, although it is believed that an alteration in The Nigella sativa belongs to family Ranunculacae,
the intestinal immune system contributes to the commonly known as black cumin seed known to
inflammation that occurs. IBD is characterized by an have a great medicinal importance possessing many
up-regulation in the synthesis and release of different medicinal properties particularly in Greco-Arab,
pro inflammatory mediators, including reactive Unani-Tibb and Ayurvedic system of medicine. The
oxygen and nitrogen metabolites, eicosanoids, seeds have claimed to have several traditional
platelet-activating factor and cytokines. All of these medicinal properties [4]. Recently, the seeds have been
mediators contribute to the pathogenic cascade that reported to exhibit many pharmacological effects

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including immunomodulator[5], anticancer[6], anti- Induction of colitis

diabetic[7], anti-hypertensive[8], hepatoprotective Colitis was induced by a single intra-colonic
[9], anti-oxidant[10], anti-bacterial activity[11], anti- application of 20 mg TNBS dissolved in 35% ethanol
helminthes [12], and anti-inflammatory[12]. This using a rubber catheter of diameter 2mm. The
study is designed to evaluate different dose effect catheter was lubricated with glycerin and inserted
Extract of Nigella sativa on TNBS induced IBD in rectally into the colon through anus such that the tip
rats. was 8cm inside from the anus. The total volume was
expelled with additional air. Rats were observed for 2
MATERIALS AND METHODS weeks and sacrificed under anesthesia using
Chemicals anesthetic ether and by cervical dislocation for
Ethanolic extract of Nigella sativa (ENS) (NASC- assessment of various parameters. During and after
0076) a gift sample obtained from Madhur Pharma, TNBS administration, the rats were kept in a head-
Bangalore, India, TNBS (2,4,6-trinitrobenzene down position until they recovered from the
sulphonic acids was purchased from Sigma anesthesia for a few minutes to prevent leakage of the
Chemical, USA), Sulfasalazine (Wallace intracolonic instillation of TNBS, and they were
Pharmaceuticals, Mumbai, India). All other returned to cages.
chemicals were of analytical grade.
The dried ethanolic extract was suspended in distill Groups
water using 1% Tween 80, used for pharmacological The animals were divided into six groups, each
screening. containing six rats.
Group I animals were administered phosphate buffer
EXPERIMENTAL ANIMALS saline intra colonically. Group II were administered
Experimental animals TNBS (20mg) dissolved in 0.25ml of 35% ethanol
Adult Swiss albino mice (20- 25g) and Wistar rats [13]. Group III, IV and V were administered TNBS
(150 -200g) of either sex were used for the study. The (20mg) dissolved in 0.25ml of 35% ethanol + ENS
mice and rats were fed with standard pellet (Parnava 100mg/kg, 200mg/kg and 400mg/kg, p.o). Group VI
Agro industries Ltd. Sangali, India) and water ad were administered TNBS (20mg) dissolved in 0.25ml
libitum. The animals were maintained under standard of 35% ethanol + Sulphasalazine [13] (SSZ) 360
12-hr light / dark cycle throughout the study. The mg/kg, p.o. After 14 days of treatment, animals were
study protocol was approved by IAEC. sacrificed and the following parameters were
(No.CPCSEA/IAEC/PC-01/346) assessed, colon weight/ length ratio, morphological
index, histopathology, myeloperoxidase activity,
Acute toxicity study [14] malondialdehyde activity, reduced glutathione,
The study was performed according to the acute toxic catalase and superoxide dismutase activity and
classic method (as per CPCSEA/OECD guidelines). serum nitrate levels.
Swiss albino mice were used for acute toxicity study.
The animals were kept fasting for overnight Assessment of colitis: The colonic segments were
providing only water, after which the test drug extract placed on an ice-cold plate, cleaned of fat and
dissolved in Water was administered orally at the mesentery and blotted on filter paper. The colon was
dose of 800 mg/kg and observed for 14 days. weighed/cm length and expressed as mg/cm tissue
Animals were observed individually at least once length (CW/LR)[13].
during the first 30 min after dosing, periodically
during the first 24 h (with special attention during the Macroscopic assessment: The colon longitudinally
first 4 h) and daily thereafter for a period of 14 days. opened and scored for macroscopically visible
Once daily cage side observations included changes damage using magnifying lens according to the
in skin and fur, eyes and mucous membrane (nasal) criteria, which take into account the extent as well as
and also respiratory rate, circulatory(heart rate and the severity of colonic damage [1].
blood pressure), autonomic (salivation, lacrimation,
perspiration, piloerection urinary incontinence, and Histopathological studies: The colonic specimens
defecation) and central nervous system (ptosis, was taken from a region of the inflamed colon
drowsiness, gait, tremors and convulsion). The immediately adjacent to the gross macroscopic
toxicity study carried out as per the guidelines of damage in the distal colon of each animal and was
AOT- 421 using albino mice. The extracts were fixed in 4% buffered formaldehyde. Sections of
found to be safe till 800mg/kg. Hence we selected tissue was cut (5µm), stained with hematoxylin and
100 mg, 200 mg and 400 mg/kg dose (P.O) for eosin (H and E) and evaluated by light microscopy
pharmacological screening. for morphological changes [1].

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Biochemical parameters assessment: MPO activity, (100mg/kg, p.o) showed focal ulceration and
an indicator of polymorphonuclear leukocyte inflammation extending to muscularis propria,
accumulation, was determined by the whereas histopathology of colon administered with
tetramethylbenzidine (TMB) method, MDA, GSH, ENS (200mg/kg and 400mg/kg p.o) showed healing
catalase and superoxide SOD levels in the colonic of intestinal wall with inflammation limited only to
tissue and serum nitrate levels were determined[14- submucosa and mucosa which was comparable to
15]. standard SSZ (Figure 1-6).
The tissue Myeloperoxidase (MPO) and
Statistical analysis: All data were expressed as mean Malondialdehyde (MDA) levels are markers for
± standard error of the mean (S.E.M.). Statistical inflammatory tissue damage and lipid peroxidation.
analysis was performed using instat statistical In TNBS administered group there was significant
software. Analysis of variance (ANOVA) followed higher MPO and MDA levels compared to normal
by Tukey Multiple Comparison Test. A value of P< control rats. The tissue MPO and MDA levels in rats
0.05 was considered as the level of significance. treated with ENS 200mg & 400mg/kg p.o) showed
significantly lower when compared to TNBS
RESULTS administered group. The tissue MPO and MDA
Administration of TNBS 20mg single dose, intra levels in rats administered with SSZ showed
colonically showed significantly higher CW/LR significantly lower when compared to TNBS
value when compared to normal control rats. ENS administered group (Table 1).
treated rats showed CW/LR values significantly In TNBS administered group there was a significant
lower which was comparable to standard decrease in colonic non-enzymatic GSH content and
sulfasalazine treated group (Table 1). The enzymatic CAT, SOD defense systems compared to
morphology of colon of TNBS administered rats’ normal group. GSH, CAT and SOD in rats treated
revealed inflammatory response with presence of with ENS 200mg & 400mg/kg p.o), standard SSZ
inflammatory changes in the mucosa. ENS and SSZ showed significantly lower when compared to TNBS
treated group showed MI (macroscopic index) administered group (Table 1).
significantly lower compared to TNBS group (Table In addition, serum nitrate level was significantly
1). However, the histopathology of colon increased in TNBS colitis group when compared to
administered with TNBS showed mucosal ulceration, the normal group and significantly lower in ENS
transmural inflammation, diffuse infiltration of 200mg & 400mg/kg p.o and standard SSZ treated
inflammatory cells in the mucosa and submucosa. group compared to the TNBS administered group..
Histopathology of colon administered with ENS

Table.1 Comparison of different parameters measured in TNBS induced IBD model in rats.
ENS ENS ENS SSZ
Parameters Normal TNBS
(100mg/kg) (200mg/kg (400mg/kg) (360mg/kg)
CW/LR )
60±4.27 176±7.43a 167±10.28 114.5±5.24c 106±8.47c 93.5±10.2c
(mg cm-1)
MI
0.33±0.21 7±0.23a 6.5±0.42 1.62±0.21c 1.33±0.21c 1±0.25c
(0-10)
MPO
22.01±0.21 68.08±1.8a 64.07±1.58 43.70±1.65b 32.21±0.7c 24.8±0.11c
(U/g tissue)
MDA(nmol/g
15.89±0.49 78.32±3.5a 71.34±3.3 40.13±2.03b 27.36±1.7c 22.32±1.0c
wet tissue)
GSH (nmol/ g
1100±18.2 782±14.6 a 940±14.7 911±10.7 b 948±19.3 c 988±18.4 c
wet tissue)
SOD (U/mg
6.23±0.02 3.6±0.4a 4.01±0.2 4.41±0.8b 6.22±0.20c 8.25±0.3c
protein)
CAT (U/mg
22.6±0.22 10.42 ±0.1a 14.8±0.2 14.05±0.25 b 17.2±0.3 c 20.8±0.3 c
protein)
Serum nitrate
16.48±1.38 48.66±2.2 a 34.81±1.26 25.05±2.04 b 18.35±1.33 c 17.06±1.23 c
(μ mol/L)
Score data were expressed as mean ± S.E.M, n=6. ; (ANOVA) followed by Tukey Multiple Comparison Test
a
P<0.001 vs Normal group; bP<0.01, cP<0.001 vs TNBS group

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Histological Sections Of Colonic Mucosa

(A) (B)
Figure 1 Histological sections of colonic mucosa in normal group showing normal histology of rat colon with
intact epithelial surface. (Magnification; (a). 20X, (b). 100X)

(A) (B)
Figure 2 Histopathoological sections of colonic mucosa in TNBS group showing mucosal ulceration and
transmural inflammation most evident in the enlarged sub mucosa of rat colon. (Magnification; (a).
20X, (b). 100X)

(A) (B)
Figure 3 Histopathoological sections of colonic mucosa in ENS 100mg/kg administered rat colon showing
focal ulceration and inflammation with involvement of muscularis propria. (Magnification; (a). 20X, (b).
100X )

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(A) (B)
Figure 4 Histopathoological sections of colonic mucosa in ENS 200mg/kg administered rat colon showing
healing intestinal wall with inflammation limited to mucosa and submucosa. (Magnification; (a). 20X, (b).
100X )

(A) (B)
Figure 5 Histopathoological sections of colonic mucosa in ENS 400mg/kg administered rat colon showing
healing intestinal wall with inflammation limited to mucosa.(Magnification; (a). 20X, (b). 100X )

(A) (B)
Figure 6 Histological appearance of SSZ 360mg/kg administered rat colon showing healing intestinal wall
with reduced inflammation limited to mucosa.(Magnification; (a). 20X, (b). 100X )

DISCUSSION damage the colonic epithelium, thereby permitting

entry of the hapten (TNBS) into the lamina propria
Intracolonic administration of the TNBS in 35% where it would bind to tissue and act as an antigen
ethanol results in long-lasting chronic ulceration and [16]. Several features of this model make it attractive
inflammation of the rat colon which is an established for the study of inflammatory bowel disease.
colitis model. This model of colitis was designed Inflammation produce by TNBS in 35% ethanol is
based on the principle that the ethanol vehicle would longer lasting with significant thickening of the

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colonic wall is associated with cellular infiltration lipid peroxidation. In our study, ENS treated group
and ulcer and persisting for a long period of time showed significant reduction in malondialdehyde
[13]. levels compared to TNBS-induced colitis group
In present study there was extensive colonic which is due to the inhibition of lipid peroxidation.
mucosal and submucosal damage characterized by NO is associated with the initiation and maintenance
infiltration of inflammatory cell and ulcer formation of inflammation in IBD and that the selective
after administration of TNBS. Increase in colon inhibition of inducible NO synthase (iNOS) reduces
weight /length ratio and MI of colonic tissue in the tissue damage [20,21]. Studies indicated that a
TNBS administered ENS 100mg/kg and 200mg/kg, high nitrate level appears to be secondary to the
p.o treated group was reduced compared to group magnitude of inflammation [22]. In present study
administered TNBS alone. However the there was a significant increase in serum NO levels in
histopathology also reveals the healing process in TNBS induced colitis group compared to normal
ENS treated colonic mucosa of rat which was similar group, however there was significant decrease in
to standard SSZ. ENS and SSZ treated groups.
Myeloperoxidase is an enzyme found in Previous studies have suggested that excessive NO
neutrophils used as marker for inflammatory damage level could dilate vasculature, enhance
[17]. Myeloperoxidase is secreted by the neutrophils vasopermeability, as well as inactivate the activity of
whenever there is inflammation and therefore the antioxidases such as SOD, CAT, and GSH by means
number of neutrophils is directly co-related with of reacting with hydrosulfide group (-SH) in the
myeloperoxidase activity. Neutrophils play an enzymes[23]. In present study, ENS treated group
important role in producing superoxide anion and a showed significant increase in anti-oxidant enzymes
cascade of various reactive species leading to a very SOD, CAT and GSH compared to TNBS induced
reactive hydroxyl and peroxide radicals [18]. colitis group, suggesting its anti-oxidant activity.
Reduction in the activity of myeloperoxidase enzyme Results showed ENS acts by anti-inflammatory, anti-
can be interpreted as a manifestation of the anti- oxidant activity and lowering lipid peroxidation.
inflammatory activity of a drug [17]. In present study Hence ENS is beneficial in experimentally induced
there was an increase in MPO after administration of IBD in rats.
TNBS. ENS 200mg/kg and 400mg/kg, p.o showed
significant reduction in the activity of MPO. The CONCLUSION
reduction in the MPO activity was confirmed
histologically, since the level of leukocyte infiltration Present study reveals that ENS possesses dose
in the colonic mucosa was lower in ENS 200mg/kg dependent anti-inflammatory and anti-oxidant
and 400mg/kg, p.o treated groups compared to TNBS properties comparable to standard sulfasalazine
administered group. Hence this might be due to anti- effects. This effect is due to its anti-inflammatory,
inflammatory activity of ENS [12]. anti-oxidant activity and inhibition of lipid
Oxidative stress and its consequent lipid peroxidation peroxidation. Thus ENS has the protective effect in
could aggravate free radicals chain reactions TNBS induced colitis in rats.
disrupting the integrity of intestinal mucosa barrier,
and activate inflammatory mediators. It has been ACKNOWLEDGEMENTS
shown that colonic MDA contents increased and
colonic SOD Levels decreased both in human and The authors are thankful to Madhur Pharma Pvt Ltd,
experimental animal studies [19, 20]. The levels of Bangalore, India, for the gift sample of ethanolic
MDA were often used as an indication of oxidative extract of Nigella sativa seeds.
damage and as a marker for free radicals-induced

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