MEDICINAL

CHEMISTRY
RESEARCH

Med Chem Res

DOI 10.1007/s00044-014-1198-3

ORIGINAL RESEARCH

2-Aminobenzimidazole conjugates of NSAIDS: novel compounds

with immunomodulatory, anti-inflammatory and antioxidant
actions
Yogita Bansal Om Silakari

Received: 6 October 2013 / Accepted: 20 July 2014

Springer Science+Business Media New York 2014

Abstract 2-Aminobenzimidazole (ABZ) and guanidine

have immunomodulatory activity, whereas NSAIDs have
anti-inflammatory activity. We, therefore, developed several novel ABZ-NSAID conjugate compounds and tested
their anti-inflammatory and immunomodulatory activities.
We also measured their antioxidant and ulcerogenic properties. We found that all compounds had not only antiinflammatory activity but they also had immunomodulatory and antioxidant activities. While compound 2 had
immunostimulatory activity (humoral antibody titre, HAT
7.2 0.20 and carbon clearance index, K 0.030 0.001),
compound 9 had immunosuppressive activity (HAT
3.4 0.24 and K 0.012 0.002). These two compounds
did not produce ulcers and hence were safe to the gastric
mucosa. In addition, we used Lipinskis parameters and
found that all compounds had drug-like properties. Thus,
we have provided new evidences to use compounds 2 and 9
for the treatment of inflammatory and immunomodulatory
disorders like rheumatoid arthritis, ulcerative colitis, multiple sclerosis, osteoarthritis and cancer.
Keywords 2-Aminobenzimidazole  NSAIDs 
Immunomodulatory  Anti-inflammatory  Antioxidant 
Lipinski  Ulcerogenic

Y. Bansal  O. Silakari (&)

Molecular Modelling Lab (MML), Department of
Pharmaceutical Sciences and Drug Research,
Punjabi University, Patiala 147002, India
e-mail: omsilakari@rediffmail.com

Introduction
2-Aminobenzimidazole (ABZ), is a rigid form of guanidine
pharmacophore (Greenhill and Lue, 1993). Guanidine is an
immunomodulator as it alters the inducible form of nitric
oxide synthetase in immune cells. The guanidine derivatives stimulate dilation of blood vessels and activate leucocytes to attack tumour cells, fungi and bacteria (Hamley
and Tinker, 1995). In addition, the ABZ-substituted compounds have antiviral (Li et al., 2007), anthelmintics,
antiproliferative (Mavrova et al., 2013; Nawrocka et al.,
2004), antihypertensive (Settimo et al., 1991), H3 antagonistic (Mor et al., 2004), human glucagon receptor antagonistic (Kim et al., 2008), male contraceptive (Chen et al.,
2013) and anti-infective (Zhang et al., 2012) activities.
These compounds also inhibit chemokine receptor
(CXCR3) (Hayes et al., 2008), interleukin 2-inducible T
cell kinase (ITK) (Lo et al., 2008) and lymphocyte tyrosine
kinase (Lck) (Zhang et al., 2009), and thereby, alter the
immune system. The structureactivity relationship (SAR)
study of interleukin-2-inducible T cell kinase (ITK)
inhibitors suggests that an acyl/aroyl moiety at amino
group of ABZ is important for binding interactions in
kinase specificity pocket of ITK receptor (Cook et al.,
2009). Moreover, the clinical use of levamisole, mizoribine, methotrexate, 6-mercaptopurine, and azathioprine
(Fig. 1) for various immunomodulatory diseases such as
leukaemia and other carcinomas, rheumatoid arthritis,
organ transplants (Krensky et al., 2006) further supports
that the ABZs are important immunomodulators.
The NSAIDs (diclofenac, ibuprofen, salicylic acid,
mefenamic acid, indomethacin, flurbiprofen, naproxen,
ketoprofen, mesalamine, felbinac and salicylic acid) are
widely used to treat inflammatory disorders. These are
often prescribed with immunomodulators to treat complex

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Med Chem Res

Fig. 1 Design strategy for ABZ-NSAID conjugates

inflammation-induced progressive immune disorders, such

as rheumatoid arthritis, ulcerative colitis, multiple sclerosis, osteoarthritis and cancer (Mcinnes and Schett, 2011;
Volin and Koch, 2011; Spain et al., 2009; Kathleen
and Jurenka, 2003). The indomethacin and OK-432 reinforces the macrophage-mediated anti-tumour response in
advanced cancer cases (Ohshika, 1988).
Since NSAIDs have potent anti-inflammatory activity,
and 2-substituted ABZs have potent immunomodulatory
actions, we designed new molecules as ABZ-NSAID
conjugates that include ABZ connected through its
2-amino group with free carboxyl group of varied NSAIDs
(Fig. 1). Since the free carboxyl group of NSAID moiety is
masked and the benzimidazole nucleus is appended to the
NSAID, we expected our conjugates to be soft on gastric
mucosa as seen with the clinically available benzimidazole
derivative class of antiulcer drugs, like omeprazole, pantoprazole, lansoprazole and astemizole. Here we show the

123

design, synthesise and pharmacological evaluation of various ABZ-NSAID conjugates for their anti-inflammatory,
immunomodulatory and ulcerogenic activities.

Results and discussion

Chemistry
We have outlined the coupling of ABZ with each of the
selected NSAID (Table 1) in the presence of dicyclohexyl
carbodiimide (DCC) (211) in Fig 2. DCC is a highyielding coupling agent used for the amide/ester formations. The reaction conditions involving use of DCC as
coupling agent for formation of an amide bond as
reported by Mishra et al. (2008) were modified for synthesizing the compounds 211. All compounds were
obtained in quantitative yields, verified to be pure and

Med Chem Res

Table 1 Structures and anti-inflammatory activity of compounds 211
Compound NSAID

Anti-inflammatory response as paw volumea (%IPVb)

Structure

2 Ibuprofen

NSAID

Compound

1.19 0.06c (54.6)

1.28 0.04c (53.8)

1.27 0.16c (51.5)

1.42 0.06c (46.1)

1.66 0.19c (36.8)

1.41 0.08c (46.1)

1.67 0.11c (36.4)

1.66 0.13c (38.4)

1.46 0.21c (44.4)

1.25 0.09c (53.8)

10

6
5

H
N

CH3

H
N

12

9 11

CH3

14

16
15

20

13

18

CH3

17

19

3 Diclofenac
9

O
H
N

NH

12

13

15

Cl

14

17

HN

11

10

16

18

2
21

Cl

19
20

4 Salicylic acid
HO
6

5
4

H
N

10

11

12

NH

14

13

5 Mefenamic acid

6
5

H
N

2
4

11

10

H
N

12

13 21

CH3

14

HN

15

22

CH3

16
17
18

20
19

6 Indomethacin
18

H
N

2
4

H
N

CH3

9
8

10

11

3
19

16

12

20

14

21 22
23

15 26

13

H3CO

17

25

24

Cl

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Med Chem Res

Table 1 continued
Compound NSAID

Anti-inflammatory response as paw volumea (%IPVb)

Structure

7 Flurbiprofen

NSAID

Compound

1.27 0.14c (51.6)

1.30 0.1c (50.0)

1.93 0.27c (26.5)

1.41 0.06c (46.0)

1.27 0.13c (51.6)

1.39 0.12c (50.0)

1.19 0.16c (54.6)

1.51 0.09c (42.3)

1.26 0.15c (51.6)

1.73 0.03c (34.6)

10

H
N

H3C

H
N

F
13

16

14

N
5

12

9 11

15

19
20

22

18
17

21

8 Naproxen
10

H3C
H
N

12

H
N

14

20

15
19

13

11

21

18

16

OCH3

17

9 Mesalamine
HO
6

5
4

10 11

H
N

9
1

12

NH

13

14

NH2

10 Ketoprofen
10

H
N

H
N

11

CH3
8

13

21

23

14

16
15

22

12

20

18

17

19

11 Felbinac
H
N
HN

6
2

5
4

O
11

8
9

10

12

17

13
16

15
14

18
19

21
20

Control
a

2.63 0.02

Values are expressed as mean SD

Percent inhibition of paw volumes of compounds at 60 min

Values are statistically different with respect to control at P \ 0.05

characterised through comprehensive spectral analysis.

The IR spectra of all compounds showed a single band at
about 3,360 cm-1 in contrast to a double band at 3,434

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and 3,357 cm-1 in IR spectrum of ABZ which indicated

that the NH2 group (primary amine) in ABZ was converted into NH group (secondary amine). In addition,

Med Chem Res

Fig. 2 Synthetic scheme for the designed ABZ-NSAID conjugates

all spectra showed Amide-I and Amide-II bands at about

1,680 and 1,575 cm-1, respectively. These suggested the
formation of an amide linkage in the compounds. The
benzimidazole nucleus in the compounds was detected as
two 2 proton double doublets at d 7.67.4 and d 7.27.0.
The chemical shift values of NH protons of benzimidazole and amide proton (CONH) were confirmed
by D2O exchange experiments. Finally, the high resolution mass spectrum of each compound showed the base
peak at m/z value corresponding to the theoretical accurate mass of its parent ion (M?H?) ion.

Table 2 Immunomodulatory activity of compounds 211

Compound Immunomodulatory response
% Change Ka

HAT

0.017 0.002b

?65.3

0.032 0.006c

?88.2

7.2 0.20b,c ?38.4

0.030 0.001c

?76.4

5.6 0.24b

0.021 0.001b,c ?23.5

6.5 0.20b,c ?25.0

Control

5.2 0.24b

% Change

Levamisole 8.6 0.24

0.027 0.001c

?58.8

5.8 0.20

?11.5

0.020 0.003b

?17.6

5.7 0.24b

?9.6

0.022 0.002b

?29.4

Anti-inflammatory activity

6.4 0.20b,c ?23.0

0.024 0.001b,c ?41.1

4.2 0.24b,c -19.2

0.014 0.004b

The anti-inflammatory activity of each compound (211)

as well as the selected NSAIDs were expressed as percent
inhibition of carrageenan-induced paw oedema (Table 1).
Each compound was found more potent than or equipotent
to the parent NSAID. The inhibition of paw volume by all
compounds was in the range of 4055 %. The peak antiinflammatory response was at 60 min. Thus, the conjugation of NSAID with ABZ retained the anti-inflammatory
activity of the NSAID, and this retention of the activity in
the conjugate compounds may be attributed to their pharmacophores contributed by the NSAIDs themselves.

9
10

3.4 0.24b,c -34.6

5.8 0.20b ?11.5

0.012 0.002b,c -29.4

0.023 0.002b,c ?35.2

11

6.2 0.20b,c ?19.2

0.025 0.005c

Immunomodulatory activity
Immunomodulatory activity was evaluated as humoral
antibody titre (HAT) and carbon clearance (phagocytic)
index (K). All compounds modulated the immune system by
changing the HAT as compared to their control (Table 2).
While compounds 8 and 9 exhibited immunosuppression
(decrease in HAT with respect to control), the other compounds stimulated the immune system (increase in HAT with
respect to control). The HAT for levamisole, 2 and 9 were
8.6 0.24 (?65.3 %), 7.2 0.20 (?38.4 %) and
3.4 0.24 (-34.6 %), respectively, which suggested that
these compounds are immunomodulators and act by
changing humoral response to sheep red blood cells
(SRBCs). The phagocytic index (K) data supported the HAT
data. The K of all compounds except 8 and 9 was higher than
the control (Table 2). This indicates that compounds 8 and 9
were immunosuppressive. The maximum K was found for

?7.6

-17.6

?47.1

Carbon clearance index. Values are expressed as mean SEM

Values are statistically different with respect to levamisole at

P \ 0.05

Values are statistically different from the control at P \ 0.05

compound 2 which was almost equivalent to levamisole

while minimum for compound 9.
Thus, the NSAIDs conjugated with ABZs have both
immunomodulatory and anti-inflammatory actions. The
immunomodulatory activity of the conjugate compounds
can be attributed to the guanidine pharmacophore, which is
incorporated in the form of ABZ. However, the nature of
immunomodulation (suppression or stimulation) by the
compounds is found to depend largely on the pharmacophores contributed by NSAID. This dependence can be
attributed to the inherent weak immunostimulatory or
immunosuppressive activities exhibited by NSAIDs themselves (Koji et al., 1998; Cho, 2007). Hence, the immunostimulatory and immunosuppressive activities of
compounds 2 and 9 could be assigned to the ABZ as well
as structural components contributed by ibuprofen and
mesalamine, respectively.
Ulcerogenic and oxidative stress studies
Since compounds 2 and 9 exhibited significant antiinflammatory and immunomodulatory activities (2 being
immunostimulatory and 9 being immunosuppressive), we

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Table 3 Ulcer index (UI) and biochemical estimations for oxidative stress of compounds 2 and 9
Compound

UI

Control

0.33 0.20a

22.14 1.19a

3.72 0.23a

184.10 4.19a

85.20 5.15b

Indomethacin

Catalase (lM/mg)

3.17 0.41

7.79 0.38

a,b

TBARS (nM/mg)

30.41 1.57

a,,b

1.22 0.08

17.92 0.46

0.84 0.12a,b

16.21 0.34a,b

6.83 0.42

a,b

10.52 0.28a,b

Glutathione (lM/100 mg)

142.11 14.34a,b
161.42 8.92a,b

Values are expressed as mean SD

a

Values are statistically different with respect to indomethacin at P \ 0.05

Values are statistically different from the control at P \ 0.05

Table 4 TPSA and calculated Lipinskis parameters for the compounds 211
Compound

Log Pa

TPSAb

MWc

nONd

nOHNHe

nviolf

nrotbg

Acceptable range

B5

\160

B500

B10

B5

B1

4.832

57.781

321.424

5.960

69.808

411.292

3.129

78.009

253.261

5.385

69.808

356.429

5.355

89.020

472.932

5.418

57.781

359.404

4.747

67.015

345.402

2.181

104.032

268.276

10

4.986

74.852

369.424

11

4.528

57.781

327.387

Calculated lipophilicity

Total polar surface area (hydrophilicity)

Molecular weight

Number of hydrogen bond acceptor

Number of hydrogen bond donors

Number of violations from Lipinskis rule of five

Number of rotatable bonds

further evaluated these for acute gastric ulcerogenic effect

and in vivo oxidative stress in wistar rats. The ulcer index
of 2 and 9 were significantly lower than that of indomethacin (1.22 and 0.84 vs. 3.17) indicating that the
compounds are less ulcerogenic than indomethacin. The
masking of free carboxyl group in NSAIDs may be
responsible for the reduced ulcerogenicity of the compounds. We further tested oxidative stress-induced markers
such as glutathione (GSH) levels, catalase (CAT) activity
and lipid peroxidation in terms of thiobarbituric acid
reactive substances (TBARS) levels (Table 3) to study the
safety of these compounds. The levels of CAT and GSH
were decreased in indomethacin-treated group indicating
that indomethacin induced the oxidative stress in animals.
Conversely, the CAT and GSH levels were higher in the
groups treated with compounds 2 and 9. These suggest that
both 2 and 9 are antioxidant. Further, indomethacin
increased the lipid peroxidation as indicated by increased

123

TBARS levels. These levels were, however, reduced by 2

and 9 suggesting that these compounds exhibit protective
effect against lipid peroxidation-induced gastric damage.
Molecular properties calculations
Lipinskis rule predicts oral bioavailability of a new molecule through calculations of its molecular properties (Lipinski et al., 2001). Lipophilicity (logP) and total polar
surface area (TPSA) are two important properties for this
prediction. TPSA is closely related to the hydrogen bonding
potential of a compound (Jarrahpour et al., 2010, 2011), and
or more are
molecules with TPSA values of about 160 A
expected to have poor intestinal absorption. When we calculated the molecular properties of compounds 211 using
molinspiration property calculator (MIPC) (http://www.
molinspiration.com), we found that TPSA, molecular
weight (MW), number of H-bond acceptors (nON) and

Med Chem Res

number of H-bond donors (nOHNH) for each compound

were within the acceptable limits (Table 4) as suggested by
the software for any compound to be bioavailable. However,
compounds 3, 5, 6 and 7 were found to have lipophilicity (log
P) more than 5. As per the Lipinskis Rule, these compounds
are predicted to have less bioavailability. The other compounds in the series were found to have zero number of
violations (nviol = 0) and hence are predicted to be drugable. Further, the minimum log P value (2.1) was found for
compound 9, which is a mesalamine conjugate. Mesalamine
itself has very low log P value (0.922) which is not favourable for good bioavailability. In order to enhance its bioavailability, it is administered as varied derivatives-like
sulfasalazine (log P 3.42), olsalazine (log P 3.9) and balsalazide (log P 2.34), which are more bioavailable and are
metabolised in vivo to mesalamine (Sellin and Pasricha,
2006). Hence, conjugation of mesalamine with ABZ (compound 9) incurs an additional advantage of increase in bioavailability of mesalamine.

Conclusion
Our pharmacological evaluations support the hypothesis
that conjugation of ABZ with an NSAID not only retains or
potentiates the anti-inflammatory activity of the NSAID,
but it also confers immunomodulatory activity. In addition,
the ABZ-NSAID conjugate compounds have antioxidative
actions and are safe to gastric mucosa. This novel combination of anti-inflammatory and antioxidant activities with
immunostimulatory effects make the compounds 2, 4 and 7
important leads for their further exploration in anticancer
immunotherapy, whereas the same combination with
immunosuppressive responses makes the compounds 8 and
9 useful candidates for inflammation-mediated immune
disorders such as rheumatoid arthritis, osteoarthritis,
ulcerative colitis and other autoimmune disorders.

The chemical reactions were monitored through thinlayer chromatography (TLC) using precoated aluminium
plates (Merck, Mumbai, India) visualised in UV chamber
at short as well as long wavelengths. Column chromatography using silica gel (100200 mesh) was employed to
purify the synthesised compounds. Melting points were
recorded in open sulfuric acid bath and uncorrected.
1
H-NMR and 13C-NMR spectra were recorded on Bruker
AC 400 NMR Spectrometer (400 MHz). Chemical shifts
were reported in parts per million (d) values using tetramethylsilane as internal standard with multiplicities (br,
broad; s, singlet; d, doublet; t, triplet; q, quartet; qv, quintet;
sx, sextet; sp, septet; m, multiplet; dd, double doublet) and
number of protons in the solvent specified. The coupling
constants (J) were expressed in Hz. FT-IR spectra were
recorded on FT-IR Perkin-Elmer 1710 series using KBr
pellets. High resolution mass spectra were recorded on Micromass Q-TOF micro mass spectrometer (Waters, MA,
USA) in positive mode of electrospray ionisation (?ESI).
The solvents and reagents were dried prior to use, when
required, over KOH or anhydrous Na2SO4 or fused CaCl2.
Albino rats (150200 g) and albino mice (1822 g) of
either sex were used for the pharmacological evaluations.
The animals were housed in cages at room temperature
with a 12 h light/dark cycle and were allowed food and
water ad libitum. These were randomly allocated into
control (one), standard (one) and test (equal to number of
test compounds) groups for each activity at the beginning
of all the experiments. The study was approved by institutional animal ethical committee under CPCSEA guidelines. The dose of each compound for each activity was set
at 150 lmol/kg of body weight (4050 mg/kg on the basis
of molecular mass) based on the pilot study carried out at
various doses as well as on the basis of literature (Achar
et al., 2010; Sondhi et al., 2002). All compounds and the
reference drugs were administered p.o. or i.p. suspended in
0.5 % sodium carboxymethylcellulose solution (SCMC).
Chemistry: general procedures

Experimental
Synthesis of ABZ
The NSAIDs were procured as generous gift samples from
different pharmaceutical industries. Diclofenac, ibuprofen,
indomethacin and mefenamic acid from Crystal Pharmaceutical (Ambala, India), flurbiprofen from FDC India Ltd
(Mumbai, India), naproxen and mesalamine from Ranbaxy
Research Laboratories (Gurgaon, India) and ketoprofen
from Dr. Reddys Research Laboratory (Hyderabad, India).
Felbinac and salicylic acid were purchased from SigmaAldrich (New Delhi, India) and S. D. Fine chemicals
(Mumbai, India), respectively. All NSAIDs, procured as
gift samples, were authenticated through melting point
determination and IR spectral analyses.

It was prepared by the method reported by Leonard et al.

(1947). Briefly, the solutions of cyanogen bromide
(0.03 mol, 3.18 g) and o-phenylenediamine (0.02 mol,
2.05 g) prepared separately each in 35 mL of aqueous
methanol (50 % v/v) were mixed in a 250-mL conical flask
and stirred at room temperature for 24 h. Thereafter,
methanol was distilled off in vacuum, the solution was
cooled at room temperature and basified with aqueous
ammonia. The precipitates were filtered and recrystallised
from ethanolwater mixture to obtain ABZ as light buff
coloured crystals.

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Med Chem Res

Synthesis of ABZ-NSAID conjugates (211)

Dicyclohexyl carbodiimide (DCC) (0.1 mol, 2.06 g) was
added to a solution of the NSAID (0.1 mol) in dichloromethane (10 mL). The contents were stirred for 30 min
followed by addition of a solution of ABZ (0.1 mol, 1.3 g) in
dichloromethane (10 mL) and pyridine (5 mL). The reaction
mixture was stirred at 0 C for the first 2 h, followed by
stirring at room temperature overnight. The precipitated
dicyclohexyurea (DHU) was filtered and the solvent was
removed in vacuum. Ethyl acetate (10 mL) was added to the
residue in order to separate the product from the residual
DHU. The ethyl acetate solution was filtered, washed with
10 % aqueous solution of sodium bicarbonate followed by
distilled water and then dried with magnesium sulphate
(anhydrous). The solvent was recovered in vacuum to obtain
the crude product which was purified by normal phase column chromatography using mobile phase composed of
chloroform and methanol (varied proportions) to afford the
conjugate products as light coloured amorphous powder.
Anti-inflammatory activity
The anti-inflammatory activity of the test compounds was
evaluated by employing a 1.0 % carrageenan solution as
inflammation inducer in wistar rats by following the
method reported by Winter (1962). The test compounds
and corresponding NSAIDs were administered i.p. 30 min
before the carrageenan solution was injected into the right
hind paw of the animal. SCMC (0.5 %) was used as control. Volume of the paw was measured at 0, 30, 60, 120 and
180 min after the carrageenan injection. The anti-inflammatory activity was calculated as percent inhibition of
carrageenan-induced paw oedema using the following
formula (Chu and Kovacs, 1977).

for 10 min, plasma was discarded and the pellet of SRBCs

was washed thrice with normal saline. Finally, SRBCs
were suspended in normal saline so that the concentration
was about 1 9 108 cells in 0.1 mL of the suspension for
further experiments.
Humoral antibody titre (HAT)
The animals were administered the test compounds and
levamisole as per the dosage regimen for 7 days. On the
7th day, mice from all groups were challenged with the
SRBCs suspension (0.1 mL) i.p. Blood sample was collected from each animal by retro-orbital puncture on 14th
day and centrifuged at 2,500 rpm for 10 min to separate the
serum. The antibody titre was determined using microtitre
plates as reported in literature (Puri et al., 1994).
Carbon clearance index (K)
The mice were treated with test compounds and levamisole
for 5 days as per the dosage regimen. Carbon ink suspension
(Rotring, Zeichentusche ink, Germany) (10 lL/g of body
weight) was injected in each mice through its tail vein on the
7th day. The blood samples were withdrawn from retroorbital vein after 3 and 15 min of the ink injection. A 25 lL
of the blood sample was mixed with 0.1 % sodium carbonate
solution (2 mL), and optical density was measured using
ELISA plate reader (APR4 Microplate reader, Germany).
The K was calculated using the following equation: K = (ln
OD1 - ln OD2)/12, where OD1 and OD2 were the optical
densities at 3 and 15 min, respectively (Luck and Nussenzweig, 1969; Jayathirtha and Mishra, 2004).
Acute ulcerogenecity test

Mice were divided into different groups with 6 animals in

each group. The control group was given a solution of 0.5 %
SCMC in water (0.3 mL/mouse, p.o.). The standard and test
groups were administered levamisole and the compounds
211, respectively, once daily at a dose of 150 lmol/kg p.o.

The rats were divided into different groups each of six animals. Rats were fasted for 20 h before the drug administration. The test compounds (2 and 9) and reference compound
(indomethacin) were administered orally as a suspension
(150 lmol/kg) in 0.5 % SCMC. The control group received
the vehicle (0.5 % SCMC). Rats were fasted for 2 h, allowed
to feed for 2 h and then fasted for another 20 h. Rats were
given another two doses on the 2nd and 3rd days. On the 4th
day, rats were sacrificed, the stomach removed, opened along
with the greater curvature and rinsed with 0.9 % saline. The
stomach was then examined for the severity of ulceration
(Cioli et al., 1979) and mean ulcer score for each treatment
group was calculated and reported as ulcer index.

Preparation of sheep red blood cells (SRBCs)

Biochemical estimations for oxidative stress

Fresh blood was collected from sheep sacrificed in the local

slaughter house. The blood was centrifuged at 4,000 rpm

Glandular parts of the excised stomachs of animals subjected

to acute ulcerogenecity test were homogenised in ice cold

% oedema inhibition 100  paw volume in treated=

paw volume in control  100
Immunomodulatory activity
Dosage regimen

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Med Chem Res

phosphate buffer (pH 7.4) with a tissue homogeniser (Model

PT194, Remi Motors, New Delhi, India) for 2 min. The
homogenate was centrifuged at 800g for 10 min. The supernatant was separated and centrifuged at 12,000g for 15 min.
The clear supernatant was analyzed for the reduced glutathione (GSH) levels (Ellman, 1959), lipid peroxidation (Ohkawa
et al., 1979) and catalase levels (CAT) (Aebi, 1974).
Statistical analysis
Data are expressed as mean standard deviation (SD)/
standard error mean (SEM) of the obtained results. The
statistical analysis of the data was performed using analysis
of variance (ANOVA) (Sigmastat 3.5) followed by multiple comparison (Tukeys) test. A value of P \ 0.05 was
considered statistically significant.
Acknowledgments The authors are thankful to Crystal Pharmaceuticals (Ambala, India), FDC India Ltd (Mumbai, India), Ranbaxy
Research Laboratories (Gurgaon, India) and Dr. Reddys Research
Laboratory (Hyderabad, India) for providing the NSAIDS and other
drugs as generous gift samples.
Conflict of interest

There are no conflicts of interest.

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