CHEMISTRY
RESEARCH
ORIGINAL RESEARCH
Received: 31 October 2009 / Accepted: 3 May 2010 / Published online: 8 June 2010
Springer Science+Business Media, LLC 2010
Introduction
NSAIDs belong to one of the most commonly used therapeutically group of agents for the treatment of pain, fever
and inflammation. However, usefulness of these agents is
limited due to higher incidence of gastrointestinal (GI)
damage including gastric ulceration, perforation and their
associated complications. During the past few years, there
has been an increased interest to discover safer NSAIDs
devoid of their ulcerogenic side effects. These GI side
effects are related to the intrinsic mechanism responsible
for their desired activity. These agents exert their
S. Sawraj T. R. Bhardawaj P. D. Sharma (&)
University Institute of Pharmaceutical Sciences,
Panjab University, Chandigarh 160014, India
e-mail: pritamdevsharma@hotmail.com
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R1
R2
H3CO
OH
HO
O
N
OH
Flavonoid
Cl
1
a
R1
R2
H3CO
O
N
OH
Cl
properties of conjugates, and contribution by their corresponding promoieties (indomethacin, flavonoid). Furthermore, equimolar physical mixtures of indomethacin and
promoieties in equimolar proportion were also studied for
the antiinflammatory activity. These physical mixtures
showed comparable results to the parent indomethacin, but
lower than their corresponding conjugates (Table 1).
Antiinflammatory activity
Analgesic activity
Antiinflammatory activity was determined by using carrageenan induced rat paw edema model (Winter et al., 1962).
Carrageenan (1% w/v) was used to produce paw edema.
Edema is presented as percentage increase in right hind paw,
in comparison to the uninjected left hind paw. Percentage
change in paw volume was calculated and expressed as the
amount of inflammation. For antiinflammatory activity, the
test compounds (4, 5) were administered orally at molar
equivalent doses of indomethacin (12 mg/kg, p.o.). Both
derivatives at molar equivalent doses showed significantly
increased antiinflammatory activity as compared to that
produced by indomethacin. This increased activity may be
due to the combined effect of improved physicochemical
Pharmacological evaluation
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Table 1 Antiinflammatory activity of indomethacin, indomethacinflavonoid conjugate (4, 5) and indomethacin ? flavonoid physical mixtures
Compound
4h
Control
0.5% CMC
64.73 0.72
81.58 0.63
Indomethacin
12.0
48.35 0.56*
57.39 0.60*
Indometahcinnaringenin (4)
23.95
39.13 0.51*,#
40.46 0.82*,#
Indometahcinhespertin (5)
20.52
37.78 0.9*
,#
38.22 0.31*,#
Indomethacin ? naringenin (1 ? 2)
12 ? 9.24
44.45 0.59*
47.01 .98*
Indomethacin ? hespertin (1 ? 3)
12 ? 10.26
43.81 0.78*
45.34 0.67*
Table 2 Analgesic activity of indomethacin, indomethacinflavonoid conjugate (4, 5) and indomethacin ? flavonoid physical mixtures
Compound
Control
0.5% CMC
Indomethacin
10.0
74.17 0.90
Indometahcinnaringenin (4)
17.1
86.7 0.46*,#
Indometahcinhespertin (5)
17.49
89.43 0.59*,#
Indomethacin ? naringenin (1 ? 2)
10 ? 18.36
77.2 0.76*
Indomethacin ? hespertin (1 ? 3)
10 ? 18.78
79.15 0.54*
Table 3 Antiulcer activity of indomethacin, indomethacinflavonoid conjugate (4, 5) and indomethacin ? flavonoid physical mixtures
Compound
Control
0.5% CMC
Indomethacin
48
5.54 0.09*
0.2 0.06
Indometahcinnaringenin (4)
82
0.62 0.09*,#
Indometahcinhespertin (5)
83.98
0.54 0.16*,#
Indomethacin ? naringenin (1 ? 2)
48 ? 36.45
3.16 0.54*
Indomethacin ? hespertin (1 ? 3)
48 ? 40.47
3.08 0.72*
Antiulcer activity
The mutual prodrugs (4, 5) were screened for their ulcerogenicity in rats, using parent drug induced acute gastric
ulcerations (Cioli et al., 1979). The animals were fasted for
24 h, divided into different groups containing six animals in
each group. Control group was treated with an equal volume
of 0.5% carboxy methyl cellulose (CMC) vehicle. Animals
were sacrificed 8 h after the treatment. The stomach was
removed, opened along the greater curvature, washed with
saline, and observed for ulcers. For the acute gastric damage
evaluation, the parent drug indomethacin was used to
produce gastric ulcers. For this purpose, indomethacin
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(48 mg/kg, p.o.) was administered which produced a significant increase in ulcer index (5.54 0.09) as compared
to the control group (0.2 0.06). Both conjugates (4, 5)
showed significantly reduced gastric damage. The reduction
in ulcer index by the physical mixture of indomethacin and
flavonoids was negligible (3.16 0.54, 3.08 0.72) as
compare to their conjugates (0.62 0.09, 0.54 0.16).
This may be due to the polar nature of the antioxidants
resulting in their poor bioavailability, whereas reduction in
ulcer index by the conjugates significantly may be due to the
improved physicochemical properties and contribution by
the antioxidant promoiety, after the cleavage of the mutual
prodrug. (Table 3; Fig. 1).
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Conclusion
In this study, indomethacinflavonoid conjugates have been
designed, synthesized and evaluated as safer NSAIDs. These
conjugates exhibited potential antiinflammatory and analgesic activity with significant reduced ulcerogenicity as compare to their physical mixtures of flavonoid and indomethacin.
This may be due to improved physicochemical properties
required for enhanced bioavailability. Furthermore, indomethacin with flavonoid physical mixture did not effectively
reduce the risk of GI side effects in comparison to their corresponding conjugates. On the basis of these observations, it
can be concluded that there is advantage of giving indomethacin and flavonoid (naringenin, hespertin) in the form of
a single molecule, i.e. indomethacinflavonoid conjugates.
Experimental protocols
Chemistry
Melting points (mp) were determined on a Veego melting
point apparatus and are uncorrected. For TLC, glass plates
coated with silica gel (E. Merck) were used. The TLC
plates were activated at 110C for 30 min and visualized
by exposure to iodine vapors. Glass columns of appropriate
sizes were used. Silica gel (60120 mesh, BDH) was used
as adsorbent. IR spectra were recorded on Perkin Elmer
882 spectrometer using potassium bromide pellets.
1
HNMR and 13CNMR spectra were recorded with Bruker
AC 300F, 400 MHz spectrometer using CDCl3 or DMSOd6 as solvents, and tetramethylsilane (THF) as internal
standard. Mass spectra were obtained with Vg-11-250J
70 s mass spectrometer at 70 eV using electron ionization
(EI) sources. The synthetic reactions were monitored by
TLC. The identity of all new compounds was confirmed by
1
HNMR, 13CNMR, IR data, elemental analysis and mass
spectrometer; homogeneity was confirmed by TLC.
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Analgesic activity
References
Analgesic activity was determined by using abdominal
writhing assay. Mice were divided into six groups: (i) vehicle
(control), (ii) indomethacin (standard), (iii) indomethacin
naringenin, (iv) indomethacinhespertin, (v) indomethacin ? naringenin (physical mixture), and (vi) indomethacin ? hespertin (physical mixture). The animals were food
deprived overnight prior to the experiments. The samples
were suspended in carboxymethylcellulose (0.5%, CMC),
and administered orally (0.1 ml/10 g of body weight) at 1 h
before the administration of freshly prepared acetic acid
solution (1%, 10 ml/kg, i.p.). The number of writhes (constriction of abdomen, turning of trunk and extension of hind
limbs) for each animal was recorded during 20 min period,
beginning 3 min after the administration of acetic acid. The
average number of writhes in each group of drug-treated
mice was compared with that of control group and degree of
analgesia was expressed as % inhibition as follows:
Nt
% Inhibition 1
100
Nc
where Nc is the number of writhes in control, and Nt is the
number of writhes in drug-treated mice.
Antiulcer activity
For the study of antiulcer activity, rats were fasted overnight
and divided into six groups: animals were treated with (i)
vehicle (control), (ii) indomethacin (standard), (iii) indomethacinnaringenin, (iv) indomethacinhespertin, (v)
indomethacin ? naringenin (physical mixture), and (vi)
indomethacin ? hespertin (physical mixture). Animals
were sacrificed 8 h after the treatment. The stomach was
removed, opened along greater curvature, washed with saline, and observed for ulcers. The ulcers were scored as:
0 = no observable damage; 1 = punctiform lesion\1 mm,
3 = filiform lesion\5 mm, 4 = punctiform lesion[1 mm
or filiform lesion[ 5mm.
Statistical analysis
Statistical analysis was carried out on in vivo studies data.
The ulcer index data was subjected to student t-test
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Wolfe MM, Lichtenstein DR, Singh G (1999) Gastrointestinal
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