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MEDICINAL

CHEMISTRY
RESEARCH

Med Chem Res (2011) 20:687694


DOI 10.1007/s00044-010-9363-9

ORIGINAL RESEARCH

Design, synthesis and evaluation of novel indomethacinflavonoid


mutual prodrugs as safer NSAIDs
Shruti Sawraj Tilak R. Bhardawaj
Pritam D. Sharma

Received: 31 October 2009 / Accepted: 3 May 2010 / Published online: 8 June 2010
 Springer Science+Business Media, LLC 2010

Abstract Indomethacin suffers from the general side


effects of NSAIDs. The study aimed to retard the adverse
effects of gastrointestinal (GI) origin. Two conjugates of
indomethacin were synthesized by esterification with
flavonoids namely, naringenin and hespertin. Purified
synthesized mutual prodrugs were characterized by m.p.,
TLC, elemental analyzes, FTIR, NMR and MS. Synthesized conjugates were subjected for antiinflammatory,
analgesic and antiulcer activity. These conjugates showed
retention of antiinflammatory activity with reduced
ulcerogenic side effects. These results indicated that
indomethacinflavonoid conjugates have the potential to be
developed as safer NSAIDs.
Keywords NSAIDs  Indomethacin  Flavonoid 
Conjugates  Ulcerogenicity

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

therapeutic effects by inhibiting the activity of the enzyme


cyclooxygenase (COX), resulting in the prevention of
prostaglandins synthesis, mediators of inflammation (Vane,
1971). During recent years, it has been known that COX
exists in two isoforms, namely COX-1 and COX-2 (Hla
and Neilson, 1992; Xie et al., 1991). COX-1 is constitutive
and provides cytoprotection in the GI tract, whereas COX2 is inducible and mediates inflammation due to the formation of prostaglandins. The mucosal integrity in the
normal GI tract is primarily maintained by the prostaglandins that are derived from COX-1, and therefore,
inhibition of COX-1 rather than COX-2 is responsible for
ulcerogenic side effect (Mc Carthy, 1989; Warner et al.,
1999; Wolfe et al., 1999). Based on these observations, it
has been suggested that selective COX-2 inhibitors may act
as safer NSAIDs, devoid of ulcerogenic side effects and a
number of such selective COX-2 inhibitors have been
developed and introduced in the market for clinical use
(Xie et al., 1992; Hawkey, 1999). However, long term use
of these agents has shown some potential limitation,
including ulcer exacerbation in high risk patients, delayed
GI ulcer healing, kidney toxicity, as well as, cardiovascular
side effects (Dogne et al., 2005). These observations
indicated that safety of these agents is questionable on their
long term use and some of these agents have been withdrawn from the market (Schnitzer, 2001). Thus, the initial
enthusiasm of developing selective COX-2 inhibitors has
faded away and need for design and development of safer
NSAIDs still remain.
Recently, it has been well recognized that generation of
free radicals and various reactive oxygen species (ROS)
plays a significant role in the formation of gastric ulceration associated with NSAID therapy (Bandyopadhyay
et al., 1999; Hassan et al., 1998). Therefore, use of antioxidants might be useful in the prevention of gastric ulcers

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by scavenging of ROS or accelerating the healing of peptic


ulcers. During the past few decades, a large number of
naturally occurring compounds have been identified as
antioxidants, which are viewed as promising therapeutic
agents for treating free radical mediated diseases including
NSAID induced peptic ulcers. A large number of herbs and
spices are recognized as source of natural antioxidants, and
studies have confirmed their efficacy for the treatment of
GI ulcers (Nakatani, 2000). Thus, it has been suggested that
concomitant use of antioxidants and NSAIDs in formulated
dosage form may possibly decrease the risk of NSAIDs
induced GI side-effects (Jimenez and Alcaraz, 1988;
Repetto et al., 2002). However, there are potential advantages in giving such drugs having complementary pharmacological activities in the form of a single chemical
entity. Such agents are named as mutual prodrugs which
are designed with improved physicochemical properties
and release the parent drug at the site of action (Singh and
Sharma, 1994; Bhosle et al., 2006; Leppanen et al., 2002).
Indomethacin (1) is one of the most potent NSAIDs.
However, its use is restricted due to high incidences of
ulcerogenic side effects. Therefore, in this study, this
potential NSAID has been selected to be conjugated with
flavonoids to form indomethacinantioxidant mutual prodrugs. The salient features of the usefulness of conjugation
of flavonoids with NSAIDs are (i) flavonoids are normal
dietary constituents and are nontoxic (Nakatani, 2000), (ii)
these agents have healing affect on gastric ulcerogenicity
(Martin et al., 1998), (iii) any drug with free carboxyl group
can be derivatized into corresponding esters with flavonoid
to alter the physicochemical properties, (iv) being a nutritional substance, use of flavonoid as derivatizing group
might permit more specific target site for enzymes involved
in the terminal phase of digestion and (v) flavonoids possess
marked antiinflammatory and antiulcer activity due to its
antioxidant effect (Martin et al., 1998; Cotelle, 2001).
This work aims to synthesize indomethacinflavonoids
ester mutual prodrugs to get safer NSAIDs, devoid of
ulcerogenic side effects while retaining the antiinflammatory and analgesic activity. In this article, we report the
synthesis and pharmacological evaluation of indomethacinflavonoid mutual prodrug as safer NSAIDs.

Results and discussion


Chemistry
Scheme 1 shows the conjugation of NSAID indomethacin
(1) with flavonoids, naringenin (2), hespertin (3) leading to
the formation of indomethacinnaringenin (4), and indomethacinhespertin mutual prodrugs (5). For this purpose,
indomethacin (1) was dissolved in THF followed by the

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Med Chem Res (2011) 20:687694

addition of DCC, and stirred at room temperature for 1 h. To


this solution, the corresponding flavonoid was added along
with DMAP, and the reaction mixture was stirred at room
temperature for 24 h. After processing the reaction mixture,
desired products (4, 5) were obtained. These compounds
were purified by column chromatography, and yield
obtained was (32.933.5%). Their structures were confirmed
by the use of elemental analysis and spectral studies.
The IR spectrum of the derivative 4 showed the absorption peaks at 3388.9 and 2955.9 cm-1 characteristic of OH
and CH stretching. The peaks at 1758.1, 1658.1, and
1639.2 cm-1 showed presence of C=O (ester linkage), C=O
(indomethacin), and C=O cyclic (naringenin ring) stretching, respectively. The 1HNMR spectrum showed signal at
d 2.45 for CH3 protons (s, indomethacin ring), d 3.82 for
OCH3 protons (s, indomethacin ring), d 3.92 for CH2 (s,
indomethacin). Two signals appeared at d 2.732.78 for cis
C-3-H (dd, J = 3.2, 17.2 Hz, naringenin moiety) and at
2.963.03 for trans C-3-H (dd, J = 12.9, 17.2 Hz, naringenin moiety). A double doublet for C-2-H (J = 3.0,
12.8 Hz) appeared at d 5.345.38. Aromatic protons of
indomethacin and naringenin appeared between d 6.68 and
7.68. Two distinct ABq were observed at 7.457.48 and
7.657.68 representing para coupling of the Indomethacin
molecule. A signal appeared at d 11.97 for C-5-OH (s, naringenin moiety) (exchangeable with D2O). In 13CNMR,
signals appeared at d 168.55, 169.60, and 195.7 for C=O
(indomethacin nucleus), COO (ester linkage) and cyclic
C=O (naringenin ring). The mass spectrum of the compound
4 showed molecular ion peak at m/z 360.6 (M?, 100%) and
M ? 1 ? Na peak appeared at m/z 636.
Similarly, IR spectrum of the derivative 5 showed the
absorption peaks at 3325.9 and 2928.0 cm-1 characteristic
of OH and CH stretching. The peaks appeared at 1737.6,
1639.8, and 1625.6 cm-1 characteristic of C=O (ester
linkage), C=O (indomethacin), and C=O cyclic (hespertin
ring) stretching, respectively. The 1HNMR spectrum
showed signal at d 2.44 for CH3 protons (s, indomethacin
ring), d 3.74 for OCH3 (s, hespertin ring), d 3.82 for OCH3
protons (s, indomethacin ring), d 3.94 for CH2 (s, indomethacin ring). Two signals appeared at d 2.722.77 for cis
C-3-H (dd, J = 3.2, 17.2 Hz, hespertin ring) and at 2.95
3.02 for trans C-3-H (dd, J = 12.9, 17.2 Hz, hespertin
ring). A signal for C-2-H (dd, J = 3.0, 12.8 Hz, hespertin
ring) appeared at d 5.265.30. Aromatic protons of indomethacin and hespertin appeared between d 6.66 and 7.68.
Two distinct ABq were observed at 7.747.47 and 7.64
7.68 representing para coupling of the indomethacin molecule. A signal appeared for at d 11.97 for C-5-OH (s,
hespertin moiety) (exchangeable with D2O). In 13CNMR,
signals for C=O (indomethacin), COO of ester and cyclic
C=O appeared at d 168.55, 169.60, and 197.69. The mass
spectrum of the indomethacinhespertin 5 showed

Med Chem Res (2011) 20:687694


Scheme 1 Synthesis of
indomethacinflavonoid mutual
prodrugs. Reagent and
conditions: a THF, DCC,
DMAP, room temperature, 24 h

689
R1
R2

H3CO

OH

HO

O
N

OH

2 R1= H R2= OH , naringenin


3 R1= OH R2=OCH3, hesperetin

Flavonoid
Cl

1
a
R1

R2

H3CO

O
N
OH

Cl

4 R1= H R2= OH, indomethacin-naringenin


5 R1= OH R2=OCH3, indomethacin-hesperetin

molecular ion peak at 247.5 (M?, 100%) and the M ?


1 ? Na peak at m/z 664.

The parent drug indomethacin has been used as reference


substance.

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

For the analgesic activity, abdominal writhing assay was


performed (Koster et al., 1959). Writhing was induced by
intraperitoneal (i.p.) injection of freshly prepared acetic acid
solution (1%, 10 ml/kg, i.p.) in mice. The number of writhes
(constriction of abdomen, turning of trunk, extension of hind
limbs) due to acetic acid was expressed as a nociceptive
response. Vehicle treated control mice were given 1% acetic
acid and writhing response was noted for 20 min. Indomethacin(10 mg/kg, p.o.) as well as synthesized conjugates
at equimolar doses significantly reduced the writhing
response (Table 2). The results showed that these derivatives
(4, 5) possess analgesic activity (86.7 0.46, 89.4 0.59)
comparable to the parent drug (74.17 0.90) (Table 2).

Pharmacological evaluation

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Med Chem Res (2011) 20:687694

Table 1 Antiinflammatory activity of indomethacin, indomethacinflavonoid conjugate (4, 5) and indomethacin ? flavonoid physical mixtures
Compound

Equimolar dose (mg/kg, p.o.)

% Increase in paw volume (mean SEM)


2h

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*

* P \ 0.05 as compared to control


#

P \ 0.05 as compared to indomethacin (12 mg/kg, p.o.)

Table 2 Analgesic activity of indomethacin, indomethacinflavonoid conjugate (4, 5) and indomethacin ? flavonoid physical mixtures
Compound

Equimolar dose (mg/kg, p.o.)

% Inhibition in writhing (mean SEM)

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*

* P \ 0.05 as compared to control


#
P \ 0.05 as compared to indomethacin (10 mg/kg, p.o.)

Table 3 Antiulcer activity of indomethacin, indomethacinflavonoid conjugate (4, 5) and indomethacin ? flavonoid physical mixtures
Compound

Equimolar dose (mg/kg, p.o.)

Ulcer index (mean SEM)

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*

* P \ 0.05 as compared to control


#
P \ 0.05 as compared to indomethacin (48 mg/kg, p.o.)

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).

Med Chem Res (2011) 20:687694

691

Fig. 1 Effect on acute


administration of (a) 0.5% CMC
(control), (b) indomethacin,
(c) indomethacinnaringenin
codrug, (d) indomethacin ?
naringenin physical mixture,
(e) indomethacinhespertin
codrug and (f) indomethacin ?
hespertin physical mixture on
gastric mucosa in rats (arrows
indicate ulcers)

The results listed in Tables 1, 2 and 3 showed that these


indomethacinflavonoid conjugates (4, 5) lack GI ulcerogenic side effects with retention of antiinflammatory and
analgesic activity.

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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Solutions were routinely dried over anhydrous sodium


sulfate prior to evaporation. Naringenin and hespertin were
purchased from Sigma-Aldrich. All other reagents and
solvents were of AR grade.
General procedure for the synthesis of indomethacin
flavonoid conjugate (4, 5)
Indomethacin (3.57 g, 0.01 M) was dissolved in 25 ml of
THF followed by the addition of DCC (2.06 g, 0.01 M).
The reaction mixture was stirred at room temperature for
1 h. To this solution, flavonoid (0.01 M) and DMAP
(40 mg) was added and stirred for 24 h at room temperature. The precipitated dicyclohexyl urea was filtered off
and the solvent of the filtrate was removed under reduced
pressure. To the residue obtained, cold ether (20 ml) was
added. The ethereal solution was filtered and the filtrate
was washed with acetic acid (1%, 39 50 ml), HCl (5%,
39 50 ml), NaHCO3 (5%, 39 50 ml) and finally with
water (39 50 ml). The organic layer was dried over
anhydrous sodium sulphate, filtered, and solvent was
removed under reduced pressure to obtain crude semisolid
product, which was subjected to column chromatography
using CH2Cl2: ethyl acetate (9.9:0.1) as eluent to obtain
pure compounds 4, 5).
Indomethacinnaringenin conjugate (4)
Conjugate (4) was obtained in 33.5% yield (2.05 g,
33.5%), as slight yellow solid, mp 118.5C, Rf 0.52
(chloroform:ethyl acetate, 9:1), IR (KBr): 3388.9 (OH st),
2955.9 (CH st), 1758.1 (C=O st), 1639.2 (C=O st, cyclic),
1601.4 (C=C st), 1348.4, 1291.5 (C=COC st), 1161.1,
1074.3 (COC st) cm-1. 1HNMR (CDCl3): d 2.45 (3H, s,
C-2-CH3), 2.732.78 (1H, dd, J = 17.2, 3.2 Hz, cis-C-300 H), 2.963.03 (1H, dd, J = 17.2, 12.9 Hz, trans-C-300 -H),
3.82 (3H, s, C-5-OCH3), 3.92 (2H, s, C-3-CH2), 5.345.38
(1H, dd, J = 12.8, 3.0 Hz, C-200 -H), 5.925.93 (1H, d,
J = 2.0 Hz, C-600 -H), 5.955.96 (1H, d, J = 2.0 Hz, C-800 H), 6.686.70 (IH, dd, J = 9, 2.52, C-6-H), 6.876.89 (1H,
d, J = 9, C-7-H), 7.047.05 (1H, d, J = 2.48, C-4-H),
7.097.12 (2H, m, C-3000 -H, C-5000 -H), 7.147.43 (2H, m,
C-6000 -H, C-2000 -H), 7.457.48 (2H, m, C-30 -H, C-5-H),
7.657.68 (2H, m, C-20 -H, C-60 -H), 11.97 (1H, s, C-400 -OH,
exchangeable with D2O). 13CNMR (CDCl3): d 18.4 (C-18),
30.62 (C-9), 43.30 (C-3), 55.84(C-17), 78.51 (C-2), 95.60
(C-8), 96.93 (C-6), 102.8 (C-14), 102.99 (C-16), 111.86
(C-5a), 115.15 (C-10), 121.92 (C-3, 5), 127.42 (C-2, 6),
129.26 (C-22, 24), 129.6 (C-21, 25), 130.52 (C-12a),
130.93(C-10 ), 131.27 (C-21, 25), 133.75 (C-20), 136.25 (C16a), 136.41 (C-11), 139.53 (C-23), 150.87 (C-15), 156.18
(C-40 ), 162.94 (C-5), 164.31 (C-7), 165.35 (C-8), 168.55
(C-9a), 169.60 (C-19), 195.7 (C-4). Mass (m/z): 636

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Med Chem Res (2011) 20:687694

(M ? 1 ? Na), 584.5, 471.5, 360.6 (base peak, 100%),


301.4, 265.5, 247.5, 225.5, 202.5.
Indomethacinhespertin conjugate (5)
Conjugate (5) was obtained in 32.9% yield (2.11 g,
32.9%), as white solid, mp 206.58C, Rf 0.61 (CHCl3:ethyl
acetate, 9:1), IR (KBr): 3325.9 (OH st), 2928.2 (CH st),
1737.6 (C=O st), 1639.8 (C=O st, cyclic), 1600.1 (C=C st),
1379.1, 1269.6 (C=COC st), 1158.7, 1086.6 (COC st)
cm-1. 1HNMR (CDCl3): d 2.44 (3H, s, C-2-CH3), 2.72
2.77 (1H, dd, J = 17.2, 3.2 Hz, cis-C-300 -H), 2.953.02
(1H, dd, J = 17.2, 12.9 Hz, trans-C-300 -H), 3.74 (3H, s,
C-4000 -OCH3), 3.82 (3H, s, C-5-OCH3), 3.94 (2H, s, C-3CH2), 5.265.30 (1H, dd, J = 12.8, 3.0 Hz, C-200 -H),
5.8825.887(1H, d, J = 2.0 Hz, C-600 -H), 5.945.95 (1H,
d, J = 2.0 Hz, C-800 -H), 6.666.69 (IH, dd, J = 9,2.52,
C-6-H), 6.876.89 (1H, d, J = 9, C-7-H), 6.936.96 (1H,
d, J = 9, C-5000 -H), 7.0727.078 (1H, d, J = 2.48, C-4-H),
7.1207.125 (1H, d, J = 2.16, C-2000 -H), 7.197.22 (1H,
dd, J = 9, 2.52, C-6000 -H), 7.457.47 (2H, m, C-3-H, C-5H), 7.647.68 (2H, m, C-2-H, C-6-H), 11.97 (1H, s,
C-500 -OH, exchangeable with D2O). 13CNMR (CDCl3): d
15.23 (C-18), 30.52 (C-9), 49.10 (C-3), 56.24 (C-17), 56.7
(C-4a), 73.2 (C-2), 99.60 (C-8), 100.1 (C-6), 105.8
(C-14),106.99 (C-16),107.86 (C-5a), 112.15 (C-13),113.65
(C-10), 115.7 (C-5), 115.9 (C-2), 120.92 (C-6), 128.3
(C-12a), 129.46 (C-22, 24), 131.6 (C-21, 25), 132.52
(C-16a), 134.63 (C-1), 135.7 (C11), 134.8 (C-20), 139.25
(C-23), 143.1 (C-3), 148.1 (C-1), 155.87 (C-15), 156.18
(C-4), 162.94 (C-5), 164.31 (C-7), 165.35 (8a), 168.55
(C-9a), 169.60 (C-19), 197.69 (C-4). Mass (m/z): 664
(M ? 1 ? Na), 584.5, 471.5, 360.6, 301.4, 265.5, 247.5
(base peak, 100%), 225.5, 202.5.
Pharmacology
Animals
Sprague-Dawley (sd) rats (weighing 150200 g) of both
sex and LACCA mice (male, 25.35 g) procured from
central animal house, Panjab University, Chandigarh, India
were used. The animals were housed in plastic cages (five
rats/cage) under standard laboratory conditions and maintained on rat chow and water.
Antiinflammatory activity
Antiinflammatory activity was assessed by the carrageenan
induced rat paw edema model. Rats were divided into
six groups: (i) vehicle (control), (ii) indomethacin (standard),
(iii) indomethacinnaringenin, (iv) indomethacinhespertin,

Med Chem Res (2011) 20:687694

(v) indomethacin ? naringenin (physical mixture) and (vi)


indomethacin ? hespertin (physical mixture). The rats were
fasted for 12 h prior to test. The test compounds were suspended in carboxymethylcellulose (0.5%, CMC) and
administered orally. Control animals were given the corresponding amount of vehicle (0.5%, CMC). The compounds
were administered on molar equivalent basis of
Indomethacin.

693

(unpaired), analysis of variance (ANOVA) test, followed


by Dunnetts test for determining the levels of significance
in antioxidant studies. P \ 0.05 was considered statistically significant.
Acknowledgement The financial support from University Grant
Commission (UGC) and the research facilities provided by University
Institute of Pharmaceutical Sciences, Panjab University, India are
gratefully acknowledged.

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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