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Mediators Inflamm. 2007; 2007: 47523. Published online 2007 March 19. doi: 10.1155/2007/47523. | PMCID: PMC1852887 |
Calotropis procera Latex Extract Affords Protection against Inflammation and Oxidative Stress in Freund's Complete Adjuvant-Induced Monoarthritis in Rats Vijay L. Kumar* and Sanjeev Roy Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India Received December 1, 2006; Revised February 12, 2007; Accepted February 12, 2007. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In view of the well-established anti-inflammatory properties of latex of Calotropis procera (DL), the present study was carried out to evaluate the protective effect of its methanol extract (MeDL) against inflammation and oxidative stress in monoarthritis induced by Freund's complete adjuvant (FCA) in rats. Intra-articular injection of FCA produced inflammation of the joint with a peak effect occurring on day 4 where a maximum increase in the levels of myeloperoxidase and inflammatory mediators like PGE2, TNF-α, and nitric oxide was observed. This was associated with oxidative stress with a marked reduction in the levels of glutathione, catalase, superoxide dismutase and glutathione peroxidase and an increase in the lipid peroxidation as indicated by the higher levels of thiobarbituric acid reactive substances (TBARSs). Subsequently on day 28 the histological analysis of the joint also revealed arthritic changes. Daily treatment of rats with MeDL (50 and 500 mg/kg) and standard anti-inflammatory drug rofecoxib (20 and 100 mg/kg), produced a significant attenuation in the inflammatory response and ameliorated the arthritic changes in the joint. The protection afforded by MeDL and rofecoxib was more pronounced than that of phenylbutazone and was associated with normalization of the levels of inflammatory mediators and biochemical parameters of oxidative stress. However, the overall protection afforded by rofecoxib was better than that of MeDL. The incidence of degenerative and inflammatory joint diseases,
namely osteoarthritis and rheumatoid arthritis, is very high over
the world [ 1, 2]. Typically arthritis is a common inflammatory disorder of the joint
characterized by inflammation of the synovial membrane, pain, and
restricted joint movement. Experimentally arthritis could be
induced by various inflammagens of which Freund's complete
adjuvant (FCA) is the most commonly used agent [ 3, 4].
Intra-articular injection of FCA is known to induce inflammation
as well as immune response and to produce features that resemble
rheumatoid arthritis in humans. The acute inflammatory response
induced by FCA is associated with leukocyte infiltration, mast
cell activation, and release of cytokines and free radicals
[ 5, 6]. This process gets aggravated with macrophage
activation and secretion of bioactive products that play an
important role in tissue destruction, vascular proliferation, and
fibrosis over a period of time [ 7]. The role of cytokines like IL-1, IL-6, tumor necrosis
factor- α (TNF- α), prostaglandins (PGs), and nitric
oxide (NO) in arthritis has been well established. The
levels of these inflammatory mediators have been reported to be
high in both experimental models of arthritis and in patients
suffering from arthritis [ 8, 9]. Besides, generation of
reactive oxygen species (ROS) and other free radicals also
contribute to the pathogenesis of arthritis [ 10]. In view of
the underlying mechanisms, both nonsteroidal and steroidal
anti-inflammatory drugs are used for the management of arthritis
[ 11]. However, due to side effects associated with the
long-term use of these agents, many patients tend to use
alternative therapeutic approaches including herbal therapies that
have been considered safe and effective in alleviating chronic
pain associated with arthritis [ 12]. Calotropis procera (Ait.) R. Br., a wild growing plant of
family Asclepiadaceae, is well known for its medicinal
properties. Different parts of this plant have been reported to
exhibit anti-inflammatory, analgesic, and antioxidant properties
[ 13]. The latex of this plant produces potent
anti-inflammatory, analgesic, and weak antipyretic effects in
various animal models [ 14– 16]. Both latex and its methanol
extract (MeDL) have been shown to inhibit inflammatory cell influx
and edema formation induced by various inflammagens [ 17]. It
also improves locomotor functions in experimentally induced
monoarthritis in rats (unpublished findings). In view of these
properties, the present study was carried out to evaluate the
effect of MeDL on the levels of PGE 2, TNF- α, nitric
oxide (NO), myeloperoxidase (MPO), oxidative stress
parameters, and joint histology in FCA-induced monoarthritis in
rats. The effect of MeDL was compared with rofecoxib, a selective
COX-2 (cyclooxygenase-2) inhibitor, and phenylbutazone (PBZ) a
nonselective COX inhibitor. 2.1. Plant material and drugs The C. procera plant was identified by the Raw Materials,
Herbarium and Museum Division, National Institute of Science and
Communication, CSIR, New Delhi, where a voucher specimen is
preserved (Voucher no. PID 1739). The latex was collected from the
aerial parts of the plant growing in the wild. It was dried under
shade at ambient temperature and was soxhlated to obtain methanol
extract (MeDL) [ 18]. The MeDL was triturated with gum acacia
used as suspending agent (1 : 1) in normal saline (NS), and
administered orally to rats at doses ranging from 50 to
500 mg/kg (MeDL 50 and MeDL 500). Rofecoxib was administered
orally at 20 and 100 mg/kg doses (Rofe 20 and Rofe 100) and
phenylbutazone at a dose of 100 mg/kg (PBZ). The drugs used in
the study were obtained from Arbro Pharmaceuticals (New Delhi,
India) (rofecoxib and phenylbutazone). Freund's complete adjuvant
was obtained from Sigma-Aldrich Corporation (Bangalore, India). 2.2. Animals The study was carried out on 5-6-month-old Wistar rats of either
sex weighing 150–180 g. The rats were obtained from the
Experimetal Animal Facility of the Institute, were kept at ambient
temperature, and had free access to water and diet. The animal
experiments were carried in accordance with the guidelines of
Institutional Animal Ethics Committee. 2.3. Experimental design Monoarticular arthritis was induced in rats by injecting
0.1 mL of 0.1% FCA (Sigma Aldrich, USA) into the
intra-articular space of right ankle joint (day 0) [ 19]. The
increase in joint diameter was measured daily starting from day 0,
using a screw gauge till the time of peak inflammation (day 4),
and then it was measured every fourth day for a period
of 28 days. The rats were divided into seven groups, consisting of
six animals each for analysis of histological and biochemical
parameters. Group I: normal control; Group II: FCA control. In Group III to
Group VII, drugs were administered orally as suspension with gum
acacia in NS, 1 hour before injecting FCA on day 0 and then daily
either for 4 days or for 28 days at doses based on our earlier
studies where no observable toxic effects were seen
[ 17, 20, 21], Group III: MeDL (50 mg/kg, MeDL 50); Group
IV: MeDL (500 mg/kg, MeDL 500); Group V: rofecoxib
(20 mg/kg, Rofe 20); Group VI: rofecoxib (100 mg/kg, Rofe
100); Group VII: phenylbutazone (100 mg/kg, PBZ). 2.4. Determination of levels of oxidative stress parameters
and inflammatory mediators The levels of biochemical markers of oxidative stress and
inflammatory mediators were determined at the site of
inflammation. Animals were sacrificed at the time of peak
inflammation (day 4) and the tissue of the arthritic joint was
removed and processed for the estimation of glutathione (GSH, mg/g
tissue) [ 22], catalase (U/mg protein) [ 23], superoxide
dismutase (SOD, U/mg protein) [ 24], glutathione peroxidase
(GPx, U/mg protein) [ 25], thiobarbituric acid-reactive
substances (TBARSs) as a measure of malondialdehyde (MDA, nmol/g
tissue) [ 26], nitric oxide (NO, μM/mg tissue)
[ 27], prostaglandin E 2 (PGE 2, pg/mg tissue, R&D
Systems), tumor necrosis factor- α (TNF- α, pg/mg
tissue, Diaclone Research), and myeloperoxidase (MPO, OD/mg
tissue) [ 28] levels. 2.5. Estimation of protein The protein concentration of the samples was determined by
Bradford's method [ 29]. 2.6. Histological analysis Rats were sacrificed on day 28, the limbs were removed above the
stifle joints, degloved and fixed in 1% formaldehyde in saline.
They were decalcified in EDTA, processed for paraffin embedding,
sectioned, and stained with hematoxylin-eosin [ 30]. The
sections were examined for arthritic changes in the control as
well as in the drug-treated rats. 2.7. Statistical analysis The values are expressed as mean ± SEM of six
observations and ANOVA was used to compare the groups. The
statistical analysis was carried out by the version 10 of the SPSS
program and the values of P < .05 were considered as
statistically significant. 3.1. Effect of MeDL on joint inflammation Injection of FCA into right ankle joint of rat produced an
increase in joint diameter that was maximum on day 4 (2.17 ± 0.13 mm), and thereafter it gradually declined. Injection of
NS on the other hand produced a marginal increase in the joint
diameter on day 2 (0.04 ± 0.10 mm) that returned to normal
within 4 days (). The inhibitory effect of various drugs was evaluated on the day of
peak inflammation, that is, day 4. Oral administration of MeDL
produced a dose-dependent decrease in joint inflammation and the
increase in joint diameter was 1.59 ± 0.09 mm and 1.20 ± 0.08 mm in MeDL 50 and MeDL 500 groups against 2.17 ± 0.13 mm in FCA control (27% and 45%
inhibition). COX-2 selective inhibitor, rofecoxib, was more
effective in inhibiting joint inflammation as compared to MeDL.
The increase in joint diameter in Rofe 20 and Rofe 100 groups was
1.66 ± 0.08 mm and 0.70 ± 0.33 mm (24% and 68%
inhibition). PBZ, a nonselective COX inhibitor produced 16%
inhibition in joint inflammation with the increase in joint
diameter of 1.82 ± 0.12 mm ( Table 1). | Table 1Inhibition of joint inflammation by various drugs in FCA-induced
monoarthritis. Values given are mean ± SEM (n = 6). |
3.2. Effect of MeDL on tissue levels of inflammatory
mediators The inflammation induced by FCA was associated with an increase in
the levels of PGE2 and TNF-α. The tissue levels of
PGE2 and TNF-α were 7.35 ± 0.14 and 71.5 ± 5.00 pg/mg tissue in the FCA control as compared to 1.00 ± 0.01 and 2.50 ± 5.00 pg/mg tissue in normal control rats,
respectively. Both MeDL and rofecoxib produced a significant
decrease in the levels of PGE2 and TNF-α (P < .005).
The levels of PGE2 in MeDL 500 group were 0.6 ± 0.05, and
in Rofe 100 group were 1.00 ± 0.23, and that of TNF-α
in MeDL 500 group were 14.50 ± 15.00, and in Rofe 100 group
were 10.50 ± 5.00 pg/mg tissue, respectively. PBZ on the
other hand was not effective in reducing the tissue PGE2
levels and was only marginally effective in reducing the tissue
TNF-α levels (). FCA injection produced
a significant increase in tissue MPO activity from 0.06 ± 0.01 OD/mg tissue in normal control rats to
1.33 ± 0.11 OD/mg tissue. Treatment with MeDL and rofecoxib
significantly reduced the tissue MPO activity and their effect was
comparable in this regard. The MPO levels were 0.14 ± 0.02 and
0.09 ± 0 OD/mg tissue in MeDL 500 and Rofe 100
group, respectively. PBZ on the other hand was marginally effective in
decreasing the MPO levels as compared to FCA control (1.00 ± 0.03 versus 1.33 ± 0.11 OD/mg tissue)
(). MeDL and rofecoxib were also equieffective
in reducing the tissue NO levels in the arthritic rats
(2.0 ± 0.11 and 2.8 ± 0.10 against 5.9 ± 0.50 μM/mg tissue in FCA control). The effect of PBZ in
this regard was comparable to that of MeDL and rofecoxib (3.0 ± 0.04 μM/mg tissue) (). 3.3. Effect of MeDL on tissue levels of GSH, catalase, SOD, GPx,
and TBARS Oxidative stress associated with FCA-induced monoarthritis was
evaluated by measuring the levels of GSH, catalase, SOD, GPx, and
TBARS in the inflamed joint tissue. FCA injection into the ankle
joint markedly decreased the tissue GSH, catalase, SOD, and GPx
levels from 18.20 ± 1.10 mg/g tissue, 28.60 ± 0.15 U/mg protein, 277.70 ± 0.15 U/mg protein, and
31.40 ± 0.10 U/mg protein in normal control rats to 4.80 ± 0.40 mg/g tissue, 0.17 ± 0.02 U/mg protein, 79.90 ± 0.10 U/mg protein, and 5.97 ± 0.05 U/mg protein,
respectively. Both MeDL and rofecoxib produced a dose-dependent
increase in the level of these oxidative stress parameters. On the
other hand, FCA produced a marked increase in the levels of TBARS
from 3.50 ± 0.50 nmol/g tissue to 103.00 ± 3.00 nmol/g tissue. Both MeDL and rofecoxib produced a
dose-dependent decrease in the levels of TBARS and the effect of
these drugs was comparable. PBZ, on the other hand, produced a
marginal change in the levels of all the oxidative stress
parameters as compared to FCA control ( Table 2). | Table 2Effect of drugs on parameters of oxidative stress in
FCA-induced monoarthritis. Values given are mean ± standard error of the mean (n = 6). |
3.4. Effect of MeDL on joint histology The inflammation induced by FCA was associated with cellular
infiltration, edema, granuloma formation, and bone destruction on
day 28 (). Both MeDL 500 and Rofe 100 significantly decreased the arthritic
changes as compared to FCA control, however, rofecoxib was more
effective in this regard (Figures and
). The latex of Calotropis procera is well known for its
anti-inflammatory properties in various experimental models. It
has also been shown to afford protection against functional
impairment produced by FCA in rat model of monoarthritis. In the
present study, we have evaluated the effect of latex of C.
procera on the levels of inflammatory mediators, oxidative stress
parameters, and joint histology in FCA-induced monoarthritis model
and compared it with rofecoxib. Intra-articular injection of FCA produced a peak
inflammatory response in the joint on day 4 that is associated
with fluid exudation, neutrophil infiltration, and mast cell
activation [ 31, 32]. This was followed by a slow regression
and the joint swelling continued up to day 28 possibly due to
oil-based adjuvant and the antigenicity of mycobacterium
[ 33]. The inhibitory effect of drugs was evaluated against
FCA-induced inflammation on day 4. MeDL produced a dose-dependent
inhibition in joint inflammation that could be attributed to its
ability to inhibit cellular influx and vascular permeability
[ 17, 20]. It has earlier been shown to inhibit inflammatory
response induced by various mediators and inflammagens like
histamine, bradykinin, prostaglandins, carragenin, and compound
48/80 [ 17]. The role of various inflammatory mediators in
adjuvant-induced arthritis has been well established [ 34, 35].
In our study, rofecoxib, a selective COX-2 inhibitor, was found to
be more effective than MeDL and phenylbutazone in inhibiting the
FCA-induced joint inflammation as reported earlier by Kumar et al.
[ 21] and Francischi et al. [ 36]. Rofecoxib acts by
inhibiting COX-2 that plays an important role in an inflammatory
response. The greater efficacy of rofecoxib could be attributed to
its better distribution at the site of inflammation as suggested
for other COX-2 inhibitors [ 37]. Further, rofecoxib was also
found to be more effective as compared to MeDL in inhibiting cell
influx and bone destruction as revealed by histological analysis.
The inhibitory effect of MeDL and rofecoxib on cell influx was
further substantiated by their ability to decrease tissue MPO
activity that has been used as an index of granulocyte
infiltration. It is interesting to note that PBZ produced only a
marginal decrease in tissue MPO activity. The inability of PBZ to
inhibit cellular influx has also been reported by Meacock and
Kitchen [ 38] and Arya and Kumar [ 17]. The neutrophilic recruitment at the site of inflammation has been
reported to involve TNF- α production that induces
the synthesis of LTB4, a well-known chemoattractant and
prostaglandins that plays a key role in the pathogenesis of
inflammatory diseases. Elevated levels of TNF- α and
prostaglandins have been reported in arthritic patients and in
experimentally induced arthritis [ 39, 40]. In our study, both
MeDL and rofecoxib produced a marked reduction in the tissue
levels of TNF- α and PGE 2. However, PBZ was ineffective
in reducing the levels of PGE 2 though it produced a significant
decrease in tissue TNF- α levels. A marked reduction in the
levels of PGE 2 brought about by MeDL was comparable to that of
rofecoxib and suggests that like rofecoxib, MeDL might be
inhibiting COX-2. Earlier, the MeDL was shown to inhibit
inflammation induced by PGE 2 [ 17]. The role of NO has been well established in an inflammatory
response. As the inflammatory response progresses, large
quantities of NO are generated through the induction of
iNOS (inducible nitric oxide synthase) that reacts with
superoxide anion to form peroxynitrate, a potent oxidizing
molecule capable of eliciting lipid peroxidation. Lipid
peroxidation is the oxidative deterioration of polyunsaturated
lipids to form radical intermediates that bring about cellular
damage. MDA, a major end product of this reaction, is an index of
lipid peroxidation and has been estimated as TBARS [ 41]. In
our study, both MeDL and rofecoxib brought down the tissue levels
of NO and TBARS. Besides, the infiltrating cells also
generate reactive oxygen species and free radicals that bring
about destruction of the inflamed joint. As a result, the
scavenging enzyme SOD that leads to the formation of hydrogen
peroxide is utilized and its activity is reduced in arthritic
rats. The hydrogen peroxide thus generated is decomposed by
catalase and glutathione peroxidase. Excessive production of lipid
hydroperoxide may also contribute to decreased activity of GPx in
arthritic condition [ 42]. Beside enzymatic antioxidants, the level of glutathione, a nonenzymatic reducing agent that traps
free radicals and prevents oxidative stress, is also decreased in
arthritis [ 43]. Both MeDL and rofecoxib maintained the
oxidative homeostasis, and the levels of GSH and activities of
catalase, SOD, and GPx were comparable to the control animals. The
antioxidant properties of rofecoxib and latex of C.
procera have also been reported earlier [ 44, 45]. Thus, present study shows that the latex of C. procera
markedly reduces cell influx, release of mediators, and oxidative
stress associated with arthritic condition, and therefore has the
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