Autonomic & Autacoid

Pharmacology
Autonomic and Autocoid
Pharmacology Protective effect of proteins derived from
Calotropis procera latex against acute
2015, 35, 1–8

inflammation in rat

V. L. Kumar1, B. Guruprasad1, P. Chaudhary1, S. M. A. Fatmi1, R. S. B. Oliveira2 &

M. V. Ramos3
1
Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110 029, India, 2Centro
Correspondence:


Universit
ario Est
acio de S
a Via Corpus Rua Eliseu Uchoa Becco, n°600 - Bairro Agua Fria CEP: 60810-270, Fortaleza,
V. L. Kumar Cear
a, Brazil, 3Departmento de Bioquimica de Biologia Molecular, Universidade Federal do Ceara, Campus do Pici, Cx.
Postal 6033, Fortaleza-Ce Brasil, CEP, 60451-970, Brazil

Summary
1. The non-dialysable proteins present in the latex of plant Calotropis procera possess anti-inflamma-
tory and analgesic properties.
2. The aim of this study was to evaluate the effect of latex proteins (LP) on the level of inflammatory
mediators, oxidative stress markers and tissue histology in the rat model of carrageenan-induced acute
inflammation. This study also aimed at evaluating the anti-inflammatory efficacy of LP against differ-
ent mediators and comparing it with their respective antagonists.
3. Paw inflammation was induced by subplantar injection of carrageenan, and the effect of LP was
evaluated on oedema volume, level of TNF-a, PGE2, myeloperoxidase, nitric oxide, reduced glutathi-
one, thiobarbituric acid-reactive substances and tissue histology at the time of peak inflammation.
4. Paw inflammation was also induced by histamine, serotonin, bradykinin and PGE2, and the inhibi-
tory effect of LP against these mediators was compared with their respective antagonists at the time of
peak effect.
5. Treatment with LP produced a dose-dependent inhibition of oedema formation, and its anti-inflam-
matory effect against carrageenan-induced paw inflammation was accompanied by reduction in the lev-
els of inflammatory mediators, oxidative stress markers and normalization of tissue architecture.
6. LP also produced a dose-dependent inhibition of oedema formation induced by different inflamma-
tory mediators, and its efficacy was comparable to their respective antagonists and more pronounced
than that of diclofenac.
7. Thus, our study shows that LP has a potential to be used for the treatment of various inflammatory
conditions where the role of these mediators is well established.

Keywords: Calotropis procera, inflammation, mediators, latex proteins

contains proteins (LP) exhibiting these properties

Introduction
Calotropis procera (Ait) R.Br., a laticiferous wild
growing plant of subfamily Asclepiadaceae, has
been used in traditional medicine as a treatment
for various diseases (Wealth of India: Raw materi-
als. III, 1992). The plant oozes out milky latex
when injured or when its aerial parts are plucked.
The latex thus released has been shown to exhibit
inflammatory properties on accidental exposure
(Kumar & Arya, 2006). Interestingly, the aqueous
extract of the dried latex when administered orally
has been shown to inhibit inflammatory response
elicited by various inflammagens (Arya & Kumar,
2005). Not only the latex has been shown to com-
Ó 2015 prise of orally active constituents that exhibit anti-
John Wiley & Sons Ltd inflammatory and analgesic properties, but it also
when administered parenterally (Kumar & Basu,
1994; Dewan et al., 2000; Soares et al., 2005;
Alencar et al., 2006). In recent studies, these pro-
teins have been shown to exhibit various pharma-
cological properties, of which their ameliorating
effect on inflammatory hyperalgesia and functional
limitations of arthritis in the rat model is notewor-
thy (Kumar et al., 2011, 2014). The previous stud-
ies have also shown that the LP fraction of latex is
free of poly-isoprene rubber-like material and its
administration in various short- and long-term
studies did not produce any toxic effects (Alencar
et al., 2004; Kumar et al., 2014).
Acute inflammation is a transient response of the
tissue to injury and is characterized by redness,
heat, oedema and pain (Ricciotti & FitzGerald,

doi: 10.1111/aap.12022 1

Autonomic and Autocoid 2011). It is initiated by the release of various pre-
Pharmacology
formed mediators such as histamine, serotonin and
2015, 35, 1–8
TNF-a and is sustained and amplified through the
synthesis and release of products of proteolytic cas-
cade-like bradykinin, prostaglandins and an array
of pro-inflammatory cytokines (Medzhitov, 2008).
The orchestrating effect of these mediators on the
microcirculation sets into action various vascular
and cellular events that result in exudation and
efflux of leucocytes at the site of inflammation
(Funk, 2001). Further, these mediators not only
contribute to the inflammatory response but they
also produce an algogenic effect associated with it
(Dray, 1995). It is well established that inhibiting
either the release or action of these mediators could
ameliorate acute inflammation and associated
hyperalgesia. Various antihistaminic, non-steroidal
and steroidal drugs have been shown to suppress
acute inflammatory response elicited by various
phlogistic agents (Kester et al., 2012). Acute inflam-
mation induced in the rat paw by carrageenan rep-
resents a classical model that has been extensively
used to study inflammation and to develop anti-
inflammatory drugs (Ichitani et al., 1997). In this
study, the effect of LP was evaluated on the levels
of inflammatory mediators, oxidative stress markers
and tissue histology in carrageenan-induced paw
inflammation. Further, the protective effect of LP
was also evaluated against inflammation induced
by various inflammatory mediators and its efficacy
was compared with their respective antagonists.
Methods
Isolation of latex proteins
The plant Calotropis procera growing in Fortaleza,
Brazil, was identified by a taxonomist, and a vou-
cher specimen (No. 32663) was deposited at Prisco
Bezerra Herbarium of the Universidade Federal do
Cear
a, Brazil. The latex collected from aerial parts
of this plant in equal volume of distilled water was
centrifuged to separate the rubbery pellet that was
discarded. The supernatant was dialysed against
distilled water, and the non-dialysable fraction
comprising of soluble proteins was freeze-dried
(LP). The details of separation and protein profile
of LP have been reported earlier (Ramos et al.,
2009). This fraction was reconstituted in normal
saline (NS) and administered intravenously in rats.
Experimental animals
The experiments were carried out on Wistar rats
of either sex weighing between 120 and 150 g.
Animals were kept at ambient temperature and
had free access to food and water. All the experi-
ments were carried out in accordance with the
Ó 2015 guidelines of Institutional Animal Ethics Commit-
John Wiley & Sons Ltd tee following due approval.
V. L. KUMAR ET AL.
Drugs, chemicals and reagents
All chemicals used in the study were of analytical
grade and were procured from Qualigens. The
ELISA kits for measuring the levels of TNF-a and
PGE2 were purchased from Cayman Chemical
Company, USA, and Gen-Probe, France, respec-
tively. Histamine and serotonin were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Brady-
kinin and bradyzide were kindly provided by
Bachem (Switzerland) and Novartis (UK), respec-
tively, PGE2 by Astra Zeneca (Bangalore, India),
chlorpheniramine by Glenmark Pharmaceuticals
Ltd. (Pune, India), cyproheptadine by Merind
India Ltd. (Baroda, India) and diclofenac by
Arbro Pharmaceuticals (New Delhi, India).
Induction of paw oedema by different
inflammagens
Oedema was induced in the hind paw of rats by
subplantar injection of 0.1 ml of carrageenan
(0.5%), histamine (0.1%), serotonin (0.002%),
bradykinin (0.001%) or PGE2 (0.004%) in nor-
mal saline (NS). For each inflammagen, a control
group was included along with other groups
where rats were treated with two different doses
of LP, a standard anti-inflammatory drug, dic-
lofenac and respective antagonist (n = 6 per
treatment group, Winter et al., 1962). In the case
of bradykinin-induced inflammation, all the ani-
mals were pretreated with captopril (5 mg kg
1
given subcutaneously) 1 h earlier to prevent its
degradation (Miyamoto et al., 2000). The paw
volume was measured up to a fixed mark on the
lateral malleolus using plethysmography before
inducing inflammation (0 h reading) and then at
different time intervals after injecting an inflam-
magen. The oedema volume at each interval was
calculated by taking the difference from 0-h
reading. The time of peak inflammatory response
was determined for each inflammagen. LP was
administered at the doses of 5 and 25 mg kg
1
by intravenous route 30 min before injecting
the inflammagen based on our previous stud-
ies (Ramos et al., 2009). At the time of peak
inflammation with each inflammagen, the inhibi-
tory effect of LP was compared with that of
standard anti-inflammatory drug, diclofenac
(D10, 10 mg kg
1) and respective antagonists
(antihistaminic chlorpheniramine 3 mg kg
1,
CPM3; antiserotonergic cyproheptadine 3 mg
kg
1, CPH3; antibradykinin–bradyzide 5 mg
kg
1, BZ5) administered orally 1 h before induc-
ing inflammation.
In the case of carrageenan-induced inflamma-
tion, a normal control (NC) group was
included, the rats were killed at the time of
peak inflammation (3 h after injection), and the
paws were removed and stored at
80 °C for
2

Autonomic and Autocoid biochemical analysis. A separate set of similar
Pharmacology groups was included for histological analysis
2015, 35, 1–8
where the dissected paws were preserved in
buffered formalin.
Biochemical estimation
The estimation of reduced glutathione (GSH) was
carried out using the method of Ellman (1959).
Briefly, the tissue was homogenized in 5% tri-
chloroacetic acid (10% w/v) and centrifuged at
3000 g for 10 min. The resulting supernatant was
used for the assay by adding 5-50 dithiobis 2
nitrobenzoic acid at alkaline pH, and the absor-
bance of the yellow-coloured thionitrobenzoic
acid was read at 412 nm. GSH levels were deter-
mined from a standard curve and expressed as
lg g
1 tissue.
A 10% homogenate of tissue prepared in
0.15 M KCl was used to determine the levels of
thiobarbituric acid-reactive substances (TBARS,
an index of lipid peroxidation) by the method of
Ohkawa et al. (1979) where the reaction was car-
ried out at 95 °C under acidic conditions. After
terminating the reaction with a mixture of buta-
nol and pyridine, the absorbance of the organic
layer was read at 532 nm. TBARS levels were
determined from a standard curve plotted using
1,1,3,3- tetraethoxypropane and expressed as
nM g
1 tissue.
Myeloperoxidase (MPO) was extracted by
homogenizing the tissue in 50 mM phosphate buf-
fer containing 0.5% hexadecyltrimethylammoni-
um bromide (pH 6.0) and freezing and thawing it
three times. After centrifugation, the supernatant
was mixed with tetramethylbenzidine and H2O2
followed by termination of reaction with H2SO4.
The levels of MPO were measured using molar
extinction coefficient after taking absorbance at
450 nm and expressed as mM g
1 tissue by the
method of Bradley et al. (1982) with slight modi-
fication.
Nitrite levels were measured as an index of NO
production where the supernatant obtained after
centrifuging the tissue homogenate was mixed
with Griess reagent (1% sulphanilamide and
0.1% naphthylethylenediamide in 5% H3PO4)
and the absorbance was read at 550 nm. The
nitrite levels were determined from a standard
curve plotted using NaNO2 and expressed as
lM g
1 tissue (Ikeda et al., 1996).
Estimation of TNF-a and PGE2
A 10% homogenate of tissue was prepared in a
buffer containing 137 mM NaCl, 2.7 mM KCl,
100 mM Na2HPO4 and 2 mM KH2PO4 (pH 7.4)
and centrifuged at 10 000 g at 4 °C for 10 min.
Ó 2015 The tissue levels of TNF-a and PGE2 were mea-
John Wiley & Sons Ltd
ANTIINFLAMMATORY EFFECT OF C. PROCERA LATEX PROTEINS
sured by sandwich ELISA kits and expressed as
ng g
1 tissue.
Histological analysis
The tissue was kept in 10% EDTA for decalcifica-
tion, embedded in paraffin, sectioned and stained
with haematoxylin-eosin. These sections were
examined for inflammatory changes, and the
drug-treated groups were compared with control
groups.
Statistical analysis
The results are reported as mean  SEM of six
observations. Comparison of control with drug-
treated groups was carried out with a one-way
ANOVA followed by post hoc analysis using SPSS,
version 11.5 (SPSS Inc. Chicago, IL, USA). A
value of P < 0.05 was considered statistically
significant.
Results
Effect of LP on carrageenan-induced paw
inflammation and mediator release
Subplantar injection of carrageenan induced
inflammation of the paw where the oedema vol-
ume was 0.42  0.03 ml. Intravenous administra-
tion of LP at 5 and 25 mg kg
1 doses produced a
dose-dependent inhibition in oedema formation
(49 and 65% inhibition), while diclofenac pro-
duced 55% inhibition at 10 mg kg
1 dose follow-
ing oral administration.
The effect of LP was evaluated on the levels of
inflammatory mediators in the paw tissue. The
inhibition in paw inflammation with LP was asso-
ciated with dose-dependent reduction in the tissue
levels of TNF-a. The levels of TNF-a in LP5
and LP25 groups were 8.06  0.10 and
7.03  0.15 ng g
1 tissue, respectively, against
10.95  0.18 ng g
1 tissue in carrageenan con-
trol group (Fig. 1a). Like TNF-a, tissue levels of
PGE2 and MPO also increased following carra-
geenan injection. Treatment with LP produced a
dose-dependent reduction in the tissue levels of
both PGE2 and MPO, and its inhibitory effect
was comparable to that of diclofenac (Fig. 1b,c).
Injection of carrageenan in the paw produced a
marked increase in the tissue levels of nitrite, a
measure of NO production, from
253.19  9.97 lM g
1 tissue in normal control to
480.70  22.06 lM g
1 tissue in carrageenan
control. A significant reduction in tissue levels of
nitrite was obtained following treatment with LP.
The levels of nitrite in LP5 and LP25 groups were
308.33  6.10 and 266.80  5.43 lM g
1 tissue,
respectively. The effect of LP on tissue nitrite
3
 V. L. KUMAR ET AL.

Autonomic and Autocoid (a) (b) 100

Pharmacology 12
2015, 35, 1–8
10 80

g–1 tissue)
TNF-α (ng g–1 tissue)
**
8 ** **
** 60 **
**
6

PGE (ng
40

2
4

20
2

0 0
NC _ LP 5 LP 25 D10 NC _ LP 5 LP 25 D10

Carrageenan Carrageenan
(c)
(d)
100
600

80 ** 500
MPO (mM g–1 tissue)

Nitrite (μM g–1 tissue)

** **
400 **
60
**
300 **
40
200
20
100

0 0
NC _ LP 5 LP 25 D10 NC _ LP 5 LP 25 D10
Carrageenan Carrageenan
Figure 1 Effect of LP on the tissue levels of TNF a (a), PGE2 (b), MPO (c) and Nitrite (d) in carrageenan induced paw
inflammation. Values are given as mean  SEM (n = 6). NC: Normal control; LP5: LP 5 mg kg
1; LP25: LP 25 mg kg
1; D10:
Diclofenac 10 mg kg
1. **P < 0.01 vs. carrageenan control.

level was more pronounced than that of diclofe-
nac (Fig. 1d).
Effect of LP on tissue levels of GSH and TBARS
Oedema formation induced by carrageenan was
associated with altered oxidative milieu where the
levels of GSH decreased and that of TBARS
increased as compared to normal controls. Treat-
ment with LP produced a dose-dependent increase
in the tissue levels of GSH to 239.16  12.27 and
393.33  5.57 lg g
1 tissue in LP5 and LP25
groups, respectively, against 202.50  5.73 lg g
1
tissue in carrageenan control. The TBARS levels in
LP5 and LP25 groups were 121.83  7.44 and
111.16  12.81 nM g
1 tissue, respectively, as
compared to 216.66  16.46 nM g
1 tissue in
carrageenan control group (Fig. 2a,b).
Effect of LP on histological changes associated
with carrageenan-induced inflammation
Subplantar injection of carrageenan produced
peak inflammatory response at 3 h where subcu-
taneous oedema and infiltration of neutrophils
Ó 2015 were observed. Treatment with LP maintained
John Wiley & Sons Ltd the tissue architecture and inhibited oedema
formation and cellular influx. The protective
effect of LP as revealed by histological analysis
was more pronounced than that of diclofenac
(Fig. 3).
Effect of LP on oedema formation induced by
different mediators
Subplantar injection of various mediators pro-
duced a marked increase in paw volume. A time
course study revealed that histamine, serotonin
and bradykinin produced a peak inflammatory
response at 30 min with oedema volume of
0.23  0.01, 0.46  0.03 and 0.35  0.02 ml,
while PGE2 produced a peak effect at 1 h with
oedema volume of 0.32  0.01 ml. Treatment of
rats with LP produced a dose-dependent inhibi-
tion of paw inflammation induced by all the
mediators. The inhibitory effect of LP25 against
paw inflammation induced by histamine was
comparable to that of CPM3 (70 vs. 67% inhibi-
tion) and against serotonin was comparable to
that of CPH3 (63 vs. 65% inhibition), respec-
tively. Like inflammation induced by biogenic
amines, the bradykinin-induced inflammation was
also inhibited by LP in a dose-dependent manner.
The oedema volume in LP5 and LP25 groups was

4
 ANTIINFLAMMATORY EFFECT OF C. PROCERA LATEX PROTEINS

Autonomic and Autocoid (a) (b)

Pharmacology 250
500
2015, 35, 1–8

TBARS (nM g–1 tissue)

400 ** 200

GSH (μg g–1 tissue)

300 150 **
* ** **
*
200 100

100 50

0 0
NC _ LP5 LP25 D10 NC _ LP5 LP25 D10
Carrageenan Carrageenan
Figure 2 Effect of LP on the tissue levels of GSH (a) and TBARS (b) in carrageenan induced paw inflammation. Values are given
as mean  SEM (n = 6). NC: Normal control; LP5: LP 5 mg kg
1; LP25: LP 25 mg kg
1; D10: Diclofenac 10 mg kg
1. *P < 0.05
**P < 0.01 vs. carrageenan control.

(a) (b)

(c) (d)

Figure 3 Effect of LP on paw tissue histology. Normal control (a); carrageenan control (b); LP 25 mg kg
1 (c); Diclofenac
10 mg kg
1 (d). Scale bar = 100 lm.

0.27  0.02 and 0.18  0.01 ml (23 and 49%
inhibition), respectively, against 0.13  0.00 ml
in bradyzide-treated group (63% inhibition). LP
also produced a marked inhibition of inflamma-
tion induced by PGE2, and oedema volumes in
LP5 and LP25 groups were 0.15  0.01 and
0.08  0.00 ml (54 and 76% inhibition), respec-
Ó 2015 tively, against 0.11  0.00 ml (66% inhibition)
John Wiley & Sons Ltd in D10 group. The anti-inflammatory efficacy of
LP against different mediators was comparable to
their respective antagonists and more pronounced
than that of diclofenac (Fig. 4a–d).
Discussion
The latex of Calotropis procera is well known for
its multifarious properties of which its anti-
inflammatory property is noteworthy. It has been

5
 V. L. KUMAR ET AL.

Autonomic and Autocoid (a) (b)

Pharmacology 0.5 0.5
2015, 35, 1–8
0.4 0.4 **

Edema Volume (ml)

Edema Volume (ml)

**
0.3 0.3

0.2 0.2 ** **

0.1 ** ** ** 0.1

0 0
_ LP 5 LP 25 CPM3 D10 _ LP 5 LP 25 CPH3 D10
Histamine Serotonin
(c) (d)
0.5 0.5

0.4 0.4

Edema Volume (ml)

Edema Volume (ml)

0.3 ** 0.3

0.2
** 0.2
** **
** **
0.1 **
0.1

0
0 _ LP 5 LP 25 D10
_ LP5 LP25 BZ5 D10

Bradykinin PGE2

Figure 4 Effect of LP on paw edema induced by histamine (a), serotonin (b), bradykinin (c) and PGE2 (d). Values are given as
mean  SEM (n = 6). LP5: LP 5 mg kg
1; LP25: LP 25 mg kg
1; CPM3: chlorpheniramine 3 mg kg
1, CPH3: cyproheptadine
3 mg kg
1, BZ5: bradyzide 5 mg kg
1, D10: Diclofenac 10 mg kg
1. **P < 0.01 vs. respective control.

shown to inhibit inflammation in various experi- the production of pro-inflammatory prostaglan-

mental models due to the presence of several con-
stituents as revealed by the efficacy of its different
extracts (Kumar & Basu, 1994; Alencar et al.,
2004; Arya & Kumar, 2005). The latex comprises
of rubber fraction and soluble proteins that con-
stitute 84 and 9% of its dry mass, respectively
(Ramos et al., 2007). The proteins present in the
latex could be separated from other constituents
of the latex by dialysis (Alencar et al., 2006). The
LP fraction exhibits both anti-inflammatory and
analgesic properties and has been demonstrated
to ameliorate inflammatory hyperalgesia in Fre-
und’s Complete Adjuvant induced arthritis
(Kumar et al., 2011, 2014). In the present study,
the effect of LP was evaluated on the levels of
various endogenous mediators and oxidative
stress markers at the site of inflammation.
Besides, the effect of LP was also evaluated
against various inflammagens to understand its
mechanism of action. The study was carried out
in paw oedema model that has been widely used
to investigate anti-inflammatory properties of
drugs. The inflammatory response in this model is
sustained through the release of prostaglandins
and nitric oxide. NO is a potent vasodilator that
increases vascular permeability and results in
Ó 2015 oedema formation through changes in local blood
John Wiley & Sons Ltd flow. Besides, it has also been shown to increase
dins in various in vitro, ex vivo and in vivo stud-
ies (Salvemini et al., 1996). Treatment of rats
with LP produced a dose-dependent reduction in
the levels of PGE2 and NO at the site of inflam-
mation. These changes were also accompanied by
a marked reduction in the level of TNF-a. TNF-a
is a pro-inflammatory cytokine that is implicated
both in the establishment and in the perpetuation
of the inflammatory process including neutrophil-
ic infiltration (Rocha et al., 2006). The exagger-
ated recruitment of neutrophils may account for
tissue destruction via the production of reactive
oxygen species (ROS) that along with various
inflammatory mediators namely TNF-a, IL-1b
and prostaglandins bring about activation of tran-
scription factor NF-jB (Schreck & Baeuerle,
1994; Perkins, 2007). Further, ROS generated
during inflammation has a role in endothelial cell
damage, increased microvascular permeability,
formation of chemotactic factors, recruitment of
neutrophils and lipid peroxidation. It has been
shown that pharmacological interventions that
reduce the generation or effects of ROS are
known be beneficial inflammatory conditions (Cu-
zzocrea et al., 2001). In the present study, treat-
ment with LP not only inhibited neutrophil
infiltration but it also restored oxidative homoeo-
stasis as evident from the reduction in the levels of

6
 ANTIINFLAMMATORY EFFECT OF C. PROCERA LATEX PROTEINS

Autonomic and Autocoid MPO and normalization of markers of oxidative
Pharmacology stress namely GSH and TBARS. The anti-oedema-
2015, 35, 1–8
tous and anti-infiltrative property of LP was also
substantiated by histological analysis of the paw.
To evaluate the efficacy of LP against inflam-
mation induced by various inflammatory media-
tors and to compare its efficacy with their
respective antagonists, a time course study was
carried out. It revealed that subplantar injection
of early mediators namely histamine, serotonin
and bradykinin produced peak inflammation at
30 min. The release of these mediators is associ-
ated with the early phase of carrageenan-induced
inflammatory response, while the late phase of
this response is mediated through the release of
prostaglandins (Di Rosa & Willoughby, 1971; Di
Rosa et al., 1971). Carrageenan has been
reported to elicit a time-dependent increase in
PGE2 production that reaches its peak at 3 h and
remains elevated thereafter (Salvemini et al.,
1996). However, PGE2 when injected into the
paw produced a peak inflammatory response at
1 h. Treatment of rats with LP produced a dose-
dependent inhibition of paw oedema induced by
all the inflammagens included in this study. It
was found to be equipotent against inflammation
induced by histamine, serotonin and carrageenan
where it produced approximately 60–70% inhibi-
tion. The inhibitory effect of LP25 was compara-
ble to that of the respective antagonists namely
chlorpheniramine and cyproheptadine in case of
inflammation induced by biogenic amines, sug-
gesting thereby that it has antihistaminic and anti-
serotonergic properties. Besides biogenic amines,
LP also inhibited the oedema formation induced
by bradykinin that has been reported to involve
its action on B1 and B2 receptors (Campos &
Calixto, 1995). The B2 receptor has been
reported to interact in a synergistic manner with
several inflammatory mediators, and the inhibi-
tory effect of bradyzide, a B2 receptor antagonist,
was more pronounced than that of LP against
bradykinin-induced inflammation (Burgess et al.,
2000). LP was most effective against PGE2-
induced oedema formation where it produced
75% inhibition, and its effect was more pro-
nounced than that of diclofenac. It is important
to note that the temporal interaction of different
mediators plays a key role in sustaining an
inflammatory response and LP was found to be
effective against inflammation induced by all the
inflammagens. Earlier such a property was
reported in the non-protein constituents of the
latex (Arya & Kumar, 2005; Kumar & Roy,
2007).
Thus, the present study shows that LP exhibits
anti-inflammatory properties by inhibiting various
pro-inflammatory mediators and cellular influx
and by maintaining oxidative homoeostasis at the
site of inflammation. By inhibiting various media-
tors, LP alleviates not only inflammation but also
the associated pain as shown earlier in the mon-
oarthritis model (Dray, 1995; Kumar et al.,
2011). Findings of present study suggest that the
latex proteins have a therapeutic potential for use
in the treatment of various inflammatory
conditions.

BRADLEY, P.P., PRIEBAT, D.A., reperfusion injury. Pharmacol.

References
ALENCAR, N.M., FIGUEIREDO, I.S., VALE,
M.R., BITENCURT, F.S., OLIVEIRA, J.S.,
RIBEIRO, R.A. & RAMOS, M.V.(2004).
Anti-inflammatory effect of the
latex from Calotropis procera in
three different experimental
models: peritonitis, paw oedema
and hemorrhagic cystitis. Planta
Med., 70, 1144–1149.
ALENCAR, N.M., OLIVEIRA, J.S.,
MESQUITA, R.O., LIMA, M.W., VALE,
M.R., ETCHELLS, J.P., FREITAS, C.D. &
RAMOS, M.V. (2006). Pro- and
anti-inflammatory activities of the
latex from Calotropis procera
(Ait.) R.Br. are triggered by
compounds fractionated by
dialysis. Inflamm. Res., 55, 559–
564.
ARYA, S. & KUMAR, V.L. (2005). Anti-
inflammatory efficacy of extracts
of latex of Calotropis procera
against different mediators of
Ó 2015 inflammation. Mediators
John Wiley & Sons Ltd Inflamm., 2005, 228–232.
CHRISTENSEN, R.D. & ROTHSTEIN, G.
(1982). Measurement of
cutaneous inflammation:
estimation of neutrophil content
with an enzyme marker.
J. Invest. Dermatol., 78,
206–209.
BURGESS, G.M., PERKINS, M.N., RANG,
H.P.et al. (2000). Bradyzide, a
potent non-peptide B(2)
bradykinin receptor antagonist
with long-lasting oral activity in
animal models of inflammatory
hyperalgesia. Br. J. Pharmacol.,
129, 77–86.
CAMPOS, M.M. & CALIXTO, J.B. (1995).
Involvement of B1 and B2
receptors in bradykinin-induced
rat paw ooedema. Br. J.
Pharmacol., 114, 1005–1013.
CUZZOCREA, S., RILEY, D.P., CAPUTI,
A.P. & SALVEMINI, D. (2001).
Antioxidant therapy: a
new pharmacological
approach in shock,
inflammation, and ischemia/
Rev., 53, 135–159.
DEWAN, S., SANGRAULA, H. & KUMAR,
V.L. (2000). Preliminary studies
on the analgesic activity of latex
of Calotropris procera. J.
Ethnopharmacol., 73, 307–311.
DI ROSA, M. & WILLOUGHBY, D.A.
(1971). Screens for anti-
inflammatory drugs. J. Pharm.
Pharmacol., 23, 297–298.
DI ROSA, M., GIROUD, J.P. &
WILLOUGHBY, D.A. (1971). Studies
on the mediators of the acute
inflammatory response induced in
rats in different sites by
carrageenan and turpentine.
J. Pathol., 104, 15–29.
DRAY, A. (1995). Inflammatory
mediators of pain. Br. J.
Anaesth., 75, 125–131.
ELLMAN, G.L. (1959). Tissue
sulfhydryl groups. Arch.
Biochem. Biophys., 82, 70–77.
FUNK, C.D. (2001). Prostaglandins
and leukotrienes: advances in

7

Autonomic and Autocoid eicosanoid biology. Science, 294,
Pharmacology 1871–1875.
2015, 35, 1–8 ICHITANI, Y., SHI, T., HAEGGSTROM, J.Z.,
SAMUELSSON, B. & HOKFEL, T.T.
(1997). Increased levels of
cyclooxygenase-2 mRNA in the
rat spinal cord after peripheral
inflammation: an in situ
hybridization study.
NeuroReport, 8, 2949–2952.
IKEDA, U., KANBE, T. & SHIMADA, K.
(1996). Adrenomedullin increases
inducible nitric oxide synthase in
rat vascular smooth muscle cells
stimulated with interleukin-1.
Hypertension, 27, 1240–1244.
KESTER, M., KARPA, K.D. & VRANA, K.E.
(2012). Inflammatory disorders.
In: Elsevier’s Integrated Review
Pharmacology, 2nd edn, pp. 161–
172. Saunders, Philadelphia, pp.
161–72.
KUMAR, V.L. & ARYA, S. (2006).
Medicinal uses and
pharmacological properties of
Calotropis procera. Recent Prog.
Med. Plants, 11, 73–88.
KUMAR, V.L. & BASU, N. (1994). Anti-
inflammatory activity of the latex
of Calotropis procera. J.
Ethnopharmacol., 44, 123–125.
KUMAR, V.L. & ROY, S. (2007).
Calotropis procera latex extract
affords protection against
inflammation and oxidative stress
in Freund’s complete adjuvant-
induced monoarthritis in rats.
Mediators Inflamm., 2007,
47523.
KUMAR, V.L., CHAUDHARY, P., RAMOS,
M.V., MOHAN, M. & MATOS, M.P.
(2011). Protective effect of
proteins derived from the latex of
Calotropis procera against
inflammatory hyperalgesia in
monoarthritic rats. Phytother.
Res., 25, 1336–1341.
Ó 2015
John Wiley & Sons Ltd
KUMAR, V.L., CHAUDHARY, P.,
OLIVEIRA, R.M. & RAMOS, M.V.
(2014). Calotropis procera latex
proteins ameliorate functional
limitations associated with
adjuvant induced inflammation in
rat. Musculoskelet. Biol., 1, 1.
MEDZHITOV, R. (2008). Origin and
physiological roles of inflamm-
ation. Nature, 454, 428–435.
MIYAMOTO, A., MATSUYAMA, T.,
ISHIGURO, S. & NISHIO, A. (2000).
Captopril increases the affinity of
bradykinin receptor binding sites
in bovine coronary arterial
endothelial cells. Jpn. J.
Pharmacol., 84, 82–85.
OHKAWA, H., OHISHI, N. & YAGI, K.
(1979). Assay for lipid peroxides
in animal tissues by thiobarbituric
acid reaction. Anal. Biochem., 95,
351–358.
PERKINS, N.D. (2007). Integrating
cell-signalling pathways with NF-
kappaB and IKK function. Nat.
Rev. Mol. Cell Biol., 8, 49–62.
RAMOS, M.V., AGUIAR, V.C., MELO, V.M.,
MESQUITA, R.O., SILVESTRE, P.P.,
OLIVEIRA, J.S., OLIVEIRA, R.S.,
MACEDO, N.M. & ALENCAR, N.M.
(2007). Immunological and
allergenic responses induced by
latex fractions of Calotropis
procera (Ait.) R.Br. J.
Ethnopharmacol., 111, 115–122.
RAMOS, M.V., OLIVEIRA, J.S.,
FIGUEIREDO, J.G. et al. (2009).
Involvement of NO in the
inhibitory effect of Calotropis
procera latex protein fractions on
leukocyte rolling, adhesion and
infiltration in rat peritonitis
model. J. Ethnopharmacol., 125,
387–392.
RICCIOTTI, E. & FITZGERALD, G.A.
(2011). Prostaglandins and
inflammation. Arterioscler.
V. L. KUMAR ET AL.
Thromb. Vasc. Biol., 31, 986–
1000.
ROCHA, A.C., FERNANDES, E.S.,
~
QUINTAO, N.L., CAMPOS, M.M. &
CALIXTO, J.B. (2006). Relevance of
tumour necrosis factor-alpha for
the inflammatory and nociceptive
responses evoked by carrageenan
in the mouse paw. Br. J.
Pharmacol., 148, 688–695.
SALVEMINI, D., WANG, Z.Q., WYATT, P.S.,
BOURDON, D.M., MARINO, M.H.,
MANNING, P.T. & CURRIE, M.G.
(1996). Nitric oxide: a key
mediator in the early and late
phase of carrageenan-induced rat
paw inflammation. Br. J.
Pharmacol., 118, 829–838.
SCHRECK, R. & BAEUERLE, P.A. (1994).
Assessing oxygen radicals as
mediators in activation of
inducible eukaryotic transcription
factor NF-kappa B. Methods
Enzymol., 234, 151–163.
SOARES, P.M., LIMA, S.R., MATOS, S.G.
et al. (2005). Antinociceptive
activity of Calotropis procera
latex in mice. J.
Ethnopharmacol., 99, 125–129.
WEALTH OF INDIA: RAW MATERIALS. III
(1992). CSIR PUBLICATION AND
INFORMATION DIRECTORATE, NEW
DELHI, PP. 78–84.
WINTER, C.A., RISLEY, E.A. & NUSS, G.W.
(1962). Carrageenin-induced
oedema in hind paw of the rat as
an assay for antiiflammatory
drugs. Proc. Soc. Exp. Biol.
Med., 111, 544–547.
(Received 21 October 2014
Revised 07 January 2015
Accepted 14 January 2015)