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Chandrappa CP et al.
World Journal of Pharmacy and Pharmaceutical Sciences
Volume 2, Issue 5, 3991-3997.
Research Article
ISSN 2278 4357
Article Received on
05 August 2013,
Revised on 25 August 2013,
Accepted on 30 September
2013
ABSTRACT
The anti-inflammatory activity of ethanol extract of stem of Carmona
retusa was investigated for in-vitro anti-inflammatory activity by
human red blood cell membrane stabilization method, heat induced
hemolysis and proteinase inhibitory activity. The anti-inflammatory
*Correspondence for
Author:
* Chandrappa CP
Department of Biotechnology,
Shridevi Institute of
11.33g/ml in the methods used. IC50 values were calculated for all the
India.
chandrappacp@gmail.com
activity of the sample was comparable with that of the standard diclofenac. The results
obtained in the present study suggest that Carmona retusa can be a potential source of antiinflammatory agents.
Key words: Carmona retusa, anti-inflammatory activity, HRBC, IC50.
INTRODUCTION
Inflammation is a local response of living mammalian tissues to the injury. It is a body
defense reaction in order to eliminate or limit the spread of injurious agents. There are
various components to an inflammatory reaction that can contribute to the associated
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Chandrappa CP et al.
symptoms and tissue injury and pain. Even though most of the synthetic anti-inflammatory
drugs are available in the market, due to their well-known side effects, toxic effects and
production cost, presently people are search for natural anti-inflammatory drugs without any
adverse effects
[1]
began by Gujral and his associates in 1956 and they screened a number of plants for their
anti-arthritic effects. Subsequently, various workers from different laboratories in India have
made significant contribution [2]. Carmona retusa leaf decoction is being used to treat cough
and stomach ache, root as antidote and also reported antibacterial, anti-inflammatory, antianalgesic and antidiabetic activity of this plant [3, 4].
MATERIALS AND METHODS
The Human Red Blood Cell (HRBC) membrane stabilization method
Fresh whole human blood (5 mL) was collected and transferred to the centrifuged tubes
containing Heparin or EDTA or Sodium citrate to prevent clotting. The tubes were
centrifuged at 3000 rpm for 10 min and were washed three times with equal volume of
normal saline. The volume of the blood was measured and reconstituted as 10% v/v
suspension with normal saline [5].
The reaction mixture consists of 1.0 mL of test sample of different concentrations (50g
400 g) in normal saline and 0.5 mL of 10% HRBC suspension, 1 ml of 0.2 M phosphate
buffer, 1 ml hyposaline were incubated at 370 C for 30 min and centrifuged at 3,000 rpm for
20 min and the hemoglobin content of the supernatant solution was estimated
spectrophotometrically at 560 nm. Diclofenac was used as standard and a control was
prepared without extracts. The percentage of HRBC hemolysis and membrane stabilization or
protection was calculated by using the following formula [6].
% Hemolysis = (Optical density of Test sample / Optical density of Control) X 100
% Protection = 100 [(Optical density of Test sample / Optical density of Control) X
100]
Membrane stabilization test by heat induced hemolysis
The reaction mixture in heat induced hemolysis consists of 1.0 mL of test sample of different
concentrations (50g 400 g) in normal saline and 1.0 mL of 10% RBC suspension, instead
of test sample, only saline was added to the control. Diclofenac sodium was taken as a
standard drug. All the tubes containing reaction mixture were incubated in a water bath at
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Chandrappa CP et al.
560C for 30 min. After incubation, the tubes were cooled under running tap water. The
reaction mixture was centrifuged at 2500 rpm for 5 min and the absorbance of the
supernatants was taken at 560 nm. The experiment was performed in triplicates. The
percentage of heat induced HRBC hemolysis and membrane stabilization or protection was
calculated by using the following Formula [7]:
% Hemolysis = (Optical density of Test sample / Optical density of Control) X 100
% Protection = 100 [(Optical density of Test sample / Optical density of Control) X
100]
Proteinase inhibitory activity
The reaction mixture contains 1.0 mL of test sample of different concentrations (50g 400
g), 1.0 mL of 20 mM TrisHCl buffer (pH 7.4) containing 0.06 mg trypsin, and the mixture
was incubated at 370C for 5 min and then 1.0 mL of 0.8% (w/v) casein was added. The
mixture was incubated for an additional 20 min. 2 ml of 70% perchloric acid was added to
terminate the reaction. Cloudy suspension was centrifuged and the absorbance of the
supernatant was read at 210 nm against buffer as blank. Diclofenac sodium was used as
standard drug. The experiment was performed in triplicate. The percentage inhibition of
proteinase inhibitory activity was determined [8].
RESULTS AND DISCUSSION
The human red blood cell (HRBC) membrane stabilization method
The ethanol extract of the stem of Carmona retusa was studied for in vitro anti-inflammatory
activity by HRBC membrane stabilization method. Among all the concentrations 400 g/ml
showed significant anti-inflammatory activity and 55.72% protection of HRBC in hypotonic
solution. Results were compared with standard diclofenac which showed 66.18% protection
and presented in Table 1. IC50 values were calculated and found to be 30.31g/ml and
40.74g/ml for plant sample and standard diclofenac respectively.
The plant extract exhibited membrane stabilization effect by inhibiting hypotonicity induced
lyses of RBC membrane. The RBC membrane is analogous to the lysosomal membrane and
its stabilization implies that the extract may as well stabilize lysosomal membranes.
Stabilization of lysosomal membrane is play an important role in limiting the inflammatory
response by preventing the release of lysosomal constituents of activated neutrophil such as
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Chandrappa CP et al.
bactericidal enzymes and proteases, which cause further tissue inflammation and damage
upon extra cellular release [9].
Table 1. Hypotonicity induced HRBC membrane stabilization test of alcholic extract of
Carmona retua and diclofenac.
Concentrations of
% protection
Hemolysis
50
71.07195
28.92805
58.33333
41.66667
100
62.11454
37.88546
53.70778
46.29222
200
55.98385
44.01615
48.78855
51.21145
300
53.45081
46.54919
43.42878
56.57122
400
44.27313
55.72687
33.81057
66.18943
30.313
40.743
sample/diclofenac
(g/ml)
% of
% protection
% of
of the
diclofenac
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Chandrappa CP et al.
Table 2. Heat induced HRBC membrane stabilization test of ethanol extract of Carmona
retua and diclofenac.
Concentrations of
sample/diclofenac
of % protection %
of
% protection
of
Hemolysis
50
84.56044
15.43956
73.95604
26.04396
100
66.42857
33.57143
66.48352
33.51648
200
61.26374
38.73626
55.82418
44.17582
300
49.94505
50.05495
47.41758
52.58242
400
43.62637
56.37363
34.28571
65.71429
19.622
25.130
(g/ml)
the
diclofenac
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Chandrappa CP et al.
of % of inhibition
sample/diclofenac
inhibition the of
(g/ml)
sample
diclofenac
50
11.33333
46.70175
100
28.52632
50.70175
200
39.92982
56.87719
300
49.61404
66.10526
400
61.75439
73.36842
14.535
45.496
the
CONCLUSION
Based on the above results it is suggest that the alcoholic extract of Carmona retusa posses
antiinflammatory activity studied by in vitro assays. Antiinflammatory activity may be due to
the presence of many phytochemical in the extract. However, further studies are required to
identify the lead molecule in the extract and to study the action of mechnasim.
ACKNOWLEDGMENT
Authors are thankful to Dr. M.R.Hulinaykar, Managing Trustee, Sri Shridevi Charitable
Trust (R.) and Dr. K.Sukumaran, Principal, Shridevi Institute of Engineering and
Technology, Tumkur for providing all facilities. Authors are also thankful to Dr.
P.Sharanappa, Associate Professor, Department of Studies in Biosciences, Hemagangothri,
University of Mysore, Hassan, Karnataka for the identification of our plant.
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