WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Chandrappa CP et al.
World Journal of Pharmacy and Pharmaceutical Sciences
Volume 2, Issue 5, 3991-3997.
Research Article
ISSN 2278 4357

IN VITRO ANTI-INFLAMMATORY ACTIVITY OF CARMONA

RETUSA (VAHL.)
Chandrappa CP1, Govindappa M1*, Anil Kumar NV2 and Channabasava R1
1

Department of Biotechnology, Shridevi Institute of Engineering and Technology, Tumkur

572106, Karnataka, India.

Department of Chemistry, Manipal Institute of Technology, Manipal University, Karnataka,

India.

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

activity of Carmona retusa found maximum in human red blood cell

Author:

membrane stabilization method, heat induced hemolysis and proteinase

* Chandrappa CP

inhibitory activity as 55.72%, 56.37% and 61.75% respectively at

Department of Biotechnology,
Shridevi Institute of

400g/ml concentration. Minimum anti-inflammatory activity has

Engineering and Technology,

shown at 50g/ml concentration as 28.92g/ml, 15.43g/ml and

Tumkur 572106, Karnataka,

11.33g/ml in the methods used. IC50 values were calculated for all the

India.

methods and found to 30.31, 19.62 and14.53g/ml for human red

chandrappacp@gmail.com

blood cell membrane stabilization method, heat induced hemolysis and

proteinase inhibitory activity respectively. The anti-inflammatory

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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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]

. A systemic study of anti-inflammatory effects of Indian medicinal plants

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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World Journal of Pharmacy and Pharmaceutical Sciences

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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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

of the sample 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

IC50 Value (g)

30.313

40.743

sample/diclofenac
(g/ml)

% of

% protection

% of

of the
diclofenac

Membrane stabilization test by heat induced hemolysis

The mechanism of anti-inflammatory activity of ethanol extract of Carmona retusa was
studied by RBCs membrane stabilization at various concentrations. All the concentrations
were effectively inhibiting the heat induced hemolysis. These results provide evidence for
membrane stabilization as an additional mechanism of their anti-inflammatory effect. This
anti-inflammatory effect may possibly inhibit the release of lysosomal content of neutrophils
at the site of inflammation [10].The percentage of hemolysis and protection of ethanol extract
is represented in Table 2.
The maximum percentage protection was recorded as 56.37 and minimum percentage 15.43
at the concentrations of 400g/ml and 50g/ml from plant extract respectively. The
maximum percentage protection was recorded 65.71 and minimum percentage 26.04 at the
concentrations of 400g/ml and 50g/ml from ascorbic acid respectively. IC50 Values were
calculated for plant extract and ascorbic acid as 19.62g/ml and 25.13g/ml respectively.
The plant extract exhibited the presence of cardiac glycosides are known to possess potent
anti-inflammatory activities [10].

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World Journal of Pharmacy and Pharmaceutical Sciences

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

of the sample 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

IC50 Value (g)

19.622

25.130

(g/ml)

the

diclofenac

Proteinase inhibitory activity

Ethanol extract of C.retusa have exhibited proteinase inhibitory activity at various
concentrations significantly. The maximum inhibition of extract was observed at 400g/ml
concentration as 61.75% and minimum at 50g/ml concentration as 46.70% respectively and
standard ascorbic acid has shown maximum inhibitory activity at 400g/ml concentration as
73.36% and minimum at 50g/ml concentration as 11.33% respectively. IC50 values for plant
extract and ascorbic acid were calculated by regression line and are as follows 14.53g/ml
and 45.49 (Table 3).
Neutrophils are most important source for proteinases which carries in their lysosomal
granules and are involved in arthritic reactions.
Proteinases have been implicated in arthritic reactions. It was already reported that leukocytes
proteinase play important role in the development of tissue damage during in inflammatory
reactions and significant level of protection was provided by proteinase inhibitors. Recent
studies have shown that many flavonoids and related polyphenols contributed significantly to
the antinflammatory activities of many plants [10]. Due to the presence of bioactive
compounds such as flavonoids, saponins, phenols, tannins and cardiac glycosides in the
extract may contribute in its anti-inflammatory activity.

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World Journal of Pharmacy and Pharmaceutical Sciences

Table 3. Proteinase inhibitory activity of alcholic extract of Carmona retua and

diclofenac sodium.
Concentrations of %

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

IC50 Value (g)

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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World Journal of Pharmacy and Pharmaceutical Sciences

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