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International Journal of Pharmacy and Pharmaceutical Science Research

Universal Research Publications. All rights reserved

ISSN: 2249-0337
Original Article
Nitric Oxide radical scavenging assay of bioactive compounds present in methanol
Extract of Centella asiatica
Dharmendra Singh*1, Manish Mishra2, Monika Gupta, Poonam Singh1, Abhishek Gupta1, Rajeev Nema1
1
Department of Biotechnology, CMBT, Bhopal, MP, India-462003
2
Ecosystem Management and Tech. Forestry, IIFM, Bhopal
Email- [email protected] phone: +919098793083
Received 02 July 2012; accepted 25 July 2012
Abstract
Centella asiatica L. has been used as a medicinal herb for thousands of years in India, China, Sri lanka, Nepal and Madagascar.
Centella asiatica is one of the chief herbs for treating skin problems, to heal wounds, for revitalizing the nerves and brain cells,
hence primarily known as a "Brain food" in India. In India, it is described under the name of Mandukaparni and used in
Ayurveda medicine. Methanol extract of Centella asiatica was screened to evaluate free radical scavenging effect. The extract
exhibited significant reducing power and free radical scavenging effect on nitric oxide radicals. This antioxidant activity was
concentration dependent which were compared with standard antioxidants such as ascorbic acid. The highest free radical
scavenging effect of the extract was observed at concentration of 200 µg/ml and the IC50 value of extract was 442. 68 µg/ml.
© 2011 Universal Research Publications. All rights reserved
Key words: Nitric oxide scavenging, Centella asiatica, Methanol Extract, Antioxidant activity.

Introduction important bio regulatory molecule, which has a number of

Oxygen is an important element for life. All living systems physiological effects including control of blood pressure,
have evolved to survive in the presence of molecular oxygen. neural signal transduction, platelet function, antimicrobial
Oxidative properties of oxygen play a vital role in many and antitumor activity (5). NO also show toxic property after
biological phenomena. It can also aggravate the damage reaction with oxygen and superoxide radicals. The reaction
within the cell by oxidative events (1). Free radicals are products are able to cause much cellular damage (6). The
highly reactive compounds, they are chemical species Centella asiatica, a creeping plant has long, prostrate,
associated with an odd or unpaired electron and can be filiform, slender stems with long internodes and rooting at
formed when oxygen interacts with certain molecules. They nodes. This have simple, long petiole leaves, small,
are neutral, short lived, unstable and highly reactive to pair inconspicuous, clusters and purple to white-green flowers, 3-
with the odd electron and finally achieve stable configuration. 6 in number are arranged in umbels arising from the axils of
Once formed these highly reactive radicals can start a chain the leaves. The fruit is 8 mm long, ovoid, hard with strongly
reaction. Free radicals generated by these reactions are thickened pericarp (7). Traditionally the plants are used as
capable of attacking the healthy cells of the body, causing medicine due to their antioxidant activity and play important
them to lose their structure and function (2). Free radicals role to alleviate many diseases. The present study aims to
were previously reported as being capable of damaging a lot spectrophotometeric quantification of nitric oxide radical
of cellular components such as proteins, lipids and DNA (3). scavenging activity in the methanol extract of Centella
Numerous physiological and biochemical processes in the asiatica.
human body may produce oxygen centered free radicals and Material & Method
other reactive oxygen species as byproducts. Overproduction 1. Sample collection and processing:
of such free radicals can cause oxidative damage to The plant material of Centella asiatica was collected from
biomolecules, eventually leading to many chronic diseases the Vanita and Dhanvantari nursery of Bhopal and
such as cancer, diabetes, aging etc. (4). Nitric Oxide is an identified taxonomically by Dr. Manish Mishra, Ecosystem

International Journal of Pharmacy and Pharmaceutical Science Research 2012; 2(3) 42-44
42
Management & Tech. Forestry, Indian Institute of Forest
Management, Bhopal (MP). The leaves of the plants were
properly washed with tap water and then rinsed with distilled
water. The rinsed leaves were shed dried and crushed to
obtain powder.
2. Extraction:
A quantity of 100g of the dried powder of Centella asiatica
extracted with 95% methanol (Merck) using soxhlet. The
soxhletion with methanol was done for a week to obtained
extract. After that, the Extract was evaporated in water bath at
50oC to obtained crude for antioxidant assay (8).
3. Scavenging of nitric oxide radicals:
Sodium nitroprusside in aqueous solution at physiological pH Figure-1 Nitric oxide scavenging assay of ascorbic acid and
spontaneously generates Nitrite oxide which interacts with methanol extract.
oxygen to produce Nitrite ions, which can be measured at
550nm by spectrophotometer in the presence of Griess Beside triterpenoids and essential oils, Centella asiatica has
reagent (9). also been reported to contain numerous flavonoids, including
3.1 Reagents and chemicals: quercetin, kaempferol, catechin, rutin, and naringin, some of
5mM Sodium Nitroprusside, Griess reagent (1% sulphonil which are major contributors in particular to the antioxidative
amide, 0.1% N 1-naphthylethylenediamine, 2% activity of Centella asiatica (15). It has been recently
orthophosphoric acid), Phosphate buffer (pH- 7.4). indicated to show antilipid peroxidative and free radical
3.2 Procedure:- scavenging activities (16). Figure-1 and table 1 illustrates a
Plant extract was dissolved in distilled water for this significant decrease in the NO radical due to the scavenging
quantification. Sodium Nitroprusside (5mM) in standard ability of extract and ascorbic acid. The methanol extract
phosphate buffer saline (0.025m, pH 7.4) was incubated with showed maximum activity of 50% respectively at 200 µg /ml;
different concentration (100-400µg/ml) of methanol extract whereas ascorbic acid at the same concentration exhibited
and tubes were incubated at 29oC for 3 hours. Control 47.3% inhibition.
experiment without the test compounds but with equivalent Conclusion:
amount of buffer was conducted in an identical manner. After This study suggests that the methanol extract of Centella
3 hours incubated samples were diluted with 1 ml of Griess asiatica plant have many bioactive compounds which show
reagents. The absorbance of the colour developed during antioxidant activity. The plant extract might be helpful in
diazotization of Nitrite with sulphanilamide and its preventing and slowing the progress of various oxidative
subsequent coupling with Napthylethylenediaminehydro- stress induced diseases. It might be useful for the
chloride was observed at 550nm on spectrophotometer. Same development of newer and more potent natural antioxidants.
procedure was done with ascorbic acid which was standard in The methanol extract of Centella asiatica exhibited
comparison to methanol extract. Calculated the % inhibition significant antioxidant activity compare to ascorbic acid. The
by formula and plot graph in compared to standard. findings of the present study suggest that Centella asiatica
3.3 Formula: could be a potential source of natural antioxidant that could
% inhibition = [O.D.of control - O.D. of Test/O.D. of control] Χ 100
have great importance as therapeutic agents in preventing or
slowing the progress of oxidative stress related degenerative
Result & Discussion: diseases. These results might be helpful for providing the
Nitric oxide (NO) is a potent pleiotropic inhibitor of platform for researchers and pharma companies for the
physiological processes such as smooth muscle relaxation, development of precious medicines which will be helpful for
neuronal signaling, and inhibition of platelet aggregation and treatment of oxidative stress generated diseases.
regulation of cell mediated toxicity (10). In addition to Acknowledgment: The authors express gratitude IIFM and
reactive oxygen species, nitric oxide is also implicated in CMBT Bhopal for facility and kind support.
inflammation, cancer and other pathological conditions (11,
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Source of support: Nil; Conflict of interest: None declared

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