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IN VITRO ANTIOXIDANT ACTIVITY OF THE

METHANOLIC EXTRACTS OF LEAF AND FRUIT
OF CALAMUS ROTANG LINN

Article January 2012

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J. Expt. Biosci. 3(2):33-36, July 2012 ISSN 2223-9626 (Online), ISSN 2077-3358 (Print)

IN VITRO ANTIOXIDANT ACTIVITY OF THE METHANOLIC EXTRACTS OF LEAF AND

FRUIT OF CALAMUS ROTANG LINN.
Aparna Shil1*, Mahbubul Kabir Himel1, Abul Khair1, M. Nur Alam2 and A.F.M. Jamal Uddin3
* Corresponding author, E-mail:aparna@juniv.edu

Abstract
Plants can be the primary source of various traditional medicines that play an important role in
health care. The present study was designed to investigate the antioxidant activity of the crude
methanolic extracts of leaves and fruits of Calamus rotang Linn. using four in vitro methods
namely, total Antioxidant activity, Flavonoid and phenol content and DPPH scavenging activity
of the sample plant extract. Gallic acid equivalent phenolic content was highest in the fruit
extract of the Calamus rotang. On the other hand, quercetin equivalent flavonoid content was
highest in the leaf extract of the plant. The IC50 value of leaf and fruit obtained by DPPH
method was comparable to that of standard antioxidant. These were 387.95 g/ml and 142.01
g/ml for leaf and fruit, respectively whereas ascorbic acid showed the value of 25.826 g/ml,
representing a considerable choice of option for further studies. However, leaf extract of C.
rotang showed highest antioxidant activity irrespective of the methods used.
Key words: Antioxidant, Flavonoid content, Phenolics content and IC50 value

Introduction
It is now well established that plants are the traditional sources of almost all raw materials for medicine.
According to Ghani (1998), plants which synthesize and accumulate some secondary metabolites like
alkaloids, sterols, terpenes, flavonoids, saponins, glycosides, cyanogenics, tannins, resins, lactones,
quinines, volatile oils etc, contain minerals and vitamins which possess medicinal properties. Natural
antioxidants have been considered as a very important source of preventive medicine from the last
twenty years. Some research have shown positive correlation between increased dietary intake of
natural antioxidants and reduced coronary disease and cancer mortality, as well as longer life
expectancy (Halliwell, 2007; Rios et al., 2009). For this reason, scientists search new sources of natural
antioxidants in plants. In this research, four in vitro methods (determination of total phenol,
determination of flavonoid content, determination of total antioxidant capacity and DPPH scavenging
activity) were used for the evaluation of antioxidant activity. Total phenolic compound assay and total
flavonoid content assay determines only the amount of total phenol and total flavonoid, respectively in
the crude extracts but does not necessarily lit up the total antioxidant activity of the constituents present
in the methanolic extract. This is because the constituents present in the extract other than phenol and
flavonoid might be responsible for the antioxidant activity too. However, the result of total antioxidant
capacity assay is more important for the determination of antioxidant property of an extract. The
present research work has been designed for the existing antioxidant activity along with DPPH
scavenging activity in two parts (leaf and fruit) of Calamus rotang Linn., a frequent growing shrub in
Bangladesh. Calamus rotang (rattan palm or climbing palm) is a plant belonging to the family
Arecaceae. It is an indigenous plant of south-west Asia. The basal section of the plant grows vertically
for ten meters and horizontally for about two hundred meters or more. Its fruit can be consumed fresh
or prepared into pickles and eaten with food. Its tender shoots are used as antihelminthic by tribal
people (Kagyung et al,. 2010). Its leaf juice is used for eye problem (Basumatary, 2004). The presence
of a saponin in the stem, an alkaloid in the leaves and a flavonoid in the root of C. rotang is used in
convulsions and cramps (Khare, 2004). This previous ideas has led to do this research work.

1
Dept. of Botany, Jahangirnagar University, 2Dept. of Pharmacy, Jahangirnagar University, 3Dept. of Horticulture, Sher-e-Bangla
Agriculture University.
33
Aparna Shil et al.

Materials and Methods

Calamus rotang was collected from the Botanical Garden of Jahangirnagar University Campus, Savar,
Dhaka and identified by the Taxonomist of the Department of Botany, JU, Savar, Dhaka. Freshly
collected roots and stems were sliced and sun-dried, and then dried in an oven at 70C temperature to
ensure fine grinding. After grinding, the powdered plant materials were processed for 8 hours using a
Soxhlet apparatus with methanol to get methanolic extracts. After that the ready extract was used as
plant extract stock. 1, 1-diphenyl-2-picryl-hydrazyl (DPPH), Ascorbic Acid, Quercetin and Gallic Acid
were obtained from Sigma Chemical Co (MO, USA). Folin-Ciocalteu Reagent (FCR) and Griess
Reagent was purchased from Merck, Germany. All other chemicals and reagents were of analytical
grade. Determination of total phenol was done by using Folin-Ciocalteu reagent (Folin & Ciocalteu,
1927). Plant extracts were diluted ten times with distilled water (0.5ml of 1:10g/ml), was taken in a test
tube and Folin-Ciocalteu reagent (5ml,1:10 diluted with distilled water) and aqueous Na 2CO3 (4mL,1M)
was mixed with it. The mixtures were allowed to stand for 15 minutes and the total phenols were
determined by colorimetry at 765 nm. The standard curve was prepared using 0, 50, 100, 150, 200, 250
mg/L solutions of Gallic acid in methanol: water (50:50, v/v). Total phenol values of the respective
extracts were expressed in terms of Gallic acid equivalent (GAE) mg/g of dry mass of the extract. The
result was calculated from the regression equation of the calibration curve (y=0.009x + 0.370,
r2=0.998). Aluminium chloride colorimetric method developed by Chang et al.2002, was used for the
flavonoids determination. The plant extract (0.5ml of 1:10g/ml) in methanol was taken in a test tube
and mixed with 1.5 ml of methanol, 0.1 ml of 10% Aluminium chloride, 0.1 ml of 1 M potassium
acetate and 2.8 ml of distilled water and then kept at room temperature for 30 minutes. The absorbance
of the reaction mixture was measured at 415 nm. Quercetin solutions of different concentrations (12.5
to 100 g/mL in methanol) were used for the preparation of calibration curve. The antioxidant capacity
was determined by using the method developed by Preito et al., 1999. The assay relies on the reduction
of M0 (V1) to M0 (V) by the activity of plant extract followed by formation of a green phosphate/Mo (V)
complex at acid pH. The antioxidant capacity is expressed as ascorbic acid equivalent (AAE). 0.3mL of
plant extract was mixed with 3 mL of reagent solution (0.6M sulfuric acid, 28 mM sodium phosphate
and 4 mM ammonium molybdate) and the mixture was incubated for 90 minutes at 95C temperature.
The solutions were then cooled down to room temperature and the absorbance of the aqueous solution
of each was measured at 695 nm against a blank. Total antioxidant capacity of the extract was measured
from the calibration curve prepared from the concentration versus optical density of ascorbic acid.
DPPH radical scavenging activity of C. rotang leaf and fruit was investigated by the method developed
by Manzocco et al., 1998. The sample extract (0.2 ml) was diluted with methanol and 2 ml of prepared
DPPH solution (0.5 mM) was added in it. The absorbance was measured at 517 nm, just after 30
minutes incubation. Ascorbic acid was used as a reference or standard antioxidant in this method. After
that the percentage of the DPPH radical scavenging activitywas calculated.
Results and Discussion
Total phenolic compound assay
The amount of total phenolics in the crude methanolic extract of Calamus rotang leaf and fruit was
14.53 and 14.69 mg/g gallic acid equivalent, respectively by using Folin-Ciocalteu Assay (Table-1).
The plant phenolic compounds are one of the important groups of compounds that play major role as
primary antioxidants or free radical scavengers. Redox properties are the main determinants of the
antioxidant activity of the phenolics and these redox potential play an important role in absorption and
neutralization of free radicals, repress singlet and triplet oxygen/free oxygen or destroying peroxides
(Osawa, 1994). Vegetables and fruits are important sources of polyphenolic compounds and it is
suggested that having 1g per day antioxidant-rich diet have important inhibitory impact on mutagenesis
and carcinogenesis in humans (Tanaka, 1988).

34
J. Expt. Biosci. 3(2):33-36, July 2012 ISSN 2223-9626 (Online), ISSN 2077-3358 (Print)

Flavonoid content assay

Content of flavonoid was measured by using the regression equation ((y=0.009x-0.0036) which was
prepared using quercetin as standard, and also, total flavonoid content is represented as quercetin
equivalent (QE). For Calamus leaf it was 2.68 mg/g (QE), while Calamus root gave 2.37 mg/g (QE)
(Table-1). The free reactive radicals like singlet oxygen, superoxide, peroxyl radical, hydroxyl radicals
and peroxynitrite are responsible for maximum damage of cells. Flavonoids are the secondary
metabolites that play an important role in protecting cells from the destructive effects of these hyper
reactive oxygen species. There is an inverse correlation between flavonoid-rich diet intake and
mortality due to coronary heart disease. Epidemiological studies have shown that flavonoid can resist
the incidence of heart attacks or coronary diseases up to a considerable rate.
Table 1. Phenolic and flavonoid content of methanolic extracts of bark and root of C. rotang
Methanolic extract Total phenolic content (GAE mg g -1) Total flavonoid content (QE mg g -1)
Leaf of C. rotang 14.53 2.68
Fruit of C. rotang 14.69 2.37

Total antioxidant assay

Calibration curve prepared from absorption spectrum at 695 nm of different Ascorbic Acid grades and
used to determine the total antioxidant capacities of the methanolic extracts of leaf and root of C.
rotang. The obtained amounts of antioxidant were 37.076 and 19.69 mg/g for leaf and root respectively
and these were expressed as Ascorbic acid equivalent.
Table 2. Antioxidant activity of the extracts of leaf and fruit of C. rotang
Antioxidant activity Extract
Leaf Fruit
-1
Total antioxidant capacity (in terms of AAE) per 37.076 mg g 19.69 mg g-1
gram of dried extract
DPPH Scavenging activity (in terms of IC50) per 387.948 g ml-1 142.01 g ml-1
mL of solution (5 g l-1) of extract

DPPH scavenging activity

The IC50 values of the methanolic extracts of C. 120.0
Ascorbic
rotang leaves and fruits were 387.948 and 142.01 Acid
g/ml respectively, whereas ascorbic acid showed the 100.0 Leaf
Percent DPPH Scavenging

value of 25.826 g/ml which were significant enough Fruit

80.0
to be considered for further research. Fig.1 shows the
amount of extract needed for 50 % inhibition (IC50). 60.0
DPPH (1,1-diphenyl-2-picryl-hydrazyl) react with
appropriate reducing agents to be stabilized and this 40.0
assay is dependent on the ability of DPPH to be
decolorized in the presence of antioxidants (Sadhu et 20.0

al., 2003). This diamagnetic molecule contains an

0.0
uneven electron, which determines the absorbance at
0.0 0.5 1.0 1.5 2.0 2.5 3.0
517 nm and also appearance of apparent deep purple Log Cocentration (microgram/mL)
color.
Fig. 1. Comparative DPPH Scavenging
activity

35
 Aparna Shil et al.

In presence of antioxidant molecule, DPPH accepts electron from the former and changes color
stoichometrically depending on the amount of electrons received from antioxidants, and that can be
determined by measuring the changes in absorbance (Rice-Evans et al., 1997).

Calamus rotang is an easy growing plant that can be cultivated in the low and unused lands of the
country. Besides, it requires very little attention for its growth and vigorously multiplication itself. The
present findings represent the importance of this common plant in respect of its high antioxidant value,
presence of secondary metabolites and DPPH scavenging activity and thus, deserve further
investigations for extended knowledge and application of the plant. IC 50 value suggests that research
should be continued to find the importance of this plant in anti-cancer activities.

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