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,
ferrous ion chelating, reducing power and ABTS
+
radical scavenging activities. The
total phenolics and flavonoids content of the extract were found to be 16.95g of
GAE/100mg extract and 3.72g of QE/100mg extract respectively. The present study
revealed that the methanolic leaf extract of this speices showed potent in vitro
antioxidant activities and so it may be useful for their nutritional and medicinal
properties.
196-204 | JRPS | 2013 | Vol 2 | No 2
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www.plantsciences.info
Journal of Research in
Plant Sciences
An International Scientific
Research Journal
Authors:
Vishnu Chandran R
1
,
Nisha Raj
1
, Jamuna S
2
and Paulsamy S
2
*.
Institution:
1.Department of
Biotechnology, SAS SNDP
Yogam College,
Konni, Kerala.
2. Department of Botany,
Kongunadu Arts and
Science College,
Coimbatore, Tamil Nadu.
Corresponding author:
Paulsamy S.
Email:
paulsami@yahoo.com
Web Address:
http://plantsciences.info
documents/PS0059.pdf.
Dates:
Received: 25 Apr 2013 Accepted: 07 May 2013 Published: 10 May 2013
Article Citation:
Vishnu Chandran R, Nisha Raj, Jamuna S and Paulsamy S.
Quantification of total phenolics and flavonoids and evaluation of in vitro antioxidant
properties of methanolic leaf extract of Tarenna asiatica - an endemic medicinal plant
species of Maruthamali hills, Western Ghats, Tami Nadu.
Journal of Research in Plant Sciences (2013) 2(2): 196-204
Original Research
Journal of Research in Plant Sciences
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An International Scientific Research Journal
INTRODUCTION
Phytochemicals are naturally occurring
compounds of plant kingdom, such as medicinal plants,
vegetables, fruits, that work with nutrients and fibers to
act against diseases or more specifically, provides
protection against diseases (Chopra et al., 1984).
Secondary metabolites of plants are the responsible
chemical compounds for the medicinal properties of the
respective plant species. Free radicals formed in human
body are playing important role in different pathological
conditions like tissue injury, inflammation, and
neurodegenerative diseases. Antioxidants are very
important chemicals which can protect the human body
from these damages caused by free radicals. (Halliwell
and Gutteridge, 1989). However, synthetic antioxidants
are not much used for this carcinogenic effect (Buxiang
and Fukuhara, 1997; Hirose, 1998). Therefore,
investigation of natural antioxidants of plant origin is
getting more attention (Kaur and Kapoor, 2001; Vinson,
2001).
Tarenna asiatica L. (Rubiaceae) is a large shrub
to small tree and is endemic to the Southern Western
Ghats of Agasthyamalai, Anaimalai, Palani hills, Niligirs
and Bababudangiri hills and distributed in Peninsular
India, Malaysia and Sri Lanka. Fruit juice is applied on
the eye lids to arrest infection. Malayali tribal in
Javvadhu hills use T. asiatica for marriages, festivals and
worship due to its aesthetic value (Ravikumar and Vijaya
Sankar, 2003). The fruits are eaten by crows. The wood
is used as fuel wood. It is a strong and used as a crobar
also in Coimbatore district. Due to its medicinal
properties the current study was aimed at to screen the
phytochemicals, quantification of total phenolics and
flavonoids and in vitro antioxidant activities such as
DPPH
was
added to these tubes and shaken vigorously. The tubes
were allowed to stand for 20 min at 27C. The control
was prepared as above but without the test extract and
methanol was used for the baseline correction. Changes
in the absorbance of the samples were monitored at
517nm. Results were compared with the activity of the
standard, BHT. The per cent DPPH
discolouration of the
samples was calculated using the following formula:
DPPH radical scavenging activity (%) = [(Control OD
Sample OD/Control OD] X 100
Antioxidant activity of the methanolic leaf
extract of the study species was expressed in IC
50
[the
microgram of extract to scavenge 50% of the DPPH
radicals] which was determined by linear regression.
Lower the IC
50
value, greater antioxidant activity.
Ferrous ion chelating assay
Ferrous ion chelating activity was determined
according to the method of Sing and Rajini (2004).
100L of 2mM FeSo4 and 300L of 5mM
ferrozine were mixed with different concentration of
samples (50-250g/mL3). The mixture was allowed to
equilibrate for 10 min before measuring the absorbance.
The ability of the sample to chelate ferrous ion was
calculated by the formula of inhibition percentage as
employed for DPPH
C for
20minutes. 1ml of 10% TCA was added to the mixture,
which was then centrifuged at 3000rpm for 1min. Finally
2mL of the supernatant solution were mixed with equal
volume of distilled water. Absorbance was measured at
700nm after the addition of 0.5mL of 1% FeCl
3
.
Ascorbic acid was used as positive control for
comparison.
Total antioxidant activity
It was performed according to the modified
method of Siddhuraju and Manian, (2007). The ABTS
+
radical cation (ABTS
+
) was generated by a reaction of 7
mmol/L ABTS
+
and 2.45 mmol/L potassium persulfate
after incubation for 16 h at laboratory temperature in
dark. Blue green ABTS
+
was formed at the end of this
period. Prior to assay, the solution was diluted in ethanol
Chandran et al.,2013
199 Journal of Research in Plant Sciences (2013) 2(2): 196-204
Plant
extract
Phytochemical compounds
Alkaloids Flavanoids Glycosides Steroids Phenols Tannins Saponins Terpenoids Triterpenoids
Leaf ++ ++ +++ - + + - - -
Table 1. Preliminary qualitative phytochemical analysis of methanolic leaf extract of Tarenna asiatica.
+ Presence of compounds; - Absence of compounds.
Plant extract Extraction yield (%)
Total phenolic content
[GAE (g/100mg)]
Total flavanoid content
[QE (g/100mg)]
Leaf 29.8 16.950.18 3.720.03
Table 2. Extraction yield, total phenolics and flavonoids content of methanolic leaf extract of Tarenna asiatica.
Values were performed in triplicates and represented as mean SD.
GAE - Gallic Acid Equivalent, QE - Quercetin Equivalent.
Chandran et al.,2013
Journal of Research in Plant Sciences (2013) 2(2): 196-204 200
(about 1:89 v/v) and equilibrated at 30C to obtain an
absorbance of 0.7000.02 at 734 nm, the wavelength of
maximum absorbance in the visible region. The stock
solution of the sample extracts in ethanol was diluted
such that, after introduction of a 10 L aliquot of each
dilution into the assay, they produced between 20 - 80%
inhibition of the blank absorbance. After the addition of
1.0mL of diluted ABTS
+
solution to 10L of sample
extracts or Trolox standards (final concentration 0-15
M) in ethanol, absorbance was recorded at 30C,
exactly 30 min after the initial mixing. Appropriate
solvent blanks were also run in each assay. Triplicates
were maintained for the experiments and the per cent
inhibition of the blank absorbance at 734 nm was plotted
as a function of Trolox concentration (Re et al., 1999).
The unit of total antioxidant activity (TAA) was defined
as the concentration of Trolox having the equivalent
antioxidant activity expressed as mol/g sample extracts
on dry weight basis.
STATISTICAL ANALYSIS
The triplicates were maintained for all tests and
the data were modified to ANOVA followed by DMRT.
Significance at 5% level was calculated.
RESULTS AND DISCUSSION
Phytochemical analysis
Preliminary qualitative phytochemical analysis
This profile of phytochemicals in the leaf part of
T. asiatica can provide ideas about the synthesis of
certain bioactive compounds for the manufacturing of
some useful drugs as reported by Yakubu et al. (2005)
for Fadogia agrestis.
Quantitative phytochemical analysis
The percentage yield of crude methanolic leaf
extract is 29.8. The total phenolics and flavonoids
S. No.
Sample
concentration
(g/ml)
% of inhibition
IC
50
value of leaf
extract
IC
50
value of BHT
1. 50 33.200.32
a
231.480.54 34.7400.26
2. 100 42.050.09
b
3. 150 52.960.31
c
4. 200 53.650.23
cd
5. 250 68.260.43
e
Table 3. DPPH
. The
percentage of inhibition was increased with the
increasing concentration of the sample from 50-250g/
mL (Table 3). The IC
50
value of the extract was 231.48
which is comparable to that of the standard, BHT.
DPPH
radical increased
sharply with the increasing concentration of the samples
and stands to a certain extent (Jamuna et al., 2012;
Karthika et al, 2012) and hence are said to be strongly
dependent on the extract concentration.
Ferrous ion metal chelating assay
Metal ion chelating activity may be due to the
chelating agents, which form sigma bonds with the metal
and effective as secondary antioxidants because they
reduce the redox potential, there by the oxidized form of
the metal ion (Gulcin et al., 2004). The results of
antioxidant activity of the methanolic leaf extract of T.
asiatica based on metal chelating activity are given in
Table 4. The percentage of metal chelating activity was
determined to be sample concentration dependent and it
was increased with the increase of concentration of
extract from 50-250g/mL. The percentage of inhibition
of the metal chelating was varying from 48.68% at 50g/
mL to 66.66% at 250g/mL. The IC
50
values of the
methanolic leaf extract of the study species was
152.90g/mL and it was comparable to that of the
standard drug, EDTA. Similar trend of metal ion
scavenging activity was observed in the species,
Cyperus rotundus (Shajiselvin and Kottai Muthu, 2011;
Thambiraj and Paulsamy, 2012).
Reducing power assay
It is based on the principle that the substances of
reduction potential react with potassium ferricyanide
(Fe
+++
) to form potassium ferrocyanide (Fe
++
) which
reacts with ferric chloride and form ferrous complex that
has an absorption maximum at 700nm. Generally, the
level of reducing power of a compound is one of the
indicators to show the degree of efficiency of its
antioxidant property. Change in yellow colour in to
various shades of green and blue colour in the present
test for the phenolic leaf extract T. asiatica indicates the
presence of antioxidant property for this species. Fig. 1
shows the reductive capabilities of methanolic leaf
Figure 1. Reducing power activity of methanolic leaf
extract of Tarenna asiatica.
Chandran et al.,2013
Journal of Research in Plant Sciences (2013) 2(2): 196-204 202
extract of T. asiatica when compared to the standard,
ascorbic acid. As responded for DPPH
and ferrous ion
chelating activities, the reducing power also increased
with the increase of concentration of the samples. The
extract of T. asiatica showed the highest reducing ability
(absorbance 0.493) for 250g/mL. However, the activity
was lesser than the standard, ascorbic acid (absorbance
1.393).
ABTS
+
cation radical scavenging activity
The decolorization of the ABTS
+
, through
measuring the reduction of the radical cation as the
percentage inhibition of absorbance at 734nm (Re et al.,
1999). ABTS
+
was generated by (Wolfenden et al.,
1982) incubating ABTS
+
chromophore through the
reaction. The reduction of the 2,2-azinobis (3-
ethylnenzo thiazoline sulphonate) radical cation
(ABTS
+
) has widely used to measure the antioxidant
capacity of natural extract (Cai et al., 2004; Cai et al.,
2006). The presence of chemical compounds in the tested
extracts that inhibit the potassium persulfate activity may
reduce the production of ABTS
+
. This study reports that
the methanolic leaf extract of T. asiatica have highest
antioxidant activity, 3186 21.10molTE/g extract
(Table 8). It shows that the methanolic leaf extract of T.
asiatica possessed the highest ABTS
+
scavenging
capacity.
Quantitative phytochemical analysis indicated
that the plant extract contains significant amounts of
phenolics and flavonoids compounds such as total
phenolic and flavanoids. These classes of secondary
metabolites are playing major role for antioxidant and
free radical scavenging effect (Sreeram et al., 2005; Gill
et al., 1999; Hazra et al., 2008). The antioxidant activity
of phenolic compounds is mainly due to their redox
properties, which play an important role in neutralizing
free radicals, quenching singlet and triplet oxygen and
flavanoids are wide spread in all natural compounds and
posses a broad spectrum of biological activities. Due to
the presence of phytochemicals in general and phenolics
and flavonoids in large amount in particular, this species,
T. asiatica may has effective free radical scavenging
activity.
CONCLUSION
On the basis of the results obtained in the present
study, it was concluded that the methanolic leaf extract
of T. asiatica possess the significant antioxidant activity.
These findings suggest that this plant is a potential
source of natural antioxidant. However, further studies
are needed to understand the mechanism of action extract
and to isolate the compounds responsible for such
activities.
ACKNOWLEDGMENT
The authors are gratefully thankful to
Dr. M. Aruchami, Secretary and Director, Kongunadu
Arts and Science College, Coimbatore for permitting to
do the work in the Botany Department of his institute
under consultancy scheme. They sincerely
acknowledge Dr. S. Paulsamy, Associate Professor in
Botany, Kongunadu Arts and Science College for
offering his expertise to carry out the work.
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