The document summarizes a study that quantified the total phenolics, flavonoids, and evaluated the in vitro antioxidant properties of the methanolic leaf extract of Tarenna asiatica, an endemic medicinal plant from Western Ghats, India. Key findings include: - The leaf extract showed the presence of phytochemicals like phenols, flavonoids, tannins, and triterpenoids. - Total phenolic content was 16.95μg of GAE/100mg extract and flavonoid content was 3.72μg of QE/100mg extract. - The extract exhibited potent antioxidant activity in various in vitro assays like DPPH, ferrous ion

1. 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.
Keywords:
Tarenna asiatica, total phenolics and flavonoids, antioxidant activities.
ABSTRACT:
The present study was aimed at to determine the preliminary phytochemical
screening, quantification of total phenolics and flavonoids and to evaluate the
antioxidant activities of an endemic medicinal plant species, Tarenna asiatica. The
antioxidant activity of methanolic leaf extract of this species was assessed by DPPH•
,
ferrous ion chelating, reducing power and ABTS•+
radical scavenging activities. The
total phenolics and flavonoids content of the extract were found to be 16.95µg of
GAE/100mg extract and 3.72µg 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 R1
,
Nisha Raj1
, Jamuna S2
and Paulsamy S2
*.
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
JournalofResearchinPlantSciences An International Scientific Research Journal

2. 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•
, ferrous ion chelating, reducing power and
ABTS•+
radical scavenging activities.
MATERIALS AND METHODS
Collection of plant material
The fresh leaves of Tarenna asiatica were
collected from the Maruthamalai hills, Coimbatore
district, Tamil Nadu, India. Collected plant materials
were washed thoroughly in tap water, shade dried and
then homogenized to fine powder and stored in air tight
bottles.
Preparation of plant extracts
About 50g of powdered plant materials were
extracted (50g/250mL) in a soxhlet extractor for 8 to 10
hours, by using methanol solvent. Then the extract was
evaporated to dryness.
Phytochemical Screening
A portion of the concentrated extracts were used
for the screening tests, both qualitative analysis and
quantitative analysis using standard methods.
Qualitative analysis of phytochemical constituents
Alkaloids
1mL of extract was added with 2-3 drops of
Dragendorff’s reagents. An orange red precipitate was
produced with Dragendorff’s reagent would indicate the
presence of alkaloids (Ciulci, 1994).
Preparation of Dragendorff’s reagent: Hundred
gram tartaric acid was dissolved in 400 mL water. To
this, 8.5 g basic bismuth nitrate was added and the
solution was shaken for 2 hours. 200 mL of 40 %
potassium iodide was then added, and the solution was
shaken vigorously. After 24 hours, the solution was
filtered.
Flavonoids
To 4mL of the extract, a piece of magnesium
ribbon was added followed by concentrated HCl drop
wise. A colour ranging from crimson to magenta
indicated the presence of flavonoids (Sofowora, 1993).
Saponins
1mL of extract was 5mL of distilled water and
shaken well. A persistent froth that lasted for at least 15
minutes indicated the presence of saponin (Brain and
Chandran et al.,2013
197 Journal of Research in Plant Sciences (2013) 2(2): 196-204

3. Turner, 1975).
Tannins
2mL of extracts were diluted with distilled water
and added with 2-3 drops of 5% Fecl3. Appearance of
blue-back or green black color indicates the presence of
tannins (Mace and Gorbach, 1963; Ciulci 1994).
Steroids
2mL of methanolic leaf extract were poured in
test tube and evaporated to dryness. The residues were
dissolved in acetic anhydride followed by the addition of
chloroform. Conc.H2SO4 was added to the sides of the
test tube. Formation of brown ring at the interface of the
two liquids and a violet colour in the supernatant layer
indicated the presence of steroids (Ciulci, 1994).
Phenols
Five mL of the concentrated extract was taken
and 2mL of neutral ferric chloride solution was added.
Appearance of violet colour indicates the presence of
phenols (Krishnamoorthy, 1988).
Glycosides
Small amount of methanolic leaf extract was
dissolved in 1mL of water followed by the addition of
NaOH. Appearance of yellow colour indicates the
presence of glycosides (Harborne, 1984).
Terpenoids
Methanolic leaf extracts of the study species
taken in a test tube. To this, some pieces of tin and 3
drops of thionyl chloride were added. Formation of violet
or purple colour indicates the presence of terpenoids
(Trease and Evans, 1996).
Triterpenoids
10mg of the extract was dissolved in 1mL of
chloroform and 1mL of acetic anhydride was added
following the addition of 2mL of conc.H2SO4. Formation
of reddish violet colour indicates the presence of
triterpenoids (Harborne, 1984).
Quantitative analysis of phytochemical constituents
Total phenolic content
The total phenolic content of plant extracts was
determined using Folin-Ciocalteu reagent according to
the procedure described by Siddhuraju and Becker
(2003). In this method, 20µg of the extract (dissolved in
the respective solvent) was taken in a test tube and made
up to the volume of 1.0mL with distilled water. Then
0.5mL of freshly prepared Folin-ciocalteu phenol reagent
(1:1 with water) and 2.5mL of 20% sodium carbonate
solution were added sequentially in each tube. The
mixtures were agitated and left in the dark at laboratory
temperature for 40min for the development of colour.
The absorbance was recorded at 725 nm against the
reagent blank using a spectrophotometer. A calibration
curve of gallic acid was constructed, and linearity was
obtained in the range of 10-50µg/mL. Using the standard
curve, the total phenol content of the extract was
calculated and expressed as gallic acid equivalent (GAE)
mg/g extract.
Total flavonoids content
The total flavonoids content was estimated as per
the modified method adopted by Zhshen et al. (1999).
1mL of leaf extract after proper dilution added with 2
mL of distilled water and then 0.15mL of sodium nitrite
solution and incubated for 6 minutes. Then, 0.15mL of
10% aluminium chloride solution was added and allowed
to stand for 6 min. Finally 2mL of 4% sodium hydroxide
was made upto 5mL with distilled water and mixed well.
At room temperature, after 15 minutes the absorbance
was measured at 510nm. By using standard curve with
quercetin, the total flavonoids content was estimated.
Mean of the triplicates was expressed as (QE) mg/g
extract on a dry weight basis.
In vitro antioxidant activity
DPPH▪
radical scavenging activity
The antiradical scavenging efficiency was
assessed using the DPPH▪
method as described by Blois
(1958). In this method commercially available methanol
soluble stable free radical DPPH▪
was used. In its radical
form, DPPH▪
has an absorption band at 515nm, which
disappears upon reduction by an antioxidant compound
Chandran et al.,2013
Journal of Research in Plant Sciences (2013) 2(2): 196-204 198

4. or a radical species. For the photometric assay, different
volumes of the extracts were taken in different test tubes.
The volume was adjusted to 100μL with methanol.
5.0mL of 0.1mM methanolic solution of DPPH▪
was
added to these tubes and shaken vigorously. The tubes
were allowed to stand for 20 min at 27°C. 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 IC50 [the
microgram of extract to scavenge 50% of the DPPH▪
radicals] which was determined by linear regression.
Lower the IC50 value, greater antioxidant activity.
Ferrous ion chelating assay
Ferrous ion chelating activity was determined
according to the method of Sing and Rajini (2004).
100μL of 2mM FeSo4 and 300μL of 5mM
ferrozine were mixed with different concentration of
samples (50-250μg/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•
radical scavenging activity. EDTA
was used as positive controls for comparison.
Reducing power assay
Total reducing power was determined as per the
method of Yildirim et al. (2001). Different
concentrations of methanolic leaf extract of T. asiatica
(50-250μg/mL) were mixed with 1mL of 0.2M sodium
phosphate buffer of 6.6pH and 1mL of 1% potassium
ferric cyanide. Then it was incubated at 50°
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% FeCl3.
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 (µg/100mg)]
Total flavanoid content
[QE (µg/100mg)]
Leaf 29.8 16.95±0.18 3.72±0.03
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.

5. Chandran et al.,2013
Journal of Research in Plant Sciences (2013) 2(2): 196-204 200
(about 1:89 v/v) and equilibrated at 30°C to obtain an
absorbance of 0.700±0.02 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 μL 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 10μL of sample
extracts or Trolox standards (final concentration 0-15
μM) in ethanol, absorbance was recorded at 30°C,
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 μmol/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
(µg/ml)
% of inhibition
IC50 value of leaf
extract
IC50 value of BHT
1. 50 33.20±0.32a
231.48±0.54 34.74±00.26
2. 100 42.05±0.09b
3. 150 52.96±0.31c
4. 200 53.65±0.23cd
5. 250 68.26±0.43e
Table 3. DPPH•
radical scavenging activity of methanolic leaf extract of Tarenna asiatica.
Values were performed in triplicates and represented as mean ± SD.
Mean values followed by different superscript in a column are significantly different (p<0.05).
Butylated Hydroxyl Toluene (BHT).
S. No.
Sample
concentration
(µg/ml)
% of inhibition
IC50 value of leaf
extract
IC50 value of
EDTA
1. 50 48.68±0.12a
152.90±0.04 120.77±0.05
2. 100 49.43±0.34ab
3. 150 58.80±0.13b
4. 200 61.04±0.45c
5. 250 66.66±0.15d
Table 4. Ferrous ion chelating activity of methanolic leaf extract of Tarenna asiatica.
Values were performed in triplicates and represented as mean ± SD.
Mean values followed by different superscript in a column are significantly different (p<0.05).
Ethylene Diamine Tetra Acetic acid (EDTA).

6. Chandran et al.,2013
201 Journal of Research in Plant Sciences (2013) 2(2): 196-204
content of methanolic leaf extract were 16.95µg of
GAE/100mg and 3.7295µg of QE/100mg (Table 2).
Phenolic compounds are known as powerful chain
breaking antioxidants and they may contribute directly to
antioxidative action (Lu et al., 2011). Flavanoids possess
a broad spectrum of chemical and biological activities
including radical scavenging properties (Prasad et al,
2009).
In vitro antioxidant activity
DPPH•
radical scavenging assay
DPPH•
is a relatively stable free radical. The
assay is based on the measurement of the scavenging
ability of antioxidants towards the stable free radicals
DPPH•
(Halliwell and Gutteridge, 2007). Free radical
scavenging capacities of methanolic leaf extract was
measured by their ability to trap free radical DDPH•
. The
percentage of inhibition was increased with the
increasing concentration of the sample from 50-250µg/
mL (Table 3). The IC50 value of the extract was 231.48
which is comparable to that of the standard, BHT.
DPPH•
test provides information on the reactivity of test
compounds with a stable free radical. Because of its,
addition of electron to 2, 2-Diphenyl-Picryl Hyrazyl
radical (DPPH•
) gives a strong absorption band at 517nm
in visible spectroscopy. The efficacies of antioxidants are
often associated with their ability to scavenge stable free
radicals (Krishnaraju et al., 2009). It has been shown that
the scavenging effects on the 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 50-250µg/mL. The percentage of inhibition
of the metal chelating was varying from 48.68% at 50µg/
mL to 66.66% at 250µg/mL. The IC50 values of the
methanolic leaf extract of the study species was
152.90µg/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.

7. 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 250µg/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 ± 21.10μmolTE/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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