Journal of Applied Pharmaceutical Science 02 (07); 2012: 149-154

ISSN: 2231-3354
Received on: 12-07-2012
Screening of in vitro antioxidant activity of
Revised on: 18-07-2012
Accepted on: 23-07-2012
methanolic leaf and root extracts of Hypochaeris
DOI: 10.7324/JAPS.2012.2722
radicata L. (Asteraceae)

Jamuna, S., S. Paulsamy and K. Karthika

ABSTRACT

The aim of present study was to estimate the total phenolic and flavonoid contents
and to investigate in vitro antioxidant potential of methanolic leaf and root extracts of the herb,
Hypochaeris radicata L. (Asteraceae). Antioxidant activity was assessed by using 2,2-
diphenyl-1-picryl-hydrazyl (DPPH•) assay, reducing power activity, [2,2’-azino-bis(3-
ethylbenzthiazoline-6-sulphonic acid)] ABTS•+ assay and ferrous ion chelating activity. Here,
butylated hydroxytoluene (BHT), ascorbic acid (ASA), trolox and EDTA were used as
standard antioxidants. The total phenolic and flavonoid contents were also determined and
expressed in gallic acid and quercetin equivalent respectively. The results of the study indicate
Jamuna, S., S. Paulsamy that the methanolic extracts of the leaf and root of H. radicata posses significant scavenging
and K. Karthika activity against DPPH• (97.99% for leaf and 96.44% for root at 250μg/ml each) and ferrous
Department of Botany, ions chelating activity (38.69% for leaf and 40.52% for root at 5000μg/ml each), reducing
Kongunadu Arts and Science College, power activity (1.38 absorbance at 600µg/ml for leaf, 0.45 absorbance at 700 µg/ml for root)
Coimbatore, India
and free radical scavenging activity (ABTS•+) (2706.73 for leaf and 2028.37μmol for root
TE/g). The free radical scavenging and antioxidant activities may be attributed to the presence
of adequate phenolic (gallic acid content is 125.5µg/10mg in leaf and 133.06µg/10mg in root)
and flavonoid compounds (105.76µg/2mg in leaf and 55.16µg/2mg in root). This study
revealed that the methanolic extracts of both leaf and root of H. radicata has demonstrated
significant antioxidant activity.

Keywords: Hypochaeris radicata L., total phenolic, flavonoids, DPPH• assay, reducing power
activity, ABTS•+ assay, ferrous ion chelating activity.

INTRODUCTION

Antioxidant compounds in food play an important role as a health protecting factor.

Primary sources of naturally occurring antioxidants are whole grains, fruits and vegetables. The
main characteristic of an antioxidant is its ability to trap free radicals. Highly reactive free radicals
and oxygen species can initiate degenerative diseases. Antioxidant compounds like phenolic
acids, polyphenols and flavonoids are commonly found in plants have been reported to have
multiple biological effects, including antioxidant activity (Brown and Rice-Evans, 1998).
Currently, the possible toxicity of synthetic antioxidants has been criticized. Thus interest in
For Correspondence
S. Paulsamy natural antioxidant, especially of plant origin has greatly increased in recent years (Jayaprakash
Email: paul sami@[Link]
_
and Rao, 2000).
 Journal of Applied Pharmaceutical Science 02 (07); 2012: 149-154

Free radicals arising from metabolism or environmental reagent according to the procedure reported by Singletion et al.,
sources interact continuously in biological systems and their (1999). About 500µl (20mg/ml) of plant sample was added to 25ml
uncontrolled generation correlates directly with molecular level of of distilled water and 1ml of Folin-Ciocalteu reagent (1:10). Then
many diseases (Huang et al., 2005). Lots of research has clearly this mixture was kept at room temperature for 3 minutes, after then
showed that free radicals would damage nearby structures 1.5ml of 2% sodium bicarbonate was added, soon after vortexting
including DNA, proteins or lipids. Radical scavenging the reaction mixture for 1 hour at room temperature, the
antioxidants are particularly important in antioxidative-defence in absorbance was measured at 760nm. All the tests were performed
protecting cells from the injury of free-radical (Youwei et al., in triplicates and the results were averaged. The concentration of
2008). Plants are the good source of biologically active total phenolic compounds in methanolic leaf and root extracts was
compounds known as phytochemicals. The phytochemicals have determined as microgram of gallic acid equivalent by using an
been found to act as antioxidants by scavenging free radicals and equation that was obtained from the standard gallic acid graph (10-
may have therapeutic potential for free radical associated disorders 300 µg/ml).
(Hausladen and Stamer, 1999). It is well known that free radicals
are the major cause of various chronic and degenerative diseases, Determination of total flavonoids content
such as coronary heart disease, inflammation, stroke, diabetes The aluminium chloride colorimetric assay was used for
mellitus and cancer (Scalbert et al., 2005). Therefore, it is total flavonoids determination, as described by Zhishen et al.
important to assess antioxidant activity of the plants used in the (1999). 100µl (20mg/ml) of the extract was mixed with 2.5 ml of
herbal medicine either to elucidate the mechanism of their distilled water and 300µl of 5% sodium nitrate. Then, it was
pharmacological action or to provide information on antioxidant incubated at room temperature for 5 minutes and 300µl of 10%
activity of these herbal plants (Abdul et al., 2012). aluminium chloride, 2ml of 1M sodium hydroxide and 1ml of
Hypochaeris radicata L. of Asteraceae family an one such distilled water were added. Then, absorbance of the reaction
plant species distributed in high hills of Nilgiris, the Western mixture was measured at 512nm, along with the standard,
Ghats, India at temperate climate is expected to have antioxidant quercetin and blank. The total flavonoids content was determined
property as it is being used by the local communities for adding as microgram, quercetin equivalent by using the standard,
freshness and activeness (Paulsamy et al., 2008). Therefore to have quercetin graph, obtained by comparing the calibration curve
the scientific validation on antioxidant properties, the leaves and prepared from a reference solution containing quercetin (10-
roots of this species were taken for the present study. The 300µg/ml).
methanolic extracts of both parts of H. radicata were used to
investigate the antioxidant activity in terms of free radical In vitro antioxidant activity
scavenging activity (DPPH•), reducing power activity, ABTS•+ The free radical scavenging activity of the methanolic leaf
assay and ferrous ion chelating activity. and root extracts of the study species, H. radicata was determined
by using various in vitro assays such as DPPH• assay, reducing
MATERIALS AND METHODS power assay and ABTS•+ assay and ferrous ion chelating activity.
Collection and identification of plant material
Free radical scavenging activity (DPPH•)
The plant material was collected from Kattabettu,
The free radical scavenging activity of methanolic extract
(2100ms above msl), the Nilgiris, Western Ghats, India. The plant
of H. radicata was measured by using 2, 2-diphenyl-1-picryl-
was authenticated by Dr. P. Sathyanarayana, Botanical Survey of
hydrazyl (DPPH•) method of Blois (1958). 0.2mM solution of
India, TNAU Campus, Coimbatore. The voucher number is
DPPH• in methanol was prepared and 100μl of this solution was
BSI/SRC/5/23/2010-11/Tech/153.
added to various concentrations of methanolic leaf and root
extracts at the concentrations of 50, 100, 150, 200 and 250μg/ml.
Preparation of plant extracts
After 30 minutes, absorbance was measured at 517nm. Butylated
Fresh plant material was washed under running tap water,
hydroxytoluene (BHT) was used as the reference material. All the
air dried and powdered. About 50g of coarsely powdered plant
tests were performed in triplicate and percentage of inhibition was
materials (50g/250ml) were extracted in a soxhlet extractor for 8 to
calculated by comparing the absorbance values of the control and
10 hours, sequentially with petroleum ether, chloroform, ethyl
test samples.
acetate, methanol and water. The extracts obtained were then
concentrated and finally dried to a constant weight. Dried extracts
Percentage of inhibition = Abscntrl-Abstest x 100
were kept at 200C until further test were carried out. For stock
Abscntrl
solutions, 10mg/ml of methanolic leaf and root extracts were
dissolved in dimethyl sulffoxide (DMSO).
The scavenging reaction between DPPH• and an antioxidant, H-A
can be written as
Determination of total phenolics content
The total soluble phenols present in the methanolic leaf (DPPH•) + (H-A) DPPH-H + (A•)
and root extracts were determined by using the Folin-Ciocalteu (Purple) (Yellow)
 Journal of Applied Pharmaceutical Science 02 (07); 2012: 149-154

Reducing power activity Range Test (DMRT) at (p<0.05) significant level. (Statsoft Inc,
Reducing power assay was determined according to the Tulsa, USA).
method of Yildirim et al., (2001). Different concentrations of
methanolic extracts of leaf (200, 300, 400, 500 and 600μg/ml) and RESULTS AND DISCUSSION
root (300, 400, 500, 600 and 700μg/ml) of the study species were
Free radical is a molecule with an unpaired electron and is
mixed with 1ml of 200mM sodium phosphate buffer (pH 6.6) and
involved in bacterial and parasitic infections, lung damage,
1ml of 1% potassium ferriccyanide followed by incubation at 500C
inflammation, reperfusion injury, cardiovascular disorders,
for 20 minutes. After adding 1ml of 10% trichloro acetic acid, the
atherosclerosis, aging and neoplastic diseases (Roy et al., 1994).
mixture was centrifuged at 3000 rpm for 10 minutes. The
They are also involved in autoimmune disorder like rheumatoid
supernatant was taken out and mixed with 2ml of distilled water
arthritis etc. (Rao et al., 2004). Our results demonstrated that the
and 0.5ml of 1% ferric chloride. After incubation for 10 minutes,
methanolic extracts of leaf and root of H. radicata possess free
the absorbance was measured at 700nm. Higher absorbance of the
radical scavenging activity in vitro models like DPPH•, ABTS•+,
reaction mixture indicates reductive potential of the extracts (Yang
reducing power activity and ferrous ion chelating assays.
et al., 2002, Rajeshwar et al., 2005, Koksal et al., 2011). All the
tests were performed in triplicates and ascorbic acid was used as
Total phenolics content
reference standard.
Phenolic compounds are known as powerful chain
breaking antioxidant (Shahidi and Wansundeara, 1992) and they
Free radical scavenging activity (ABTS•+)
are very important plant consistuents because of their scavenging
The total antioxidant activity of the samples was
ability, which is due to their hydroxyl groups (Hatano et al., 1989).
measured by [2, 2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic
In methanolic leaf and root extracts of H. radicata, the total
acid)] ABTS•+ radical cation decolorization assay according to the
phenolic content was found to be 125.5µg/10mg and
method of Re et al. (1999). ABTS•+ was produced by reacting
133.06µg/10mg respectively in terms of gallic acid equivalent
7mM ABTS+ aqueous solution with 2.4mM potassium persulfate in
(Table 1). In addition it has been determined that the highest
the dark for 12-16 hours at room temperature. The radical was
extraction yield was found in root extract.
stable in this form for more than two days when stored in the dark
at room temperature. Prior to assay, this solution was diluted in Table. 1: Extraction yield, total phenolics and flavonoids content of methanolic leaf
ethanol (about 1:89 v/v) and equilibrated at 300C to give an and root extracts of Hypochaeris radicata.
absorbance of 0.7000±0.02 at 734 nm. Then, 2ml of diluted Extraction yield Total phenolic Total flavonoid
(MeOH) content content
ABTS•+ solution was added to the sample concentration at 20μl Sample [Gallic acid [Quercetin
(1mg/ml). After 30 minutes of incubation at room temperature, the % yield (W/W) equivalent equivalent
(µg/10mg)] (µg/10mg)]
absorbance was recorded at 734nm and percentage of inhibition Leaf 18.4 125.5 ± 0.5 105.76 ± 0.75
was calculated. Trolox was used as a reference standard. Root 25.6 133.06 ± 0.6 55.16 ± 0.76
Triplicates were performed. 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).
Ferrous ion chelating activity
The chelating of ferrous ions by leaf and root methanolic Total flavonoids content
extracts of H. radicata was estimated by the method of Singh and Flavonoids are a group of polyphenolic compounds,
Rajini (2004). The different concentrations of methanolic extracts which exhibit several biological effects such as anti-inflammatory,
(1000, 2000, 3000, 4000 and 5000μg/ml separately for leaf and anti-hepatotoxic, anti-ulcer, anti-allergic, anti-viral and anti-cancer
root) were mixed with 100μl of 2mM ferrous sulphate solution and activities (Umamaheswari et al., 2008). They are capable of
300μl of 5mM ferrozine. The mixture was incubated at room effectively scavenging the reactive O2 species because of their
temperature for 10 minutes. The absorbance of the solution was phenolic hydroxyl groups and so they are potent antioxidants also
measured at 562nm. Ethylene diammine tetra acetate (EDTA) was (Cao et al., 1997). The total flavonoids content of methanolic leaf
used as standard. All the tests were performed in triplicate and and root extracts of H. radicata was determined to be
percentage of inhibition was calculated by using this formula, 105.76µg/2mg and 55.165µg/2mg respectively inturms of
quercetin equivalent (Table 1).
Percentage of inhibition = Abscntrl-Abstest X 100
Abscntrl Free radical scavenging activity (DPPH•)
DPPH• is one of the free radicals widely used for testing
Statistical analysis preliminary radical scavenging activity of the plant extract
The statistical comparison among the groups were (Bhuiyan et al., 2009). Scavenging of DPPH• radical is related to
performed with one way analysis of variance (ANOVA) test using the inhibition of lipid peroxidation (Rekka and Kourounakis 1991).
a statistical package program SPSS 10 and the significance of the DPPH• is usually used as a substance to evaluate the antioxidant
difference between means was determined by Duncan’s Multiple activity (Tara Chand et al., 2012). Antioxidants either transfer an
 Journal of Applied Pharmaceutical Science 02 (07); 2012: 149-154

electron or a hydrogen atom to DPPH•, thus neutralizing its free Free radical scavenging activity (ABTS•+)
radical character (Pan et al., 2008). DPPH• test, which is based on The decolorization of the ABTS•+, through measuring the
the ability of DPPH•, a stable free radical, to decolorize in the reduction of the radical cation as the percentage inhibition of
presence of antioxidants, is a direct and reliable method for absorbance at 734nm (Re et al., 1999). ABTS•+ was generated by
determining radical scavenging action (Raquibul Hasan et al., incubating ABTS•+ chromophore through the reaction (Wolfenden
2009). The DPPH• assay has been largely used as a quick, reliable et al., 1982). The presence of specific chemical compounds in the
and reproducible parameter to search the in vitro general extracts of H. radicata may inhibit the potassium persulfate
antioxidant acitivity of pure compounds as well as plant extracts activity and hence reduced the production of ABTS•+. This study
(Koleva et al., 2002). The reducing capacity of compounds could reports that the methanolic leaf extract of H. radicata has highest
serve as indicator of potential antioxidant property (Meir et al., antioxidant activity (2706.73 μmol/g) than that of its root counter
1995). In the present study, the percentage of scavenging effect on part (2028.37μmol/g) (Table 4).
the DPPH• radical was concomitantly increased with the increase
in the concentration of both leaf and root methanolic extracts from Ferrous ion chelating activity
50 to 250 µg/ml. The percentage of inhibition was existing from Iron is essential for life because it is required for oxygen
54.91 at 50µg/ml to 98.36 at 300 µg/ml for leaf extract and for root transport, respiration and activity of many enzymes. However, it is
extracts, they were 23.27 at 50µg/ml and 96.72 at 250 µg/ml an extremely reactive metal and catalyzes oxidative changes in
(Table 2). From the results it is known that the species, H. lipids, proteins and other cellular components (Smith et al., 1992).
radicata possess hydrogen donating capabilities for methanolic The metal chelating ability of the methanolic leaf and
leaf extract and does scavenging free radicals. Furthermore, it was root extracts was measured by the formation of ferrous ion-
noticed that the leaf extract has more pronounced scavenging ferrozine complex. Ferrozine combines with ferrous ions forming
activity than that of the standard, BHT (Table 2). a red coloured complex which absorbs at 562n m (Yamaguchi et
Table. 2: Free radical scavenging activity (DPPH) of methanolic leaf and root
al., 2000). It was reported that the chelating agents which forms σ
extracts of Hypochaeris radicata. bond with a metal, are effective as secondary antioxidants, because
Sample Leaf extract Root extract BHT they reduce the redox potential there by stabilizing the oxidized
S.
concentration % of % of
No. % of inhibition form of the metal ion (Duh et al., 1999). Iron binding capacity in
(µg/ml) inhibition inhibition
1. 50 54.21a ± 0.66 23.4a ± 0.33 36.24a ± 0.31 terms of percent inhibition of the methanolic extract of H. radicata
2. 100 90.86b ± 0.37 56.52b ± 0.38 42.21b ± 0.38
3. 150 93.25c ± 0.62 75.49c ±0.45 49.39c ±0.34 at 5000µg/ml was higher for root (40.52%) than the leaf (38.96%)
4. 200 96.3d ±0.51 95.36d ± 0.47 42.16d ± 0.40 (Table 5). However, it was not comparable to that of the reference
5. 250 97.99e ± 0.36 96.44e ± 0.33 57.15e ± 0.24
standard, EDTA.
BHT was used as reference standard.
Values were performed in triplicates and represented as mean ± SD.
Mean values followed by different superscript in a column are significantly CONCLUSION
different (p<0.05).

Reducing power activity Searching plant sources may bring new natural products
Reducing power activity is often used to evaluate the into pharmaceutical, cosmetic and food production. An in vitro
ability of natural antioxidant to donate electron (Yildirim et al., antioxidant study provides scientific evidence to prove the
2000, Dorman et al., 2003). Many reports have revealed that there traditional claims to the Asteraceae member, H. radicata. On the
is a direct correlation between antioxidant activities and reducing basis of the results obtained in the present study, it was concluded
power of certain plant extracts (Duh, 1998; Duh et al., 1999; that the methanolic leaf and root extracts of this species possess
Yildirim et al., 2000). The reducing power activity of methanolic significant antioxidant activity.
leaf and root extracts of H. radicata increased consistently with the Presence of adequate amount of phenol and flavonoid
increase in the volume of extract from 200µg to 600µg for leaf and compounds may account for this fact. So these findings of present
300µg to 700µg for root. When compared with the root extract study suggest that this plant have a potential source of natural
(0.433 at 700µg/ml), leaf extract showed higher absorbance (1.092 antioxidant. Further studies are warranted for the isolation and
at 600µg/ml). It is known further that the reducing power activity characterization of antioxidant compounds, and also in vivo studies
of leaf extract was far higher than the standard, ascorbic acid are needed for understanding their mechanism of action as
(Table 3). antioxidants.

Table. 3: Reducing power activity of methanolic leaf and root extracts of Hypochaeris radicata.
Sample Leaf extract Ascorbic acid Ascorbic acid
S. Sample concentration Root extract (Absorbance
concentration (Absorbance at (Absorbance at (Absorbance at
No. (µg/ml) at 700nm)
(µg/ml) 700nm) 700nm) 700nm)
1. 200 0.67a ± 0.44 0.39a ± 0.04 300 0.16a ± 0.01 0.55b ± 0.03
2. 300 0.71b ± 0.02 0.55b ± 0.03 400 0.18a ± 0.01 0.64c ± 0.04
3. 400 0.78c ± 0.01 0.64c ± 0.04 500 0.22b ± 0.02 0.86d ± 0.03
4. 500 1.05de ± 0.01 0.86d ± 0.03 600 0.36c ± 0.02 0.98e ± 0.02
e e
5. 600 1.38 ± 0.54 0.98 ± 0.02 700 0.45d ± 0.02 1.09f ± 0.04
Ascorbic acid was used as reference standard.
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).
 Journal of Applied Pharmaceutical Science 02 (07); 2012: 149-154

Table. 4: ABTS activity of methanolic leaf and root extracts of Hypochaeris radicata.
Sample Total antioxidant activity (µmol TE/g extract)
Leaf 2706.79 ± 2.21
Root 2028.37 ± 3.15
Total antioxidant activity (µmol equivalent trolox).
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).

Table. 5: Ferrous ion chelating activity of methanolic leaf and root extracts of Hypochaeris radicata.
Leaf extract Root extract EDTA
S. No. Sample concentration (µg/ml)
% of inhibition % of inhibition % of inhibition
a
1. 1000 34.68 ± 0.48 30.49a ± 0.37 56.28a ± 0.19
2. 2000 36.34 b ± 0.28 31.57b ± 0.09b 71.55b ± 0.33
3. 3000 37.02bc ± 0.46 32.60c ± 0.26 83.46c ± 0.20
4. 4000 37.59d ± 0.35 34.68d ± 0.45 93.19d ± 0.21
5. 5000 38.69e ± 0.23 40.52e ± 0.34 96.69e ± 0.15
EDTA, reference standard.
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).

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