Screening of in vitro antioxidant activity of methanolic leaf and root extracts of Hypochaeris radicata L . ( Asteraceae )

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 2,2diphenyl-1-picryl-hydrazyl (DPPH) assay, reducing power activity, [2,2’-azino-bis(3ethylbenzthiazoline-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 that the methanolic extracts of the leaf and root of H. radicata posses significant scavenging activity against DPPH (97.99% for leaf and 96.44% for root at 250μg/ml each) and ferrous ions chelating activity (38.69% for leaf and 40.52% for root at 5000μg/ml each), reducing power activity (1.38 absorbance at 600μg/ml for leaf, 0.45 absorbance at 700 μg/ml for root) 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.


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 natural antioxidant, especially of plant origin has greatly increased in recent years (Jayaprakash and Rao, 2000).
Free radicals arising from metabolism or environmental sources interact continuously in biological systems and their uncontrolled generation correlates directly with molecular level of many diseases (Huang et al., 2005).Lots of research has clearly showed that free radicals would damage nearby structures including DNA, proteins or lipids.
Radical scavenging antioxidants are particularly important in antioxidative-defence in protecting cells from the injury of free-radical (Youwei et al., 2008).
Plants are the good source of biologically active compounds known as phytochemicals.The phytochemicals have been found to act as antioxidants by scavenging free radicals and may have therapeutic potential for free radical associated disorders (Hausladen and Stamer, 1999).It is well known that free radicals are the major cause of various chronic and degenerative diseases, such as coronary heart disease, inflammation, stroke, diabetes mellitus and cancer (Scalbert et al., 2005).Therefore, it is important to assess antioxidant activity of the plants used in the herbal medicine either to elucidate the mechanism of their pharmacological action or to provide information on antioxidant activity of these herbal plants (Abdul et al., 2012).
Hypochaeris radicata L. of Asteraceae family an one such plant species distributed in high hills of Nilgiris, the Western Ghats, India at temperate climate is expected to have antioxidant property as it is being used by the local communities for adding freshness and activeness (Paulsamy et al., 2008).Therefore to have the scientific validation on antioxidant properties, the leaves and roots of this species were taken for the present study.The methanolic extracts of both parts of H. radicata were used to investigate the antioxidant activity in terms of free radical scavenging activity (DPPH • ), reducing power activity, ABTS •+ assay and ferrous ion chelating activity.

Collection and identification of plant material
The plant material was collected from Kattabettu, (2100ms above msl), the Nilgiris, Western Ghats, India.The plant was authenticated by Dr. P. Sathyanarayana, Botanical Survey of India, TNAU Campus, Coimbatore.The voucher number is BSI/SRC/5/23/2010-11/Tech/153.

Preparation of plant extracts
Fresh plant material was washed under running tap water, air dried and powdered.About 50g of coarsely powdered plant materials (50g/250ml) were extracted in a soxhlet extractor for 8 to 10 hours, sequentially with petroleum ether, chloroform, ethyl acetate, methanol and water.The extracts obtained were then concentrated and finally dried to a constant weight.Dried extracts were kept at 20 0 C until further test were carried out.For stock solutions, 10mg/ml of methanolic leaf and root extracts were dissolved in dimethyl sulffoxide (DMSO).

Determination of total phenolics content
The total soluble phenols present in the methanolic leaf and root extracts were determined by using the Folin-Ciocalteu reagent according to the procedure reported by Singletion et al., (1999).About 500µl (20mg/ml) of plant sample was added to 25ml of distilled water and 1ml of Folin-Ciocalteu reagent (1:10).Then this mixture was kept at room temperature for 3 minutes, after then 1.5ml of 2% sodium bicarbonate was added, soon after vortexting the reaction mixture for 1 hour at room temperature, the absorbance was measured at 760nm.All the tests were performed in triplicates and the results were averaged.The concentration of total phenolic compounds in methanolic leaf and root extracts was determined as microgram of gallic acid equivalent by using an equation that was obtained from the standard gallic acid graph (10-300 µg/ml).

Determination of total flavonoids content
The aluminium chloride colorimetric assay was used for total flavonoids determination, as described by Zhishen et al. (1999).100µl (20mg/ml) of the extract was mixed with 2.5 ml of distilled water and 300µl of 5% sodium nitrate.Then, it was incubated at room temperature for 5 minutes and 300µl of 10% aluminium chloride , 2ml of 1M sodium hydroxide and 1ml of distilled water were added.Then, absorbance of the reaction mixture was measured at 512nm, along with the standard, quercetin and blank.The total flavonoids content was determined as microgram, quercetin equivalent by using the standard, quercetin graph, obtained by comparing the calibration curve prepared from a reference solution containing quercetin (10-300µg/ml).

In vitro antioxidant activity
The free radical scavenging activity of the methanolic leaf and root extracts of the study species, H. radicata was determined by using various in vitro assays such as DPPH • assay, reducing power assay and ABTS •+ assay and ferrous ion chelating activity.

Free radical scavenging activity (DPPH • )
The free radical scavenging activity of methanolic extract of H. radicata was measured by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH • ) method of Blois (1958).0.2mM solution of DPPH • in methanol was prepared and 100μl of this solution was added to various concentrations of methanolic leaf and root extracts at the concentrations of 50, 100, 150, 200 and 250μg/ml.After 30 minutes, absorbance was measured at 517nm.Butylated hydroxytoluene (BHT) was used as the reference material.All the tests were performed in triplicate and percentage of inhibition was calculated by comparing the absorbance values of the control and test samples.

Percentage of inhibition = Abs cntrl -Abs test x 100 Abs cntrl
The scavenging reaction between DPPH • and an antioxidant, H-A can be written as

Reducing power activity
Reducing power assay was determined according to the method of Yildirim et al., (2001).Different concentrations of methanolic extracts of leaf (200, 300, 400, 500 and 600μg/ml) and root (300, 400, 500, 600 and 700μg/ml) of the study species were mixed with 1ml of 200mM sodium phosphate buffer (pH 6.6) and 1ml of 1% potassium ferriccyanide followed by incubation at 50 0 C for 20 minutes.After adding 1ml of 10% trichloro acetic acid, the mixture was centrifuged at 3000 rpm for 10 minutes.The supernatant was taken out and mixed with 2ml of distilled water and 0.5ml of 1% ferric chloride.After incubation for 10 minutes, the absorbance was measured at 700nm.Higher absorbance of the reaction mixture indicates reductive potential of the extracts (Yang et al., 2002, Rajeshwar et al., 2005, Koksal et al., 2011).All the tests were performed in triplicates and ascorbic acid was used as reference standard.

Free radical scavenging activity (ABTS •+ )
The total antioxidant activity of the samples was measured by [2, 2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] ABTS •+ radical cation decolorization assay according to the method of Re et al. (1999).ABTS •+ was produced by reacting 7mM ABTS + aqueous solution with 2.4mM potassium persulfate in the dark for 12-16 hours at room temperature.The radical was 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 ethanol (about 1:89 v/v) and equilibrated at 30 0 C to give an absorbance of 0.7000±0.02at 734 nm.Then, 2ml of diluted ABTS •+ solution was added to the sample concentration at 20μl (1mg/ml).After 30 minutes of incubation at room temperature, the absorbance was recorded at 734nm and percentage of inhibition was calculated.Trolox was used as a reference standard.Triplicates were performed.

Ferrous ion chelating activity
The chelating of ferrous ions by leaf and root methanolic extracts of H. radicata was estimated by the method of Singh and Rajini (2004).The different concentrations of methanolic extracts (1000, 2000, 3000, 4000 and 5000μg/ml separately for leaf and root) were mixed with 100μl of 2mM ferrous sulphate solution and 300μl of 5mM ferrozine.The mixture was incubated at room temperature for 10 minutes.The absorbance of the solution was measured at 562nm.Ethylene diammine tetra acetate (EDTA) was used as standard.All the tests were performed in triplicate and percentage of inhibition was calculated by using this formula, Percentage of inhibition = Abs cntrl -Abs test X 100 Abs cntrl

Statistical analysis
The statistical comparison among the groups were performed with one way analysis of variance (ANOVA) test using a statistical package program SPSS 10 and the significance of the difference between means was determined by Duncan's Multiple Range Test (DMRT) at (p<0.05) significant level.(Statsoft Inc, Tulsa, USA).

RESULTS AND DISCUSSION
Free radical is a molecule with an unpaired electron and is involved in bacterial and parasitic infections, lung damage, inflammation, reperfusion injury, cardiovascular disorders, atherosclerosis, aging and neoplastic diseases (Roy et al., 1994).They are also involved in autoimmune disorder like rheumatoid arthritis etc. (Rao et al., 2004).Our results demonstrated that the methanolic extracts of leaf and root of H. radicata possess free radical scavenging activity in vitro models like DPPH • , ABTS •+ , reducing power activity and ferrous ion chelating assays.

Total phenolics content
Phenolic compounds are known as powerful chain breaking antioxidant (Shahidi and Wansundeara, 1992) and they are very important plant consistuents because of their scavenging ability, which is due to their hydroxyl groups (Hatano et al., 1989).In methanolic leaf and root extracts of H. radicata, the total phenolic content was found to be 125.5µg/10mgand 133.06µg/10mg respectively in terms of gallic acid equivalent (Table 1).In addition it has been determined that the highest extraction yield was found in root extract.

Total flavonoids content
Flavonoids are a group of polyphenolic compounds, which exhibit several biological effects such as anti-inflammatory, anti-hepatotoxic, anti-ulcer, anti-allergic, anti-viral and anti-cancer activities (Umamaheswari et al., 2008).They are capable of effectively scavenging the reactive O 2 species because of their phenolic hydroxyl groups and so they are potent antioxidants also (Cao et al., 1997).The total flavonoids content of methanolic leaf and root extracts of H. radicata was determined to be 105.76µg/2mgand 55.165µg/2mg respectively inturms of quercetin equivalent (Table 1).

Free radical scavenging activity (DPPH • )
DPPH • is one of the free radicals widely used for testing preliminary radical scavenging activity of the plant extract (Bhuiyan et al., 2009).Scavenging of DPPH • radical is related to the inhibition of lipid peroxidation (Rekka and Kourounakis 1991).DPPH • is usually used as a substance to evaluate the antioxidant activity (Tara Chand et al., 2012).Antioxidants either transfer an electron or a hydrogen atom to DPPH • , thus neutralizing its free radical character (Pan et al., 2008).DPPH • test, which is based on the ability of DPPH • , a stable free radical, to decolorize in the presence of antioxidants, is a direct and reliable method for determining radical scavenging action (Raquibul Hasan et al., 2009).The DPPH • assay has been largely used as a quick, reliable and reproducible parameter to search the in vitro general antioxidant acitivity of pure compounds as well as plant extracts (Koleva et al., 2002).The reducing capacity of compounds could serve as indicator of potential antioxidant property (Meir et al., 1995).In the present study, the percentage of scavenging effect on the DPPH • radical was concomitantly increased with the increase in the concentration of both leaf and root methanolic extracts from 50 to 250 µg/ml.The percentage of inhibition was existing from 54.91 at 50µg/ml to 98.36 at 300 µg/ml for leaf extract and for root extracts, they were 23.27 at 50µg/ml and 96.72 at 250 µg/ml (Table 2).From the results it is known that the species, H. radicata possess hydrogen donating capabilities for methanolic leaf extract and does scavenging free radicals.Furthermore, it was noticed that the leaf extract has more pronounced scavenging activity than that of the standard, BHT (Table 2).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).

Reducing power activity
Reducing power activity is often used to evaluate the ability of natural antioxidant to donate electron (Yildirim et al., 2000, Dorman et al., 2003).Many reports have revealed that there is a direct correlation between antioxidant activities and reducing power of certain plant extracts (Duh, 1998;Duh et al., 1999;Yildirim et al., 2000).The reducing power activity of methanolic leaf and root extracts of H. radicata increased consistently with the increase in the volume of extract from 200µg to 600µg for leaf and 300µg to 700µg for root.When compared with the root extract (0.433 at 700µg/ml), leaf extract showed higher absorbance (1.092 at 600µg/ml).It is known further that the reducing power activity of leaf extract was far higher than the standard, ascorbic acid (Table 3).

Free radical scavenging activity (ABTS •+ )
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 incubating ABTS •+ chromophore through the reaction (Wolfenden et al., 1982).The presence of specific chemical compounds in the extracts of H. radicata may inhibit the potassium persulfate activity and hence reduced the production of ABTS •+ .This study reports that the methanolic leaf extract of H. radicata has highest antioxidant activity (2706.73μmol/g) than that of its root counter part (2028.37μmol/g)(Table 4).

Ferrous ion chelating activity
Iron is essential for life because it is required for oxygen transport, respiration and activity of many enzymes.However, it is an extremely reactive metal and catalyzes oxidative changes in lipids, proteins and other cellular components (Smith et al., 1992).
The metal chelating ability of the methanolic leaf and root extracts was measured by the formation of ferrous ionferrozine complex.Ferrozine combines with ferrous ions forming a red coloured complex which absorbs at 562n m (Yamaguchi et al., 2000).It was reported that the chelating agents which forms σ bond with a metal, are effective as secondary antioxidants, because they reduce the redox potential there by stabilizing the oxidized form of the metal ion (Duh et al., 1999).Iron binding capacity in terms of percent inhibition of the methanolic extract of H. radicata at 5000µg/ml was higher for root (40.52%) than the leaf (38.96%) (Table 5).However, it was not comparable to that of the reference standard, EDTA.

CONCLUSION
Searching plant sources may bring new natural products into pharmaceutical, cosmetic and food production.An in vitro antioxidant study provides scientific evidence to prove the traditional claims to the Asteraceae member, H. radicata.On the basis of the results obtained in the present study, it was concluded that the methanolic leaf and root extracts of this species possess significant antioxidant activity.
Presence of adequate amount of phenol and flavonoid compounds may account for this fact.So these findings of present study suggest that this plant have a potential source of natural antioxidant.Further studies are warranted for the isolation and characterization of antioxidant compounds, and also in vivo studies are needed for understanding their mechanism of action as antioxidants.

Table . 1
: Extraction yield, total phenolics and flavonoids content of methanolic leaf and root extracts of Hypochaeris radicata.

Table . 3
: Reducing power activity of methanolic leaf and root extracts of Hypochaeris radicata.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).

Table . 4
: ABTS activity of methanolic leaf and root extracts of Hypochaeris radicata.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).Ferrous ion chelating activity of methanolic leaf and root extracts of Hypochaeris radicata.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).