Food Chemistry 115 (2009) 631634

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Superoxide and hydroxyl radical scavenging actions of botanical extracts

of Wagatea spicata
Geetha Samak a,*, Revathi P. Shenoy b, S.M. Manjunatha b, K.S. Vinayak c
a

DVS College of Arts and Science, Sir M.V. Road, Shimoga, Karnataka 577201, India
Department of Biochemistry, Kasturba Medical College, MAHE, Manipal, Karnataka 576119, India
c
Department of Applied Botany, Kuvempu University, Shankaraghatta, Karnataka 577451, India
b

a r t i c l e

i n f o

Article history:
Received 26 September 2008
Received in revised form 30 October 2008
Accepted 19 December 2008

Keywords:
Wagatea spicata
Free radical
Superoxide
Hydroxyl
Phenolics
Flavonoid

a b s t r a c t
Wagatea spicata, candy corn plant, a woody legume shrub, widespread medicinal plant found in Western
Ghat of India has signicant abilities to scavenge highly reactive free radicals. Shade dried leaf, bark and
ower powder of this plant has been extracted with water and fractionated with different solvents.
Extracts and their solvent fractions were found to be good scavengers of superoxide and hydroxyl radicals. Free radical scavenging action of W. spicata is due to its rich phenolic and avonoid contents. Bark
and leaf extracts showed signicant scavenging action against superoxide radicals, where as ower
extracts efciently inhibited hydroxyl radicals.
2008 Elsevier Ltd. All rights reserved.

1. Introduction
Reactive Oxygen Species (ROS) have been implicated in more
than 100 diseases from Malaria to Haemorrhagic shock to AIDS
(Alho & Leinonen, 1999). Atmospheric pollutants and radiations
have become the main sources of free radicals. Oxidative stress
causes various forms of tissues damage and inammation, and
plays an important role in the development of several degenerative
changes in cells and tissues, which ultimately lead to several
degenerative disorders. Bodily defenses are not completely efcient in preventing on going oxidative damage to DNA, lipids and
proteins. Dietary antioxidants, vitamins, avonoids, plant phenolics, herbal formulations and Ayurvedic preparations are very
essential in protecting against oxidative stress (Weiss & Landauer,
2000). Antioxidant supplementation constitutes important defence
against variety of diseases and environmental stresses.
Wagatea spicata Wt. Syn. Moullva spicata (Dalz) (Family: Fabaceae) is commonly known as the candy corn plant, a small prickly,
woody ornamental plant with long aming spikes. This legume
shrub is one of the most widespread medicinal plants in the Western Ghat forests of India. The whole plant possesses medicinal
properties, and is useful in the treatment of various ailments such
as fever, cough, gastrointestinal disorders, skin ailments etc., as re-

* Corresponding author. Tel.: +91 8182 278455/265566.

E-mail address: [email protected] (G. Samak).
0308-8146/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.12.078

ported by local people as they are using regularly roots, leaves and
bark of the plant. Bark is used as an application for skin diseases.
Root extract of W. spicata has a high phagocytic co-efcient and
was found to be effective against skin infections (Behl & Tripathi,
1975).
In the present study, the free radical scavenging efcacy of plant
material was evaluated with respect to superoxide and hydroxyl
radicals. The aqueous extract of leaf, bark and ower of W. spicata
were subjected to different solvent fractionation. Crude aqueous
extracts were analysed for phenolic and avonoid contents.

2. Materials and methods

2.1. Plant material
W. spicata plant materials, leaves, bark and owers were collected soon after monsoon season from Western Ghats region of
India.
2.2. Chemicals
Thiobarbituric acid (TBA) from Sigma Chemical Company (St.
Louis, MO). Phenazine methosulphate, deoxyribose, reduced nicotinamide adenine dinucleotide (NADH), gallic acid, quercitin and
other chemicals were of analytical grade purchased from SRL Research chemicals, India.

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G. Samak et al. / Food Chemistry 115 (2009) 631634

2.3. Preparation of aqueous extract

Leaves, bark and owers of W. spicata were collected, dried in
shade and powdered. The powder was used for extraction. 100 g
of plant material powder was reuxed with 750 ml of double distilled water (DDW) for 1 h at 7580 C, cooled and ltered. This
was repeated in three trials; extracts were pooled and evaporated
using a Lyophiliser (Geetha, Kedlaya, & Vasudevan, 2003). The extract yield was 16% in leaf, 30% in bark and 35% in ower.

mixed with FolinCiocalteaus reagent (5 ml, 1:10 diluted with distilled water) followed by addition of aqueous Na2CO3 (4 ml, 1 M).
After incubating the reaction mixture at RT for 15 min and total
phenols were determined colourimetrically at 765 nm. The standard curve of gallic acid monohydrate was prepared using 0, 50,
100, 150, 200, 250 mg L 1. The total phenolic content of the extracts was expressed in terms of mg gallic acid equivalent (GAE)/
g dry weight of the plant extract.
2.8. Estimation of the total avonoid contents in the Wagatea extracts

2.4. Fractionation of extract

2.5. Superoxide scavenging activity

Superoxide scavenging activities of the compounds were determined by monitoring the competition of those with NBT for the
superoxide anion generated by the PMSNADH system (Liu, Ooi,
& Chang, 1997). Superoxide radicals were generated in 1 ml
20 mM TrisHCl buffer pH 8.0 containing 0.05 mM nitroblue tetrazolium (NBT), 0.01 mM phenazine methosulphate (PMS) and test
compounds were preincubated for 2 min. The reaction was initiated by the addition of 0.078 mM NADH. Blue chromogen, formed
due to NBT reduction, was read at 560 nm. Results were expressed
as percentage of inhibition of superoxide radicals.
2.6. Hydroxyl radical scavenging activity
Hydroxyl radicals were measured by the deoxyribose method
(Halliwell, Gutteridge, & Aruoma, 1987). Hydroxyl radicals generated by ferric-ascorbateEDTAH2O2, which attacks on deoxyribose to form products called thiobarbituric acid reactive
substances (TBARS), which upon heating with TBA at low pH yield
pink chromogen. The hydroxyl scavenger, when added, competes
with deoxyribose for hydroxyl radicals and decreases TBARS formation and pink chromogen (Elizabeth & Rao, 1990). The reaction
mixture containing 3 mM deoxyribose, 0.1 mM ferric chloride,
0.1 mM EDTA, 0.1 mM ascorbic acid and 2 mM H2O2 in 20 mM
phosphate buffer pH 7.4 was added to various concentrations of
test compounds. After incubating for 30 min at 37 C, the reaction
mixture was added to 0.5 ml of 5% trichloroacetic acid and 0.5 ml
of 1% TBA to yield a nal volume 3 ml. The reaction mixture was
kept in a boiling water bath for 30 min, cooled and the absorbance
was measured at 532 nm. Scavenging action was expressed as
inhibitory concentration of test compound required to produce
50% inhibition of hydroxyl radicals i.e. IC50 values.
2.7. Estimation of the total phenolic contents in the Wagatea extracts
Phenol in alkaline medium reacts with phosphomolybdic acid
of FolinCiocalteaus reagent producing blue colour complex. The
amount of total phenolics in the extracts was determined by FolinCiocalteaus reagent (McDonald, Prenzler, Autolovich, & Robards, 2001). A dilute plant material extract 0.5 ml of
1:0.5 mg ml 1) or gallic acid (standard phenolic compound) was

An aluminium chloride colourimetric method was used for

avonoids determination (Chang, Yang, Wen, & Chern, 2002). Each
extract (0.5 ml of 1:0.5 mg ml 1) in methanol were separately
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. It remained at room temperature for 30 min; the absorbance of the
reaction mixture was measured at 415 nm with a double beam
Genesis UV Spectrophotometer. The calibration curve was prepared by preparing quercetin solutions at concentrations 12.5 to
100 g ml 1 in methanol.
3. Results and discussion
3.1. Bark and leaf extracts of W. spicata are potent scavenger of
superoxide radicals
W. spicata leaf, bark and ower extracts showed a dose dependent inhibition of superoxide radicals (Fig. 1). Crude bark extract
was found to be a better scavenger (IC50 at 15 lg) than leaf and
ower extracts (Table 2). Further methanol and water fraction of
bark extract contained the active inhibitor of superoxide (IC50 at
13 lg and 14 lg, respectively). The crude extract of leaf showed
better scavenging action than its fractions, where as water and
methanol fractions of ower showed better scavenging action
(IC50 at 30 lg and 20 lg, respectively) than the crude extract.
IC50 values of all these compounds were greater than that of ascorbic acid where IC50 was achieved at 5 lg concentration (Geetha
et al., 2003).
3.2. Flower and bark extracts of W. spicata are potent scavenger of
hydroxyl radicals
The ability of W. spicata leaf, bark and ower extracts and their
fractions to scavenge hydroxyl radicals was measured by studying
competition between deoxyribose and test compounds for hydroxyl radical generated from ferric-ascorbateEDTAH2O2 system.
Hydroxyl radicals attack deoxyribose starting a set of reactions,

% of inhibition of superoxide radical

Leaf, bark and ower aqueous extracts of W. spicata were re-extracted with organic solvents ranging from polar to non-polar in
succession. They were successively re-extracted with petroleum
ether (6080 C), diethyl ether, ethyl acetate, methanol and water
(Geetha et al., 2003; Suffness & Douros, 1979). The nal yield of
solvent free fractions of the aqueous extract of leaf, bark and ower
was measured. No fractions yielded in petrol ether and diethyl
ether solvent. Solvent fractions of ethyl acetate, methanol and
water solvents were present with respect to leaf and bark, only
methanol and water fractions in ower extract. Relative proportions of fractions are given in Table 1.

100

Leaf

Bark

Flower

75
50
25
0
5

12.5

25

50

Extract concentration g/ml

Fig. 1. Super oxide radical scavenging action of aqueous extract of leaf, bark and
ower material of W. spicata. (Values are mean SEM of three separate
experiments.)

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G. Samak et al. / Food Chemistry 115 (2009) 631634

which eventually results in TBARS formation. When a molecule

scavenges hydroxyl radicals the TBARS formation is decreased.
There is a concentration dependant inhibition of hydroxyl radical
by W. spicata extracts and their fractions (Fig. 2). The crude ower
extract was found to be a better scavenger than bark and leaf extracts (IC50 at 1.5 lg concentration) (Table 3). Among the fractions,
ethyl acetate and methanol fractions of leaf and bark extracts
inhibited TBARS formation at very low concentration with IC50 values at 0.05 lg, 0.5 lg, 0.5 lg and 1.2 lg, respectively.

Table 1
Relative proportion of solvent fractions yielded during fractionation of crude leaf,
bark and ower extracts of W. spicata expressed as percentage of total amount of
crude extract used for the fractionation (average of two trials).
Solvent fractions

Leaf

Bark

Flower

Petrol ether
Diethyl ether
Ethyl acetate
Methanol
Water
Undissolved

0
0
0.65
60.1
26.1
13.15

0
0
0.75
64.45
25.9
8.9

0
0
0
33.7
38.9
27.4

3.3. Flower extract of Wagatea contains very high amount of total

phenolics
The amount of total phenolics of the extracts was expressed in
terms of gallic acid equivalents. The amount of total phenolics varied widely in Wagatea plant materials. Flower extracts had relatively high values of phenolic content (226.73 mg gallic acid
equivalent/g of dry weight of crude extract) (Table 4). Bark contained 127.3 mg gallic acid equivalent/g of dry weight of crude
extract.

Table 2
Super oxide radical scavenging action of W. spicata aqueous extract of leaf, bark and
ower and their fractions expressed as IC50 values (concentration in micrograms (lg/
ml) needed for 50% inhibition).
Extract

Leaf

Bark

Flower

Crude extract
Ethyl acetate fraction
Methanol fraction
Water fraction

28 0.64
125 0.4
75 0.5
50 2.4

15 2.4
40 1.3
13 0.4
14 3

55 1.23

20 0.3
30 0.3

3.4. Leaf extract of Wagatea contains more avonoid content

% of inhibition of hydroxyl radical

The total amount of avonoids in the extracts was expressed in

terms of quercetin equivalents. The amount of total avonoids varied widely in Wagatea plant materials (Table. 4). The leaf extract
had relatively high amounts of avonoids (12.7 mg quercetin
equivalent/g of dry weight of crude extract. Bark contained
8.8 mg quercetin equivalent/g of dry weight of crude extract),
but the ower extract contained the lowest value of avonoid content (4.5 mg quercetin equivalent/g of dry weight of crude extract).
The present study reveals that W. spicata extracts and their fractions are potent scavenger of deleterious free radicals, such as O
and OH , at very low concentrations. Superoxide is the rst reduction product of molecular oxygen, a highly toxic radical, the most
abundantly produced in all aerobic cells by several enzymatic
and non-enzymatic pathways, attacks a number of biological molecules and leads to unfavourable alterations of biomolecules
including DNA (Waris & Alam, 2004). It also forms an important
source of other deleterious radicals such as hydroxyl and hydroperoxides, which initiate free radical chain reactions (Halliwell &
Gutteridge, 1990).
Free radical scavenging action of W. spicata can be attributed to
its rich phenolic and avonoid contents. The phenolic rich ower
extract brought signicant inhibition of hydroxyl radicals whereas
phenolic and avonoid rich bark was found to be good scavenger of
superoxide radicals. Ethyl acetate and methanol fractions of bark
and leaf extract efciently scavenged hydroxyl radicals at very
low concentrations.

100

Leaf

Bark

Flower

75
50
25
0
1.5

16

Extract concentration (g/ml)

Fig. 2. Hydroxyl radical scavenging action of aqueous extract of leaf, bark and
ower material of W. spicata. (Values are mean SEM of three separate
experiments.)

Table 3
Hydroxyl radical scavenging action of W. spicata aqueous extract of leaf, bark and
ower and their fractions expressed as IC50 values (concentration in micrograms (lg/
ml) needed for 50% inhibition).
Extract

Leaf

Bark

Flower

Crude extract
Ethyl acetate fraction
Methanol fraction
Water fraction

8.7 0.06
0.05 0.4
0.5 0.4
8.33 0.7

4.6 0.6
0.5 0.1
1.2 0.5
16.67 0.03

1.5 0.8

20 0.007
16.67 0.03

Table 4
Total phenolic and avonoid content of leaf, bark and ower extracts of W. spicata
(values are mean SEM of three separate experiments).
Plant material

Total phenolics

Total avonoids

Leaf
Bark
Flower

47 3
127 1
227 2

13 1
91
40

The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. In addition, they have
a metal chelating potential (Rice-Evans, Miller, Bolwell, Bramley, &
Pridham, 1995). The presence of conjugated ring structures and hydroxyl groups allows phenolics to actively scavenge free radicals;
carboxylic acid groups inhibit lipid peroxidation and are also
known for their ability to inhibit prooxidant enzymes. (Decker,
1995).
Naturally occurring Polyphenols and avonoids have been
shown to prevent lipid peroxidation, LDL oxidation, development
of Atherosclerosis and heart disease. (Fuhrman, Rosenblat, Hayek,
Coleman, & Aviram, 2000; Geetha, Kedlaya, & Vasudevan, 2004;
Hertog, Feskens, Holiman, Katan, & Kromhout, 1993; Samak, Rao,
Kedlaya, & Vasudevan, 2007). In several studies it was concluded
that plant avonoids which show antioxidant activity in vitro also
function as antioxidants in vivo (Geetha et al., 2004; Shimoi, Masuda, Shen, Furugori, & Kinae, 1996). From the present study it is evident that W. spicata extracts and fractions are good scavengers of
reactive free radicals. Many antioxidants similar to ascorbate and
phenolic compounds, possess prooxidant properties (Narla & Rao,
1995). But W. spicata extracts and their fractions are free from such
prooxidant properties.

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G. Samak et al. / Food Chemistry 115 (2009) 631634

Crude extracts of fruits, herbs, vegetables, cereals, and other

plant materials rich in phenolics are increasingly of interest in
the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food
(Marja et al., 1999). In recent years there is a growing interest in
antioxidant supplements for the prevention of many diseases. In
this context W. spicata can be exploited for its impressive free radical scavenging activities. Ingestion of alcohol-free red wine or a
phenolic compound mixture extracted from red wine has been
shown to improve the antioxidant status of plasma in humans
(Carbonneau, Leger, Descomps, Michel, & Monnier, 1998; Serani,
Maiani, & Ferro-Luzzi, 1998). Hence phenolic rich Wagatea can be
explored in different directions to tackle several oxidative stress
induced ailments.
Acknowledgements
This work is supported by University Grant Commission of India
UGC Minor Research Project under X plan.
Prof. Sudharkar Nayak, Head of the Department of Biochemistry, KMC, MAHE, Manipal, Karnataka, India is thankfully acknowledged for supporting this work in his department.
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