LWT 39 (2006) 10591065

Antioxidant activity of the roots of Decalepis hamiltonii

(Wight & Arn.)
Anup Srivastava, Shereen R. Harish, T. Shivanandappa

Department of Food Protectants and Infestation Control, Central Food Technological Research Institute, Mysore 570020, India
Received 19 May 2005; received in revised form 8 July 2005; accepted 8 July 2005
Abstract
Tuberous roots of Decalepis hamiltonii (Dh) are consumed in southern India as pickles and beverage for their health benets. The
antioxidant potential of the roots was measured using various in vitro assays. Among the extracts, the methanolic and aqueous
extracts showed high antioxidant activity measured as scavenging of DPPH, superoxide and hydroxyl radicals. Both the aqueous
and methanolic extracts inhibited microsomal lipid peroxidation and exhibited strong reducing power and metal chelating activity.
The antioxidant activity did not correlate with the phenolic content of the extracts. These results demonstrate the antioxidant
potency of the root extracts which could be the basis for its alleged health promoting potential of Dh. The roots of Dh could serve as
a new source of natural antioxidants or nutraceuticals with potential applications to reducing the level of oxidative stress and related
health benets.
r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Decalepis hamiltonii; Lipid peroxidation; Free radical scavenging; Reducing power; Metal ion chelation; Phenolic content
1. Introduction
Free radicals have been implicated in many diseases
such as cancer, atherosclerosis, diabetes, neurodegen-
erative disorders and aging (Halliwell & Gutteridge,
1999; Yu, 1994). The value of nutraceuticals in food has
long been recognized for their health benets (Klein,
Sato, Meguid, & Miyata, 2000). Green tea (Cao, Soc,
& Prior, 1996; Chen & Ho, 1995), red wine (Kinsella,
Frankel, German, & Kanner, 1993) and ginseng (Fu &
Ji, 2003; Kitts, Wiejewickreme, & Hu, 2000) are known
to have benecial effects on the prevention or progres-
sion of diseases related to oxidative stress on account of
their high antioxidant activity. It is believed that higher
intake of antioxidant rich food is associated with
decreased risk of degenerative diseases particularly
cardiovascular diseases and cancer (Ames, Shigenaga,
& Hagen, 1993; Joseph et al., 1999). Vegetables contain
several antioxidant nutrients in addition to vitamin C, E
and carotenoids which contribute to their total anti-
oxidant capacity (Wang, Cao, & Prior, 1997). Several
studies have shown that plant derived antioxidant
nutraceuticals scavenge free radicals and modulate
oxidative stress-related degenerative effects (Thatte,
Bagadey, & Dahanukar, 2000). Nutraceuticals are
becoming widely incorporated in functional food owing
to their therapeutic effects in enhancing the well-being.
There is a great deal of interest in newer natural
bioactive molecules with health promoting potential.
Decalepis hamiltonii (Dh) (family: Asclepediaceae)
grows wild in the forests of peninsular India. Its tubers
are consumed as pickles and as a juice for its alleged
health promoting properties. However, the roots of Dh
have not been investigated for their health promoting
potential. Earlier studies have shown that the roots
contain aldelydes, inositols, saponins, amyrins and lupeols
ARTICLE IN PRESS
www.elsevier.com/locate/lwt
0023-6438/$30.00 r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2005.07.005

Corresponding author. Tel.: +91 8212513210;

fax: +91 8212517233.
E-mail address: tshivanandappa@yahoo.com (T. Shivanandappa).
(Murti & Sheshadri, 1940, 1941a, b) as well as volatile
compounds such as 2-hydroxy-4methoxybenzaldehyde,
vanillin, 2-phenyl ethyl alcohol, benzaldehyde, and
others (Nagarajan, Rao, & Gurudutt, 2001). The roots
have also been used as a substitute for Hemidesmus
indicus in ayurvedic preparations of ancient Indian
medicine (Nayar, Shetty, Mary, & Yoganarshimhan,
1978). This paper reports the antioxidant potential of
the aqueous and methanolic extracts of the Dh root
employing various in vitro assay systems, such as
inhibition of lipid peroxidation (LPO), DPPH/super-
oxide/hydroxyl radical scavenging, reducing power and
metal chelating activity.
2. Materials and methods
2.1. Chemicals
Butylated hydroxylanisole (BHA), nitroblue tetrazo-
lium (NBT), 1,1-diphenyl-2-picrylhydrazyl (DPPH),
phenazine methosulphate (PMS), thiobarbituric acid
(TBA), bovine serum albumin (BSA) and ethylenedia-
mine tetra-acetic acid (EDTA) were purchased from M/s
Sigma Chemicals Co. (St. Louis, MO). Nicotinamide
adenine dinucleotide-reduced (NADH), trichloroacetic
acid (TCA), deoxyribose, ascorbic acid and other
chemicals were purchased from M/s Sisco Research
Laboratories, Mumbai, India. All reagents were analy-
tical grade.
2.2. Preparation of root powder and extraction
Tuberous roots (10 kg) of Dh were purchased from the
local suppliers. They were washed with water and then
crushed with a roller to separate the inner woody core
and the outer eshy layer. The eshy portion was
collected, dried at 40 1C in a hot air oven and then nely
powdered. The powder (1.9 kg) was used for extraction.
Sequential extraction of the root powder was done
with different solvents with increasing polarity i.e.
hexane, chloroform, ethyl acetate, acetone, methanol
and water. A total of 50 g of root powder was extracted
in 0.5 l of the solvent in glass conical ask on a shaker
for 24 h at room temperature. The extract was ltered
with Whatman paper no. 1 and dried by ash
evaporation/lyophilization.
The methanolic extract was prepared by soxhlet
extraction in methanol. A 100 g of root powder was
extracted (1 l) at 50 1C for 24 h. Methanol was ash
evaporated and the extract weighed (28 g). The aqueous
extract was prepared by homogenizing the root powder
(100 g) in 1 l of warm water (50 1C) and allowed to stand
for 24 h, ltered with Whatman paper no. 1 and the
ltrate was lyophilized and weighed (22 g).
2.3. DPPH radical scavenging assay
DPPH radical scavenging activity was done according
to Yamaguchi, Takamura, Matoba, and Terao (1998).
Briey, 1 ml of DPPH solution (0.1 mmol/l, in 95%
ethanol (v/v)) was incubated with different concentra-
tions of the extract. The reaction mixture was shaken
and incubated for 20 min at room temperature and the
absorbance was read at 517 nm against a blank. The
radical scavenging activity was measured as a decrease
in the absorbance of DPPH and calculated using the
following equation:
Scavenging effect %
1 A
Sample517 nm
=A
Control 517 nm
100.
2.4. Superoxide radical scavenging assay
The superoxide radical scavenging ability of the
extracts was measured by the method of Nishikimi,
Rao, and Yagi (1972). The reaction mixture contained
different concentrations of the extract, PMS (0.1 mmol/
l), NADH (1 mmol/l) and NBT (1 mmol/l) in phosphate
buffer (0.1 mol/l, pH 7.4), was incubated at room
temperature for 5 min and the color was read at
560 nm against a blank. The scavenging effect was
calculated using the equation described as in the case of
DPPH.
2.5. Hydroxyl radical scavenging assay
The reaction containing different concentrations of
the extract was incubated with deoxyribose (10 mmol/l),
H
2
O
2
(10 mmol/l), FeCl
3
(5 mmol/l), EDTA (1 mmol/l)
and ascorbic acid (5 mmol/l) in potassium phosphate
buffer (50 mmol/l, pH 7.4) for 60 min at 37 1C (Halliwell,
Gutteridge, & Cross, 1987). The reaction was terminated
by adding TCA (5 g/100 ml water) followed by the
addition of TBA (0.2 g/100 ml water) and boiled in water
bath for 15 min. The absorbance of the color was
measured at 535 nm against the reagent blank and the
inhibition of the oxidation of deoxyribose was calcu-
lated with respect to the control.
2.6. Inhibition of microsomal lipid peroxidation
Liver excised from adult male Wistar rats, was
homogenized (20 g/100 ml Tris buffer) in 0.02 mol/l, tris
buffer (pH 7.4). Microsomes were isolated by the
calcium aggregation method (Kamath & Rubin, 1972).
100 ml of liver microsomal suspension (0.5 mg protein)
was incubated with 1 mmol/l each of FeSO
4
and
ascorbic acid with or without extract in a total volume
of 1 ml in 0.1 mol/l phosphate buffer (pH 7.4). After
incubation at 37 1C for 60 min, the reaction mixture was
boiled with TBA (0.67 g/100 ml water) for 15 min.
ARTICLE IN PRESS
A. Srivastava et al. / LWT 39 (2006) 10591065 1060
Formation of TBA reactive substances (TBARS) was
calculated from the absorbance at 535 nm (Buege &
Aust, 1978). BHA was used as the positive control.
2.7. Measurement of reducing power
The reducing power of the extracts was measured by
incubating the reaction mixture (1 ml) containing the
extract in phosphate buffer (0.2 mol/l, pH 6.6) with
potassium ferricyanide (1 g/100 ml water) at 50 1C for
20 min. The reaction was terminated by adding TCA
solution (10 g/100 ml water), centrifuged at 3000 rpm for
10 min and the supernatant was mixed with ferric
chloride (0.1 g/100 ml water), the absorbance measured
at 700 nm (Yen & Chen, 1995). Increased absorbance of
the reaction mixture indicated increased reducing
power.
2.8. Metal ion chelating assay
The Fe
2+
-chelating ability of the extract was mea-
sured by the ferrous iron-ferrozine complex at 562 nm
(Decker & Welch, 1990). The reaction mixture contain-
ing FeCl
2
(2 mmol/l) and ferrozine (5 mmol/l) along with
extracts was adjusted to a total volume of 0.8 ml with
methanol, mixed and incubated for 10 min at room
temperature. The absorbance of the mixture was read at
562 nm against a blank. EDTA was used as positive
control. The ability of the extract to chelate ferrous ion
was calculated using the equation described for DPPH.
2.9. Total phenolic content
Total phenolic content was estimated by FolinCio-
calteau method (Singleton & Rossi, 1965). To 6.0 ml
double distilled water, a 0.1 ml sample and 0.5 ml
FolinCiocalteau reagent was mixed followed by the
addition of 1.5 ml Na
2
CO
3
(20 g/100 ml water) and the
volume was made up to 10.0 ml with distilled water.
After incubation for 30 min at 25 1C, the absorbance was
measured at 760 nm and the phenolic content was
calculated with a guaicol standard and expressed as
guaicol equivalents.
Microsomal protein estimation was done by the
method of Lowry, Rosenbrough, Farr, and Randall
(1951) using BSA as the standard.
2.10. Statistical analysis
Data were expressed as mean7S.E. of three separate
experiments and subjected to the analysis of variance
(Po0:05) using the computer programme Excel and
Statistica software (1999).
3. Results and discussion
3.1. Antioxidant activity of the extracts
Antioxidant activity of the sequential extracts are
presented in Table 1. Among the extracts, maximum
antioxidant activity was shown by the methanolic and
aqueous extracts and therefore, they were chosen for
further study. The results indicate the choice of the
solvent for obtaining the extract with high antioxidant
activity.
3.2. DPPH radical scavenging activity
A high radical scavenging activity was observed in
both the aqueous and methanolic extracts in a
concentration dependent manner. The aqueous extract
was slightly more active than the methanolic extract
(IC
50
0.29 and 0.36 mg/ml, respectively) (Table 2 and
Fig. 1). Proton-radical scavenging action is an impor-
tant attribute of antioxidants, which is measured by
DPPH radical scavenging assay. DPPH, a protonated
radical, has characteristic absorbance maxima at 517 nm
which decreases with the scavenging of the proton
radical (Yamaguchi et al., 1998). Hydrogen-donating
ability of the antioxidant molecule contributes to its free
radical scavenging nature (Chen & Ho, 1995).
3.3. Scavenging of superoxide radical
Superoxide radical scavenging activity was shown
by both aqueous and methanolic extracts and was
ARTICLE IN PRESS
Table 1
Antioxidant activity
a
of the sequential extracts of D. hamiltonii
Solvent Extract yield (g) DPPH radical scavenging (%) Superoxide radical scavenging (%) LPO (% inhibition)
Hexane 3.78 3.670.4 0 43.372.8
Chloroform 4.76 36.0672.6 0 64.773.6
Ethyl acetate 1 47.5173.1 13.772.1 71.474.2
Acetone 1.19 55.4773.6 27.273.4 76.575.1
Methanol 12.95 69.7774.2 49.974.6 68.873.9
Water 8.36 73.5474.7 86.274.8 6073.3
a
Antioxidant activity was assayed at a concentration of 1 mg/ml for all the extracts.
A. Srivastava et al. / LWT 39 (2006) 10591065 1061
concentration dependent, with an IC
50
value of 0.53 and
2.12 mg/ml, respectively (Table 2 and Fig. 2). Aqueous
extract was markedly a more potent scavenger of
superoxide anion than the methanolic extract. Super-
oxide radicals are generated during the normal physio-
logical process mainly in mitochondria. Although
superoxide anion is by itself a weak oxidant, it gives
rise to the powerful and dangerous hydroxyl radicals as
well as singlet oxygen both of which contribute to the
oxidative stress (Dahl & Richardson, 1978; Meyer &
Isaksen, 1995). Therefore superoxide radical scavenging
by antioxidants has physiological implications.
3.4. Hydroxyl radical scavenging activity
Both aqueous and methanolic extracts of D. hamilto-
nii displayed hydroxyl radical scavenging activity. IC
50
values for the aqueous and methanolic extract were 1.84
and 3.95 mg/ml, respectively (Table 2 and Fig. 3). The
hydroxyl radical is an extremely reactive free radical
formed in biological systems and has been implicated as
a highly damaging species in free radical pathology,
capable of damaging biomolecules of the living cells
(Gordon, 1990; Halliwell, 1991; Hochestein & Atallah,
1988). Hydroxyl radical has the capacity to cause DNA
strand breakage, which contributes to carcinogenesis,
mutagenesis and cytotoxicity (Aruoma, Halliwell, &
Dizdaroglu, 1989). In addition, this radical species is
considered as one of the quick initiators of the LPO
process, abstracting hydrogen atoms from unsaturated
fatty acids (Kappus, 1991).
3.5. Inhibition of microsomal lipid peroxidation
As shown in Fig. 4, both aqueous and methanolic
extracts were equipotent in inhibiting LPO of liver
microsomes. IC
50
values for the aqueous and methanolic
extract were 0.51 and 0.46 mg/ml, respectively (Table 2
and Fig. 4). LPO has been broadly dened as the
oxidative deterioration of polyunsaturated lipids
(Kappus, 1991). Initiation of a peroxidation sequence
in a membrane or polyunsaturated fatty acid is due to
abstraction of a hydrogen atom from the double bond in
the fatty acid. The free radical tends to stabilize by a
molecular rearrangement to produce a conjugated diene,
which then readily reacts with oxygen molecule to give a
peroxy radical (Jadhav, Nimbalkar, Kulkarni & Mad-
havi, 1996). Peroxy radicals can abstract a hydrogen
ARTICLE IN PRESS
Table 2
Antioxidant activity of the extracts of D. hamiltonii roots
Extracts IC
50
(mg/ml)
DPPH Superoxide Hydroxyl
radical
Microsomal
LPO
Metal
chelation
Aqueous 0.29 0.53 1.84 0.51 5.49
Methanolic 0.36 2.12 3.95 0.46 32.49
100
75
D
P
P
H

r
a
d
i
c
a
l

s
c
a
v
e
n
g
i
n
g

(
%
)
50
25
0
0 0.4
Concentration of extract (mg/ml)
0.8 1.2
Fig. 1. DPPH scavenging effect of D. hamiltonii root extracts.
(Control absorbance 0.74) KAqueous extract, Methanolic
extract.
100
75
S
u
p
e
r
o
x
i
d
e

r
a
d
i
c
a
l

s
c
a
v
e
n
g
i
n
g

(
%
)
50
25
0
0 1.4 0.7
Concentration of extract (mg/ml)
2.8 3.5 2.1
Fig. 2. Superoxide radical scavenging by D. hamiltonii root extracts.
(Control, DA
340 nm
/min 0.1022) K Aqueous extract, Metha-
nolic extract.
100
75
H
y
d
r
o
x
y
l

r
a
d
i
c
a
l

s
c
a
v
e
n
g
i
n
g

(
%
)
50
25
0
0 6 3
Concentration of extract (mg/ml)
9
Fig. 3. Hydroxyl radical scavenging by D. hamiltonii extracts. (Control
absorbance 0.67) K Aqueous extract, Methanolic extract.
A. Srivastava et al. / LWT 39 (2006) 10591065 1062
atom from another molecule to give lipid hydroper-
oxide, R-OOH. A probable alternative fate of peroxy
radicals is to form cyclic peroxides; these cyclic
peroxides, lipid peroxides and cyclic endoperoxides
fragment to aldehydes such as malondialdehyde
(MDA) and polymerization products. MDA and 4-
hydroxy nonenal are the major break down products of
LPO. MDA is usually taken as a marker of LPO and
oxidative stress (Janero, 1990).
3.6. Reducing power
The reducing power of D. hamiltonii extracts was
concentration-dependent (Table 2 and Fig. 5). Metha-
nolic extract was being slightly more active than the
aqueous extract. It is believed that antioxidant activity
and reducing power are related (Duh, 1998; Duh, Tu, &
Yen, 1999; Tanaka, Kuie, Nagashima, & Taguchi,
1988). Reductones inhibit LPO by donating a hydrogen
atom and thereby terminating the free radical chain
reaction (Yen & Chen, 1995).
3.7. Metal ion chelating activity
The ferrous ion-chelating effect was shown by both of
aqueous and methanolic extracts of Dh with IC
50
values
of 5.49 and 32.49 mg/ml, respectively (Table 2 and
Fig. 6). Iron is known to generate free radicals through
the Fenton and HaberWeiss reaction (Halliwell &
Gutteridge, 1990). Metal ion chelating activity of an
antioxidant molecule prevents oxyradical generation
and the consequent oxidative damage. Metal ion
chelating capacity plays a signicant role in antioxidant
mechanism since it reduces the concentration of the
catalysing transition metal in LPO (Duh et al., 1999). It
is reported that chelating agents, which form s-bonds
with a metal, are effective as secondary antioxidants
since they reduce the redox potential thereby stabilizing
the oxidized form of the metal ion (Gordon, 1990).
3.8. Total phenolic content
Phenolics content in the methanolic extract of Dh was
higher than that of the aqueous extract (21.473.7 and
13.872.4 mg guaicol equivalents/g, respectively). Anti-
oxidant activity of the plant extract is often associated
with the phenolic compounds present in them. Hydro-
gen donating property of the polyphenolic compounds is
responsible for the inhibition of free radical induced
LPO (Yen, Duh, & Tsai, 1993). In our study, there
seemed to be little correlation between the phenolic
content and antioxidant activity of the extracts since
aqueous extract with lower phenolic content showed
higher antioxidant activity. However, it is known that
nonphenolic antioxidants could also contribute to the
antioxidant activity of an extract (Harish & Shivanan-
dappa, 2005; Mariko, Hassimotto, Genovese, & Lajolo,
2005).
In order to characterize antioxidant activity of a plant
extract, it is desirable to subject it to a battery of tests
ARTICLE IN PRESS
100
75
L
P
O

i
n
h
i
b
i
t
i
o
n

(
%
)
50
25
0
0 0.8 0.6 0.4 0.2
Concentration of extract (mg/ml)
1
Fig. 4. Inhibition of liver microsomal lipid peroxidation by D.
hamiltonii extracts. (Control value 6.5 n moles MDA/mg protein)
K Aqueous extract, Methanolic extract.
0.6
0.4
R
e
d
u
c
i
n
g

p
o
w
e
r
0.2
0
0 4 3 2 1
Concentration of extract (mg/ml)
5
Fig. 5. Reducing power of D. hamiltonii extracts. K Aqueous
extract, Methanolic extract.
100
80
60
M
e
t
a
l

c
h
e
l
a
t
i
o
n

(
%
)
40
20
0
0 60 45 30 15
Concentration of extract (mg/ml)
75
Fig. 6. Metal ion chelating effect of D. hamiltonii extracts. (Control
absorbance 0.70) K Aqueous extract, Methanolic extract.
A. Srivastava et al. / LWT 39 (2006) 10591065 1063
that evaluates the range of activities such as scavenging
of the reactive oxygen species, inhibition of membrane
LPO and metal ion chelation. Antioxidant-rich plant
extracts serve as sources of nutraceuticals that alleviate
the oxidative stress and therefore prevent or slow down
the degenerative diseases (Ames et al., 1993; Cao et al.,
1996; Fu & Ji, 2003; Joseph et al., 1999; Kitts et al.,
2000). This study is the rst to report the antioxidant
activity of the roots of Dh which may be associated with
their alleged health benets. The broad range of
antioxidant activity of the extracts indicates the
potential of the roots as a source of natural antioxidants
or nutraceuticals with potential application to reduce
oxidative stress with consequent health benets. Our
efforts are underway to isolate and identify the
antioxidant molecules in the roots of Dh and study
their health promoting potential and mammalian safety.
Acknowledgements
The authors wish to thank the Director of the institute
for his keen interest in this study. The rst three authors
acknowledge the Council for Scientic and Industrial
Research, New Delhi for awarding the research fellow-
ships.
References
Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1993). Oxidants,
antioxidants and the degenerative diseases of aging. Proceedings of
the National Academy of Sciences of the United States of America,
90, 79157922.
Aruoma, O. I., Halliwell, B., & Dizdaroglu, M. (1989). Iron ion-
dependent modication of bases in DNA by the superoxide radical-
generating system hypoxanthine/xanthine oxidase. Journal of
Biological Chemistry, 264, 1302413028.
Buege, J. A., & Aust, S. T. (1978). Microsomal lipid peroxidation.
Methods in Enzymology, 52, 302310.
Cao, G. H., Soc, E., & Prior, R. L. (1996). Antioxidant capacity of
tea and common vegetables. Journal of Agricultural and Food
Chemistry, 44, 34263431.
Chen, C. W., & Ho, C. T. (1995). Antioxidant properties of
polyphenols extracted from green tea and black tea. Journal of
Food Lipids, 2, 3546.
Dahl, M. K., & Richardson, T. (1978). Photogeneration of superoxide
anion in serum of bovine milk and in model systems containing
riboavin and amino acids. Journal of Dairy Science, 61, 400407.
Decker, E. A., & Welch, B. (1990). Role of ferritin as a lipid oxidation
catalyst in muscle food. Journal of Agricultural and Food
Chemistry, 38, 674677.
Duh, P. D. (1998). Antioxidant activity of burdock (Arctium lappa
Linne): its scavenging effect on free radical and active oxygen.
Journal of the American Oil Chemists Society, 75, 455465.
Duh, P. D., Tu, Y. Y., & Yen, G. C. (1999). Antioxidant activity of
water extract of Harng Jyur (Chrysanthemum morifolium Ramat).
Lebensmittel Wissenschaft und Technologie, 32, 269277.
Fu, Y., & Ji, L. L. (2003). Chronic ginseng consumption attenuates
age-associated oxidative stress in rats. Journal of Nutrition, 133,
36033609.
Gordon, M. H. (1990). The mechanism of antioxidant action in vitro.
London: Elsevier Applied Science.
Halliwell, B. (1991). The biological toxicity of free radicals and other
reactive oxygen species. In O. I. Aruoma, & B. Halliwell (Eds.),
Free radicals and food additives (pp. 3757). Oxford: Oxford
University Press.
Halliwell, B., & Gutteridge, J. M. (1999). Free radicals in biology and
medicine. Oxford: Oxford University Press.
Halliwell, B., & Gutteridge, J. M. C. (1990). Role of free radicals and
catalytic metal ions in human disease: an overview. Methods in
Enzymology, 186, 185.
Halliwell, B., Gutteridge, J. M. C., & Cross, C. E. (1987). The
deoxyribose method: a simple test tube assay for determination
of rate constants for reactions of hydroxyl radicals. Analytical
Biochemistry, 165, 215219.
Harish, R., & Shivanandappa, T. (2005). Antioxidant activity and
hepatoprotective potential of Phyllanthus niruri. Food Chemistry, in
press.
Hochestein, P., & Atallah, A. S. (1988). The nature of oxidant and
antioxidant systems in the inhibition of mutation and cancer.
Mutation Research, 202, 363375.
Jadhav, S. J., Nimbalkar, S.S., Kulkarni, A.D., & Madhavi, D. L.
(1996). Lipid oxidation in biological and food systems. In: D. L.
Madhavi, S. S. Deshpande, & D. K. Salunke (Eds.), Food
antioxidants. New York: Marcel Dekker.
Janero, D. (1990). Malondialdehyde and thiobarbituric acid-reactivity
as diagnostic indices of lipid peroxidation and peroxidative tissue
injury. Free Radical Biology and Medicine, 9, 515540.
Joseph, J. A., Shukitt-Hale, B., Denisova, N. A., Bielinski, D., Martin,
A., McEwen, J. J., & Bickford, P. C. (1999). Reversals of age-
related declines in neuronal signal transduction, cognitive, and
motor behavioural decits with blueberry, spinach, or straw-
berry dietary supplementation. Journal of Neuroscience, 19,
81148121.
Kamath, S. A., & Rubin, E. (1972). Interaction of calcium with
microsomes: A modied method for the rapid isolation of rat liver
microsomes. Biochemical Biophysical Research Communication, 49,
5259.
Kappus, H., (1991). Lipid peroxidationMechanism and biological
relevance. In: O. I. Aruoma, & B. Halliwell (Eds.), Free radicals and
Food Additives. London:Taylor and Francis.
Kinsella, J. E., Frankel, E., German, B., & Kanner, J. (1993). Possible
mechanisms for the protective role of antioxidants in wine and
plant foods. Food Technology, 4, 8589.
Kitts, D. D., Wiejewickreme, A. N., & Hu, C. (2000). Antioxidant
properties of a North American ginseng extract. Molecular and
Cellular Biochemistry, 203, 110.
Klein, C., Sato, T., Meguid, M. M., & Miyata, G. (2000). From food
to nutritional support to specic nutraceuticals: A journey across
time in the treatment of disease. Journal of Gastroenterology, 35,
16.
Lowry, O. H., Rosenbrough, N. J., Farr, A. L., & Randall, R. J.
(1951). Protein measurement with the folinphenol reagent. Journal
of Biological Chemistry, 193, 265275.
Mariko, N., Hassimotto, M., Genovese, I. M., & Lajolo, F. M. (2005).
Antioxidant activity of dietary fruits, vegetables and commercial
frozen pulps. Journal of Agricultural and Food Chemistry, 53,
29282935.
Meyer, A. S., & Isaksen, A. (1995). Application of enzymes as food
antioxidants. Trends Food Science Technology, 6, 300304.
Murti, P. B. R., & Sheshadri, T. R. (1940). A study of the chemical
components of Decalepis hamiltonii (Makali Veru). Proceedings of
Indian Academy of Sciences, 13, 221232.
Murti, P. B. R., & Sheshadri, T. R. (1941a). A study of the chemical
components of Decalepis hamiltonii. Proceedings of Indian Academy
of Sciences, 13, 339403.
ARTICLE IN PRESS
A. Srivastava et al. / LWT 39 (2006) 10591065 1064
Murti, P. B. R., & Sheshadri, T. R. (1941b). A study of the chemical
components of Decalepis hamiltonii (Makali Veru). Proceedings of
Indian Academy of Sciences, 14, 9399.
Nagarajan, S., Rao, L. J. N., & Gurudutt, K. N. (2001). Chemical
composition of the volatiles of Decalepis hamiltonii (Wight & Arn.).
Flavour and Fragrance Journal, 16, 2729.
Nayar, R. C., Shetty, J. K. P., Mary, Z., & Yoganarshimhan
(1978). Pharmacognostical studies on the root of Decalepis
hamiltonii Wt. and Arn. and comparison with Hemidesmus
indicus (L.) R. Br.*. Proceedings of Indian Academy of Sciences,
87, 3748.
Nishikimi, M., Rao, N. A., & Yagi, K. (1972). The occurence of
superoxide anion in the reaction of reduced phenazine methosul-
phate and molecular oxygen. Biochemical Biophysical Research
Communication, 46, 849864.
Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics
with phosphomolybdicphosphotungstic acid reagents. American
Journal of Enology and Viticulture, 16, 144158.
Tanaka, M., Kuie, C. W., Nagashima, Y., & Taguchi, T. (1988).
Applications of antioxidative Maillard reaction products from
histidine and glucose to sardine products. Nippon Suisan Gakkaishi,
54, 14091414.
Thatte, U., Bagadey, S., & Dahanukar, S. (2000). Modulation of
programmed cell death by medicinal plants. Molecular and Cellular
Biochemistry, 46, 199214.
Wang, H., Cao, G., & Prior, R. L. (1997). Oxygen radical absorbing
capacity of anthocyanins. Journal of Agricultural and Food
Chemistry, 45, 304309.
Yamaguchi, T., Takamura, H., Matoba, T., & Terao, J. (1998). HPLC
method for evaluation of the free radical-scavenging activity of
foods by using 1,1-diphenyl-2-picrylhydrazyl. Bioscience Biotech-
nology Biochemistry, 62, 12011204.
Yen, G. C., Duh, P. D., & Tsai, C. L. (1993). The relationship between
antioxidant activity and maturity of peanut hulls. Journal of
Agricultural and Food Chemistry, 41, 6770.
Yen, G. C., & Chen, H. Y. (1995). Antioxidant activity of various tea
extracts in relation to their antimutagenicity. Journal of Agricultur-
al and Food Chemistry, 43, 2732.
Yu, B. P. (1994). Cellular defenses against damage from reactive
oxygen species. Physiological Reviews, 76, 139162.
ARTICLE IN PRESS
A. Srivastava et al. / LWT 39 (2006) 10591065 1065