antioxidant potential of selected vegetables commonly used...
TRANSCRIPT
Indian Journal of Natural Products and Resources
Vol. 1(3), September 2010, pp. 306-313
Antioxidant potential of selected vegetables commonly used in diet
in Asian subcontinent
R N Gacche*, V N Kabaliye, N A Dhole and A D Jadhav
School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded-431 606, Maharashtra, India
Received 11 September 2009; Accepted 2 February 2010
In the present study eleven different fruits and leaves of commonly used vegetables in diet in Asian subcontinent have
been evaluated for antioxidative constituents and free radical scavenging activities. Fifty per cent ethanolic extracts of
Abelmoschus esculentus (Linn.) Moench (fruits), Trigonella foenum-graecum Linn. (leaves), Spinacia oleracea Linn.
(leaves), Brassica oleracea Linn. var. capitata and B. oleracea var. botrytis (leaves, inflorescence and young stems),
Coriandrum sativum Linn. (seeds and leaves), Capsicum annuum Linn. var. grossum (Willd.) Sendt. (fruits), Cucurbita
maxima Duch. (fruits), Cyamopsis tetragonoloba Linn. (fruits), Anethum graveolens Linn. (fruits and seeds), Solanum
melongena Linn. (fruits) were tested for the determination of free radical scavenging potentials and quantification of
antioxidant agents such as ascorbic acid and phenolic compounds. Amongst the tested vegetables the sample of B. oleracea
var. botrytis has shown (67.2%) the highest 1, 1-Diphenyl-2-picryl hydrazine (DPPH radical) scavenging potential while B.
oleracea var. capitata was found to be the most effective (58.4%) inhibitor of lipid peroxidation. The extract of S. oleracea
(43.9%) and S. melongena (32.8%) were found to be effective in ferrous ion chelating abilities. The maximum amount
(25.60mg/100g) of vitamin-C was found in A. esculentus while the amount of total phenolics was noted maximum (13.30
mg/g) in C. maxima. With some exceptions the activity profiles of all other samples were found to be good to moderate.
Keywords: Antioxidant, Ascorbic acid, Free radical scavenging activity, Total phenolics, Vegetables.
IPC code; Int. cl.8 A23L 1/00, A61K 36/00, A61P 39/06
Introduction Intake of fast food and sedentary life style has been
proved to be an incompatible combination leading to
obesity and adverse health effects in majority of the
European countries1. Moreover in the midst of fast
growing world and changing life style typically of “I
don't have much time for anything else” it has become
essential to revive, re-strengthen and focus the
importance of intake of fruit and leafy vegetables in
daily diet for health amelioration. Clinical studies
have revealed that increased intake of fresh fruits and
vegetables in daily diet lower the risk of many human
diseases especially degenerative aliments linked to
ageing process. Consumption of leafy vegetables is a
major source of vitamins and micro-nutrients needed
for the normal functioning of the body physiology.
Vegetables serve as a good source of antioxidants
apart from nutritive contribution of carbohydrates,
proteins and lipids. There is a vast body of literature
accumulated in the recent years suggesting the role of
leafy vegetables in health management and especially
lowering the risk of chronic human ailments such as
cancer, cardiovascular disease and other age related
disorders2. The generation of free radicals is a vital
phenomenon of the normal metabolism of human
body. Varieties of free radicals are generated as a
byproduct of cellular functions. Normally all these
free radicals are neutralized by the enzymatic and
nonenzymatic antioxidants present as an inbuilt
antioxidant mechanism in the body and also by the
dietary antioxidants supplemented in daily diets
through fruits and vegetables3. Green leafy vegetables
are rich sources of nutrients which form a major class
of vegetable groups that have been called as “natures
anti-aging wonders”4.
The major phytochemicals
found in fruit and leafy vegetable involved in
stabilization of free radicals includes flavonoids,
coumarins, tannins and other phenolic compounds.
From the studies it has been indicated that these
phytochemicals, especially the polyphenols have high
free radical scavenging activities, which help to
reduce the risk of chronic age related neurological
degenerative diseases5,6
. Most of these
phytochemicals may help to protect cells from
oxidative damage induced by free radicals and
thereby help to reduce the oxidative stress7.
——————
*Correspondent author: E-mail: [email protected];
Phone: 02462-229242 Extn. 195; Fax. 02462-229225
GACCHE et al.: ANTIOXIDANT POTENTIAL OF VEGETABLES
307
A brief overview of the nutritional and medicinal
attributes of the selected vegetables is summarized
below. Abelmoschus esculentus (Linn.) Moench fruits
are nutritious, emollient and demulcent. Seeds are
stimulant and antispasmodic. The decoction of
immature capsules is used as demulcent, anodyne,
diuretic, anticatarrhal and also effective in dysentery,
ardor urine, dysuria and gonorrhoea8. The young plant
and aromatic leaves of Trigonella foenum-graecum
Linn. are widely used as leafy vegetable. The leaves
have cooling properties and are useful in external and
internal swelling and burns. The seeds also promote
the growth of hair and prevent hair falling9. Spinacia
oleracea Linn. commonly used as leafy vegetable is a
good source of minerals, Vitamin B complex,
ascorbic acid, carotenoids, flavonoids, apocyanin and
p-coumaric acid10
. Recently it has been reported that
the contents of this spinach are helpful in reducing
radiation induced oxidative stress and also improves
learning and memory in mice11
. Cabbage, Brassica
oleracea Linn. var. capitata is a common vegetable
and it is included in the diet of those patients
particularly suffering from fistula and liver troubles12
.
Cabbage is a rich source of ascorbic acid and also
contains several trace elements. The green type of
cabbage is more nutritive and popular. It is rich
source of ascorbic and pantothenic acid (11µg/g fresh
weight). It also contains malic and citric acid in the
ratio of 3:2, in addition to this, small amount of oxalic
and succinic acids are also present13
. The
inflorescence and young stems of Cauliflower, B.
oleracea var. botrytis are succulent and generally
used as boiled vegetable or as a salad13
.
Traditionally the seeds and leaves of the
Coriandrum sativum Linn. are used as a spice for
imparting flavour in several Asian food preparations.
The juice of whole plant is beneficial in erythema.
The strong decoction in milk with little sugar is very
effective in dyspnea, flatulence, indigestion and
bleeding piles. The fruits basically act as tonic,
aphrodisiac, stimulant, antibilious, antihelmintic,
carminative, and aromatic. Coriander water is very
useful in indigestion and many other bowel problems.
The dried fruit decoction is helpful in bilious
complaints, indigestion, intestinal troubles, flatulence,
sore throat, vomiting and cataract, used as eye wash in
chronic conjunctivitis8. The fruits of Capsicum
annuum Linn. var. grossum (Willd.) Sendt. popularly
known as Shimla mirch is commonly used as fruit
vegetable in India. It is good source of ascorbic acid
and polyphenols, the amount varying depends upon
cultivar, stage of maturity and season. The cut fruits
are eaten raw in salad and cooked in many ways13
.
The fruit pulp of Cucurbita maxima Duch. is widely
used on boils, carbuncles, ulcers, etc. The dried fruit
pulp has applications in haemorrhages. The fruits are
largely used as vegetable and seed oil act as nervine
tonic9. Generally younger tender pods of Cyamopsis
tetragonoloba Linn. are very nutritious as green
vegetable. The gum extracted from seed flour is very
viscous at low concentration and is used in several
food preparations, cosmetics, paper, textile industries,
etc. Seeds are highly valued as a cattle feed12
. The
fruits and seeds of Anethum graveolens Linn. are used
as antihelmintic, antipyretic, aromatic, diuretic,
emmenagogue, galactogogue, stimulant and also
beneficial in colic acid hiccup. The leaves soaked in
warm oil are applied locally to abscesses and boils to
hastern suppuration8. The fruits of Solanum
melongena Linn. are used to cure toothache. It is also
recommended as an excellent remedy for those
suffering from liver complaints9. The fruits are rich in
Vitamin B2 hence it shows antiglossitic property. The
fruits are effective in liver complaints, reduce fat and
cause mild sleep8. In the present investigation an
attempt has been made to demonstrate the free radical
stabilizing and medicinal properties of vegetables
frequently used in the numerous food preparations in
Asian subcontinent.
Materials and Methods The selected fresh vegetables (Plate 1) were
purchased from the local vegetable market at Nanded
(MS) India. 1, 1-Diphenyl-2-picryl hydrazine (DPPH)
and Rutin hydrate were obtained from Sigma-Aldrich
Inc., USA. Ferrozine was purchased from Himedia
Laboratories Pvt. Ltd., Mumbai. All other reagents
and solvents of analytical grade were used and
obtained from commercial sources. Preparation of alcoholic extract of selected vegetables
Soxhlet extraction method was employed for
preparation of 50% alcoholic extracts of selected
vegetables. A sample of (powdered) each was
extracted for 6 hours. The collected solvent extract
was evaporated, dried and stored at 4°C for further
use. DPPH radical scavenging activity
The DPPH radical scavenging activity was
measured by using method reported by Kato et al14
.
INDIAN J NAT PROD RESOUR, SEPTEMBER 2010
308
Plate 1 Selected vegetables studied
GACCHE et al.: ANTIOXIDANT POTENTIAL OF VEGETABLES
309
DPPH radical (10-4
M in absolute ethanol) solution
was mixed with equal amount of vegetable extract
(1mg/ml) solution. After 20 min incubation period
absorbance was measured at 517 nm. Glutathione
(1mM) was used as a standard reference compound.
Per cent inhibition was calculated by using formula
as,
100Sample of O.D.
Sample of O.D.1
activity scavenging
radical DPPH %×−=
O.D.= Optical density
Assay for inhibition of diene conjugates
For the inhibition assay of diene conjugates
(hydroperoxides), RBC membrane solution was
prepared as per the method of Dodge et al15
. The
assay and inhibition assay of membrane diene
conjugates was carried out using a method reported
by Buege & Aust16
. In to the isolated membrane
solution (1ml), 5ml of chloroform: methanol (2:1)
was mixed and centrifuged at 3600 rpm for 15 min for
separation of two phases. The chloroform layer was
dried by evaporation at 45°C in water bath. The
resultant lipid residue was dissolved in 1.5ml of
cyclohexane and absorbance was measured at 234 nm
against cyclohexane as a blank. Ascorbic acid (1mM)
was used as reference compound.
100Sample of O.D.
Sample of O.D.1
inhibition
on peroxidati lipid %×−=
Ferrous ion chelating activity (FICA)
The ferrous ion chelating activity of the selected
samples was measured by using method of Decker &
Welch17
. Three ml of individual extract (1mg/ml) was
mixed with 0.3 ml of 2mM FeCl2, 0.6 ml of 5mM
Ferrozine solution and reaction mixture was kept for
10 min incubation at room temperature and
absorbance was measured at 562 nm. α-Tocopherol
(1mM) was used as a standard compound.
100Sample of O.D.
Sample of O.D.1
activity
cheltingion ferrous %×−=
Estimation of Vitamin C content
The amount of vitamin C was determined by using
method described by Sadasivam and Manickam18
.
The principle of the method is that the 2, 6-
dichlorophenol indophenol dye is reduced to a
colourless leuco-base by ascorbic acid and the latter is
oxidized to dehydroascorbic acid. The end point of
this titration is appearance of pink colour.
Estimation of total phenolics
The total phenol content in the selected samples
was estimated by using method of Bray and Thorpe19
.
The principle underlying the reaction mechanism is
that, in alkaline medium phenol reacts with Folin-
Ciocalteau reagent which results in the production of
blue coloured complex having maximum absorption
at 660 nm. Rutin hydrate (500 µg/ml) was used as
standard phenolic compound for preparation of
standard curve. The concentration of total phenols
was expressed in mg/g in rutin equivalent.
Results and Discussion
The results summarized in Table 1 indicate that the
selected samples were found to interact with DPPH
radicals and thereby stabilize their hyperactivity.
Amongst the tested herbs the extract of B. oleracea
var. botrytis (67.2%) was found to be more effective
as DPPH radical scavenger, while the minimum effect
was observed in case of S. oleracea (20.4%). The
other samples were found to be in a range of 62.4-
25.7% as compared to Glutathione (60.72%), a
standard free radical stabilizing agent (Fig. 1). The
results of the inhibition of formation of
hydroperoxides show that, all the selected vegetables
have a potential to inhibit lipid peroxidation
especially the effect was more predominant in case of
B. oleracea var. capitata (58.4%), A. graveolens
(56.9%) and C. sativum (54.9%), whereas the activity
of all other samples was found to be in a moderate
range of 45.3 to 13.8% (Fig. 2). The ferrous ion
chelating activity was noted higher in S. oleracea
(43.9%) followed by S. melongena (32.8%). Except
the samples of A. esculentus (26.5%), C. maxima
(22.3%) and T. foenum-graecum (16.8%), the
remaining samples did not react with ferrous ions
(Fig. 3).
The sample of A. esculentus was found to possess
the highest (25.60 mg/100g) amount of vitamin C,
while all other samples contained a significant amount
of ascorbic acid in a range of 20.70-5.63 mg/100g of
sample. The amount of total phenol in rutin equivalent
was found maximum in C.maxima (13.30 mg/g) and
S. melongena (10.41 mg/g) whereas the remaining
vegetables have considerable amount of total phenols
in a range of 8.7-2.6 mg/g of the sample.
DPPH is a stable free radical and it has been widely
used to evaluate the free radical scavenging ability of
different dietary antioxidants. The principle of the
assay is that the antioxidant reacts with DPPH radical
and converts into corresponding hydrazines. The fall
INDIAN J NAT PROD RESOUR, SEPTEMBER 2010
310
in extinction is correlated with the potential of
antioxidant to scavenge free radicals20
. Proton radical
scavenging action is an important mechanism
of oxidation. DPPH has a proton free radical and
gives strong absorption at 517 nm (Purple colour).
The presence of sufficient amount of vitamin C
and the phenolic compounds in the selected
vegetables can be a cause for reduction of DPPH into
hydrazine.
Lipid peroxidation is a key process in many
pathological events. The oxidation of unsaturated
fatty acids present in biological membranes result into
formation and spread of lipid radicals. The uptake of
oxygen by these radicals, the rearrangement of double
bonds in unsaturated lipids results ultimately in the
destruction of membranes and produces a products
such as malondialdehyde, which is well known
mutagenic and carcinogenic in activity. The formation
of hydroperoxides is one of the intermediate in lipid
peroxidation process. Oxidative stress and free
radicals have been considered as one of the agent for
recruiting the process of lipid peroxidation. The
hydroperoxide inhibition activity shown by the
selected samples can be attributed with
phytochemicals especially polyphenols which have
been described as chain breaking agents in lipid
peroxidation21
.
Transitional metals especially iron has the ability to
generate free radicals from peroxides by Fenton
reaction22
. Fe++
has capability to induce production of
oxyradicals and lipid peroxidation and hence samples
showing Fe++
reduction in the Fenton reaction are
considered to protect cellular damage induced by
oxidative stress23
. In general vegetable extracts
Table 1 Profile of DPPH radical scavenging activity, inhibition of lipid peroxidation, ferrous ion chelating activities (FICA), Vitamin C
and total phenols of selected vegetables
S. No. Botanical & common names of the
vegetables/Family
DPPH radical
scavenging
activity (%)
Inhibition of lipid
peroxidation
(%)
FICA (%)
Vitamin C
(mg/100g)
Total phenols
(mg/g)
1. Abelmoschus esculentus (Linn.)
Moench (Lady’s Finger), Malvaceae
43.8 ± 0.40
13.8 ± 0.45 26.5 ± 0.24 25.60 ± 0.41 5.35 ± 0.15
2. Trigonella foenum-graecum Linn.
(Fenugreek), Fabaceae
25.7 ± 0.34 22.0 ± 0.52 16.8 ± 0.32 20.70 ± 0.62 6.36 ± 0.25
3. Spinacia oleracea Linn.
(Spinach), Chenopodiaceae
20.4 ± 0.48 29.4 ± 0.62 43.9 ± 0.61 15.36 ± 0.60 2.60 ± 0.18
4. Brassica oleracea Linn. var.
capitata (Cabbage), Brassicaceae
42.4 ± 0.52 58.4 ± 0.42 NR 12.28 ± 0.45 3.00 ± 0.12
5. Brassica oleracea Linn. var. botrytis
(Cauliflower), Brassicaceae
67.2 ± 0.46 18.1 ± 0.38 NR 5.63 ± 0.67 4.99 ± 0.14
6. Coriandrum sativum Linn.
(Coriander), Apiaceae
62.4 ± 0.55 54.9 ± 0.52 NR 20.48 ± 0.60 2.20 ± 0.22
7. Capsicum annuum Linn. var. grossam
(Willd.) Sendt.
(Capsicum), Solanaceae
25.0 ± 0.47 33.6 ± 0.42 NR 20.48 ± 0.52 4.20 ± 0.26
8. Cucurbita maxima Duch.
(Bottle gourd), Cucurbitaceae
40.7 ± 0.62 40.3 ± 0.58 22.3 ± 0.60 15.36 ± 0.85 13.30 ± 0.15
9. Cyamopsis tetragonoloba Linn.
(Clusterbean)
51.6 ± 0.57 45.3 ± 0.39 NR 15.36 ± 0.56 5.79 ± 0.10
10. Anethum graveolens Linn.
(Dill), Apiaceae
44.6 ± 0.42 56.9 ± 0.48 NR 10.24 ± 0.54 8.70 ± 0.21
11. Solanum melongena Linn.
(Brinjal), Solanaceae
51.6 ± 0.45 16.4 ± 0.91 32.8 ± 0.30 20.48 ± 0.86 10.41 ± 0.18
12. Glutathione 60.72 ± 0.48 ND ND ND ND
13. α-Tocopherol ND ND 60.7 ± 0.42 ND ND
14. Ascorbic acid ND 53.83 ±0.44 ND ND ND
The result presented here are the mean ± SD
NR-No reaction under experimental conditions, ND- Not determined.
GACCHE et al.: ANTIOXIDANT POTENTIAL OF VEGETABLES
311
interfere with the formation of ferrous and ferrozine
complex, suggesting that it possesses chelating
activity and captures ferrous ion before ferrozine.
The importance of vitamin C as an antioxidant is
indispensable in biological system. This vitamin is
reputed for scavenging the harmful free radicals
produced in the body and also enhance the antioxidant
defense mechanism in body. It has been reported that
a dose of 1g of vitamin C per day is sufficient to
counteract the ill effects of low density lipoprotein24
.
Recent studies have shown that intake of sufficient
amount of vitamin C is highly protective to prevent
stroke and heart attack25
. Consuming diet rich in
vitamin C from fruits and vegetables provides
protection against cancer26
.
Numerous studies have described antioxidant
potential and free radical scavenging abilities of plant
phenolics. Phytochemicals especially plant
polyphenols contains major group of compounds that
act as a primary source of antioxidants27
.
Fig. 1—DPPH radical scavenging activity of selected vegetables
Fig. 2—Inhibition of lipid peroxidation (%) of selected vegetables
INDIAN J NAT PROD RESOUR, SEPTEMBER 2010
312
Polyphenolic compounds such as flavonoids,
anthocyanins, tannins and other phenolic substances
have been reported to scavenge variety of free
radicals which including oxy-radicals such as
hydroxyl radicals28
, superoxide anion radicals29
, lipid
peroxy radicals30
, etc. Besides the role of plant
phenolics as antioxidants, the group polyphenols have
been used for prevention of cancer and heart
diseases31
.
Conclusion
The dietary intake of vegetables can be useful in
the management of oxidative stress and age related
human ailments. Moreover in the present context of
busy and stressful life style trend all over the world, it
has become necessary to focus and inculcate the
medicinal importance of fruits and vegetables in order
to minimize the risk of life taking episodes especially
the cardiac arrest/failures. However, further in vivo
studies are also required to investigate the antioxidant
potential of individual component and its quantity in
plant sample.
Acknowledgements
Authors are thankful to Swami Ramanand Teerth
Marathwada University, Nanded (M.S.) for financial
assistance (BCUD/MIN.UNI/2008-2009/6534) and
Director, School of Life Sciences, S.R.T.M.
University, Nanded for providing the necessary
facilities during this work.
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