antioxidant potential of selected vegetables commonly used...

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


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


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.


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


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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antioxidant potential of selected vegetables commonly used...

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