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A Study of Antioxidant Properties of Some Varieties ofGrapes (Vitis vinifera L.)Vinayak V. Kedage a , Jai C. Tilak b , Ghanasham B. Dixit a , Thomas P. A. Devasagayam b &Minal Mhatre ca Department of Botany, Shivaji University, Kolhapur, Maharashtra, 416 004, Indiab Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai,400 085, Indiac Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai,400 085, IndiaPublished online: 16 Jan 2007.

To cite this article: Vinayak V. Kedage , Jai C. Tilak , Ghanasham B. Dixit , Thomas P. A. Devasagayam & Minal Mhatre(2007) A Study of Antioxidant Properties of Some Varieties of Grapes (Vitis vinifera L.), Critical Reviews in Food Science andNutrition, 47:2, 175-185, DOI: 10.1080/10408390600634598

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Critical Reviews in Food Science and Nutrition, 47:175–185 (2007)

Copyright C©© Taylor and Francis Group, LLC

ISSN: 1040-8398

DOI: 10.1080/10408390600634598

A Study of Antioxidant Properties

of Some Varieties of Grapes

( Vitis vinifera L.)

VINAYAK V. KEDAGEDepartment of Botany, Shivaji University, Kolhapur 416 004, Maharashtra, India

JAI C. TILAKRadiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085, India

GHANASHAM B. DIXITDepartment of Botany, Shivaji University, Kolhapur 416 004, Maharashtra, India

THOMAS P. A. DEVASAGAYAMRadiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085, India

MINAL MHATRENuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India

Grapes (Vitis vinifera L.) are a major fruit crop in the world. Grapes seem to confer health benefits due to their antioxidantactivity. We have evaluated the antioxidant potential of 11 grapes varieties from India and nearby Asian countries. The assaysemployed involve different levels of antioxidant action like ferric reducing antioxidant power, radical scavenging by 1,1-diphenyl-2-picrylhydrazyl, ferrylmyoglobin/2,2′-azobis-3-ethylbenzthiazoline-6-sulfonic acid, oxygen radical absorbancecapacity (ORAC), and inhibition of lipid peroxidation. The total phenolic and flavonoids contents were also estimated. Ourstudy indicates that cv. Mango is the most potent followed by Sharad Seedless. Ethanolic extracts were found to be moreeffective than aqueous extracts. Cv. Sharad Seedless, Mango, and Manikchaman also had high ORAC values. Their HPLCanalysis showed the presence of various antioxidant polyphenols. In conclusion our studies identified some varieties of grapeswith high antioxidant activities and showed that their high antioxidant potential may be due to their phenolic and flavonoidcontents.

Keywords grapes, antioxidant, phenolics, flavonoids, lipid peroxidation, ORAC, HPL, TBARS, TEAC

INTRODUCTION

Health is of prime importance in ones life. Consumption of

adequate amounts of fruits and vegetables is considered essen-

tial for a healthy life. A recent article on “new food pyramid”

recommends 2 to 3 servings of fruits daily for achieving opti-

mum health.1 A higher intake of fruits and vegetables has been

correlated with a lower incidence of major human illnesses like

cardiovascular diseases (CVDs) and cancer.2−4 In this respect

Address correspondence to Minal Mhatre, Nuclear Agriculture and Biotech-

nology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India.

E-mail: mmhatre@magnum.barc.ernet.in

“grapes” (Vitis vinifera L.) and other dietary constituents de-

rived from it like “grape juice” and “wine” have attracted a

great deal of attention in recent years. Certain populations from

Europe have a lower incidence of CVD and the resultant mor-

tality. This protective effect seems to be due to their dietary

habits that include a high consumption of wine, grapes, and

grape juice. Polyphenolics present in the grapes and its prepara-

tions seem to be responsible, on account of their potent antiox-

idant activities.5,6 The composition and properties of grapes

have been extensively investigated. Reports indicate that grapes

contain large amounts of phenolic compounds,7−9 which play

an important role in human health, such as lowering of human

low-density lipoprotein.10,11 It has been demonstrated that wine

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176 V. V. KEDAGE ET AL.

and other products derived from grapes have high antioxidant

capabilities.12 A large number of polyphenols and flavonoids

such as p-coumaric acid, cinnamic acid, caffeic acid, ferulic

acid, vannilic acid, catechin, epicatechin, quercetin, proantho-

cyanidins, besides trihydroxy stilbenes such as resveratrol and

polydatin have been reported from grapes. In addition to these,

viniferin, a potent antifungal agent, and anthocyanins, which

are strong antioxidants that inhibit platelet aggregation are also

present.6

In developing countries, CVD is emerging as a major health

problem in recent years. This is considered to be due to change

in life style and dietary habits. This is especially more so among

the urban population and the affluent rural families. The change

in dietary habits mainly pertains to higher intake of carbohy-

drates, dairy products, and higher calories.13,14 Among the fruits

consumed by this population, grapes is an important ingredient.

Unlike in the European population, wine does not form a major

constituent of grapes consumed. Grapes is a major fruit crop in

India and other parts of Asia. Several varieties are also culti-

vated. The area under grape cultivation and amount of grapes

produced are very high. Grapes is one of the world’s largest fruit

crops, which approximates an annual production of 58 million

metric tons.15 It is one of the important commercially irrigated

fruit crops in India and its cultivation occupies 40,000 ha of

land and productivity is fairly high as compared to the other

grape growing countries in the world.16 More than fifteen grape

varieties are cultivated in India and nearby countries, mainly

Thompson Seedless and its clones (Tas-E-Ganesh, Sonaka and

Manikchaman), Sharad Seedless, Flame Seedless, and Anab-

E-Shahi, which are considered the ruling grape varieties. Vitis,

cv. Mango, cv. Sharad Seedless are black grapes and cv. Flame

Seedless, cv. Kalisahebi are red grapes, while others have green

fruits. They are categorized depending upon their fruit color and

seedless ness (Table 1). The color of the berries also may indicate

the amount of polyphenols present. Thompson Seedless with its

clones alone occupies 55% of the total land under cultivation of

grapes.17

To our knowledge, there are no detailed studies on the antiox-

idant abilities of different grapes varieties in India and nearby

Asian countries besides the possible mechanisms in relation to

their polyphenolic composition. In the present study, we have

determined the antioxidant potential of 11 different varieties of

grapes predominant in the Asian region using assays pertaining

to different levels of antioxidant action. To explain the possi-

ble differences observed, we have measured the total phenolic

and total flavonoid contents of berry extracts by biochemical

Table 1 Categories of Grapes varieties and their characteristics

Colored, seeded Banglore Blue, Kalisahebi, Banglore Purple, Mango

Colored, seedless Sharad Seedless, Flame Seedless

White, seeded Anab-E-Shahi, Dilkhush (Clone of Anab-E-Shahi),

Raosahebi

White, seedless Thompson Seedless and its clones (Tas-E-Ganesh,

Sonaka, and Manikchaman), Arkavati,

Maru Seedless, H5 hybrid.

methods. HPLC analysis of three most potent varieties was also

carried out to identify their phenolic compositions.

MATERIALS AND METHODS

Reagents and Standards

Ascorbic acid, aluminum chloride, 2,2′-azobis-3-

ethylbenzthiazoline-6-sulfonic acid (ABTS) diammonium

salt, β-phycoerythrin, 1,1′-diphenyl-2-picrylhydrazyl (DPPH),

ethylene diamine tetra acetic acid (EDTA), ferric chloride,

Folin-Ciocalteu reagent, hydrogen peroxide, myoglobin,

potassium ferricyanide, potassium phosphate (monobasic and

dibasic), sodium carbonate, trolox (6-hydroxy-2,5,7,8-tetra-

methylchroman-2-carboxylic acid), 1,1,3,3-tetraethoxypropane,

2,4,6-tripyridyl-s-triazine (TPTZ), 2-thiobarbituric acid (TBA),

and trichloroacetic acid were purchased from Sigma Chemical

Co., U.S.A. Glacial acetic acid (HPLC grade) and methanol

(HPLC grade) were purchased from Merck. 2.2′-Azobis

(2-amidinopropane) dihydrochloride (AAPH) (=2.2′-azobis(2-

methylpropionamidine) dihydrochloride), Trolox (6-Hydroxy

2,5,7,8 tetramethyl chroman 2-carboxylic acid) was from

Aldrich Chemical Co., U.S.A. Other chemicals used in our

studies were of the highest quality commercially available

from local suppliers. Berries of different grape varieties

were obtained from different areas of grape cultivation in

Maharashtra.

Sample Preparation

Mature berries from 11 different grape varieties viz.

Thompson Seedless, Sonaka, Tas-E-Ganesh, Manikchaman, H5

Hybrid, Sharad Seedless, Flame Seedless, Mango, Kalisahebi,

Anab-E-Shahi and Raosahebi were collected from different

fields of Maharashtra. Berries (10 g, with skin) of each vari-

ety were crushed in 50 ml of solvent and filtered through a

muslin cloth. The extracts were condensed to 10 ml by using

rotary evaporator. The condensed extracts were kept at −20◦C

and for the assays 10% (w/v) extracts (diluted with distilled wa-

ter) were used. For the preparation of ethanolic and aqueous

extracts solvents used were 70% ethanol and double distilled

water, respectively.

Quantitative Determination of Total Phenolsand Total Flavonoids

The total phenolic contents of both ethanolic and aque-

ous extracts were measured using a modified Folin-Ciocalteu

method.18 The measurement was compared to a standard curve

of gallic acid concentrations and expressed as milligrams of

gallic acid equivalents per g fresh weight of grapes. Flavonoid

contents of both ethanolic and aqueous extracts were also

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ANTIOXIDANT PROPERTIES OF GRAPES 177

measured.19 The values obtained were compared to a standard

curve of quercetin concentrations and expressed as milligrams

of quercetin equivalents per g fresh weight.

Radical Scavenging Assays

DPPH scavenging effect was carried out with different

extracts.20 In this method, a commercially available, stable free

radical—DPPH, soluble in methanol, was used. In its radical

form, DPPH has an absorption maxima at 515 nm, which disap-

pears on reduction by an antioxidant compound. The calibration

curve was plotted with % DPPHSCAVENGED versus concentration

of the standard antioxidants (L-ascorbic acid and Trolox). In

the ferrylmyoglobin/ABTS+ spectrophotometric assay, the in-

hibition of radical formation by the extracts was determined by

using the ferrylmyoglobin/ABTS+ protocol.21 The calibration

curve was plotted with lag time in seconds versus concentration

of the standard antioxidants (L-ascorbic acid and Trolox). The

ferric complex reducing ability of the extracts was measured

by ferric reducing antioxidant power (FRAP) assay22. The cal-

ibration curve was plotted with absorbance at 595 nm versus

concentration of FeSO4 in the range of 0–1 mM (both, aque-

ous and ethanolic solutions). Then the concentration of FeSO4

was plotted against concentrations of the standard antioxidants

(L-ascorbic acid and Trolox).

Isolation of Mitochondrial Fraction from Rat Liver

Three months old female Wistar rats (weighing about

250 ± 20 g) were used for the preparation of mitochondria. In

brief, rat livers were excised and homogenized in 0.25 M sucrose

containing 1 mM EDTA. The homogenate was centrifuged at

3000 × g for 10 min, to remove cell debris and the nuclear frac-

tion. The supernatant was centrifuged at 10,000 × g for 10 min

to sediment mitochondria. The mitochondrial pellet was washed

thrice with 50 mM KPO4 buffer, pH 7.4, to remove sucrose.23

All the experiments were carried out at 4◦C. Protein was es-

timated and pellets were suspended in the above buffer at the

concentration of 10 mg protein/ml.

Exposure of Rat Liver Mitochondria to Oxidative Stress

Oxidative damage was induced by ascorbate-Fe2+-system as

described previously.24 Incubations were carried out at 37◦C

in a shaker-water bath. After the incubation, samples were

boiled with TBA reagent for 30 minutes. The pink color of

thiobarbituric acid reactive substances (TBARS) formed were

estimated at 532 nm spectrophotometrically as malondialde-

hyde equivalents after accounting for appropriate blanks. Mal-

ondialdehyde standard was prepared by the acid hydrolysis of

tetraethoxypropane.

Oxygen Radical Absorbance Capacity (ORAC) Assay

The cv. Sharad Seedless, cv. Mango and cv. Manikchaman

extracts showed high antioxidant properties in earlier assays,

hence these three varieties were chosen for a more specific as-

say relevant to food materials, the ORAC assay. 1% ethanolic

extracts of these varieties were selected and assessed for in-

hibition of β-phycoerythrin damage by peroxyl radicals gen-

erated by thermal decomposition of azo initiator APPH. The

fluorescence was recorded after every 5 min, till the last read-

ing was less than 5% of the zero min reading. ORAC val-

ues were calculated in terms of μmoles trolox/g of fresh

weight.25

Statistical Analysis

Data are presented as mean ± SE. (standard error). Coef-

ficient of correlation was calculated for intra-group variations.

Significance of inter-group differences was determined by analy-

sis of variance (ANOVA). A p value of p < 0.05 was considered

statistically significant.

HPLC Chromatographic Analysis of Grape Extracts

Ethanolic extracts of three different varieties of grapes viz.,Sharad Seedless. Mango, and Manikchaman were used for

the determination of compositional differences as analyzed by

HPLC. 10% ethanolic extracts (diluted with double distilled wa-

ter) were used for analysis. These extracts were centrifuged at

15,000 rpm at 4◦C for 20 min. Supernatants were collected and

filtered through 2 μ filter. The filtered extracts were used for

the HPLC analysis. The standard phenolic acids and flavonoids

used for the HPLC analysis were gallic acid, catechin, caffeic

acid, hydrocaffeic acid, o-coumaric acid, p-coumaric acid, rutin

and quercetin.

The qualitative analysis of the polyphenol contents was

performed using a Waters HPLC system (Waters/Millipore,

Milsford, MA, USA) consisting of a model 515 pump, a model

2487 dual wavelength absorbance detector and a model 717 au-

tosampler. The separation of the polyphenols was conducted in

a C18 column (Delta Pak), 5 μ, 3.9 × 150 mm, 300 A◦. Flow

rate was set to 0.75 ml/min. A sample volume of 20 μl was

injected to the column using a Waters 717 auto sampler. The

two solvents used to make the gradient were (A) 25% methanol

in 1% acetic acid, and (B) 75% aqueous methanol in 1% acetic

acid. The solvent gradient in volumetric ratios of solvents A and

B was as follows: 0–30 min, 100 A/0 B; 30-45 min, 82 A/18

B; 45–65 min, 72 A/28 B; 65–85 min, 60 A/40 B; > 85 min, 0

A/100 B. Dual wavelength (280 nm and 360 nm) was used to

detect the eluent.26,27

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178 V. V. KEDAGE ET AL.

RESULTS AND DISCUSSION

Exposure to adverse pathophysiological states causes en-

hanced generation of the reactive oxygen species (ROS) result-

ing in oxidative stress.28 ROS may cause degenerative human

diseases such as cancer, heart diseases and cerebrovascular dis-

eases through multiple mechanisms.29 There are reports of in-

creases in prevalence of coronary heart disease (CHD) in India

and other developing countries, with a steady increase in the

number of patients with acute myocardial infarction.30 It has

been shown that people of Indo-origin may be more prone to

CHD due to metabolic syndrome comprising of resistance to

insulin-mediated glucose uptake, serum triglycerides, low lev-

els of HDL cholesterol etc.31 Various antioxidants may prevent

and/or improve different diseased states. Natural products espe-

cially derived from dietary components such as fruits and veg-

etables yield rich dividends in terms of potential benefits in con-

trolling diseases. Polyphenolic antioxidants such as flavonoids

occur naturally in vegetables, fruits and beverages such as tea

and wine. Their intake was significantly and inversely associated

with mortality from CHD and also showed an inverse correla-

tion with incidence of myocardial infarction.32 Consumption of

grapes also can help. Hence we have estimated antioxidant abil-

ities of Indian grape varieties and their chemical compositions.

Antioxidant Activity

For measuring antioxidant activity in vitro, we have

used different methods corresponding to different levels of

antioxidant action such as 1) Ferric reducing antioxidant

power (FRAP) assay; 2) Inhibition of radical formation by

ferrylmyoglobin/ABTS+ assay; 3) Radical scavenging by using

DPPH and ORAC assays; and 4) inhibition of lipid peroxidation

by measuring TBARS in rat liver mitochondria. The activities

in ethanolic extracts are expressed as μg/ml of Trolox equiva-

lent antioxidant capacity (TEAC) per gram of fresh weight of

grapes and those in aqueous extracts are expressed as μg/ml of

Ascorbic acid equivalent antioxidant capacity (AEAC) per gram

of fresh weight of grapes.

In all the tests employed, it was found that ethanolic ex-

tracts are more effective than aqueous extracts, possibly due

to the presence of polyphenols and flavonoids. Since dietary

sources contain both oil-soluble and water-soluble compounds,

two types of extracts have been prepared i.e. ethanolic and

aqueous, extracting oil-soluble and water-soluble antioxidants

respectively.

Figure 1 represents ferric reducing capacity obtained by using

FRAP assay. In Fig. 1a, the highest ferric reducing capacity was

found for the ethanolic extract of cv. Mango in terms of trolox

concentrations followed by cv. Sharad Seedless; while among

the aqueous extracts (Fig. 1b) Flame Seedless, Manikchaman

had significant ferric reducing capacities as compared to other

grape varieties.

Figure 2 presents data on ferrylmyoglobin/ABTS+ assay.

Among the ethanolic extracts (Fig. 2a) and aqueous extracts

(Fig. 2b). The cv. Mango possessed the highest antioxidant ac-

tivity with 1.642 and 0.228 μg/ml Trolox and ascorbic acid

equivalents respectively representing very high ability of radi-

cal inhibition.

In the data on DPPH radical scavenging assay as shown

in Fig. 3, among the ethanolic extracts (Fig. 3a) the black cv.

Mango was found to be the most potent radical scavenger with

other black cv. Sharad Seedless and a red cv. Flame Seedless,

with 0.227, 0.212, and 0.151 μg/ml of TEAC with the 82.32%,

76.83%, and 54.76% scavenging respectively. TEAC values of

the various extracts ranged between 0.022 to 0.135μg/ml TEAC.

Among the aqueous extracts, (Fig. 3b) cv. Mango (0.076 μg/ml

of AEAC), cv. H5 hybrid (0.061 μg/ml AEAC) and cv. Raosa-

hebi (0.046 μg/ml of AEAC) showed significant radical scav-

enging abilities.

Table 2 presents data on the inhibitory effects of the ethano-

lic and aqueous extracts of 11 grape cultivars against lipid per-

oxidation induced by ascorbate-Fe2+ in rat liver mitochondria.

In the ethanolic extracts cv. Sharad Seedless was found to be

the most effective and gave 74.13% protection, followed by cv.

Mango and cv. Kalisahebi that yielded 70.15% and 69.65% pro-

tection respectively. Among the aqueous extracts cv. Thompson

Seedless was the most effective and gave 84.87% protection

followed by cv. Sharad Seedless (70.59% protection) and cv.

Mango (59.66% protection).

From the above assays performed to determine the antiox-

idant activities of grape extracts; we have selected three most

potent varieties of grapes i.e. cv. Sharad Seedless, cv. Mango

and cv. Manikchaman for ORAC assay, a standard assay for

the determination of antioxidant potential of foodstuffs.33 In

this assay, as shown in Table 3, it was found that cv. Sharad

Seedless has the highest ORAC value (46.81 μmoles of Trolox

equivalent/g of fresh wt) with greatest ability to scavenge per-

oxyl radicals followed by cv. Manikchaman (45.33 μmole of

Trolox equivalent /g of fresh wt.) and cv. Mango (43.69 μmole

of Trolox equivalent/g of fresh wt.). The ORAC scores of the

grapes extracts used in our study are much higher than the re-

ported ORAC values for wet matter of some other fruits like

strawberry, 15.36; plum, 9.49; orange, 7.50; red grape, 7.39 and

white grape, 4.46 μm/g.33

Determination of Total Phenolic and Flavonoid Contentsby Biochemical as well as HPLC Analysis

All the 11 varieties of grapes were analyzed for total phenol

and total flavonoid contents and some select varieties by HPLC

to identify different phenolics and flavonoids such as gallic

acid, catechin, caffeic acid, hydrocaffic acid, O-coumaric acid,

p-coumaric acid, rutin, and quercetin. As shown in Table 4, in

the total phenolic content assay the contents in ethanolic extracts

of the varieties differ significantly among the samples (with the

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ANTIOXIDANT PROPERTIES OF GRAPES 179

Table 2 Effect of ethanolic and aqueous extracts of different grapes cultivars on lipid peroxidation in rat liver mitochondria

Ethanolic extracts Aqueous extracts

Serial no. Cultivars/Treatment TBARS (nmoles/mg protein) % Protection TBARS (nmoles/mg protein) % Protection

1 Control 1.56 ± 0.15 — 3.17 ± 0.58 —

2 Damage 12.72 ± 2.13 — 9.78 ± 1.00 —

3 Thompson Seedless 7.22 ± 1.03 49.25 4.17 ± 0.63 84.87

4 Sonaka 8.39 ± 0.06 38.81 8.84 ± 1.83 14.29

5 Shard Seedless 4.45 ± 0.24 74.13 5.11 ± 1.11 70.59

6 Flame Seedless 10.17 ± 0.76 22.89 6.00 ± 0.09 57.14

7 Anab-E-Shahi 12.06 ± 3.73 5.97 8.50 ± 2.18 19.33

8 Kalisahebi 4.95 ± 0.15 69.65 8.50 ± 1.67 19.33

9 Mango 4.89 ± 0.40 70.15 5.83 ± 0.83 59.66

10 Tas-E-Ganesh 10.48 ± 5.19 20.06 7.72 ± 0.94 31.09

11 Manikchman 12.17 ± 0.59 4.98 8.39 ± 1.60 21.01

12 H5 Hybrid 8.89 ± 0.29 34.33 5.45 ± 0.58 65.55

13 Raosahebi 9.34 ± 2.89 30.35 7.45 ± 0.30 35.29

Lipid peroxidation was measured as formation of thiobarbituric acid reactive substances (TBARS). Peroxidation (damage) was induced by ascorbate-Fe2+-system.

Values represent mean ± SE of three different experiments.

Figure 1 Represents data on ferric complex reducing ability by FRAP assay of 10% ethanolic and 10% aqueous extracts of 11 grape cultivars. Figure 1a represents

ferric complex reducing ability of ethanolic extracts in terms of equivalent concentrations of trolox (μg/ml), which is an ethanol soluble standard antioxidant and

in Figure 1b ferric complex reducing ability by aqueous extracts in terms of equivalent concentrations of ascorbic acid (μg/ml), which is a water-soluble standard

antioxidant. Values are mean ± SE of three different experiments each.

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180 V. V. KEDAGE ET AL.

Figure 2 Represents data on inhibition of radical formation by using the ferrylmyoglobin/ABTS+ assay of 10% ethanolic and 10% aqueous extracts of 11 grape

varieties. Figure 2a represents inhibition of radical formation by ethanolic extracts in terms of equivalent concentrations of trolox (μg/ml) and in Figure 2b inhibition

of radical formation by aqueous extracts in terms of equivalent concentrations of ascorbic acid (μg/ml). Values are mean ± SE of three different experiments

each.

values ranging between 11.205 to 84.561 mg gallic acid/g of

fresh wt.) as well as in the aqueous extracts (11.216 to 45.392

mg gallic acid /g of fresh wt.).

The total flavonoid contents of the samples derived from

11 different grape varieties were given in Table 4. The to-

Table 3 ORAC values of ethanolic extracts of three most potent varieties

ORAC value (μ mole

Serial no. Cultivar TE∗ /g fresh weight.)

1. Sharad Seedless 46.81 ± 1.45

2. Mango 43.69 ± 0.57

3. Manikchaman 45.33 ± 0.73

∗TE: Trolox equivalent value. Values are represented as mean ± SE of three

different experiments.

tal flavonoid contents of ethanolic extracts vary from 0.331

to 4.678 mg/g of fresh wt. Data from Table 4 indicates

that differences in total phenol and total flavonoids content

among the varieties may depend on the fruit color. From the

HPLC analysis of black grape varieties cv. Sharad Seedless

and cv. Mango besides the green variety cv. Manikchaman

as presented in Fig. 4, it is evident that they contain dif-

ferent types of flavonoids. The first two varieties i.e. cv.

Sharad Seedless and cv. Mango have the phenolic com-

pounds gallic acid, catechin, hydrocaffeic acid, O–coumaric

acid, and rutin, while in the green variety, cv. Manikchaman

shows the presence of gallic acid, catechin, hydrocaffeic acid,

and rutin possibly as their active components in the berry

extracts.

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ANTIOXIDANT PROPERTIES OF GRAPES 181

Table 4 Total phenolic and total flavonoid contents of aqueous and ethanolic extracts of grape berries

Phenolic contents (mg of Gallic acid Flavonoid contents (mg of Quercetin

equivalents/ g fresh weight) equivalents/ g fresh weight)

Cultivars Ethanolic extracts Aqueous extracts Ethanolic extracts Aqueous extracts

Thompson Seedless 18.550 ± 0.41 25.579 ± 0.74 0.331 ± 0.055 1.421 ± 0.084

Sonaka 11.205 ± 0.02 11.216 ± 1.06 0.428 ± 0.094 0.324 ± 0.015

Sharad Seedless 64.842 ± 0.68 15.567 ± 0.51 3.507 ± 0.408 1.350 ± 0.185

Flame Seedless 54.246 ± 0.78 17.556 ± 0.33 2.078 ± 0.459 1.146 ± 0.081

Anab-E-Shahi 17.661± 0.14 19.099 ± 0.37 1.016 ± 0.061 0.806 ± 0.062

Kalisahebi 18.713 ± 0.20 16.269 ± 0.64 0.702± 0.100 1.069 ± 0.027

Mango 84.561 ± 0.85 45.392 ± 0.73 4.678 ± 0.109 1.418 ± 0.013

Tas-E-Ganesh 26.807 ± 0.46 17.673 ± 0.25 0.738 ± 0.112 1.075 ± 0.024

Manikchaman 39.977 ± 0.51 20.947 ± 0.33 1.577 ± 0.203 0.570 ± 0.064

H5 Hybrid 48.865 ± 0.48 21.415 ± 0.24 1.591 ± 0.138 1.503 ± 0.061

Raosahebi 32.304 ± 0.22 37.930 ± 1.18 1.404 ± 0.241 0.997 ± 0.043

Total phenolic contents are expressed as milligrams of gallic acid equivalents per g fresh weight of grape. Total flavonoid contents are expressed as milligrams of

quercetin equivalents per g fresh weight of grape. Values represent mean ± SE of three different experiments.

Figure 3 Represents data on DPPH Radical scavenging assay of 10% ethanolic and 10% aqueous extracts of 11 grape varieties. Figure 3a represents DPPH

scavenging effect of ethanolic extracts in terms of equivalent concentrations of Trolox (μg/ml) and in Figure 3b. DPPH radical scavenging effect of aqueous extracts

in terms of equivalent concentrations of ascorbic acid (μg/ml). Values are mean ± SE of three different experiments each.

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182 V. V. KEDAGE ET AL.

Figure 4 Represents HPLC chromatograms recorded on the samples of ethanolic extracts of (a) cv. Sharad Seedless, (b) cv. Mango and (c) cv. Manikchaman at

280 nm, and (d) cv. Sharad Seedless, (e) cv. Mango and (f) cv. Manikchaman at 360 nm. GA—Gallic acid, C—Catechin, HC—Hydrocaffeic acid, CA—Caffeic

acid, O-CA—O-coumaric acid, Rutin and Quercetin were determined as standard antioxidants in these extracts. 10% ethanolic extracts (diluted with double distilled

water) were used for analysis. These extracts were centrifuged at 15,000 rpm at 4◦C for 20 min. Supernatants were collected and filtered trough 2 μ filter.

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ANTIOXIDANT PROPERTIES OF GRAPES 183

Figure 5 Relationship between total phenolic content and antioxidant activity in terms of a) FRAP assay, b) ferrylmyoglobin/ABTS assay and c) DPPH assay

of different ethanolic extracts of grapes.

Correlation between the Polyphenol Contentsand Antioxidant Activity

From the above data, it was seen that different varieties pos-

sess varying degrees of antioxidant potential in different assays.

This is due to the contribution to antioxidant activity by differ-

ent phenolic and flavonoid compounds present in black, red, and

green grape varieties. The presence of some standard antioxi-

dants was confirmed by HPLC analysis of the extracts from three

selected varieties. To determine the possible correlation between

total phenolic and total flavonoid contents and the respective

antioxidant activities of the grape extracts, we calculated coeffi-

cient of correlation for TEAC values from three different assays

such as FRAP, ABTS, and DPPH assays (Fig. 5).

When the total phenolics are correlated to the TEAC values

obtained by FRAP, ferrylmyoglobin/ABTS and DPPH assays, a

significant correlation was found, with the coefficients of corre-

lation 0.775, 0.99, and 0.982, respectively, for the ethanolic ex-

tracts. Similarly a significant correlation was observed between

the flavonoid content of ethanolic extracts and antioxidant ac-

tivities. The coefficients of correlation were found to be 0.775,

0.947, and 0.952 for FRAP, ABTS, and DPPH assays respec-

tively. However, only a marginal correlation was found between

total phenolic/flavonoid contents and antioxidant activities of

aqueous extracts of grape with coefficient of correlation > 0.3

for all varieties. The TEAC values from FRAP, ferrylmyoglobin

and DPPH assays were tested by analysis of variance (ANOVA)

and found to be statistically significant (P < 0.05).

Phenolic compounds in wine and grapes are linked to the

cardioprotective effect mediated through their antioxidant ac-

tivity. The phenolic compounds in fresh grapes and commercial

grape juices may also be beneficial in the prevention of coronary

heart disease as they also have strong antioxidant activity toward

human LDL oxidation in vitro.34 Interaction of grape pheno-

lics with peroxyl/alkoxyl radicals prevents lipid peroxidation

in terms of decreased formation of conjugated dienes. Thus in

“French Paradox” there is a lower rate of CHD despite consump-

tion of fats, which is normally correlated with high risk of heart

attacks. This “paradox” is partly due to higher consumption of

red wine, derived from grapes. Phenolic antioxidants in grapes

and wines prevent LDL oxidation and decrease platelet aggrega-

tion. Grape juice reduces oxidative stress and DNA damage as

studied by comet assay.35 Dealcoholized red wine or grape juice

decreases atherosclerosis by antioxidant mechanisms entirely

due to polyphenols.36 Higher concentration of phenolics were

reported in red wine grape varieties where the anthocyanins were

the most abundant phenolic compounds.37 Similarly they found

that the black and red cultivars are having higher phenolics and

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184 V. V. KEDAGE ET AL.

flavonoid concentrations than the green cultivars. Red wine con-

tains more phenols than white wine and thus prevents atheroscle-

rosis efficiently.38 Considerable differences were observed in

the polyphenolic content of red and white grape varieties. A

good correlation is found to exist between the total polyphenol

content and their antioxidant power.39 Red grape juice contains

flavonoids such as catechin, epicatechin, quercetin, resveratrol,

caffeic acid, and anthocyanin whereas green grape juice contains

malvidine, cinnamic acid, and low amount of catechin.26,40,41

Flavonoids of red wine and grape juice, have a role in the inhibi-

tion of in vitro platelet activity in a canine model of arterial steno-

sis with intimal damage and periodic thrombosis.42 Grape seed

anthocyanidines are anti-carcinogenic and chemopreventive and

protect against oxidative stress-induced DNA damage and apop-

totic cell death.43 Prevalence of cataract and adult-onset of di-

abetes is higher in India and other tropical countries.44 Grape

seed procyanidines possess antidiabetic properties and prevent

progression of cataract.45 Hence grape varieties like cv. Mango

and cv. Sharad Seedless may help in the prevention/management

of such ailments.

CONCLUSIONS

In conclusion our studies show that among the different va-

rieties of grapes examined, cv. Mango, cv. Sharad (black) and

cv. Manikchaman (green) have the most potent antioxidant ac-

tivities. Their antioxidant activities, assayed at different levels

correlate well with their chemical composition in terms of total

phenolics and flavonoids. These varieties of grapes, if consumed

in adequate amounts, may confer health benefits, especially in

populations prone to CVD. Further studies on LDL oxidation

using these varieties may corroborate the above statement.

ABBREVIATIONS

AA, ascorbic acid; ABTS, 2,2′-azobis-3-ethyl-

benzthiazoline-6-sulfonic acid; AEAC, ascorbic acid equivalent

antioxidant capacity; DPPH, 1,1′-diphenyl-2-picrylhydrazyl;

FRAP, ferric reducing antioxidant power; HPLC, high per-

formance liquid chromatography; ORAC, oxygen radical

absorbance capacity; TEAC, Trolox equivalent antioxidant

capacity; Trolox, 6-Hydroxy 2,5,7,8 tetramethyl chroman

2-Carboxylic Acid

REFERENCES

[1] Willet, W.C., and Stampfer, M.J. 2003. Rebuilding the food pyramid. Sci.Am., 52–59.

[2] Block, G., Patterson, B., and Subar, A. 1992. Fruits, vegetables, and cancer

prevention: a review of the epidemiological evidence. Nutr. Cancer, 18:1–

29.

[3] Surh, Y.J. 2003. Cancer chemoprevention with dietary phytochemicals.

Nature Rev. Cancer, 3:768–780.

[4] Brandt, K., Christesen, L.P., Hansen-Moller, J., Hansen, S.L., Haraldsdottir,

J., Jespersen, L., Purup, S., Kharazmi, A., Barkholt, V., Frokiaer, H., and

Kobaek-Larsen, M. 2004. Health promoting compounds in vegetables and

fruits: A systematic approach for identifying plant components with impact

on human health. Trends Food Sci. Technol., 15:384–393.

[5] Stein, J.H., Keevil, J.G., Wiebe, D.A., Aeschlimann, S., and Folts, J.D.

1999. Purple grape juice improves endothelial function and reduces the

susceptibility of LDL cholesterol to oxidation in patients with coronary

artery disease. Circulation, 100:1050–1055.

[6] Escarpa, A., and Gonzalez, M.C. 2001. An overview of analytical chemistry

of phenolic compounds in foods. Crit. Rev. Anal. Chem., 31:57–139.

[7] Somers, T.C., and Ziemelis, G. 1985. Spectral evaluation of total pheno-

lic components in Vitis vinifera: grapes and wines. J. Sci. Food Agric.,36:1275–1284.

[8] Macheix, J.J., Fleuriet, A., and Billot, J. 1990. Fruit phenolics. Boca Raton

FL: CRC Press.

[9] Ricardo-da-Silva, J.M., Rosec, J.P., Bourzeix, M., and Heredia, N. 1990.

Separation and quantitative determination of grape and wine procyanidins

by HPLC. J. Sci. Food Agric., 53:85–92.

[10] Frankel, E.N., Waterhouse, A.L., and Kinsella, J.E. 1993. Inhibition of

human LDL oxidation by resveratrol. Lancet, 341:1103–1104.

[11] Tussedre, P.L., Frankel, E.N., Waterhouse, A.L., Peleg, H., and German,

J.B. 1996. Inhibition of in vitro human LDL oxidation by phenolic antiox-

idants from grapes and wines. J. Sci. Food Agric., 70:55–61.

[12] Alonso, A.M., Guillen, D.A., Barroso, C.G., Puertas, B., and Gracia, A.

2002. Determination of antioxidant activity of wine byproducts and its

correlation with polyphenolic content. J. Agric. Food. Chem., 50:5832–

5836.

[13] Reddy, K.S. 2004. Cardiovascular disease in non-western countries. N.Engl. J. Med., 350: 2438–2440.

[14] Popkin, B.M., Horton, S., Kim, S., Mahal, A., and Shuigao, J. 2001. Trends

in diet, nutritional status and diet-related noncommunicable diseases in

China and India: The economic costs of the nutrition transition. Nutr. Rev.,59:379–390.

[15] FAO Production Year Book. 1997. Statistics No. 51. Rome. Food and Agri-

culture organization of the United Nations.

[16] Patil, S.G. 1999. Grape beeding for disease resistance. Drakshvrutta Sou-venir, 171–192.

[17] Chadha, K.L. 2001. Grapes. In: Chadha, K.L., Ed. Handbook of Horti-culture. Indian Council of Agricultural Research, New Delhi, India. pp.

182–188.

[18] Wolfe, K., Wu, X., and Liu, R.H. 2003. Antioxidant activity of apple peels.

J. Agric. Food Chem., 51: 609–614.

[19] Luximon-Ramma, A., Bahorun, T., Soobrattee, M.A., and Aruoma, O.I.

2002. Antioxidant activities of phenolic, proanthocyanidin and flavonoid

components in extracts of Cassia fistula. J. Agric. Food Chem., 50:5042–

5047.

[20] Aquino, R., Morelli, S., Lauro, M.R., Abdo, S., Saija, A., and Tomaino,

A. 2001. Phenolic constituents and antioxidant activity of an extract of

Anthurium versicolor leaves. J. Natl. Prod., 64:1019–1023.

[21] Alzoreky, N., and Nakahara, K. 2001. Antioxidant activity of some edi-

ble Yemeni plants evaluated by Ferrylmyoglobin/ABTS assay. Food Sci.Technol. Res., 7:141–144.

[22] Pulido, R., Bravo, L., and Saura-Calixto, F. 2000. Antioxidant activity of

dietary polyphenols as determined by a modified Ferric Reducing Antiox-

idant Power assay. J. Agric. Food Chem., 46:3396–3402.

[23] Devasagayam, T.P.A. 1986. Lipid peroxidation in rat uterus. Biochem. Bio-phy. Acta, 876:507–514.

[24] Devasagayam, T.P.A. 1986. Senescence associated decrease of NADPH-

induced lipid peroxidation in rat liver microsomes. FEBS Lett., 205:246–

250.

[25] Cao, G., and Prior, R.L. 2002. Measurement of oxygen radical absorbance

capacity in biological samples. Meth. Enzymol., 299:50–62.

[26] Yilmaz, Y., and Toledo, R.T. 2004. Major flavonoids in grapes seeds and

skins: antioxidant activity of catechin, epicatechin and gallic acid. J. Agric.Food Chem., 52:255–260.

Dow

nloa

ded

by [

Aub

urn

Uni

vers

ity]

at 0

0:27

23

Sept

embe

r 20

13

ANTIOXIDANT PROPERTIES OF GRAPES 185

[27] Oszmianski, J., and Lee, C.Y. 1990. Isolation and HPLC determination of

phenolic compounds in red grapes. Am. J. Enol. Vitic., 39:259–262.

[28] Sies, H. 1986. Biochemistry of oxidative stress. Angew. Chem. Int. Ed.Engl., 25:1058–1071.

[29] Yoshikawa, T., Toyokuni, S., Yamamoto, Y., and Naito, Y. 2000. In: FreeRadicals in Chemistry Biology and Medicine, OICA International, London.

[30] Krishnaswami, S. 1998. Observations on serial changes in coronary artery

disease in Indians. Curr. Sci., 74:1064–1068.

[31] Bhatnagar, D. 1998. The metabolic basis of increased coronary risk at-

tributed to people from the Indian subcontinent. Curr. Sci., 74:1087–1094.

[32] Hertog, M.G.L., Feskens, E.J.M., Hollman, P.C.H., Katan, M.B., and

Kromhout, D. 1993. Dietary antioxidant flavonoids and risk of coro-

nary heart disease: The Zutphen Elderly Study. Lancet., 342:1007–

1011.

[33] Lachnicht, D., Brevard, P.B., Wagner, T.L., and DeMars, C.E. 2002. Dietary

oxygen radical absorbance capacity as a predictor of bone mineral density.

Nutr. Res., 22:1389–1399.

[34] Frankel, E.N., and Mayer, A.S. 1998. Antioxidants in grapes and grape

juices and their potential health effects. Pharmaceutic. Biol., 36:14–20.

[35] Park, Y.K., Park, E., Kim, J.S., and Kang, M.H. 2003. Daily grape juice

consumption reduces oxidative DNA damage and plasma free radical levels

in healthy Koreans. Mutation Research, 529:77–86.

[36] Vinsen, J.A., Teufel, K., and Wu, N. 2001. Red wine, dealcoholized red

wine and especially grape juice inhibit atherosclerosis in a hamster model.

Atherosclerosis, 156:67–72.

[37] Yi, O.S., Meyer, A.S., and Frankel, E.N. 1997. Antioxidant activities of

grape extracts in a lecithin liposome extracts. J. Am. Oil Chemist’s Soc.,74:1301–1307.

[38] Fragopoulou, E., Antonopoulou, S., Nomikoz, T., and Demopoulos, C.A.

2003. Structure elucidation of phenolic compounds from red/white wine

with antiatherogenic properties. Biochem. Biophys. Acta, 1632:90–99.

[39] Borbalan, A.M.A., Zorro, L., Guillen, D.A., and Barroso, C.G. 2003.

Study of the polyphenol content of red and white grape varieties by liq-

uid chromatography-mass spectrometry and its relationship to antioxidant

power. J. Chromatography, 1012:31–38.

[40] Simonetti, P., Gardana, C., and Pietta, P. 2001. Caffeic acid as biomarker

of red wine intake. Meth. Enzymol., 335:122–130.

[41] Peng, Y., Chu, Q., Liu, F., and Ye, J. 2004. Determination of phenolic

constituents of biological interest in red wine by capillary electrophoresis

with electrochemical detection. J. Agric. Food Chem., 52:153–156.

[42] Folts, J.D. 1998. Antithrombotic potential of grape juice and red wine for

preventing heart attacks. Pharmaceutic. Biol., 36:21–27.

[43] Bagchi, D., Bagchi, M., Stohs, S., Ray, S.D., Sen, C.K., and Preuss, H.G.

2002. Cellular protection with proanthocyanidins derived from grape seeds.

Ann. N. Y. Acad. Sci., 957:260–270.

[44] Balasubramanian, D. 1997. Cataract—Where do we stand? Indian J.Opthal., 45:5–6.

[45] Yamakoshi, J., Saito, M., Kataoka, S., and Tokutake, S. 2002. Procyanindin-

rich extract from grape seeds prevents cataract formation in hereditary

cataractous (ICR/f) rats. J. Agric. Food Chem., 50:4983–4988.

Dow

nloa

ded

by [

Aub

urn

Uni

vers

ity]

at 0

0:27

23

Sept

embe

r 20

13