comparative antioxidant capacities of phenolic compounds measured by various tests

Food Chemistry xxx (2009) xxx–xxx

ARTICLE IN PRESS

Contents lists available at ScienceDirect

Food Chemistry

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

Analytical Methods

Evaluation of spectrophotometric methods for antioxidant compoundmeasurement in relation to total antioxidant capacity in beverages

Jessica Tabart a, Claire Kevers a,*, Joël Pincemail b, Jean-Olivier Defraigne b, Jacques Dommes a

a University of Liège, Plant Molecular Biology and Biotechnology Unit, B22, Sart Tilman, B-4000 Liege, Belgiumb University of Liège, CREDEC, Pathology Tower, B23, Sart Tilman, B-4000 Liege, Belgium

a r t i c l e i n f o

Article history:Received 22 October 2008Received in revised form 27 July 2009Accepted 14 October 2009Available online xxxx

Keywords:AntioxidantFlavonolsFlavonoidsAnthocyaninsFlavanolsAscorbic acid

0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.10.031

* Corresponding author. Tel.: +32 43 66 38 46; fax:E-mail addresses: [email protected]

(C. Kevers), [email protected] (J. Pincemail)(J.-O. Defraigne), [email protected] (J. Dommes).

Please cite this article in press as: Tabart, J., et aantioxidant capacity in beverages. Food Chemist

a b s t r a c t

The validity of different colorimetric methods used to quantify various families of antioxidant com-pounds was evaluated with standard compounds. The colorimetric tests for global evaluation of flavo-noids, anthocyans, and flavanols were found generally unreliable, as reactions could be different forindividual compounds within a family (anthocyanins or flavonols or flavan-3-ols) and not specific toone family. In the flavonoid test, for example, flavonols reacted very well, anthocyanins did not react,and flavanons reacted only slightly. The same methods were applied also to beverages known for theirantioxidant content (apple, orange, grape, and vegetable juices, ice tea, and red wine) and the data werecompared with the results of HPLC analysis of specific compounds. The values obtained in a colorimetrictest were generally higher than the sum of the values obtained for the corresponding individualcompounds by HPLC analysis, mainly because other compounds can interfere with the colorimetric tests.For example, in wine, anthocyanin concentrations obtained by colorimetric test were 9068 ± 1407 lmol/100 ml (mean ± SEM), higher than the sum of the six main anthocyanidins detected by HPLC, only41 lmol/100 ml. The relative antioxidant capacity values determined for beverages on the basis of color-imetric tests could exceed by far the values previously measured in radical-scavenging tests (for instance,the antioxidant capacity attributable to anthocyans in wine on the basis of the colorimetric test was 50times higher than the total antioxidant capacity measured by the ORAC assay). In conclusion, colorimetrictests for flavonoids, anthocyans, and flavanols appeared generally unreliable for estimating their contentand thus the antioxidant capacity reliable to these compounds.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The study of phytochemicals as bioactive food components is ofmajor interest. Ecological and epidemiological studies indicate thathigh consumption of fruits and vegetables has protective effectsagainst degenerative diseases (e.g. cancer, cardiovascular disease)in which development oxidative stress – resulting in enhanced freeradical activity – plays a key role (Arts & Hollman, 2005). Inaddition to supplying nutrients such as mineral salts, trace ele-ments, fibers, folates, etc., diets rich in fruits and vegetables supplyantioxidants that can limit oxidative damage to lipids, DNA, andproteins and which may be largely responsible for the benefits as-cribed to such diets. Although much of the antioxidant capacity ofa fruit or vegetable can be attributed to compounds such asvitamins C and E, carotenoids, and glutathione, high antioxidantactivities have also been recorded for members of the large poly-

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+32 43 66 38 72.(J. Tabart), [email protected], [email protected]

l. Evaluation of spectrophotomry (2009), doi:10.1016/j.foodch

phenol family (which includes flavonoids such as flavones, isoflav-ones, flavanones, anthocyanins, catechin, and isocatechin),frequent components of the human diet (Tabart, Kevers, Pincemail,Defraigne, & Dommes, 2009; Villano, Fernandez-Pachon, Troncoso,& Garcia-Parrilla, 2005).

Mullen, Marks, and Crozier (2007) suggest that protective ef-fects against a number of degenerative diseases could be enhancedby regular consumption of a combination of juices rich in pheno-lics, so as to provide an abundance and diversity of such com-pounds. In addition to juices derived from purple grapes,grapefruit, cranberries, and apples, red wine and green tea are alsoviewed as exceptional dietary sources of various classes of phenols,including benzoic and cinnamic acid derivatives, flavan-3-ols,flavonols, and anthocyanins (Bravo, 1998).

The phenolic compound content of plant materials can be deter-mined either by separation methods (e.g. high-performance liquidchromatography, gas chromatography, capillary zone electropho-resis) as a set of individual substances or by spectrophotometricassays detecting specific groups of chemically similar reactivecompounds. Chromatographic determination is very precise,accurate, and informative, but identifying all phenolic compounds

etric methods for antioxidant compound measurement in relation to totalem.2009.10.031

http://dx.doi.org/10.1016/j.foodchem.2009.10.031

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https://www.researchgate.net/publication/8089851_Polyphenols_and_disease_risk_in_epidemiologic_disease?el=1_x_8&enrichId=rgreq-910aa093-34c9-4fd3-9fe8-dbee3b354253&enrichSource=Y292ZXJQYWdlOzIyMjg0MzA4NTtBUzoyMTI2NzIzNTczNzYwMDJAMTQyNzcxNjQ0OTkwMQ==

2 J. Tabart et al. / Food Chemistry xxx (2009) xxx–xxx

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in a specific plant material can be daunting in terms of time con-sumed and analysis costs (Stratil, Klejdus, & Kuban, 2006).

One aim of the present study was to test the validity of existingcolorimetric methods for evaluating various families of antioxidantcompounds, mainly phenolics, using standard antioxidant com-pounds (flavonoids, anthocyanins, catechins, ascorbic acid, andglutathione). Another was to apply the same methods to severalbeverages (apple, orange, grape, and vegetable juices, ice tea, andred wine) and to compare the results with those obtained by HPLCanalyses. We have further sought to relate the measured antioxi-dant levels in beverages to previously established antioxidantcapacity values (Tabart et al., 2009), with a view to determininghow different polyphenol families contribute to the total antioxi-dant capacity.

2. Materials and methods

2.1. Materials

Chemicals. Standards of flavonols and anthocyanins were ob-tained from Extrasynthese (Genay, France). Flavanons, flavan-3-ols, phenolic acids, ascorbic acid, glutathione, and Trolox [(±)6-hy-droxy-2,5,7,8-tetramethylchromane-2-carboxylic acid] were pur-chased from Sigma Chemical Co. (St. Louis, MO).

Beverages. Apple and orange juices (Minute Maid�, Coca-ColaEnterprises, Brussels, Belgium), purple grape juice (No. 1, Carre-four, Brussels, Belgium), vegetable juice V8 (Cambells Foods Bel-gium, Puurs, Belgium), ice green tea� (Lipton, Unilever Belgium,Brussels, Belgium), red wine (Merlot 2004, Vallé des Rappel, Chile)were tested.

2.2. Sample preparation

Stock standard solutions (1 mmol) in methanol were preparedfor each standard and appropriate dilutions were done for eachtype of measurement, with the specific solvent used for polyphenolquantification (colorimetric tests, HPLC).

The various beverages were centrifuged (Beckman CoulterAvanti JE) at 17,000g for 15 min and at 20 �C. The resultingsupernatants were used directly as final samples except for wine,in which case alcohol was removed by evaporation under reducedpressure at 38 �C and the volume adjusted with distilled wateras proposed by Fernandez-Pachon, Villano, Troncoso, and Garcia-Parrilla (2006) to avoid ethanol interference.

The extracts were hydrolyzed before measurement of flavonolaglycons and anthocyanins, as proposed by Häkkinen, Kärenlampi,Jeinonen, Mykkänen, and Torronen (1998). In short, extracts wereincubated for one hour in the presence of 1.2 M HCl, MeOH(50%), and 300 mg/L ascorbic acid.

2.3. Colorimetric tests

Total flavonoid content was measured as in Lamaison and Carnat(1990). Appropriately diluted extracts (1 mL) were mixed with1 mL reagent (AlCl3�6H2O, 2% in methanol). The absorbance at430 nm (spectrophotometer Kontron Unikon 931) was measured10 min later. Quercetin was used as standard, and results are ex-pressed in lmol quercetin equivalents (QE) per mmol standardor per 100 mL beverage.

Total anthocyan quantification was performed by the pH-differ-ential method (Nielsen, Haren, Magnussen, Dragsted, & Rasmus-sen, 2003). The extract was diluted in a pH 1.0 solution (0.1 MHCl, 25 mM KCl) and in a pH 4.5 solution (0.4 M CH3COONa). Theabsorbance of each mixture was then measured at 535 and700 nm against distilled water. The value (Abs535 � Abs700)pH1.0 �

Please cite this article in press as: Tabart, J., et al. Evaluation of spectrophotomantioxidant capacity in beverages. Food Chemistry (2009), doi:10.1016/j.foodch

(Abs535 � Abs700)pH4.5 corresponds to the absorbance due toanthocyans. Calculation of anthocyan concentrations was basedon a cyanidin 3-glucoside (molar extinction coefficient 25.965cm�1, molecular weight 449.2 g mol�1). Results are expressed inlmol cyanidin 3-glucoside equivalents per mmol standard or per100 mL beverage.

Total flavanol content was evaluated by the vanillin assay(Nakamura, Tsuji, & Tonogai, 2003), in which one molecule of van-illin reacts with one molecule of flavanol to produce a red chromo-phore. The conversion was monitored as an increase in theabsorbance at 500 nm. One volume of sample diluted in methanolwas mixed with 2.5 volumes of vanillin (1% in methanol) and 2.5volumes of HCl (9 M in methanol). The mixture was incubatedfor 20 min at 35� before analysis. For each sample, a blank wasused in which the vanillin solution was replaced with methanolalone. A standard curve was constructed with catechin.

Reduced ascorbic acid (AA) was measured by the 2,6-dichloroin-dophenol (DCIP) method of the Association of Vitamin Chemists(1961). In this assay, briefly, each molecule of vitamin C convertsa molecule of DCIP into a molecule of DCIPH2, and this conversionis monitored as a decrease in the absorbance at 520 nm. A standardcurve was prepared with a series of known AA concentrations. Onemilliliter of diluted sample (in 5% metaphosphoric acid) or ascorbicacid calibration solution was mixed with 500 lL 10% metaphos-phoric acid. With 600 lL of this mixture were mixed 300 lL citricacid, citrate buffer (pH 4.15) and 300 lL DCIP (0.1 mg mL�1). Opti-cal density blanching was used; for each sample, the blank valuewas determined after addition of 60 lL AA (1 mg mL�1) so as tomeasure the inference due to the sample color. Results are ex-pressed in lmol AA per mmol standard or per 100 mL beverage.

Reduced glutathione (GSH) was oxidized with 5,50-dithio-bis (2-nitrobenzoic acid) (DTNB) to yield oxidized glutathione (GSSG) and2-nitro-5-thiobenzoic acid (TNB). GSSG was reduced to GSH by ac-tion of the highly specific glutathione reductase and NADPH. TNBformation was monitored as the rate change in absorbance at412 nm and was proportional to total glutathione (GSH + GSSG).Oxidized glutathione was determined after removal of reducedglutathione with N-ethylmaleimide. GSH was determined by sub-tracting GSSG from total glutathione (Franck et al., 1998).

2.4. Separation and measurement of compounds by HPLC

Analyses of extracts were performed in a liquid Elite LachromMerck Hitachi chromatograph equipped with an L2450 photodiodearray detector (sampling period: 400 ms, spectral bandwidth:4 nm). Separation was carried out with a LiChroCART steel car-tridge (250 mm � 4.6 mm) (Merck) filled with 5-lm RP 18 parti-cles and maintained thermostatically at 30 �C (for flavonols andflavan-3-ols) or 40� (for anthocyanidins).

For flavonol aglycone analysis of hydrolyzed extracts, the mobilephase was a linear gradient of water–acetonitrile (50:50, adjustedto pH 1.8 with perchloric acid) and water–acetonitrile (95:5, ad-justed to pH 1.8 with perchloric acid), at a flow rate of 1.2 mL/min as previously described by Tabart, Kevers, Pincemail, Defraig-ne, and Dommes (2006). Spectra were recorded at 365 nm.

For anthocyanidin analysis of hydrolyzed extracts, the mobilephase was a gradient of water–acetonitrile–formic acid (87:3:10to 40:50:10) at a flow rate of 0.8 mL/min as previously describedby Dutruc-Rosset (2003). Spectra were recorded at 518 nm.

For flavan-3-ol analysis, the mobile phase described by Sharma,Gulate, Ravindranath, and Kumar (2005) was modified. It was com-posed of acetonitrile (90%), orthophosphoric acid (0.1%) and water.A flow gradient was used: 0.4 mL min�1 for 3 min, a linear decreaseto 0.3 mL min�1 at 10 min and to 0.2 mL min�1 at 13 min, a steadystate to 25 min, followed by a linear increase to 0.4 mL min�1 at35 min. Spectra were recorded at 230 nm.



https://www.researchgate.net/publication/222843085_Comparative_antioxidant_capacities_of_phenolic_compounds_measured_by_various_tests?el=1_x_8&enrichId=rgreq-910aa093-34c9-4fd3-9fe8-dbee3b354253&enrichSource=Y292ZXJQYWdlOzIyMjg0MzA4NTtBUzoyMTI2NzIzNTczNzYwMDJAMTQyNzcxNjQ0OTkwMQ==

J. Tabart et al. / Food Chemistry xxx (2009) xxx–xxx 3

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All samples were prepared in triplicate and each sample analy-sis was performed in triplicate. All results presented are the means(±SEM). One-way analysis of variance (ANOVA-1) was used todetermine significant differences between each standard com-pound or each beverage in specific spectrophotometric tests. ANO-VA-1 was also applied to determine significant difference betweenthe flavonoid content in various beverages tested by HPLC. Differ-ences at P < 0.05 were considered significant. Statistical analysiswas performed using Microsoft Excel (Microsoft, Roselle, IL).

3. Results

3.1. Measurement of standard compounds by colorimetric tests

Antioxidant compounds (flavonols, anthocyanins, flavanons,flavan-3-ols, several phenolic acids, ascorbic acid, and reduced

Fla

0

500

1000

1500

2000

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Q K M

Q-R K-G C D V

Pel

Peo

Pet

C-G

Flavonols Anthocyanins

µmol

EC

a/m

mol

An

0

500

1000

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2000

Q K M

Q-R K-G C D V

Pel

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µmol

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-G/m

mol

Fla

0200400600800

10001200

Q K M

Q-R K-G C D V

Pel

Peo

Pet

C-G


µmol

EQ

/mm

ol a a

a

a

a a

a

aaaa b,a

b

b b,d

b b b

c

Fig. 1. Detection of standard compounds (1 mmol) determined by the flavonoid (A, lmcolorimetric tests (error bars represent +SEM). AA: ascorbic acid; C: cyanidin; C-G: cyanCa: catechin; CA: chlorogenic acid; D: delphinidin; D-G: delphinidin-3-O-glucoside; EAgallate; GA: gallic acid; GCa: gallocatechin; Gl: reduced glutathione; HE: hesperidin; Kpelargonidin; Peo: peonidin; Pet: petunidin; Q: quercetin; Q-R: rutin; V: malvidin. Lettercompounds illustrated within graph.


glutathione) were evaluated by colorimetric assays purporting tomeasure specifically the total flavonoid, anthocyan, flavanol, ascor-bic acid, or glutathione content. As shown in Fig. 1, assay perfor-mances varied according to the compound studied.

Flavonoids. The reactions of the tested standards are illustratedin Fig. 1A. Flavonols reacted well in this test described by Lamaisonet al. (1990). The reaction was quite similar for three aglycons(quercetin, kampferol and myricetin) and the quercetin glycoside(rutin). Kaempferol-3-O-glucoside was less reactive. Slight reactiv-ity (less than 10% of that observed with quercetin) was seen withthe flavanons, two phenolic acids (chlorogenic acid and ellagicacid), and glutathione. Other groups measured did not cross-reactwith this test.

Anthocyans. The various anthocyanins tested reacted differentlywith this method (Fig. 1B). Among the anthocyanidins, the stron-gest reaction was observed with delphinidin and malvidin. This

vanols

C-R

C-G

a

D-G HE NA

Ca

ECa

EGC

a

GC

a

EGC

aG GA

CA EA AA Gl

Flavanons Flavan-3-ols Phenolicacids

Others

thocyans

C-R

C-G

a

D-G HE NA

Ca

ECa

EGC

a

GC

a

EGC

aG GA

CA EA AA Gl


Others

vonoids

C-R

C-G

a

D-G HE NA

Ca

ECa

EGC

a

GC

a

EGC

aG GA

CA EA AA Gl


Others

A

C

B

a

aaaa b,ab,ab,a

b,d

b

c c c c c

d d d d d

d

d

c c c

e

ol EQ/mmol), anthocyan (B, lmol EC-G/mmol), and flavanol (C, lmol ECa/mmol)idin-3-O-glucoside; C-Ga: cyanidin-3-O-galactoside; C-R: cyanidin-3-O-rutinoside;: ellagic acid; ECa: epicatechin; EGCa: epigallocatechin; EGCaG: epigallocatechin

: kaempferol; K-G: kaempferol-3-O-glucoside; M: myricetin; NA: naringenin; Pel:s (a, b, c, d and e) indicates a significant difference determined by (P < 0.05) between




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response was more than twice as high as the difference of colora-tion observed with cyanidin, pelargonidin, and peonidin. Petunidinwas not detected. The glycosides of cyanidin reacted more stronglythan cyanidin, whereas the glycoside of delphinidin reacted lessstrongly than delphinidin. The other standards tested did not react,apart from a very slight reaction of the flavanon standards(amounting to less than 0.5% of the reactivity of cyanidin-3-O-glucoside).

Flavanols. With this assay (Fig. 1C), flavan-3-ols showed thestrongest reaction. Epicatechin showed higher reactivity than cat-echin, epigallocatechin, and epigallocatechin gallate. Among theother compounds, the intensity of the reaction varied from 0 (quer-cetin and some anthocyanins) to 20% of the intensity of the cate-chin reaction. For the other flavonols, the flavanons, the phenolicacids, ascorbic acid, and glutathione, the reaction intensity was be-tween 10% and 20% of that of catechin.

Ascorbic acid. Only ascorbic acid was significantly detected withthe specific method used. A very weak reaction was also observedwith myricetin (less than 0.5% of the intensity of the ascorbic acidreaction (data not shown)).

Glutathione. The method used was very specific to reduced glu-tathione. No reaction was recorded with any other standard tested(data not shown).

3.2. Measurement of antioxidant compounds in beverages bycolorimetric tests

Apple, orange, grape, and vegetable juices, ice green tea, and redwine were tested for their content in various antioxidant com-pounds by the different colorimetric methods described above(Fig. 2).

Flavanols

0

50100

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juice

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ejui

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erlot)

µmol

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Flavonoids

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30

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juice

Oran

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ejui

ce

Vegeta

ble ju

ice

Ice Tea

Gree

n

Wine (M

erlot)

µmol

EQ

/100

mL

A

C

Fig. 2. Evaluation of total flavonoid (A, lmol EQ/100 mL), anthocyan (B, lmol EC-G/100 mvarious beverages by colorimetric tests (error bars represent +SEM). X10 on (B) indicate


Flavonoids. A high flavonoid content was observed in red wine(Fig. 2A), three times higher than for ice tea and orange juice. Prac-tically no flavonoids were detected in apple, grape, or vegetablejuice.

Anthocyans. A high anthocyan content was measured in redwine, at least 10 times higher than those recorded in the other bev-erages (Fig. 2B). Grape juice also showed a significant anthocyancontent, exceeding the level recorded for apple juice and ice greentea. No anthocyans were found in orange or vegetable juice.

Flavanols. As observed for anthocyans, red wine showed a highflavanol content, 10 times higher than those of grape juice and icegreen tea (Fig. 2C). No flavanols were detected in apple, orange, orvegetable juice.

Ascorbic acid. All the beverages tested contained ascorbic acid,but orange juice showed the highest level, followed by ice greentea and red wine (Fig. 2D). The ascorbic acid content of the apple,grape, and vegetable juices was lower.

Glutathione. This compound was not found in any beverage(data non shown).

3.3. Separation and measurement of antioxidant compounds

The aglycons of flavonols and anthocyanins were analyzed byHPLC after hydrolysis of the beverage. Flavan-3-ols were also sep-arated by HPLC and measured in each beverage.

Flavonols. Quercetin was detected only in vegetable juice andred wine, in similar amount (Fig. 3). Red wine also showed a sim-ilar concentration of kaempferol. Myricetin was not detected inany beverage.

Anthocyanidins. Some anthocyanidins were detected in alltested beverages, at various levels (Fig. 3). Red wine showed the

Ascorbic acid

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Apple

juice

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/100

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Anthocyans

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juice

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erlot)

µmol

EC

-G/1

00 m

L X 10

B

D

L), flavanol (C, lmol ECa/100 mL) and ascorbic acid (D, lmol AA/100 mL) content ins that anthocyan content of wine must be multiply by 10.



Flavonols

0

10

20

30

40

50

Apple juice

Orange juice

Grape juice

Vegetablejuice

Ice TeaGreen

Wine(Merlot)

nmol

/100

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Q

K

M

Anthocyanidins

0

2

4

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8

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Apple juice

Orange juice Grape juice

Vegetablejuice

Ice TeaGreen

Wine (Merlot)

µmol

/100

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D

Pet

C

Peo

Pel

V

Flavan-3-ols

0

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Apple juice

Orange juice

Grape juice

Vegetablejuice

Ice TeaGreen

Wine(Merlot)

µmol

/100

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Ca

ECa

EGCa

GCa

EGCaG

Fig. 3. HPLC analyses of flavonol aglycons (A, nmol/100 mL), anthocyanidins (B, lmol/100 mL) and flavan-3-ols (C, lmol/100 mL) in some beverages (error bars represent+SEM).


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highest concentrations of all anthocyanidins except peonidin. Allthe anthocyanidins (except pelargonidin) were found in grapejuice, at levels considerably lower than in red wine. In the otherbeverages, all anthocyanidin contents were very low, but in eachcase delphinidin and petunidin were the most abundant.

Flavan-3-ols. The five species measured were present in icegreen tea and red wine, most levels being higher in the former thanin the latter (except the epicatechin and gallocatechin levels, whichwere the same in both beverages) (Fig. 3). Only gallocatechin wasfound in grape, vegetable, and orange juice. In orange juice, epigal-


locatechin was also present. None of these flavan-3-ols was de-tected in apple juice.

4. Discussion

The colorimetric methods here used to quantify ascorbic acid(DCIP method) and glutathione proved to be very specific. Antho-cyan quantification by the pH-differential method also appearsvery specific to anthocyans and has been used to detect them




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(Gonzales-Rodriguez, Perez-Juan, & Luque de castro, 2002), but dif-ferent anthocyans respond very differently. In contrast, the methodof Lamaison et al. (1990) for the measurement of flavonoids ap-pears adequate only for flavonols. Some other flavonoids, such asflavanons, could also be detected but with lower sensitivity.

Anthocyans and flavan-3-ols were not detected. It is worthmentioning that the color intensifies with a higher degree ofhydroxylation as a result of Al3+ complexation (Adelmann et al.,2007). Among the standards tested, the flavan-3-ols reacted verywell with vanillin (total flavanol colorimetric test), but a lot ofother compounds, such as flavanons, also reacted to a lesser extent(Sarkar & Howarth, 1976). Other polyphenolics reacting in the van-illin–HCl assay include procyanidins and dihydrochalcone becauseof their structure (a single bond at the 2.3 position and free meta–ortho-hydroxy groups on the ring, Nakamura et al., 2003; Sarkar &Howarth, 1976). Other structural factors influencing the reactionsensitivity include the degree of polymerization of proanthocyani-din (sensitivity increases with increasing polymerization) and thedegree of esterification of flavan-3-ols (sensitivity decreases withincreasing esterification) (Nakamura et al., 2003; Sun, Ricardo-da-Silva, & Spronger, 1998).

Separation by HPLC followed by absorbance measurement isneeded to evaluate concentrations of the various compoundscorrectly. A comparison of Figs. 2 and 3 reveals great differencesbetween the results obtained by colorimetric tests and HPLC anal-ysis. Although the flavonoid test used here detects mainly flavo-nols, the levels of flavonols separated and measured by HPLCwere too low to account for the results of this test, which must

Table 1The present measurements realized (content, columns 2 and 3) of each compoundfound in wine by colorimetric tests (a) or by HPLC (b) were converted into theirrelative antioxidant capacity (column 4–7) by using the antioxidant value previouslyobtained (Tabart et al., 2009) for each standard of phenolic compounds by twomethods: DPPH and ORAC (lmol ETrolox/100 mL) assays. This calculation permits anevaluation of the contribution of the different compounds measured to the totalantioxidant capacity of wine (per 100 ml). Nd, not detectable, –, not determined.

Compounds Content(lmol/100 mL)

Calculated values of relativeantioxidant capacity

(DPPH lmolET/100 mL)

(ORAC lmolET/100 mL)

(a) (a) + (b) (a) (a) + (b)

Glutathione nd (a) nd nd nd ndAscorbic acid 84.86 (a) 46 46.49 43 42.67Flavonoids (Q) 24.86 (a) 22 – 101 –Anthocyans (C-G) 9068.56 (a) 5831 – 64160 –Flavanols (Ca) 290.71 (a) 235 – 2245 –

Flavonols (3)Quercetin 0.04 (b) – 0.04 – 0.16Myricetin nd (b) – nd – ndKampferol 0.04 (b) – 0.03 – 0.25

Anthocyanidins (6)Delphinidin 8.86 (b) – 13.30 – 32.86Petunidin 9.03 (b) – 5.81 – 63.91Cyanidin 9.71 (b) – 5.29 – 41.52Peonidin 0.62 (b) – 0.50 – 3.35Pelargonidin 5.29 (b) – 2.60 – 30.08Malvidin 7.71 (b) – 11.10 – 43.96

Flavan-3-ols (5)Catechin 4.17 (b) – 3.37 – 32.17Epicatechin 2.44 (b) – 2.52 – 12.13Epigallocatechin 12.34 (b) – 18.72 – 37.77Gallocatechin 13.95 (b) – 119.72 – 112.40Epigallocatechin gallate 0.47 (b) – 1.75 – 1.56

Total calculated 6134 231.24 66,999 454.79

Total antioxidantcapacity measured(Tabart et al., 2009)

663 1100


thus have detected additional compounds (unknown or nottested). In wine (Table 1), for instance, the total flavonoid contentmeasured was 25 lmol/100 ml, as opposed to only 0.08 lmol/100 ml flavonols (sum of relevant flavonol aglycons: quercetinand kampferol) detected by HPLC analysis. For anthocyans like-wise, the colorimetric test yielded higher values than HPLCanalysis. For wine (Table 1), the total anthocyan content was9069 lmol/100 ml according to the colorimetric test, but only41 lmol/100 ml anthocyanidins (sum of the six main anthocyani-dins detected by HPLC) were found. For flavanols a major differ-ence was again observed, but by HPLC we separated only the freeflavan-3-ols. The colorimetric test is not specific to these free com-pounds; tannins can also be detected (Nakamura et al., 2003).

In a previous paper (Tabart et al., 2009), the antioxidant capac-ity of the different standards and beverages used in the presentstudy was determined by two different assays: one measuringscavenging of the 2,2-diphenyl-1-picryhydrazil (DPPH) radicaland one measuring the oxygen radical-scavenging capacity(ORAC). With these values of the antioxidant capacity of standardcompounds, we have converted the present measurements of eachcompound found in beverages (by colorimetric tests or by HPLC)into antioxidant capacity. Thus, by integrating the antioxidantcapacities of polyphenolic standard compounds with the presentmeasurements of specific compounds by colorimetric tests andHPLC analyses, we have tried to determine how each type of com-pound contributes to the total antioxidant capacity of beverages.The results of this conversion (amount to antioxidant capacity)were indicated in Tables 1 and 2 for wine, orange juice, and tea,respectively.

The results based on the colorimetric tests are quite surprising.In wine (Table 1), ascorbic acid (46 and 43 lmol ET/100 ml mea-sured, respectively, by DPPH and ORAC assays) and flavonoids(22 and 101 lmol ET/100 ml measured, respectively, by DPPHand ORAC assays) can together account for 10 (DPPH assay) or13% (ORAC assay) of the antioxidant capacity, depending on themethod used to determine this capacity. Anthocyans alone (in C-G equivalents) seem to account for an antioxidant activity severaltimes higher than the capacity determined by either method. Thisis also the case when the flavanol results are converted to an anti-oxidant capacity on the basis of the ORAC assay. Addition of thecalculated values gives abnormally high results as compared tothose previously measured by the DPPH or ORAC assay. SpecificHPLC analysis of different compounds (flavonols, anthocyanidins,flavan-3-ols) present in wine, on the other hand, shows that 35%or 41% of the total wine antioxidant capacity (as determined,respectively, by the DPPH or ORAC assay) can be attributed to fla-van-3-ols, anthocyanins, and ascorbic acid (the most active com-pounds), followed by flavonols. In red wine, Fernandez-Pachon,Villano, Garcia-Parrilla, and Troncoso (2004) have shown that50% of the total radical-scavenging activity of red wines (ABTS,DPPH methods) can be attributed to polymeric phenolic com-pounds. Phenolic acids and some other compounds (Fernandez-Pachon et al., 2006) such as proanthocyanidins (Cimino, Sulfaro,Trombetta, Saija, & Tomaino, 2007) can explain the difference.

In orange juice (Table 2), the ascorbic acid content can explain50% of the antioxidant capacity measured by the DPPH assay, butonly 13% of that measured by the ORAC assay. The two methods,based on different mechanisms (DPPH: reducing capacity andORAC: peroxyl radical-scavenging capacity) can be used to evalu-ate the antioxidant capacity of compounds or beverages, but itwas known that the different methods could give very differentresults, sometime widely divergent (Tabart et al., 2009). Bycolorimetric assays, only flavonoids were detected while by HPLCanalysis, anthocyanidins and flavan-3-ols were detected also, thusaccounting for part of the antioxidant capacity. In all, ascorbicacid, anthocyanidins, and flavan-3-ols can explain 85% of the



Table 2Conversion of the present measurements into their relative antioxidant capacity(column 4–7) as explained in Table 1 and evaluation of the contribution of thedifferent compounds measured to the total antioxidant capacity of orange juice andice green tea. The relative antioxidant capacity corresponding to the concentrationobtained for each flavonol, anthocyanidin and flavan-3-ol was calculated and theresults were summed per family. Nd, not detectable, –, not determined.

Compounds Contentlmol/100 mL

Calculated values of relativeantioxidant capacity

(DPPH lmolET/100 mL)

(ORAC lmolET/100 mL)

(a) (a) + (b) (a) (a) + (b)

Orange juiceAscorbic acid 178.14 97 97.37 89 89.36Flavonoids (Q) 8.64 8 – 35 –Anthocyans (C-G) nd nd – nd –Flavanols (Ca) nd nd – nd –Flavonols (3) nd – nd – ndAnthocyanidins (6) 0.62 – 0.74 – 3.01Flavan-3-ols (5) 10.76 – 67.74 – 69.29

Total calculated 105 165.85 124 161.66


192 690

Ice green teaAscorbic acid 137.40 75 74.94 69 68.77Flavonoids (Q) 6.69 6 – 27 –Anthocyans (C-G) 214.97 138 – 1521 –Flavanols (Ca) 20.30 16 – 157 –Flavonols (3) nd – nd – ndAnthocyanidins (6) 0.68 – 0.70 – 3.98Flavan-3-ols (5) 53.53 – 185.58 – 275.56

Total calculated 235 261.22 1774 348.31


497 537


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antioxidant capacity measured by the DPPH assay but only 23% ofthat measured by the ORAC assay. Flavanones (another type of fla-vonoid) and hydrocinnamic acids, not measured, are present in or-ange juice (Klimczak, Malecka, Szlachta, & Gliszczynka-Swiglo,2007) and may explain part of the difference.

For tea (Table 2) as for wine, there is a discrepancy between thehigh level of anthocyans measured by the colorimetric test and thelow anthocyanidin levels measured by HPLC analysis. The differentspecificity of the anthocyanins (anthocyanidins and glycolysedforms) for the colorimetric test (Fig. 1) could partially explainedthis discrepancy while in HPLC analysis, only the aglycon forms(after hydrolysis) were measured. This discrepancy leads to a muchhigher calculated ORAC-based antioxidant capacity value than pre-viously measured in ice green tea. In contrast, the colorimetric testyielded a flavanol level lower than the summed flavan-3-ol levelsdetermined by HPLC analysis. Tea is known for its high contentin flavan-3-ols and for their high antioxidant capacity (Sultanaet al., 2008).

In conclusion, colorimetric tests for flavonoids, anthocyans, andflavanols appear generally unreliable for estimating the antioxi-dant capacity. Compounds of the same type (anthocyanins, flavo-nols, or flavan-3-ols) react differently in some tests. In theflavonoid test, for example, flavonols reacted very well, anthocya-nins failed to react, and flavanons reacted only slightly. Other col-orimetric tests (for anthocyans and flavanols) show a lack ofspecificity for a single type.

The Folin–Ciocalteu method can be used to assay the total phe-nolic compound content but it also measures the total reducingcapacity of a sample. Moreover, this method is not specific to phe-nolic compounds. Many non-phenolic compounds, such as ascorbic


acid (Tabart et al., 2009) and saccharides present in fruits and veg-etables, can reduce the Folin–Ciocalteu reagent (Stratil, Klejdus, &Kuban, 2007). When HPLC analysis was applied to beverages, thelevels obtained by summing the levels of individual compoundswere very different from those determined by colorimetry, gener-ally much lower. The values given by the colorimetric tests weregenerally too high, suggesting that other compounds can react inthe tests. Likewise, the relative antioxidant capacity values deter-mined for beverages on the basis of colorimetric tests could exceedby far the values actually measured in radical-scavenging tests (forinstance, the antioxidant capacity attributable to anthocyans inwine on the basis of the colorimetric test was 50 times higher thanthe total antioxidant capacity measured by the ORAC assay).

Investigations into the antioxidant capacity of foods should takeinto account the chemical natures and concentrations of the vari-ous antioxidants present in each food matrix, since the total anti-oxidant capacity of a food may reflect the combined activities ofdiverse antioxidants rather than being attributable to any particu-lar phenolics. Yet the complexity of the food composition makes itcostly and inefficient to separate each antioxidant compound andto study it individually. It is also necessary to consider the possibil-ity of synergistic or antagonistic interactions among antioxidantcompounds in a food mixture. For instance gallic acid and epigallo-catechin gallate, major antioxidants in tea, show strong antioxi-dant activity but can also act as pro-oxidants (Johnson & Loo,2000).

It is important to have the best analytical tools for determiningthe antioxidant content of any food matrix, as the agro-food indus-try will soon have to provide strong scientific evidence in supportof allegations regarding the health benefits conferred by foods con-taining various antioxidants. The present study shows that resultsmay be significantly different according to the method of investi-gation. Scientists must therefore focus on developing standardizedmethods for antioxidant determination in food.

Acknowledgments

We thank HEPL-Rennequin Sualem and J. Bleret for technicalassistance. J.T. gratefully acknowledges the support of the Luxem-bourg Ministry of National Education. The skilful assistance of theAPE personnel (provided to CEDEVIT by the Regional Governmentof Wallonia) was greatly appreciated.

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