Stability of the total antioxidant capacity and total polyphenol content of 23

commercially available vegetable juices before and after in vitro digestion measured
by FRAP, DPPH, ABTS and FolinCiocalteu methods
Peter C. Wootton-Beard, Aisling Moran, Lisa Ryan
Functional Food Centre, Oxford Brookes University, Gipsy Lane, Oxford, UK
a b s t r a c t a r t i c l e i n f o
Article history:
Received 18 August 2010
Accepted 22 October 2010
Keywords:
Antioxidants
Polyphenols
Bioaccessibility
Vegetable juice
Beetroot
In vitro digestion
Vegetables are known to contain a wide variety of antioxidants which may provide protection against the
development of a number of disease states. Recently there has been a large increase in the number of
vegetable juices which have become commercially available. The objective of the present study was to analyse
the total antioxidant capacity of 23 commercially available vegetable juices [via Ferric Reducing Antioxidant
Power (FRAP), 2, 2-diphenyl-1-picrylhydrazyl (DPPH

), 2, 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS
+
) and FolinCiocalteu Reagent (FCR) for total polyphenols] and to determine the stability of the
antioxidant capacity following an in vitro digestion procedure using the same methods. All 23 juices were
signicant sources of antioxidants both in terms of total antioxidant capacity and total polyphenols, although
results varied considerably between the juices [13699500 mol/L (FRAP), 57.8100% inhibition of DPPH

,
10.990.7% inhibition of ABTS
+
and 4493025 g ferulic acid equivalents/mL for FCR]. Beetroot juice
displayed the highest level of total antioxidants and total polyphenols compared to the other juices which
were analysed (tomato, carrot, mixed vegetable, mixed fruit and vegetable). The antioxidant capacity of the
juices remained high throughout the in vitro digestion.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Vegetable juices are a good source of many biologically active
antioxidant compounds (Kiefer et al., 2004). Antioxidants found in
vegetable juices such as tomato, carrot, beetroot and mixed vegetable
are thought to contribute towards positive health outcomes in cases of
cardiovascular disease (Kohlmeier & Hastings, 1995), cancer (Rao &
Agarwal, 2000; van Poppel & Goldbohm, 1995; Weisburger, 1991),
and age-related degeneration (Snodderly, 1995). It is believed that
this effect is achieved by cumulative biological exposure to antiox-
idants which are able to quench the proliferation of radical oxygen
(ROS) and reactive nitrogen (RNS) species which are implicated in the
pathology of these diseases (Valko et al., 2007). Recent studies have
begun to demonstrate that a number of vegetable juices display high
total antioxidant capacity (TAC) when quantied using biochemical
assays (Giovanelli & Pagliarini, 2009; Lichtenthaler & Marx, 2005).
Such assays rely on either single electron transfer (SET) or hy-
drogen atom transfer (HAT) reaction kinetics and comparisons are
made most relevantly between assays which utilise the same type of
reaction kinetics (Huang, Ou & Prior, 2005). SET assays such as Ferric
Reducing Antioxidant Power (FRAP) (Benzie & Strain, 1996), 2,2-
diphenyl-1-picrylhydrazyl (DPPH

) (Brand-Williams, Cuvelier & Ber-

set, 1995) and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS
+
) (Ozgen, Reese, Tulio, Scheerens & Miller, 2006; Rice-Evans &
Miller, 1994) are simple, cost effective, easily interpreted and display
either reduction capacity (FRAP) or direct free radical inhibition
(DPPH

and ABTS
+
). In addition a number of studies also present a
measure of the total polyphenol content of food products in order to
draw comparisons with other similar products, and to provide more
detailed information about this sub-group of antioxidants comprising
avanoids, lignins and tannins (Bravo, 1998). Polyphenolic com-
pounds are thought to be particularly important in the pathologies of
heart disease, hypertension and age-related degeneration (Zern &
Fernandez, 2005; Zhao, 2009). Total polyphenols are commonly
quantied using the FolinCiocalteu method (Singleton, Orthofer &
Lamuela-Raventos, 1999).
Further to these investigations it is important to quantify the
proportion of the ingested antioxidants which are available for use in
the biological system. This is referred to as bioaccessibility and refers
specically to the quantity of antioxidants which are released from
the food matrix and presented to the intestinal brush border for
transport into the cell (Garrett, Failla & Sarama, 1999). An important
distinction must be made between this term and bioavailability
which refers to the quantity of antioxidants which actually pass
through the cell membrane and are available for use within the cell
Food Research International 44 (2011) 217224
Abbreviations: FRAP, Ferric Reducing Antioxidant Power; DPPH, 2, 2-diphenyl-1-
picrylhydrazyl; ABTS, 2, 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid).
Corresponding author. Tel.: +44 1865 483199; fax: +44 1865 483618.
E-mail address: lisaryan@brookes.ac.uk (L. Ryan).
0963-9969/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2010.10.033
Contents lists available at ScienceDirect
Food Research International
j our nal homepage: www. el sevi er. com/ l ocat e/ f oodr es
(Granado-Lorencio et al., 2007). There is a signicant interest in the
bioaccessibility of antioxidants from various food stuffs in order to
identify particular products which may help alleviate micronutrient
deciencies, or help treat diseases which may be exacerbated by ROS
or RNS proliferation. Several studies have presented in vitro digestion
models which simulate both the gastric and duodenal phases (Garrett,
Failla & Sarama, 2000; O'Connell, Ryan & O'Brien, 2006). Commonly
they involve the addition of digestive enzymes and bile salts at
biologically relevant pH and temperatures. TAC is measured by one or
more biochemical assays before, after the gastric phase, and again
after the duodenal phase of the digestion model in order to determine
the stability of antioxidants throughout the process.
Epidemiological evidence suggests that diet and nutrition may
have a signicant effect in the prevention of serious diseases and
potentially, a very signicant effect on wider public health (Agarwal &
Rao, 2000; Asplund, 2002; Johnson, 2004). Previously, a number of in
vitro methods have been used to determine the bioaccessibility and/or
bioavailability of individual antioxidant compounds such as carote-
noids, tocopherols, or polyphenols in order to isolate compounds
which remain stable and active throughout the digestion and ab-
sorption processes (McDougall, Fyffe, Dobson & Stewart, 2005;
O'Connell, Ryan & O'Brien, 2007; Paetau et al., 1998a; Ryan, O'Connell,
O'Sullivan, Aherne & O'Brien, 2008). No study to date has been able to
show a protective effect of an acute dose of any individual compound
in a well controlled, randomised, double blind, crossover human
intervention trial. Focus has therefore returned to the classic, long
term, epidemiological studies which showed a reduction in disease
rates over time when higher amounts of fruit and vegetables were
consumed. Measurement of TAC before and after in vitro digestion
offers a method of ranking particular food products, such as different
vegetable juices, in order of their potential protective capabilities
(Huang et al., 2005). Thus, the aims of this research were to quantify
the TAC of 23 commercially available vegetable juices and assess the
stability of their TAC after the gastric and duodenal phases of an in
vitro digestion model using the FRAP, DPPH and ABTS
+
assays as well
as the FolinCiocalteu method for total polyphenols.
2. Materials and methods
2.1. Materials
All chemicals were of analytical grade and were purchased from
Sigma Aldrich (Poole, UK). Vegetable juices were purchased fromlocal
commercial outlets (Tesco, Oxford, UK; Sainsbury's, Oxford, UK;
ASDA, Oxford, UK; Londis, Oxford, UK; Waitrose, Oxford, UK; Holland
and Barrett, Oxford, UK; The Co-operative, Oxford, UK).
2.2. Sample preparation
Twenty three vegetable juices were selected on the basis of com-
mercial availability. Both value and premium brands were selected
which included fresh, concentrated and long life varieties. The prices
of the juices ranged from 84p to 3.49/L. The juices selected included:
Big Tom spiced tomato juice (James White Drinks Ltd, Ipswich, UK),
Sainsbury's tomato juice from concentrate, Sainsbury's basic tomato
juice from concentrate (Sainsbury's Supermarkets Ltd, London, UK),
Sunraysia pure squeezed tomato juice (Sunraysia, London, UK),
Princes tomato juice from concentrate (Princes Ltd, Liverpool, UK),
Tesco tomato juice from concentrate, Tesco value tomato juice from
concentrate (Tesco, UK), Waitrose fresh pressed tomato juice, Wait-
rose long life tomato juice fromconcentrate (Waitrose, Bracknell, UK),
ASDA tomato juice fromconcentrate (ASDA stores Ltd, Leeds, UK), Del
Monte tomato juice from concentrate (Del Monte Europe Ltd, Staines,
UK), Sunpride tomato juice (Gerber Juice Co. Ltd, Somerset, UK), The
Co-operative tomato juice from concentrate with added salt (The Co-
operative, Manchester, UK), Eden organic carrot juice (Granovita, UK),
Sunraysia organic carrot juice (Sunraysia, London, UK), James White
organic beetroot juice (James White Drinks Ltd, Ipswich, UK), Eden
organic beetroot juice (Granovita, UK), James White organic vegetable
juice (James White Drinks Ltd, Ipswich, UK), Eden organic vegetable
cocktail (Granovita, UK), V8 100% vegetable juice original, V8 100%
fruit and carrot juice tropical (Campbell Foods, Rijksweg, Belgium),
Cawston Press apple and rhubarb juice, and Cawston Press apple and
beetroot juice (Cawston Press, Wokingham, UK). The time to expiry
data for all juices is provided in Table 1. In all experiments the
beverages were prepared using a standard protocol. Amber bottles
were used throughout to prevent the photodecomposition of an-
tioxidants and efforts were made to exclude oxygen contact with the
samples. All experiments were carried out on a minimum of three
separate occasions. Samples were analysed in triplicate for each
experiment.
2.3. In vitro digestion procedure
The in vitro digestion model was adapted from Ryan et al. (2008).
Vegetable juice samples were transferred to clean amber bottles and
mixed with saline (balanced salt solution) to create a nal volume of
20 mL The samples were acidied to pH 2.0 with 1 mL of a porcine
pepsin preparation (0.04 g pepsin in 1 mL 0.1 MHCl) and incubated at
37 C in a shaking water bath at 95 rpmfor 1 h. After gastric digestion,
500 l of each sample was extracted and stored at 20 C. The pHwas
then increased to 5.3 with 0.9 M sodium bicarbonate followed by the
addition of 200 L of bile salts glycodeoxycholate (0.04 g in 1 mL
saline), taurodeoxycholate (0.025 g in 1 mL saline), taurocholate
(0.04 g in 1 mL saline) and 100 L of pancreatin (0.04 g in 500 L
saline). The pH of each sample was increased to 7.4 with 1 M NaOH.
Samples were incubated in a shaking water bath (95 rpm) at 37 C for
2 h to complete the intestinal phase of the in vitro digestion process.
After the intestinal phase, 500 L of each sample was extracted and
stored at 20 C, samples were analysed within 2 weeks.
2.4. Ferric-ion reducing antioxidant power
The total antioxidant capacity of the samples, before and after the
gastric and duodenal phases of digestion, was determined using a
Table 1
Time to expiry of the tested vegetable juices.
Vegetable juice Time to expiry
(unopened)
a
Big tom spiced tomato juice 20 months
Sainsbury's tomato juice from concentrate 11 months
Sainsbury's basic tomato juice from concentrate 11 months
Sunraysia pure squeezed tomato juice 20 months
Princes tomato juice from concentrate 6 months
Tesco tomato juice from concentrate 6 months
Tesco value tomato juice from concentrate 5 months
Waitrose fresh pressed tomato juice 1 month
Waitrose long life pressed tomato juice 20 months
ASDA tomato juice from concentrate 5 months
Del Monte tomato juice from concentrate 8 months
Sunpride tomato juice 10 months
The Co-operative tomato juice from concentrate 5 months
Eden organic carrot juice 19 months
Sunraysia organic carrot juice 10 months
Eden organic beetroot juice 21 months
Beet itJames White organic beetroot juice 20 months
James White organic vegetable juice 19 months
V8 100% vegetable juice (original) 10 months
Eden organic vegetable cocktail 5 months
V8 100% fruit and carrot juice (tropical) 13 months
Cawston Press apple and rhubarb juice 14 months
Cawston Press apple and beetroot juice 13 months
a
Dates indicated represent the time remaining to expiry when the product was
purchased, not necessarily the total shelf life of the product.
218 P.C. Wootton-Beard et al. / Food Research International 44 (2011) 217224
modication of the FRAP assay of Benzie and Strain (1996). Briey
FRAP reagent was prepared from 300 mM acetate and glacial acetic
acid buffer (pH3.6), 20 mMferric chloride and 10 mM4,6-tripryridyl-
s-triazine (TPTZ) made up in 40 mM HCl. All three solutions were
mixed together in the ratio 10:1:1. The FRAP assay was performed by
warming 1 mL of dH
2
O to 37 C before adding 25 L of sample and
1 mL of reagent and incubating at 37 C for 4 min. Absorbance at
593 nm was determined relative to a reagent blank also incubated at
37 C. The total antioxidant capacity of samples was determined
against a standard of known FRAP value, ferrous sulphate (1000 M).
2.5. DPPH

radical scavenging activity

Antioxidant activities of the samples before and after the gastric
and duodenal phases of digestion were also analysed by investigating
their ability to scavenge the DPPH

free radical using a modication

of the method by Brand-Williams et al. (1995). DPPH

has an intense
violet colour with a maximum absorbance at 517 nm, but turns
colourless as unpaired electrons are scavenged by antioxidants.
Reaction mixtures containing 0.1 mL of sample and 3.9 mL of 50 M
DPPH

(prepared in methanol) were incubated in a water bath at 37 C

for 30 min. After incubation, an aliquot of sample from each test tube
was removedandplacedinto a cuvette to prevent occlusionof the light
pathway by sediment inthe juice, andthe absorbance was measuredat
517 nm. The percentage inhibition was calculated against a control
and compared to an ascorbic acid standard curve (01000 m). The
effect of digestion on the ability to scavenge the DPPH

radical was
expressed as fold change compared to the sample prior to digestion.
For accurate comparisons to be drawn a 1:5 dilution of the juices prior
to digestionwas made and testedin order that the digestedsamples be
compared to the original samples at the same dilution ratio.
2.6. ABTS
+
radical cation scavenging activity
Antioxidant activities of the samples before and after the gastric
and duodenal phases of digestion were also analysed by investigating
their ability to scavenge the ABTS
+
free radical using a modied
methodology previously reported by Ozgen et al. (2006). When
combined with an oxidant (2.45 mM potassium persulfate), ABTS
(7 mM in 20 mM sodium acetate buffer, pH 4.5) reacts to create a
stable, dark blue-green radical solution following 1216 h of
incubation in the dark (4 C). The solution was then diluted to an
absorbance of 0.70.01 at 734 nm to form the test reagent. Reaction
mixtures containing 20 L of sample and 3 mL of reagent were
incubated in a water bath at 30 C for 30 min. As unpaired electrons
are sequestered by antioxidants in the sample the test solution turns
colourless and the absorbance at 734 nm is reduced. An aliquot from
each test tube was removed and placed into a cuvette to prevent
occlusion of the light pathway by sediment in the juice before the
absorbance of each sample was measured. The percentage inhibition
was calculated against a control and compared to a Trolox standard
curve 10100 mM and expressed as fold change compared to the
sample prior to digestion. Again, digested samples were compared
with 1:5 dilutions of the original juices.
2.7. FolinCiocalteu method for total polyphenols
The supernatant (0.2 mL) was added to 1.5 mL of freshly prepared
FolinCiocalteu Reagent (FCR) (1:10 v/v with water). The mixture
was allowed to equilibrate for 5 min and then mixed with 1.5 mL of
60 g/L sodium carbonate solution. After incubation at room temper-
ature for 90 min, the absorbance of the mixture was read at 725 nm
using the respective solvent as blank. The results were expressed as g
of ferulic acid equivalents (FAE) per mL of sample.
2.8. Data analysis
All dataarepresentedas means (SEM) of at least threeindependent
experiments (n=3), eachexperiment hadaminimumof threereplicates
of each sample. For comparisons between samples, data was analysed by
ANOVA and Tukey's multiple comparison test (SPSS, version 17). A
probability of 5% or less was accepted as statistically signicant.
3. Results
3.1. FRAP analysis
The initial FRAP values of the tested vegetable juices varied
considerably (13699500 mol/L) (Table 2). Within categories,
beetroot juices displayed the highest FRAP values (83559500 mol/
L) followed by mixed fruit and vegetable juices (20606047 mol/L),
tomato juices (18433139 mol/L), mixed vegetable juices (2468
2601 mol/L) and carrot juices (13691533 mol/L). Following both
the gastric and duodenal phases of digestion, the FRAP values of all 23
juices were signicantly (Pb0.05) increased. High importance is
placed upon the post digestion values for these juices because the in
vitro digestion model gives an indication as to the availability of
vegetable juice antioxidants in a biological system, since the model is
designed to simulate in vivo digestion. Beetroot juices in particular
showed a near 2 fold increase in FRAP following the gastric phase of
digestion although this was not maintained after the duodenal phase
for Eden organic beetroot juice. This pattern was evident for the
majority of the juices, whereby the greatest increase was observed
after the gastric phase with a subsequent small decrease after the
duodenal phase, although still remaining higher than their respective
levels prior to digestion. The only exception was Sainsbury's tomato
juice from concentrate which increased a little further between
gastric and duodenal phases. Importantly the FRAP values of all the
juices remained relatively stable following both phases of digestion
and none were decreased compared to the juices prior to digestion.
Thirteen tomato juices were analysed comprising both value and
premium brands. All were long life (non-refrigerated) products with
the exception of Waitrose fresh pressed tomato juice. In comparison
this fresh juice displayed a signicantly (Pb0.05) higher FRAP value
than Waitrose long life. In most cases the value brands had a higher
reducing capacity then the premium brands in the FRAP assay.
3.2. DPPH

radical scavenging activity

DPPH

radical scavenging activity was quantied in terms of

percentage inhibition of a pre-formed free radical by antioxidants in
eachsample. There was a signicant variationinthe percentage inhibition
of the DPPH

radical by the juices (57.8100% inhibition) (Table 3). Again

beetroot juice exhibited the highest antioxidant capacity with both juices
fully inhibiting the radical. Tomato juices (67.388.5%), carrot juices
(57.882.2%), mixed fruit and vegetable juices (64.489.1%) and mixed
vegetable juices (82.283.5%) exhibitedsimilar DPPH

scavenging activity.
Eden organic carrot juice (82.2%) showed greater scavenging DPPH

scavenging capacity than Sunraysia organic carrot juice (57.8%). Concur-

rently, the Sunraysia tomato juice also had a considerably lower DPPH

scavenging capacity (67.3%) than any of the other tomato juices despite
costingthemost. Interestinglytherewas a trendfor theradical scavenging
capacity to be slightly increased following the gastric phase and slightly
decreased following the duodenal phase, although there were some
exceptions to this trend. Overall, DPPH

radical scavenging capacity

remained consistently high throughout in vitro digestion.
3.3. ABTS
+
radical cation scavenging activity
ABTS activity was quantied in terms of percentage inhibition of
the ABTS
+
radical cation by antioxidants in each sample. There was a
219 P.C. Wootton-Beard et al. / Food Research International 44 (2011) 217224
signicant variation in the percentage inhibition of the juices (10.9
92.3% inhibition) (Table 4). Beetroot juice was once again the most
efcient scavenger of the radical (92.192.3% inhibition). Interestingly
both of the Cawston Press fruit and vegetable juices (apple and
rhubarb/apple and beetroot) were also particularly effective scaven-
gers of the ABTS
+
radical (90.190.7% inhibition), the other mixed
fruit and vegetable juice (V8 100% fruit and carrot juice tropical) had a
more modest inhibitory effect at 39.1%. Tomato juices ranged from
22.4% for Sunraysia (the most expensive brand) to 39.1 and 38.4% for
Tesco own brand and value juices. There was one notable exception
amongst tomato juices in that ASDA tomato juice from concentrate
inhibited the radical by 70.5%, by far the most potent of the tomato
juices. Within the mixed vegetable juices, James White organic
vegetable juice (55.9%) displayed a greater ABTS
+
scavenging
capacity than the other mixed vegetable juices (25.526.1%). Carrot
juice proved the most inefcient scavenger of the ABTS
+
radical at
10.923.9% inhibition. Following the gastric phase of the in vitro
digestion model, 18 of the juices increased their inhibitory effects
signicantly (Pb0.05). The only juice to show a signicant decrease
following the gastric phase of digestion was Tesco tomato juice from
concentrate; concurrently Sainsbury's basic tomato juice from
concentrate displayed the greatest increase at 2.06 fold. Contrary to
the results from the DPPH

assay 12 of the juices maintained this

signicant increase after the duodenal phase. Despite this nding,
beetroot juice although remaining high, showed a signicant decrease
(Pb0.05) in ABTS
+
inhibition following the duodenal phase (0.79 and
Table 3
DPPH inhibition of the tested juices before, and after the gastric and duodenal phases of in vitro digestion.
Vegetable juice DPPH (% inhibition) DPPH gastric (fold change) DPPH duodenal (fold change)
Big tom spiced tomato juice 83.50.2 0.61

0.96
Sainsbury's tomato juice from concentrate 79.51.1 1.01 0.71

Sainsbury's basic tomato juice 88.50.2 1.24

0.86

Sunraysia pure squeezed tomato juice 67.30.9 1.02 0.70

Princes tomato juice from concentrate 81.00.6 1.13

0.82

Tesco tomato juice from concentrate 81.20.5 1.29

0.85

Tesco value tomato juice from concentrate 81.80.2 1.02 0.67

Waitrose fresh pressed tomato juice 85.70.2 1.21

0.76

Waitrose long life pressed tomato juice 82.50.2 1.27

0.92

ASDA tomato juice from concentrate 83.90.4 1.25

0.94
Del Monte tomato juice from concentrate 80.40.4 1.15

0.88

Sunpride tomato juice 76.60.1 1.14

1.03
The Co-operative tomato juice 87.60.1 1.09

0.80

Eden organic carrot juice 82.20.1 0.84

0.84

Sunraysia organic carrot juice 57.81.9 0.80

0.81

Eden organic beetroot juice 1000.0 1.02 1.12

Beet itJames White organic beetroot juice 1000.0 1.23

0.69

James White organic vegetable juice 83.50.2 0.80

0.78

V8 100% vegetable juice (original) 83.10.3 1.08

0.98
Eden organic vegetable cocktail 82.20.2 1.03 0.79

V8 100% fruit and carrot juice (tropical) 81.82.3 0.74

0.97
Cawston Press apple and rhubarb juice 89.10.2 0.63

0.84

Cawston Press apple and beetroot juice 64.40.6 0.64

0.79

Pb0.05, denotes signicant increase from the DPPH percentage inhibition value prior to in vitro digestion [ANOVA, Tukey's test]. Values represent meanstandard error of the
means (SEM) of three independent experiments (n=3). Fold change refers to the fold increase or decrease in TAC of digested samples (1:5 dilution in saline) compared to a 1 in 5
saline dilution of the original juice prior to digestion.
Table 2
FRAP values for tested juices before, and after the gastric and duodenal phases of in vitro digestion.
Vegetable juice FRAP prior (mol/L) FRAP gastric (mol/L) FRAP duodenal (mol/L)
Big tom spiced tomato juice 225195 3799308

3687257

Sainsbury's tomato juice from concentrate 197121 3668298

3687327

Sainsbury's basic tomato juice 249719 389083

3743149

Sunraysia pure squeezed tomato juice 184315 284171

2638132

Princes tomato juice from concentrate 2750111 3724120

319083

Tesco tomato juice from concentrate 268374 328769

30775

Tesco value tomato juice from concentrate 2905223 3882105

3308302

Waitrose fresh pressed tomato juice 313996 5008159

3772239

Waitrose long life pressed tomato juice 241588 317287

2959174

ASDA tomato juice from concentrate 249784 3547138

328992

Del Monte tomato juice from concentrate 220240 341787

311145

Sunpride tomato juice 246328 4427541

3880112

The Co-operative tomato juice 310432 359933

3059119

Eden organic carrot juice 153311 292689

273228

Sunraysia organic carrot juice 136922 373464

391636

Eden organic beetroot juice 950027 18,639184

889092

Beet itJames White organic beetroot juice 835585 15,014146

12,397326

James White organic vegetable juice 260122 3852180

3592308

V8 100% vegetable juice (original) 257349 4206349

370479

Eden organic vegetable cocktail 246893 4157118

368916

V8 100% fruit and carrot juice (tropical) 206049 5160272

4950203

Cawston Press apple and rhubarb juice 601754 8758131

8399434

Cawston Press apple and beetroot juice 640773 8936129

8958275

Pb0.05, denotes signicant increase fromthe Ferric Reducing Antioxidant Power (FRAP) value prior to in vitro digestion [ANOVA, Tukey's test]. Values represent meanstandard
error of the means (SEM) of three independent experiments (n=3).
220 P.C. Wootton-Beard et al. / Food Research International 44 (2011) 217224
0.56 fold change respectively). Similarly both Cawston Press juices
(apple and rhubarb/apple and beetroot) decreased signicantly
(Pb0.05) after the duodenal phase. Despite these observed decreases,
both beetroot and the mixed fruit and vegetable juices still had a
higher ABTS
+
scavenging capacity than all the other juices, even if
they had increased signicantly.
3.4. Total polyphenols analysis
The total polyphenol content of each juice was quantied using the
FolinCiocalteu reagent. All 23 juices were a signicant source of
polyphenols; however the total amount varied signicantly between
juices (5143025 g FAE/mL) (Table 5). Prior to digestion results
showed that Eden organic beetroot juice contained the highest
amount of polyphenols (3025 g FAE/mL), Sunraysia organic carrot
juice contained the lowest amount of polyphenols (514 g FAE/mL).
After the gastric phase of digestion there was a signicant (Pb0.05)
increase in total polyphenol content for all 23 juices. The greatest
increase was seen in Sunpride tomato juice (a value brand) with an
increase in total polyphenol content from 668 to 1117 g FAE/mL.
Eden organic beetroot juice showed a modest increase in total
polyphenols following the gastric phase, increasing from 3025 to
3124 g FAE/mL and remained the juice with the highest polyphenol
content. After the duodenal phase of digestion, 19 of the juices
showed a further increase in total polyphenol content. The Waitrose
fresh tomato juice increased most signicantly at this stage (257 g
FAE/mL). Four of the 23 juices decreased in total polyphenol content
after the duodenal phase of digestion. The most notable of which was
once again observed for Eden organic beetroot juice, which despite
remaining high, decreased from 3124 g FAE/mL (after the gastric
phase) to 1619 g FAE/mL (after the duodenal phase).
4. Discussion
Previous publications detailing the antioxidant capacity of vege-
table juices are sparse. Some studies have presented information on
immune modulation, in vivo lipid peroxidation and carcinogenesis
following consumption of tomato and carrot juices (Bub et al., 2000;
Paetau et al., 1998b; Watzl, Bub, Briviba & Rechkemmer, 2003) but no
studies to date have provided information regarding beetroot juice,
mixed fruit and vegetable juices or mixed vegetable juices. These
studies have provided information about whole juices but no studies
to date have reported the stability of vegetable juice antioxidants
following digestion. By measuring the TAC of the juices after the in
vitro digestion it is possible to quickly and cost-effectively provide
more biologically relevant data detailing the antioxidant properties of
these juices.
Crucially the results of this study showed that antioxidant capacity
is relatively stable throughout the digestive process in the vast ma-
jority of vegetable juices. This is in contrast with the previous research
examining fruit juices which have consistently shown large decreases
in TAC post digestion (Bermudez-Soto, Tomas-Barberan & Garcia-
Conesa, 2007; McDougall, Dobson, Smith, Blake & Stewart, 2005;
McDougall, Fyffe, et al., 2005), suggesting that vegetable juices may be
a greater source of bioaccessible antioxidants than the more popular
fruit juices. In the study by Bermudez-Soto et al. (2007) the
researchers reported a reduction in various polyphenols following
similar two phase digestions of a variety of fruit juices. The fruit juices
displayed largely increased polyphenol content after the gastric phase
of digestion (similar to the current study) but fell to below pre-
digestion levels after the duodenal phase, a trend which was not
observed in the current study of vegetable juices. Ryan and Prescott
(2010) suggested that this may have been due to a structural
transformation in the polyphenols which render them undetectable
by the individual HPLC polyphenol analysis which Bermudez-Soto
et al. (2007) had used, the authors did not measure total polyphenols.
The current study shows that this does not occur in vegetable juices
since the polyphenol content was much higher after both stages of
digestion compared to the levels prior to digestion. It also highlights
the efcacy of multiple methods of analysis to prevent under-
reporting of the antioxidant content of juices.
The results of the three assays which were conducted are com-
parable in magnitude for each category of juice. There were some
exceptions to this trend whereby particular juices performed
markedly better in one or more of the assays. Overall beetroot juice
displayed the strongest radical scavenging capacity in both the DPPH

and ABTS
+
assays and also displayed the highest reducing capacity as
measured by FRAP. Beetroot juices also ranked amongst the highest
Table 4
ABTS
+
inhibition of the tested juices before, and after the gastric and duodenal phases of in vitro digestion.
Vegetable juice ABTS (% inhibition) ABTS gastric (fold change) ABTS duodenal (fold change)
Big tom spiced tomato juice 32.70.3 1.37

1.49

Sainsbury's tomato juice from concentrate 27.31.1 1.79

1.38

Sainsbury's basic tomato juice 32.90.9 2.06

1.51

Sunraysia pure squeezed tomato juice 22.40.6 1.96

1.32

Princes tomato juice from concentrate 31.80.4 1.78

0.98
Tesco tomato juice from concentrate 39.10.4 0.77

0.94
Tesco value tomato juice from concentrate 38.40.3 1.06 1.00
Waitrose fresh pressed tomato juice 26.50.8 1.33

1.65*
Waitrose long life pressed tomato juice 30.90.3 1.33

1.49

ASDA tomato juice from concentrate 70.50.5 1.42

0.96
Del Monte tomato juice from concentrate 30.01.0 1.25

1.21

Sunpride tomato juice 31.40.2 1.32

1.00
The Co-operative tomato juice 29.90.2 1.54

1.55

Eden organic carrot juice 23.90.3 1.38

1.58

Sunraysia organic carrot juice 10.90.2 0.91

0.79

Eden organic beetroot juice 92.10.2 1.14

0.56

Beet itJames White organic beetroot juice 92.30.1 1.02 0.92

James White organic vegetable juice 55.90.9 1.18

1.18

V8 100% vegetable juice (original) 25.50.3 1.09

1.09*
Eden organic vegetable cocktail 26.10.2 1.48

1.41

V8 100% fruit and carrot juice (tropical) 39.10.1 1.29

1.01
Cawston Press apple and rhubarb juice 90.70.3 1.00 0.71

Cawston Press apple and beetroot juice 90.10.3 0.93 0.70

Pb0.05, denotes signicant increase from the ABTS percentage inhibition value prior to in vitro digestion [ANOVA, Tukey's test]. Values represent meanstandard error of the
means (SEM) of three independent experiments (n=3). Fold change refers to the fold increase or decrease in TAC of digested samples (1:5 dilution in saline) compared to a 1 in 5
saline dilution of the original juice prior to digestion.
221 P.C. Wootton-Beard et al. / Food Research International 44 (2011) 217224
juices in terms of total polyphenol content. The TAC of beetroot juice
was greater than or comparable to that of pomegranate or cranberry
juice (Ryan & Prescott, 2010). Beetroot is afforded its vibrant red and
purple colourations by the betacyanin; betanin. Betacyanins are one of
the two sub-categories of betalains which may reduce LDL cholesterol
oxidation and therefore impact upon cardiovascular diseases (Escri-
bano, Pedreno, Garcia-Carmona & Munoz, 1998; Tesoriere, Allegra,
Butera & Livrea, 2004). Beetroot is also a source of many other health
promoting constituents such as folic acid, iron, magnesium, selenium,
potassium, calcium, zinc, phosphorus, sodium, biotin, niacin and -
carotene as well as vitamins A, B
6
and C. The high antioxidant capacity
of beetroot juice, together with its other nutritional value may make
beetroot juice a very positive addition to the diet.
Interesting comparisons emerged between the two beetroot juices
following FRAP analysis. The rst beetroot juice, Eden organic, is
produced by combining pure beetroot juice with lactic acid fermented
beetroot juice and ascorbic acid as a preservative. The second, James
White organic beetroot juice (Beet it), is made from 90% pressed
beetroots and 10% pressed apple juice. Initially Eden organic beetroot
juice displayeda higher FRAPvalue (9500 mol/L) thanthe James White
juice (8355 mol/L). Principally due to the higher beetroot content,
since beetroot has a higher FRAP value compared to apple juice (Ryan &
Prescott, 2010), and perhaps in part to the addition of ascorbic acid, a
powerful reducingagent initself. This trendis continuedafter thegastric
phase where the Eden organic juice increased almost 2 fold to
18,639 mol/L and the James White juice, similar in magnitude,
increased to 15,014 mol/L. Following the duodenal phase of digestion
where the pH is rst adjusted to 5.3 and then to 7.4 following the
addition of bile salts and pancreatin, both juices show a decrease in
FRAP. The James White beverage remains well above pre-digestion at
12,397 mol/L. The Eden beverage however, displayed a reducing
capacity that fell below even its pre-digestion value (8890 mol/L).
Since this result is shown consistently across three separate ex-
periments it is reasonable to assume that the antioxidants in this juice
are destroyed, inhibited, transformed or otherwise far less available
during this phase. A similar trend was observed for the ABTS
+
assay,
and for the James White juice, in the DPPH

assay. There are several

key differences between the juices, which could account for these
results. Firstly, Eden organic beetroot juice is at least partially lactic
acid fermented. Lactic acid fermentation involves the addition of
probiotic lactic acid bacteria, which consume reducing sugars in the
raw produce causing a rapid but modest acidication of the juice. This
process extends the shelf life of the juice and provides additional
nutritional value in terms of probiotic bacteria (Moraru, Bleoanca &
Segal, 2007), it has also been suggested to increase its nutritional
content in terms of phytochemicals (Hugenholtz and Smid, 2002).
Potentially, the lacto-fermentation of this particular juice induces a
structural transformation, perhaps by the action of the acidication of
the product. The pH of a substance is known to affect racemisation of
molecules, possibly creating two chiral enantiomers with different
reactivity in the respective reagents. In theory this could alter their
biological reactivity and may render the antioxidants more reactive
early in the digestive process, particularly at acidic pH in the
gastric phase and less reactive at pH 7.4 in the duodenal phase as
racemisation can increase with pH in other compounds (Jamali,
Bjornsdottir, Nordfang & Hansen, 2008). As a result there are either
fewer reactive antioxidants available at the duodenal phase or their
reducing capacity is impaired. There is also the suggestion, that
dependant on any structural changes which occur, resulting metabo-
lites may react differently across different assays. The Eden organic
beetroot juice did not showa reduction in reducing capacity following
digestion when assessed using the DPPH

radical scavenging assay.

As mentioned there was a contribution to TAC from ascorbic acid,
used as a preservative in the Eden organic beetroot juice. Eleven of the
23 juices analysed had added preservatives which have antioxidant
effects. Citric acid was present in Sainsbury's basic tomato juice, Tesco
value tomato juice, Waitrose fresh and long life tomato juices, ASDA
tomato juice, Del Monte tomato juice, Sunpride tomato juice and the
Co-operative tomato juice. Ascorbic acid was present in Eden organic
beetroot as well as Cawston Press apple and rhubarb juice. V8 fruit
and carrot juice (tropical) contained both citric acid and ascorbic acid
as preservatives. The contribution of ascorbic acid to TAC varies
greatly between products and can account for 65100% of TAC in
citrus fruits or as little as 5% in apple or pineapple (Gardner, White,
McPhail & Duthie, 2000), however since it is a biologically active
antioxidant, its contribution should be considered equally with other
antioxidant fractions. Citric acid has been reported to account for up to
25% of antioxidant activity but is not classied as a true antioxidant
prompting the recent development of assays which have selectivity
over citric acid such as the Cerium reducing antioxidant capacity
Table 5
Total Polyphenol measurement of the tested juices before, and after the gastric and duodenal phases of in vitro digestion.
Vegetable juice Polyphenols (g FAE/mL) Polyphenols gas (g FAE/mL) Polyphenols duo (g FAE/mL)
Big tom spiced tomato juice 82144 10257

113714

Sainsbury's tomato juice from concentrate 92443 122522

12519

Sainsbury's basic tomato juice 99274 11889

12497

Sunraysia pure squeezed tomato juice 57517 89216

104514

Princes tomato juice from concentrate 85236 103112

10778

Tesco tomato juice from concentrate 87233 114220

130127

Tesco value tomato juice from concentrate 93145 112632

126033

Waitrose fresh pressed tomato juice 57321 90441

116123

Waitrose long life pressed tomato juice 70413 106833

128623

ASDA tomato juice from concentrate 114033 154919

142212

Del Monte tomato juice from concentrate 97627 130011

134719

Sunpride tomato juice 69027 11176

121120

Co-operative tomato juice 93422 111316

12398

Eden organic carrot juice 60512 95615

10948

Sunraysia organic carrot juice 44911 85923

106511

Eden organic beetroot juice 302553 312445

161922

Beet itJames White organic beetroot juice 212666 249023

198532

James White organic vegetable juice 84021 11239

122019

V8 100% vegetable juice (original) 82626 116611

124110

Eden organic vegetable cocktail 62715 92416

108016

V8 100% fruit and carrot juice (tropical) 79021 125315

140714

Cawston Press apple and rhubarb juice 178252 206443

192320

Cawston Press apple and beetroot juice 198659 217365

201954

Pb0.05, denotes signicant increase fromthe total polyphenol content of the juice prior to in vitro digestion [ANOVA, Tukey's test]. Values represent meanstandard error of the
means (SEM) of three independent experiments (n=3). Gas=measurement after the gastric phase, Duo=measurement after the duodenal phase. FAE=Ferulic acid equivalents.
222 P.C. Wootton-Beard et al. / Food Research International 44 (2011) 217224
(CERAC) assay (Ozyurt, Demirata & Apak, 2010) and Copper (II)
reducing antioxidant capacity (CUPRAC) assay (Apak, Guclu, Ozyurek
& Karademir, 2004). It is difcult to quantify the specic contributions
of these organic acids to TAC without further analysis using capillary
electrophoresis (Tezcan, Gultekin-Ozguven, Diken, Ozcelik & Erim,
2009) because no information is given on the amount which is used
for each juice. It is likely that preservatives account for a small
proportion of the TAC measurements of these juices, perhaps 525% in
those containing high amounts of citric acid. Citric acid may be
benecial in reducing hydrogen peroxide (H
2
O
2
) formation once the
juices have been opened (Aoshima & Ayabe, 2007). Both long life and
fresh juices contained antioxidant preservatives.
Differences were observed between the two radical scavenging
assays (DPPH

and ABTS
+
). Both assays measure the ability of all of
the antioxidants in a sample to scavenge a pre-formed radical. The
DPPH

radical is stable at formation, DPPH in powder form is simply

dissolved in methanol and the resulting purple solution is added to
the sample. In contrast the ABTS
+
radical cation is created by reacting
ABTS (7 mM in 20 mM acetate pH 4.5) with the oxidant, potassium
persulfate (2.45 mM), the reaction proceeds during a 1216 h
incubation at a stoichiometric ratio of 1:0.5 to form the radical cation.
Previous work has shown that this method allows formation of a
radical which remains stable for several days due to the optimised pH
(Cano, Hernandez-Ruiz, Garcia-Canovas, Acosta & Arnao, 1998), in
fact in the current study the radical remained stable for a week or
more. Further fundamental differences can be observed between the
species of radical which is created in each of these methods and the
methodologies by which they are executed.
Firstly, the ABTS
+
assay is an electron transfer, end-point assay
whereby different antioxidant compounds donate one or two
electrons to reduce the radical cation. Regardless of the donating
potential of individual antioxidants they all have time to react fully
giving an accurate measurement of TAC at the end-point of the assay
(Huang et al., 2005). The DPPH

assay is based on the normal HAT

reaction that occurs between antioxidants and the peroxyl radical.
Instead of peroxyl radicals, more stable and less transient nitrogen
radicals are created, with which some antioxidants react more slowly
than they would with the peroxyl radical in a biological system
(Huang et al., 2005). Additionally, the reaction then proceeds in a
manner which bears more similarity to a SET reaction (Foti, Daquino &
Geraci, 2004). In the current study this does not seemto affect the TAC
measurement of the juices prior to digestion, since most of the juices
display a high inhibition of the radical. However following the gastric
phase of in vitro digestion, when structural transformations may occur
in the antioxidants, the TAC is relatively unchanged in the DPPH

assay
but increases signicantly (Pb0.05) for a number of the juices in the
ABTS
+
assay. Theoretically, alterations in the structure of antiox-
idants following digestion may affect their reactivity with the less
biologically relevant nitrogen radical formed in the DPPH

assay,
leading to underestimation of TAC at this time point. Similar mech-
anisms may also contribute to explaining the differences observed
between the measurements of TAC in the two assays after the
duodenal phase. A number of juices remain signicantly higher
(Pb0.05) than their pre-digestion value in the ABTS
+
assay, whilst all
of the juices show a similar or reduced TAC after the duodenal phase
in the DPPH

assay. There was a relatively weak correlation observed

between the results for DPPH

and ABTS
+
(r
2
=0.45), differences in
the rank order between the 13 tomato juices may have skewed this
statistic, weakening what may otherwise have been a stronger
correlation.
Despite these observed differences, both assays rank the juices in a
similar order, both give beetroot juice as the most effective free radical
scavenger, both rank the mixed fruit and vegetable juices second and
both rank carrot juices as the most inefcient scavenger. This in-
formation is actually more relevant to the pursuit of public health
than knowing the exact chemical reactivity of each component of a
sample in each different assay. By obtaining consistent information
about the rank order of different products, in terms of their TAC, from
multiple methodologies, accurate information about the most potent
juices can be disseminated to a wider audience.
Comparisons were also drawn between the results obtained by the
FRAP assay and those obtained for total polyphenols by FCR. Both of
these assays utilise similar SET reaction kinetics and are both es-
sentially measures of reducing capacity. Where FRAP measures the
reducing capacity of all the antioxidants in the mixture, FCR records
only those classed as polyphenolic compounds (Huang et al., 2005).
As a result previous researchers have often found very good linear
correlations between FCR and one or more SET based assays such as
FRAP or ABTS
+
(Jimenez-Alvarez et al., 2008; Molan, De & Meagher,
2009; Sreeramulu & Raghunath, 2010; Stratil, Klejdus & Kuban, 2006).
The same relationship is observed in the current study, where a very
strong positive linear correlation (r
2
=0.96) is observed between
FRAP and FCR. Additionally, there were strong, positive, linear
correlations between other assays utilising similar SET reaction
kinetics. FRAP also correlated well with ABTS
+
(r
2
=0.87), whilst
ABTS
+
showed a strong positive association to FCR (r
2
=0.89). The
methodological anomaly in these comparisons was DPPH

, which also
correlated poorly with FRAP (r
2
=0.53) and FCR (r
2
=0.50).
5. Conclusion
This research has shown that all vegetable juices may be a sig-
nicant source of antioxidants, regardless of price, storage or
processing conditions. However, there is a wide variation in TAC
between different types of juice. Beetroot juice displayed by far the
highest antioxidant capacity across all of the assays conducted, and
has an antioxidant capacity similar to, or greater than that of pome-
granate or cranberry juice. Additionally, this research has provided
the rst measurement concerning the stability of commercial
vegetable juice antioxidants following in vitro digestion. All of the
juices were either stable or enhanced in terms of TAC following in
vitro digestion, although there was a large variation in the responses
of individual juices to this procedure. This research highlights the
need for further investigation in several areas. Firstly, it is important
to consider the contribution of structurally transformed molecules
and other antioxidant metabolites to total antioxidant capacity. It is
likely that a number of methods underestimate antioxidant capacity
due to a failure to measure these. Further to this, it is important that
research aims to provide biologically relevant information on
antioxidants by providing data concerning the bioaccessibility and
bioavailability of antioxidants in a human system. Finally this study
highlights the importance of using multiple methods of analysis in the
absence of any single accepted assay for the measurement of TAC.
Acknowledgements
Part of this research was supported by the Wellcome trust (award
number WT093679MA). The authors report no conict of interest,
nancial or otherwise.
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