Food Research International 40 (2007) 15–21

www.elsevier.com/locate/foodres

Food antioxidant capacity determined by chemical methods

may underestimate the physiological antioxidant capacity
a,b a,b,¤ a,c
José Serrano , Isabel Goñi , Fulgencio Saura-Calixto
a
Nutrition and Gastrointestinal Health Unit, UCM/CSIC, 28040 Madrid, Spain
b
Departamento de Nutrición y Bromatología I, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain
c
Departamento de Metabolismo y Nutrición, Instituto del Frío, CSIC, Madrid, 28040 Madrid, Spain

Received 2 March 2006; accepted 14 July 2006

Abstract

Antioxidant capacity assays are of growing interest in the study of dietary antioxidant properties since they are able to analyse a com-
plex mixture of antioxidants and its synergistic interactions. However, most of the antioxidant capacity assays in the literature are limited
by the antioxidant extraction technique, since some antioxidants may remain associated in the extraction residues. The objective of this
work was to compare an in vitro physiological procedure for antioxidant extraction with a methanol/acetone/water extraction (chemical
procedure). Enzymatic digestions and in vitro colonic fermentations were used on solid plant foods daily consumed in the Spanish diet to
estimate the total antioxidant capacity released during the entire digestion process. The in vitro physiological procedure yielded a higher
antioxidant capacity than the chemical procedure (7000 and 900 mol trolox equivalents measured by ABTS, respectively). Our results
suggest that determination of antioxidant capacity in food chemical extracts may underestimate the real antioxidant capacity that may be
in close contact with the intestinal lumen.
© 2006 Elsevier Ltd. All rights reserved.

Keywords: Antioxidant capacity; Bioaccessibility; Spanish diet; Digestion; Colonic fermentation

1. Introduction (Beta Carotene Cancer Prevention Study Group The

Many observational epidemiological studies have shown
that a high fruit and vegetable intake is associated with a
lower cancer incidence (Poppel, 1996; Weisburger, 1991),
specially cancers from the gastrointestinal tract (Johnson,
2004). This is due in part to the dietary antioxidant content
of fruit and vegetables. Dietary antioxidants are believed to
be eVective in the prevention of oxidative stress related dis-
eases (Kaur & Kapoor, 2001). Antioxidants have thus
recently become a topic of increasing interest. However,
results of randomized trials looking at the possible preven-
tive eVect of dietary antioxidant supplementation with one
or more selected antioxidants have been contradictory
* Several studies addressing the antioxidant capacity in the
Corresponding author. Address: Departamento de Nutrición y Bro-
matología I, Facultad de Farmacia, Avda, Complutense s/n, Ciudad Univers-
itaria, 28040 Madrid, Spain. Tel.: +34 91 394 18 12; fax: +34 91 394 17 32.
E-mail address: igonic@farm.ucm.es (I. Goñi).
Alpha-Tocopherol, 1994; Lee & Park, 2003; Omenn et al.,
1996; Paolini et al., 2003).
Plant foods provide a wide variety of dietary antioxi-
dants, such as vitamins C and E, carotenoids, Xavonoids
and other phenolic compounds. The additive and synergis-
tic eVects of these antioxidants with other dietary com-
pounds (e.g. minerals) may contribute to the health beneWts
of the diet. Due to the complexity of the antioxidant com-
position in foods, the study of single antioxidant com-
pounds is costly and may be of limited value because the
possible synergistic interaction among the antioxidant com-
pounds in a food mixture is not considered. That is why
antioxidant capacity assays are attracting more interest in
the study of antioxidant properties of foods and diets.
diet have been reported (Brighenti et al., 2005; Pulido, Her-
nandez-García, & Saura-Calixto, 2003; Wu et al., 2004). Our
group recently reported the antioxidant capacity of the

0963-9969/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2006.07.010
16 J. Serrano et al. / Food Research International 40 (2007) 15–21

Table 1
Intake of plant foods in the Spanish diet
g Fresh g Edible
matter/dayc portion/day
Cereals Ricea (7%), white bread (67%), white bread sliced (5%), spaghettia (5%), biscuits (8%), croissants (8%) 221.6 221.6
Vegetables Potatoesa (40%), tomatoes (13%), tomatoes transformed (6%), onions (7%), garlic (1%), cabbagea (2%), 330.9 280.8
green beansa (2%), cucumber (2%), capsicum (4%), mushrooms (1%), lettuce (7%), asparagus (1%),
spinacha (1%), charda (1%), othersb (12%)
Nuts Almonds (12%), peanuts (16%), walnuts (16%), othersb (56%) 6.8 5.9
Fruits Oranges (24%), mandarin oranges (6%), bananas (10%), apples (13%), pears (8%), peaches (5%), apricots 265.7 200.8
(1%), strawberries (3%), melon (8%), watermelon (6%), plums (2%), cherries (1%), grapes (3%), kiwi (3%),
olives (3%), othersb (4%)
Legumes Chickpeasa (35%), beansa (31%), lentilsa (34%) 22.3 22.3
a
Boiled.
b
Vegetables: artichokea, carrot, tender pumpkina, celerya, auberginea, turnipa, leeka, pumpkina, beet roota, avocado; Nuts: hazelnuts, pistachios; Fruits:
pomegranate, mango, pineapple, grapefruit, caqui, chirimoya.
c
ConWdence level 95%, error range 2% in amount of food.

Spanish diet (Saura-Calixto & Goñi, 2006). The studies cited
above deal only with the antioxidant capacity extracted by
chemical solvents (methanol, acetone, chloroform). But
some antioxidants may remain in the residues from organic
extraction of foods (Perez-Jimenez & Saura-Calixto, 2005).
For that reason we suggest that normal antioxidant capacity
assays are limited by the extraction technique employed
(Serrano, Goñi, & Saura-Calixto, 2005a). Moreover, from a
physiological point of view, to exert their biological proper-
ties antioxidants have to be available in some extent in the
target tissue. Therefore, the biological properties of antioxi-
dants may depend on their release from the food matrix dur-
ing the digestion process (bioaccessibility) and may diVer
quantitatively and qualitatively from those produced by the
chemical extraction employed in most studies.
The objective of this work was to compare an in vitro
physiological procedure for antioxidant extraction with a
methanol/acetone/water extraction. An in vitro gastrointes-
tinal model that includes digestive enzyme treatment
(Saura-Calixto, Garcia-Alonso, Goñi, & Bravo, 2000) and
an in vitro colonic fermentation method (Goñi & Martin-
Carrón, 2002) were used to estimate the bioaccessible anti-
oxidant capacity of plant foods of the Spanish diet in the
human gut. This model was previously used in diVerent
food sources such as cereals (Perez-Jimenez & Saura-Cali-
xto, 2005) and green leafy vegetables (Serrano, Goñi, &
Saura-Calixto, 2005b) to study polyphenol and carotenoid
bioaccessibility, respectively.
2. Materials and methods
2.1. Chemicals
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-car-
boxylic acid), a water soluble analogue of vitamin E was
purchased from Sigma–Aldrich Química, SA (Madrid,
Spain). 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) was from
Fluka Chemicals (Madrid, Spain). All reagents used were of
analytical grade. The enzymes used for the digestive enzy-
matic treatment were as follows: pepsin (Merck), pancrea-
tin, lipase, -amylase (Sigma–Aldrich Química SA), and
amyloglucosidase (Roche).
2.2. Dietary information
Estimates of plant food intakes in the Spanish diet were
based on National consumption data (MAPA, 2001). These
data are obtained annually from daily budget question-
naires. Six thousand households are surveyed, along with
700 hotels and restaurants and 200 institutions such as
schools, hospitals and the armed forces (conWdence level
95%; error range 2% in amount of food). Each family daily
register by scanning the foods presents at home. Hotels, res-
taurants and institutions give information about purchases
of foods four times per year. Dietary intakes included in
Table 1 correspond to the intake of plant foods in Spain (g/
person/day).
2.3. Sample preparation
Two purchases of each individual plant food listed in the
National dietary survey (MAPA, 2001) (Table 1), were
acquired at diVerent local supermarkets. Individual items
selected in this study are representative of plant food com-
mon in the Spanish diet. The edible portion of the daily
amount consumed per capita for each plant food as eaten
was weighed and grouped into Wve duplicate samples, one
for each of the Wve types of plant foods: cereals (total:
221.6 g), vegetables (total: 280.8 g), legumes (22.3 g), nuts
(total: 5.9 g) and fruits (total: 200.8 g). These Wve samples
correspond to the total per capita daily intake of solid plant
food in the Spanish diet. Each duplicated sample was
freeze-dried, ground and stored until analysis.
2.4. Chemical procedure. Determination of antioxidant
capacity content in plant foods of the Spanish diet
The chemical procedure was employed to determine the
total content of antioxidant capacity in the diet (Fig. 1, Sec-
tion A).
 J. Serrano et al. / Food Research International 40 (2007) 15–21 17

Section A. Chemical approach

Aqueos-organic Supernatant: extractable dietary antioxidants Antioxidant

capacity
extraction
Plant foods
(Spanish diet)
n=6 Residue (non-extractable compounds)

Section B. In vitro physiological approach

In vitro enzymatic digestion

Plant foods n1 to n12
(Spanish diet) Pepsin (pH 1.5)
Pancreatin (pH 7.5)
Lipase+bile salts (pH 7.5)
α-amylase (pH 6.9)
amiloglucosidase (pH 4.75)

Centrifugation

n7 to n12 Supernatant Residue

Antioxidant capacity
analysis
Dialysis
Insoluble indigestible fraction
Soluble indigestible fraction

n1 to n6 n1 to n6

Total indigestible fraction

(soluble + insoluble)

In vitro colonic fermentation

24 h, 37 ºC, anaerobic conditions
n1 to n6

Fermentation supernatant Residue after fermentation

n1 to n6
Antioxidant capacity analysis

Fig. 1. Schematic of the methodology used to estimate the bioaccessibility of antioxidant capacity. n D number of samples for each food group.

2.4.1. Chemical extraction of antioxidants 15 s at 37 °C, using a Beckman DU-640 spectrophotometer

Original samples were extracted by shaking at room
temperature with methanol–water–HCl (50:50 v/v, pH 2,
50 mL/g sample, 60 min, room temperature; constant shak-
ing) and acetone–water (70:30 v/v, 50 mL/g sample, 60 min,
room temperature; constant shaking). After centrifugation
(15 min, 25 °C, 3000g) supernatants were combined and
used to determine extractable antioxidant capacity content
by FRAP and ABTS assays.
2.4.2. FRAP assay
For the FRAP assay (Benzie & Strain, 1996; Pulido,
Bravo, & Saura-Calixto, 2000), 900 L of the FRAP
reagent, containing TPTZ, FeCl3, and acetate buVer, was
mixed with 90 L of distilled water and 30 L of the test
sample of the blank (solvents used for extraction). Maxi-
mum absorbance values taken at 595 nm were taken every
(Beckman Instruments Inc., Fullerton, CA). The readings at
30 min were selected for calculations of FRAP values. Solu-
tions of known Trolox concentration were used for antioxi-
dant capacity equivalents.
2.4.3. ABTS assay
The antioxidant capacity was estimated in terms of radi-
cal scavenging activity following the procedure described
elsewhere (Re et al., 1999) with some modiWcation (Pulido
et al., 2003). BrieXy, ABTS radical cation (ABTS+«) was
produced by reacting 7 mmol/L ABTS stock solution with
2.45 mmol/L potassium persulphate in the dark at room
temperature for 12–16 h before use. The ABTS+« solution
was diluted with 5 mM phosphate buVered saline (pH 7.4)
to an absorbance of 0.70 § 0.02 at 730 nm. After addition of
0.1 mL of sample to 3.9 mL of diluted ABTS+« solution,
18 J. Serrano et al. / Food Research International 40 (2007) 15–21
absorbance readings were taken every 20 s using a Beckman
DU-640 spectrophotometer. The reaction was monitored
during 6 min. Inhibition of absorbance vs. time was plotted
and the area below the curve (0–6 min) was calculated.
Solutions of known Trolox concentration were used for
antioxidant capacity equivalents.
2.5. In vitro physiological procedure. In vitro gastrointestinal
model and antioxidant capacity bioaccessibility
There are two main steps in the methodology proposed
to estimate the bioaccessibility of dietary antioxidant
capacity: (a) isolation of the indigestible fraction (small
intestine bioaccessibility); and (b) colonic fermentation of
the indigestible fraction (large intestine bioaccessibility)
(Fig. 1, Section B). The indigestible fraction was previously
deWned as the part of vegetables that is not digested or
absorbed in the small intestine and reaches the colon where
it serves as a substrate for fermentative microXora (Saura-
Calixto et al., 2000). Indigestible fraction is made up of die-
tary Wbre and other compounds of proven resistance to the
actions of enzymes such as indigestible protein, resistant
starch, polyphenols and other bioactive compounds. Ana-
lytical conditions for indigestible fraction determination
are close to physiological conditions (pH, temperature, and
incubation times). The indigestible fraction is composed by
two fractions: a soluble fraction (supernatant of enzymatic
digestion) and an insoluble fraction (residue of enzymatic
digestion). The estimation of the small intestine bioaccessi-
bility of the antioxidant capacity was estimated in the
supernatants (soluble indigestible fraction) by FRAP and
ABTS assays.
In the in vitro colonic fermentation model, the total indi-
gestible fraction (soluble + insoluble) was fermented in
strict anaerobic conditions using rat cecal content as inocu-
lum. Several compounds are released from the food matrix
by the action of bacterial enzymes, while other compounds
remain in the food matrix as a part of the residue after fer-
mentation. The residue after fermentation contains com-
pounds of proven resistance to enzymatic and colonic
bacterial degradation, which probably would be excreted in
the feces, these comprise the unaccessible compounds. The
analysis of the antioxidant capacity of the supernatants
after the in vitro colonic fermentation allowed us to esti-
mates the antioxidant capacity bioaccessible in the large
intestine.
2.5.1. Determination of indigestible fraction
Twelve samples of each food group (n1 to n12) were suc-
cessively incubated with digestive enzymes. BrieXy, 300 mg
of sample was incubated with pepsin (EC 3.4.23.1, 0.2 mL
of a 300 mg/mL solution in HCl–KCl 0.2 M buVer, pH 1.5,
40 °C, 1 h, Merck 7190), pancreatin (1 mL of a 5 mg/mL
solution in phosphate buVer 0.1 M; pH 7.5, 37 °C, 6 h,
Sigma P-1750), lipase (EC 3.1.1.3, 2 mL of a 7 mg/mL solu-
tion in phosphate buVer 0.1 M; pH 7.5, 37 °C, 6 h, Sigma L-
3126), bile extract porcine (2 mL of a 17.5 mg/mL solution
in phosphate buVer 0.1 M; pH 7.5, 37 °C, 6 h, Sigma B-
8631) and -amylase (EC 3.2.1.1, 1 mL of a 120 mg/mL
solution in tris–maleate buVer 0.1 M; pH 6.9, 37 °C, 16 h,
Sigma A-3176). Then samples were centrifuged (15 min,
25 °C, 3000g) and supernatants removed. Residues were
washed twice with 5 mL of distilled water and all superna-
tants combined. Residues were stored at ¡18 °C for
in vitro colonic fermentation. Each supernatant was incu-
bated with 100 L of amyloglucosidase (EC 3.2.1.3, Roche,
102 857) for 45 min at 60 °C. Some supernatants (n7 to n12)
were stored al ¡18 °C for antioxidant capacity analysis.
Other supernatants (n1 to n6) were transferred into dialysis
tubes (12000–14000 MWCO; Dialysis Tubing Visking,
Medicell International Ltd., London, UK), and dialyzed
against water for 48 h at 25 °C (water Xow 7 L/h) to elimi-
nate digestible compounds. Dialysis retentates (n1 to n6)
were concentrated to 5 mL in a R-114 Büchy vacuum rota-
tory evaporator and then added to their correspondent
residue (insoluble indigestible fraction, n1 to n6, e.g. soluble
indigestible fraction n1 added to insoluble indigestible frac-
tion n1) and stored at ¡18 °C for in vitro colonic fermenta-
tion.
2.5.2. In vitro colonic fermentation
Male Wistar rats (body weight of 200 § 5 g) fed with
standard maintenance diets adjusted to rat nutritional
requirements (AO4, Panlab, Barcelona, Spain) were sup-
plied by the breeding Center at the Faculty of Pharmacy
(University Complutense of Madrid, Spain). Rats were
killed in a carbon dioxide chamber and fresh rat cecal
contents were used as inoculum. Ceca were removed
through abdominal midline incisions. Rat cecal contents
were scraped, weighed and added to a Xask containing
sterile anaerobic medium to give a 100 g/L inoculum. The
anaerobic medium adapted from Goering and Van Soest
(1970) contained trypticase, micromineral and macromin-
eral solutions and resazurin as anaerobic redox indicator.
The inoculum was mixed (10 min) in a Stomacher 80 Lab
Blender (Seward Medical, London, UK) and Wltered
(1 mm mesh) before use. Total indigestible fractions (n1 to
n6) were mixed with fermentation medium (8 mL, 4 °C,
16 h). Tubes were sealed with rubber caps (No. 407-0-13,
Ormacisa, Madrid, Spain). Two milliliter of inoculum was
added and the headspace rinsed with carbon dioxide
(1 min). Tubes were placed in a shaking water bath (37 °C,
24 h). Blanks containing no substrate and lactulose
(Sigma L-7877) were included in the experiment as zero
and completely fermentable substrate, respectively. All the
steps were carried out in an oxygen-free CO2 saturated
atmosphere. After incubation time, pH was measured and
NaOH 1 M was used to stop the fermentation process.
Samples were centrifuged (2500g, 10 min, 25 °C) and the
supernatants were collected and stored at ¡18 °C for anti-
oxidant capacity analysis (antioxidant capacity bioacces-
sible in the large intestine). The antioxidant capacity of
the supernatants was corrected with blanks of fermenta-
tion.
 J. Serrano et al. / Food Research International 40 (2007) 15–21
Table 2
Antioxidant capacity of plant foods in the Spanish diet (mol Trolox eq/g dry original sample)a
Food group Chemical approach Physiological approach
Methanol/acetone/water extracts Digestive enzyme treatment (bioaccessible
in the small intestine)
ABTS FRAP ABTS
Cereals 1.3 § 0.1 2.2 § 0.1 27.4 § 2.2
Vegetables 6.7 § 0.7 10.3 § 0.1 22.4 § 2.9
Legumes 6.4 § 0.5 9.0 § 0.2 36.0 § 0.7
Fruits 10.2 § 0.4 25.5 § 0.5 13.6 § 0.6
Nuts 33.6 § 0.8 44.8 § 1.4 36.9 § 1.1
a
Mean value § standard deviation, n D 6.
2.6. Statistical analysis of data
All data were reported as mean § standard deviation
from six replicates in each food group and treatment.
3. Results and discussion
The experimental design was carried out in two phases
(Fig. 1): (a) a chemical procedure to determine dietary anti-
oxidant capacity content and (b) a in vitro physiological
procedure to determine antioxidant capacity bioaccessibil-
ity (in vitro digestive enzyme and colonic fermentation
release of antioxidants from the food matrix).
3.1. Chemical approach
The chemical procedure consisted in conventional anti-
oxidant extraction with organic solvents (methanol/water/
HCl and acetone/water) followed by analysis of antioxidant
capacity by FRAP and ABTS assays.
Regarding chemical extraction of dietary antioxidants,
several extraction techniques have been reported. Metha-
nol, acetone, ethyl acetate and chloroform are the main sol-
vents used for extraction of low molecular weight
polyphenols (Nakamura, Suganuma, Kuyama, Sato, &
Ohtsuki, 1998; Rubilar et al., 2006; Vrhovsek, Rigo, Tonon,
& Maltivi, 2004). Carotenoids and vitamin E are principally
extracted with high apolar solvents (Castan, Villard, Jakob,
Puigserver, & Ajandouz el, 2005; Larsen & Christensen,
2005), while vitamin C is extracted with polar solvents (Fre-
nich, Torres, Vega, Vidal, & Bolanos, 2005). Because of the
complexity and diversity of antioxidant and food matrices,
nowadays there is no optimal method or solvent used to
extract all antioxidants present in foods. Since dietary anti-
oxidants may have diVerent solvent aYnities, some studies
analyse lipophilic and hydrophilic antioxidants separately
to assess antioxidant capacity but may not assess the syner-
gistic eVects of these two kinds of antioxidants. The chemi-
cal extraction procedure employed in this study combined
polar (methanol/water/HCl) and apolar (acetone/water)
extraction of antioxidants, and all solvents were compatible
with the antioxidant capacity assays employed in this study.
Table 2 shows the chemical procedure for determining
antioxidant capacity content (mol Trolox equivalent/g of
19
In vitro colonic fermentation (bioaccessible
in the large intestine)
FRAP ABTS FRAP
6.3 § 0.6 1.9 § 0.1 1.4 § 0.7
11.9 § 2.0 5.1 § 0.2 3.5 § 0.7
10.6 § 2.6 5.5 § 0.1 6.5 § 0.2
17.7 § 2.3 2.0 § 0.1 2.5 § 0.7
42.8 § 3.8 3.3 § 0.2 6.8 § 0.2
dry original sample) of solid plant foods from the Spanish
diet measured by FRAP and ABTS assays. DiVerent anti-
oxidant capacities were observed in diVerent food groups.
The nut group showed the highest antioxidant capacity per
gram of original sample, while the cereal group scored low-
est in both assays. Comparable results are reported in the
literature for ABTS assays in vegetables (Pellegrini et al.,
2003) and FRAP and ABTS in fruit and vegetables (Nils-
son et al., 2005). Our results followed the same pattern as
other reports in the literature.
We would stress the biological importance of these Wnd-
ings. The current knowledge of the types and quantities of
antioxidant components in foods reported elsewhere may
have little bearing on actual dietary antioxidant content
and bioavailability. The reason for this is that only a por-
tion (sometimes highly variable depending on the food
matrix, processing and storage) of these compounds are
extracted by the organic solvents employed in most extrac-
tion methods, whereas a considerable part remains in the
extraction residues, for example condensed tannins, hydro-
lysable tannins and other bound polyphenols that are
available in the gastrointestinal tract (Gonthier et al.,
2003). A physiological perspective on antioxidant capacity
bioaccessibility in an entire diet would yield more useful
information about possible health eVects of antioxidant-
rich diets.
3.2. Physiological approach
The physiological approach of the antioxidant capacity
is shown in Table 2. In the nut and legume groups, high
antioxidant capacity release from the food matrix was
achieved by digestive enzyme treatment and in vitro colonic
fermentation respectively, while in the fruit and cereal
groups the respective values were lower.
More than 90% of bioaccessible antioxidant capacity
was released from the food matrix by digestive enzymes.
This means that most antioxidants would be available in
the Wrst stage of the digestion process. However, numerous
studies have shown bioavailabilities of bioaccessible antiox-
idants in the small intestine to be very low, for example
polyphenols (CliVord, 2004), -carotene (Novotny, Kuri-
lich, Britz, & Clevidence, 2005), lycopene (Erdman, 2005)
and vitamin E (Lodge, 2005). Therefore, a major part of
20 J. Serrano et al. / Food Research International 40 (2007) 15–21

Table 3
Antioxidant capacity intake from solid plant foods in the Spanish diet (mol Trolox eq/day) determined by chemical and physiological approach
Food group Intake (g edible portion/day) Chemical approach Physiological approach
Methanol/acetone/water Bioaccessible in the small Bioaccessible in the large
extracts intestine intestine
ABTS FRAP ABTS FRAP ABTS FRAP
Cereals 221.65 33.4 367.4 4452.5 1026.4 308.5 227.3
Vegetables 280.19 271.6 417.5 775.2 413.7 176.2 120.9
Legumes 22.19 134.7 188.8 609.8 180.7 93.2 110.1
Fruits 200.6 341.5 850.4 339.4 443.4 50.0 62.5
Nuts 5.96 33.4 235.1 211.5 245.3 18.9 38.9
Total 903.6 2059.2 6388.4 2309.5 646.8 559.8
Antioxidant capacity intake from beverages in the Spanish diet (3894 and 2575 mol trolox equivalent measured by FRAP and ABTS methods, respec-
tively) (Pulido et al., 2003).

bioaccessible antioxidants in the small intestine could also
reach the colon.
The colon is another important site in the gastrointesti-
nal tract where antioxidants become available. The abun-
dant microXora in the colon plays a critical role in the
metabolism of antioxidants (Jenner, Rafter, & Halliwell,
2005; Serrano et al., 2005a). After microbial enzyme metab-
olism of any antioxidants that reach the colon, there are
three possible routes available, namely (1) absorption of
intact antioxidant through the colonic epithelium and pas-
sage into the bloodstream (Rios et al., 2003); (2) breakdown
of the original antioxidant structure into metabolites (Jen-
ner et al., 2005); or (3) simple availability in the colon where
they may counteract the pro-oxidant or toxic species pro-
duced during colonic bacterial metabolism.
The antioxidant capacity available after in vitro colonic
fermentation is shown in Table 2. Less antioxidant capacity
was released than by digestive enzymes, probably because
some of the antioxidants released from the food matrix
during colonic fermentation may served as a substrate for
bacterial metabolism.
The diVerences between physiological and chemical
extraction are more evident in the analysis of antioxidant
capacity intake. Table 3 shows the contribution of solid
plant foods to the antioxidant capacity of the Spanish diet
based on National Food Consumption data from the year
2000 (MAPA, 2001). No signiWcant changes in plant food
intake were found in further National Surveys (MAPA,
2004). The total antioxidant capacity of a whole diet must
include also the antioxidant capacity of beverages. Includ-
ing the antioxidant capacity from beverages (Pulido et al.,
2003), is estimated that about 9000 mol Trolox equivalents
of antioxidant capacity (small + large intestine bioaccesibil-
ity measured by ABTS) from solid plant foods would be
available in the gut. Cereals and beverages groups were the
major contributors to the estimated antioxidant capacity
intake.
3.3. Concluding remarks
Several diVerences were found between the chemical and
the in vitro physiological approaches. In all plant food
groups, the sum of small and large intestine bioaccessibility
yielded higher antioxidant capacity than the chemical pro-
cedure (Table 2), especially in the cereal, vegetable and
legume groups. A signiWcant part of the antioxidants con-
tained in plant foods are not analysed in most antioxidant
capacity assays, where the antioxidant extraction is incom-
plete. Further research is needed to identify the bioaccessi-
ble antioxidants that may produce signiWcant eVects for
gastrointestinal health.
Data on vitro physiological antioxidants may be useful
for the design and interpretation of epidemiological studies
on the eVects of antioxidants and vegetable foods on health.
Acknowledgments
The present research was performed under the Wnancial
support of the Spanish Comisión Interministerial de Cien-
cia y Tecnología (Project AGL2005-04769-ALI) and J. Ser-
rano wants to thank the scholarship of the Agencia
Española de Cooperación Internacional, Becas MAE. We
thank the technical assistance of Mrs. Ma. Rosa Redondo.
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