Journal of Food Composition and Analysis 22 (2009) 381–387

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Journal of Food Composition and Analysis

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Original Article

Phenolic compounds, carotenoids and antioxidant activity of three tropical fruits

Christian Mertz a, Anne-Laure Gancel a, Ziya Gunata b, Pascaline Alter a, Claudie Dhuique-Mayer a,
Fabrice Vaillant a, Ana Mercedes Perez c, Jenny Ruales d, Pierre Brat a,*
a
Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), Département PERSYST, UMR Qualisud, TA B-95/16,
F-34398 Montpellier Cedex 5, France
b
UMR Qualisud, Université Montpellier 2 place E. Bataillon, 34095 Montpellier Cedex 5, France
c
Centro Nacional de Ciencia y Tecnologı́a de Alimentos (CITA), Universidad de Costa Rica, San José, Costa Rica
d
Escuela Politécnica Nacional (EPN), Quito, Ecuador

A R T I C L E I N F O A B S T R A C T

Article history: Major compounds (i.e. phenolic compounds and carotenoids) were analysed in the extracts of the edible
Received 31 October 2007 part of three tropical fruits: the Andean blackberry, the naranjilla and the tree tomato. Ellagitannins and
Received in revised form 19 May 2008
anthocyanins were predominant in blackberries and phenolic composition can be used to differentiate
Accepted 2 June 2008
the two species studied. Similar phenolic composition occurred in red and yellow tree tomato except for
anthocyanins which were absent in the yellow tree tomato. Phenolic acids were detected in the naranjilla
Keywords:
pulp. Carotenoids were analysed in the fruits. The composition in carotenoids was similar in the two
Solanum quitoense
varieties of tree tomato and their vitamin A activity was calculated. Carotenol fatty acid esters were
Solanum betaceum
Rubus glaucus predominant. b-Cryptoxanthin esters and b-carotene were the major carotenoids. The carotenoid
Rubus adenotrichus content was high compared to literature data, providing an important high vitamin A activity. In
Phenolic compounds blackberries and naranjilla, this class of compounds was found only at trace level. Finally, ORAC values
Carotenoids were estimated in different solvent extracts and results were compared with published data in common
HPLC-MS fruits.
ORAC ß 2009 Published by Elsevier Inc.
Food composition
Food analysis

1. Introduction Blackberries are currently promoted as being a rich source of

The interest of consumers for novel foods is increasing
considerably. The new use of food ingredients and new types of
food in the European food supply is of economic interest for tropical
countries. Nevertheless, the access for bio-diverse products to the
European market needs many requirements, notably the knowledge
of their composition and nutritional value. As part of the PAVUC
project (Producing Added Value from Under-utilized tropical fruit
Crops), three tropical fruits (Andean blackberry, tree tomato and
naranjilla), native from Latin America and consumed as fruit juice or
desserts, were selected. Besides their organoleptic quality, their real
nutritional potential remained undetermined.
Polyphenols, together with carotenoids, are markers of the
nutritional quality of foods. Polyphenols are known for their
antioxidant activity as radical scavengers and possible beneficial
roles in human health, such as reducing the risk of cancer,
cardiovascular disease, and other pathologies (Bravo, 1998;
Hollman et al., 1996; Sellappan et al., 2002).
polyphenols, with ellagitannins and anthocyanins being the major
ones (Häkkinen et al., 1999; Siriwoharn and Wrolstad, 2004).
Anthocyanins were detected in the tree tomato (Bobbio et al.,
1983; Wrolstad and Heatherbell, 1974) and no data was found in
literature about the phenolic composition of naranjilla.
Carotenoids have been reported in tree tomato (Rodriguez-
Amaya et al., 1983). Except for the well-known provitamin A
activity of some carotenoids, they could be involved in protective
effects against degenerative or cardiovascular diseases (Olson,
1999) and are known for having antioxidant capacity (Olson,
1996). To exploit the nutritional potential of these fruits, their
composition in polyphenols and carotenoids together with their
antioxidant capacity were studied in this work.
2. Materials and methods
2.1. Plant materials

Two varieties (red and yellow) of tree tomato (Solanum

* Corresponding author. Tel.: +33 4 6761 65 03; fax: +33 4 6761 44 33. betaceum Cav.) and naranjilla (Solanum quitoense Lam.) were
E-mail address: brat@cirad.fr (P. Brat). harvested in Ecuador. The Andean blackberries were native from

0889-1575/$ – see front matter ß 2009 Published by Elsevier Inc.

doi:10.1016/j.jfca.2008.06.008
382 C. Mertz et al. / Journal of Food Composition and Analysis 22 (2009) 381–387
Costa-Rica (Rubus adenotrichus Schlech.) and Ecuador (Rubus
glaucus Benth.). After freezing, fruits were transported in refri-
gerated containers to our laboratory and stored at 20 8C for one to
five months until analysis.
2.2. Sample preparation
All analysed fruits were at fully ripe stage. Blackberries (10 kg)
were ground for 3 min in a Waring blender (Vorwerk, Montpellier,
France). Thirty red tree tomato and 40 yellow tree tomato fruits
were separately peeled, ground with an ultra turax in dark room
and seeds were removed by sieving. For naranjilla, 30 fruits were
sieved to afford the edible part of the fruit. The moisture contents
of Andean blackberries, tree tomato and naranjilla were
82.5  0.5%, 87.5  0.3% and 91.1  0.2%, respectively. For each fruit,
portions of the resulting pulp were immediately freeze dried for
polyphenol analysis. The remaining pulp was kept at 20 8C until
analysis (less than one month). It was used for carotenoid analysis
and determination of the antioxidant activity.
2.3. Chemicals
All solvents were of HPLC grade, purchased from Carlo Erba (Val
de Reuil, France) except of methyl tert-butyl ether (MTBE) (Sigma–
Aldrich, Steinheim, Germany). Folin-Ciocalteu reagent, sodium
chloride, anhydrous sodium sulphate, magnesium hydroxide
carbonate, potassium chloride and formic acid were purchased
from Carlo Erba (Val de Reuil, France). Chlorogenic and caffeic
acids, cyanidin 3-O-glucoside, kaempferol, b-carotene, b-crypto-
xanthin, lutein, and zeaxanthin were from Extrasynthese (Genay,
France). Ferulic acid, quercetin, ellagic acid 2,6-di-tert-butyl-4-
methylphenol (BHT) were from Sigma (L’isle d’Abeau, France).
Fluorescein and 6-hydroxy-2,5,7,8-tetramethyl-2-carboxilic acid
(Trolox) were purchased from Sigma (Steinheim, Germany). 2-20 -
azobis (2-amidinopropane) dihydrochloride (AAPH) was pur-
chased from Wako Chemicals (Richmond, USA).
2.4. Polyphenolic compounds extraction
Two grams of powder were extracted twice for 15 min with
60 mL of 70% aqueous acetone containing 2% formic acid. The
extracts were combined, filtered and concentrated under vacuum
to remove acetone (40 8C). The aqueous layer was concentrated to
yield aqueous acetone extract. An aliquot was analysed by liquid
chromatography with diode array detection and mass spectro-
metry (LC-DAD/MS). Minor phenolic compounds in blackberries
were extracted as previously described (Mertz et al., 2007).
2.5. Total phenolic content (TPC)
The total phenolic content was determined by the Folin-
Ciocalteu method optimized by George et al. (2005). 50 mL of
aqueous acetone extract were used for the quantification. Results
were expressed as mg of gallic acid equivalent (GAE) per 100 g of
dry matter (DM). Analysis was made in triplicate.
2.6. HPLC-DAD-MSn analysis of phenolic compounds
Samples were filtered through a 0.45 mm filter (Millipore). The
HPLC analysis was carried out on a Waters 2690 HPLC system
equipped with Waters 996 DAD (Waters Corp., Milford, MA) and
Empower Software (Waters). The separation was performed at
30 8C using a 250 mm  4.6 mm, 5 mm particle size, endcapped
reversed-phase Lichrospher ODS-2 (Interchim, Montluçon,
France). The injection volume was 10 mL and the detection was
carried out between 200 and 600 nm. MSn analysis was carried out
using a LCQ ion trap mass spectrometer fitted with an electrospray
interface (Thermo Finnigan, San Jose, CA, USA). The LC/MS
parameters and HPLC solvents used are described in our previous
published study (Mertz et al., 2007). Identifications were achieved
on the basis of the ion molecular mass, MSn and UV–visible spectra.
Blackberry phenolics were analysed as previously described
(Mertz et al., 2007). Naranjilla and tree tomato phenolics were
analysed using the following gradient: from 5% to 35% B in 50 min,
from 35% to 50% B in 5 min, from 50% to 80% B in 5 min, after which
the column was washed and equilibrated to the initial conditions.
The percentage of formic acid in both solvents was 2% for
anthocyanins and reduced to 0.1% for other phenolic compounds.
2.7. Quantitative analysis of polyphenols
The HPLC analysis was carried on a Dionex liquid chromato-
graph equipped with model P680 pumps, an ASI 100 autosampler
and a UVD 340U diode array detector coupled to a HP ChemStation
(Dionex, France), according to the previous published study (Mertz
et al., 2007). Gradient conditions were the same as used for
identification. Hydroxycinnamic acids in naranjilla and tree
tomato extracts were quantified at 280 nm using calibration
curves established with chlorogenic acid standard (R2 = 0.997).
Each analysis was made in triplicate.
2.8. Carotenoid extraction
Carotenoid extraction was adapted from that described by
Taungbodhitham et al. (1998). 3 g of puree was stirred for 5 min
with 80 mg of MgCO3 and 15 mL of extraction solvent (ethanol/
hexane, 4:3 (v/v), containing 0.1% of BHT as antioxidant). The
residue was separated from the liquid phase by filtration with a
filter funnel (porosity no. 2) and washed with 15 mL of ethanol and
30 mL of hexane. Organic phases were transferred in a separatory
funnel and successively washed with 50 mL of 10% sodium
chloride and 3 mL  50 mL of distilled water. The aqueous layer
was removed. The hexanic phase was dried under anhydrous
sodium sulphate, filtered and evaporated to dryness at 40 8C in a
rotary evaporator. The residue was dissolved in 500 mL of
dichloromethane and 500 mL of MTBE/methanol (80/20, v/v).
Samples were placed in amber vials before HPLC analysis.
The saponification was carried out in 10% methanolic KOH,
according to the method described by Fanciullino et al. (2006).
Analyses were carried out under red light to avoid carotenoid
degradation during extraction and saponification.
2.9. HPLC-MSn analysis of carotenoids and related fatty acid esters
The HPLC apparatus was a Surveyor plus model equipped of an
autosampler, a PDA detector and LC pumps (Thermo Electron
Corporation, San Jose, CA, USA). Carotenoids and related fatty acid
esters were analysed according to the previously published
method of Dhuique-Mayer et al. (2005). Carotenoids were
separated along a C30 column (250 mm  4.6 mm, 5 mm particle
size), YMC (EUROP, GmbH). The mobile phases were water/20 mM
ammonium acetate as eluent A, methanol/20 mM ammonium
acetate as eluent B and MTBE as eluent C. Flow rate was fixed at
1 mL/min and the column temperature was set at 25 8C. A gradient
program was performed: 0–2 min, 40% A/60% B, isocratic elution;
2–5 min, 20% A/80% B; 5–10 min, 4% A/81% B/15% C; 10–60 min, 4%
A/11% B/85% C; 60–71 min, 100% B; 71–72 min, back to the initial
conditions for reequilibration. The injection volume was 10 mL and
the detection was carried out between 250 and 600 nm. After
passing through the flow cell of the diode array detector the
 C. Mertz et al. / Journal of Food Composition and Analysis 22 (2009) 381–387
column eluate was split and 0.5 mL was directed to the ion trap
mass. Experiments were performed in positive ion mode. Scan
range was 100–2000, scan rate: 1 scan/s. The desolvation
temperature was set at 250 8C.
2.10. Quantification of carotenoids and related fatty acid esters
Carotenoids and related fatty acid esters were quantified by
HPLC using an Agilent 1100 System (Massy, France). The column
and gradient conditions were the same as used in mass spectro-
metry analysis. The injection volume was 20 mL. Absorbance was
followed at 290, 350, 400, 450 and 470 nm using an Agilent 1100
photodiode array detector. Chromatographic data and UV–visible
spectra were collected, stored and integrated using an Agilent
Chemstation plus software. Because reference standards for
carotenoid esters are not commercially available, the total amount
of carotenoid esters was calculated as b-carotene equivalents in
micrograms per 1 g of puree. For this purpose, only the areas
corresponding to peaks that disappear after alkaline treatment
were summed. Quantification of b-carotene, b-cryptoxanthin
zeaxanthin and lutein before and after saponification was achieved
using calibration curves established at 450 nm with authentic
standards, with five concentrations. Concentrations of standard
solutions were calculated by spectrophotometric measurement
dissolving standard with the appropriate solvent and using a molar
extinction coefficient (emol) (Britton et al., 1995). Purity of
standards was verified by HPLC and photodiode array detection.
Correlation coefficients ranged from 0.996 to 0.999. Limits of
detection (LOD) and quantification (LOQ) were calculated for b-
carotene, b-cryptoxanthin and lutein by preparing serial dilutions
of these compounds in mobile phase. Calibration curves and then
LOD and LOQ were determined with LOD = 3  S/a and
LOQ = 10  S/a (where S is the standard deviation of the blank
signal and a is the slope of the calibration curve).
The concentration of each carotenoid was expressed as
micrograms per 1 g of fresh weight (FW). Concentrations are
given as the mean of data of three assays. Recoveries were
determined by adding internal standard (b-apo-80 -carotenal)
before extraction for unsaponified extracts and after saponification
for saponified extracts.
The vitamin A activity was calculated using the following
formula: Vitamin A = (mg/b-carotene)/6 + (mg/b-cryptoxanthin)/
12 and was expressed as retinol equivalents (RE) per kg of fresh
weight.
2.11. Antioxidant activity
2.11.1. Preparation of extracts
Two grams of edible part of the fruits were weighted in
Eppendorf tubs and centrifuged 5 min at 14,000 rpm. The super-
natants were then recovered and constituted the crude extract
(CE). Others extracts were obtained as follows: 3 g of edible part of
the fruits were extracted during 30 min with 7 mL of acetone and
the mixtures were filtrated (Whatman, England) and washed with
10 mL of acetone/water (7:3, v/v). The filtrates were evaporated
under vacuum at 40 8C and then diluted with water in a 10 mL flask
and constituted the acetone extracts (AE). 5 mL of AEs were
washed with 2 mL  5 mL hexane. Thus, we obtained the washed
acetone extract (WAE). 3 mL of WAEs were loaded onto a 3 mL
XAD-7 column to remove the non-phenolic compounds. The
column was then washed with 2 mL  5 mL of water. Phenolic
compounds were desorbed with 2 mL  5 mL of methanol/water
(8:2, v/v) and 2 mL  5 mL of pure acetone. The eluates were
evaporated under vacuum at 40 8C and then diluted with water in a
10 mL flask and constituted the XAD-7 Extracts (XAD-7E). The
383
extraction of carotenoids was achieved as described above to
afford the Hexane extracts (HEs). The residues obtained after
evaporation of the hexanic phase were dissolved in pure acetone in
a 10 mL flask to constitute the HEs.
2.11.2. ORAC assay
ORAC assays were performed according to Huang et al. (2002).
We used with a microplate spectrofluorimeter TECAN Infinite 200
(TECAN Austria GmbH) in 96-well polypropylene plates. The
excitation and emission wavelengths were 485  9 nm and
520  20 nm, respectively. Solutions were all prepared with
75 mM Phosphate buffer (pH 7.4) except for the measurement of
the ORAC value of HEs where the buffer was replaced by acetone in
the Trolox solutions and sample solutions. Each well was filled with
160 mL of a 78.75 nM fluorescein (FL) solution (63 nM final in the
well) and 20 mL of the buffer or acetone (blank), the standard 0–
40 mM Trolox solution (0–4 mM final), or the sample (crude, acetone,
washed acetone, and hexane extracts with appropriate dilution). The
plate was incubated at 37 8C during 15 min before 20 mL of a 178 mM
AAPH solution (17.8 mM final) were added. Fluorescein and trolox
solution were daily made using a 787.5 mM and a 500 mM stock
solution, respectively, and stored in the dark at 4 8C. AAPH was made
daily and discarded within 8 h if unused. After the AAPH addition, the
fluorescence decay is measured every minute during 60 min.
The final values were calculated by using a regression equation
between the trolox concentration and the net area under the FL
decay curve. The area under the curve (AUC) and the net AUC are
calculated as follow:
 
f f f f f 60
AUC ¼ 0:5 þ 1 þ 2 þ 3 þ . . . þ 59 þ 0:5
f0 f0 f0 f0 f0
where f0 is the initial fluorescence reading at 0 min and fi is the
fluorescence reading at time i.
net AUC ¼ AUC sample  AUCblank
The relative trolox equivalent ORAC value is calculated as rela-
tive ORAC value = [(AUCsample  AUCblank)/(AUCtrolox  AUCblank)]
(molarity of trolox/molarity of the sample)
The ORAC values were expressed as micromole Trolox
equivalents per gram of fresh weight (mM TE/g FW).
2.11.3. Statistical analysis
All extractions were made in quadruplicates and four dilutions
were made for each extract. Each value was the mean of the ORAC
values from the four extractions that were the mean of the ORAC
values obtained for the four dilutions. The standard deviations
were also calculated for the four extractions. Analyses of variance
were performed with the XLSTAT 2007.6 software (Addinsoft).
Differences at P < 0.05 were considered significant.
3. Results and discussion
3.1. Analysis of phenolic compounds
The identification of phenolic compounds in blackberries was
reported in our previous study (Mertz et al., 2007). Briefly, major
compounds were anthocyanins (A1, A2, A3) and ellagitannins (E1,
E2) and other phenolic compounds were minor (data not shown).
These identifications were supported by literature data (Mullen
et al., 2002; Mullen et al., 2003; Tanaka et al., 1993).
UV–visible spectra, LC–MS, and the subsequent fragmentation
of the predominant ions in MS–MS were used to analyse aqueous
acetone extracts of tree tomato and naranjilla. Whenever possible,
chromatographic retention and literature data were used to
support the identification of the compounds. Chromatographic
384 C. Mertz et al. / Journal of Food Composition and Analysis 22 (2009) 381–387

tentatively identified as dicaffeoylquinic (DCQA) and caffeoylqui-

nic acids, respectively. Two other compounds with similar UV
spectra, present in the tree tomato extracts only, had m/z at 341
and 355 in the negative mode. They are identified as caffeoyl and
feruloyl hexose, respectively, according to literature data (Clifford
et al., 2007; Määttä-Riihinen et al., 2003; Määttä-Riihinen et al.,
2004).

3.2. Total phenolic content

Blackberries had an average concentration of TPC of 4250 mg/

Fig. 1. 15–50 min segment of LC-DAD chromatograms at 510 nm of red tamarillo
extract (gradient 2% 5–35% en 50 min ). A4: delphinidin glucosyl rutinoside; A5:
delphindin rutinoside; A6: cyaniding rutinoside; A7: pelargonidin rutinoside.
profiles of red and yellow tree tomato are very similar (data not
shown) except of the presence of anthocyanins in the red variety.
Fig. 1 shows the LC-DAD chromatogram at 510 nm of the red tree
tomato extract. Four compounds were detected with UV–visible
data characteristic of anthocyanins (Table 1). LC–MS data in the
positive ion mode showed molecular ions at m/z 773 (peak A4), 611
(peak A5), 595 (peak A6), and 579 (major peak A7). MS2 spectrum of
A4 had fragment ions at m/z 627 (M-146, loss of a rhamnosyl unit),
465 and 303 (loss of two hexosyl units). Peaks A5, A6 and A7 had
molecular ions at m/z 611, 595 and 579, respectively, with the
same fragmentation scheme (minor ion at M-146, major ion at M-
308). Thus, A4, A5, A6 and A7 were expected as being delphinidin
glucosyl rutinoside, delphinidin, cyanidin, and pelargonidin
rutinoside, respectively. Major anthocyanins A5, A6 and A7 have
been previously identified in tree tomato fruit from New Zealand
(Wrolstad and Heatherbell, 1974).
The analysis of tree tomato and naranjilla extracts revealed the
presence of two compounds with UV spectra matching with
hydroxycinnamic acids (Table 1). LC/MS experiments in the
negative mode afforded ions at m/z 515 and 353 which fragmented
to produce ion at m/z 191. According to MS and literature data
(Clifford et al., 2007; Lai et al., 2007) these compounds were
100 g DM and 6300 mg/100 g DM in R. adenotrichus and R. glaucus,
respectively. This TPC is significantly higher than those found for
naranjilla (650) and tree tomato fruits (308–570). Blackberries are
traditionally a rich source of polyphenols. It is difficult to compare
the TPC content found in this study with literature data on berries
because most of the authors did not substract interfering
compounds (e.g. reducing sugars or ascorbic acid) leading thus
to an overestimation of the TPC (George et al., 2005). Nevertheless,
values obtained are higher than those mentioned in literature
(Heinonen et al., 1998; Sellappan et al., 2002; Wada and Ou, 2002)
suggesting that these blackberries have a high antioxidant
potential. No data about total phenolic content in the literature
were found for tree tomato and naranjilla.
3.3. Content of phenolic classes
3.3.1. Blackberries
In agreement with previous papers, ellagitannins and antho-
cyanins are the major phenolic classes that characterize Rubus
species (Kähkönen et al., 2001; Määttä-Riihinen et al., 2004). The
amounts found in R. glaucus and R. adenotrichus ranged from 1000
to 3000 mg/100 g DM (data not shown).
3.3.2. Tree tomato
Pelargonidin rutinoside (115 mg/100 g DM) and delphinidin
rutinoside (33 mg/100 g DM) were the two major anthocyanins in
the red variety. As expected, this phenolic class having a typical
purple colour was absent in the yellow tree tomato. All
hydroxycinnamic acids identified were quantitatively higher in
the red tree tomato than in the yellow one (Table 2). These results
are in agreement with the TPC, which is higher in the red tree
tomato.

3.3.3. Naranjilla
Table 1 Only mono and dicaffeoyl quinic acids were quantified. The first
Identification of phenolic compounds by using their spectral characteristics in LC- one was predominant (106 mg/100 g DM) while DCQA content was
DAD, positive or negative ions in LC–MS and MSn, and previous identification dataa.
lower (5.4 mg/100 g DM). No data was found in the literature about
Tentative identification LC-DAD data (nm) LC–MS data (m/z)b the phenolic composition of this fruit.
MS MS2/MS3
3.4. Analysis of carotenoids and related fatty acid esters
Tree tomato
Delphinidin glucosyl 280, 526 773 (+) 627, 303
rutinoside (A4) 3.4.1. Qualitative analysis
Delphinidin rutinoside (A5) 280, 526 611 (+) 465, 303 The HPLC chromatograms of unsaponified extracts of tree
Cyanidin rutinoside (A6) 280, 516 595 (+) 449, 287 tomato fruit (red and yellow) indicated that carotenol fatty acid
Pelargonidin rutinoside (A7) 280, 502 579 (+) 433, 271 esters eluting after b-carotene were predominant (data not
Dicaffeoylquinic acid 292, 318 515 () 353, 191
Caffeoylquinic acid 290sh, 326 353 () 191
shown). LC/MS analysis in the positive mode revealed that esters
Caffeoyl glucose 296sh, 328 341 () 179 of b-cryptoxanthin were the major compounds, with b-cryptox-
Feruloyl glucose 298sh, 329 355 () 193 anthin esterified with myristic and palmitic acids. These results
Naranjilla
were established on the basis of LC/MS and literature data
Dicaffeoylquinic acid 292, 318 515 () 353, 191 (Breithaupt and Bamedi, 2001; Wingerath et al., 1996). Whenever
Caffeoylquinic acid 290sh, 326 353 () 191 possible, co-chromatography with authentic standards supported
a
Abbreviations: sh, maximum of the shoulder in the spectrum; (+): positive
the identifications. b-Carotene was the major free carotenoid
mode; (): negative mode. present in these unsaponified extracts. After saponification, the
b
In MS–MS, the most abundant parent ion in LC–MS is fragmented. major xanthophylls found in the tree tomato extracts were
 C. Mertz et al. / Journal of Food Composition and Analysis 22 (2009) 381–387 385

Table 2
Contentsa of phenolic compounds in tree tomato.

Phenolic compounds Red tree tomato Yellow tree tomato

Anthocyanins
Delphinidin glucosyl rutinoside (A4) 5.3  0.2c b

b
Delphinidin rutinoside (A5) 32.7  0.4
b
Cyanidin rutinoside (A6) 12.1  0.2
b
Pelargonidin rutinoside (A7) 115.0  1.3

Hydroxycinnamic acids
Dicaffeoylquinic acid 21.0  0.3 17.1  0.2
Caffeoylquinic acid 54.8  0.4 32.8  0.2
Caffeoyl glucose 9.7  0.1 3.7  0.01
Feruloyl glucose 9.8  0.1 6.3  0.05
a
The contents are expressed in milligrams standard equivalents per 100 g of dry
matter and are the average of three assays.
b
Not detected. Fig. 2. Fluorescent decay curves of fluorescein in the presence of Trolox at different
c
Standard deviation. concentrations and crude extract (CE) of red tree tomato.

b-cryptoxanthin, lutein and zeaxanthin, together with two other
unidentified compounds at m/z 601 and 585, in the positive mode.
In the unsaponified extracts, carotenol mono- and bis-fatty acid
esters were detected. Thus, according to literature data, UV
characteristics and MS analyses, lutein dimyristate, b-cryptox-
anthin myristate and palmitate, zeaxanthin dimyristate, and
zeaxanthin dipalmitate were tentatively identified (Breithaupt
et al., 2002; Khachik et al., 1988; Wingerath et al., 1996). No
structural elucidation was performed to distinguish between the
regioisomers. The chromatographic profiles of saponified and
unsaponified extracts were identical in both red and yellow tree
tomato. The only difference was the slightly higher amount of
carotenol esters (and free carotenoids after saponification) in the
red variety. In blackberries and naranjilla, b-carotene was the
major carotenoid.
3.4.2. Quantification
Quantitative analysis of tree tomato extracts was performed
before and after saponification. b-Apo-80 -carotenal was used as
internal standard for the quantitative determination of some
carotenoids and carotenol fatty acid esters. In unsaponified
extracts, carotenoid esters were major and represent 78% of the
total carotenoid content (Table 3). b-Carotene was the major free
carotenoid (4.6 and 5.1 mg/g FW in the yellow and red tree tomato,
respectively). The recovery of the internal standard without
saponification was about 93%.
After saponification, b-cryptoxanthin, lutein and zeaxanthin
were quantified using calibration curves established with the
available corresponding standards. The results are presented in
Table 3
Contentsa of carotenoids in tree tomato.
Carotenoid Yellow tree tomato
Before saponification
Zeaxanthinb 0.1  0.02d
b-Cryptoxanthin 1.1  0.1
b-Carotene 4.6  0.3
Estersc 22.6  1.0
After saponification
Lutein 0.98  0.05
Zeaxanthinb 0.59  0.02
b-Cryptoxanthin 13.5  0.1
a
Contents are expressed as mg standard equivalents per gram of fresh weight.
Results are the mean of three independent determinations. The limits of detection
(LOD) and the limits of quantification (LOQ) were 0.07 mg and 0.23 mg for b-
cryptoxanthin, 0.004 and 0.013 mg for b-carotene, 0.0051 and 0.017 mg for lutein.
b
Contents are expressed as lutein equivalents.
c
Contents are expressed as b-carotene equivalents.
d
Standard deviation.
Table 3. The recovery of the internal standard was more than 85%
and b-cryptoxanthin content was the higher with values ranging
from 14 to 16 mg/g FW. Lutein and zeaxanthin contents were lower
(1.25 and 1.7 mg/g FW, respectively, in the red tree tomato). These
values are in agreement with previous studies (Rodriguez-Amaya
et al., 1983). The carotenoid content of the tree tomatoes is higher
than that of most tropical fruits, making them nutritionally
interesting (Breithaupt and Bamedi, 2001; Kimura et al., 1991;
Rodriguez-Amaya, 1999). Vitamin A values calculated were about
2000 RE/kg FW. In blackberries and naranjilla, carotenoids were
found only at low level (data not shown).
3.5. Antioxidant activity assays
Many assays exist to evaluate the antioxidant activity but the
ORAC method remains the most utilized. It enables to determine
the antioxidant activity in both hydrophilic and lipophilic media. In
the ORAC assay, the peroxyl radical reacts with the fluorescein to
form non-fluorescent product (Prior et al., 2005). Fig. 2 shows the
fluorescent decay curves of fluorescein in the presence of Trolox at
different concentrations and in the crude diluted extract (CE) of the
red tree tomato. Out of concern for clarity, the curves of others
extract were not drawn on the Fig. 2 but appeared similar to the
curve of the red tree tomato extract (CE).
Table 4 shows the ORAC value (expressed as mmol Trolox
equivalents per gram of fresh weight) of different extracts obtained
from the edible parts of the fruits. The crude extract corresponded
to the centrifuged edible part of fruits. The acetone (AE), washed
acetone (WAE) and XAD-7 (XAD-7E) extracts were more or less
purified phenolic fractions. The hexane extract corresponded to the
fraction of carotenoids (Fig. 3).
The ORAC values of the crude extracts ranged from 6.5 to 18.7
Red tree tomato for the yellow tree tomato and the Andean blackberry, respec-
tively. These values are rather high when compared to those of
fruit juice usually consumed in Europe such as apple (ORAC
0.3  0.06
1.5  0.08 value = 1.9 mM TE/g of fresh fruit), tomato (1.6), red grape (4.0),
5.1  0.3 orange (6.8) (Wang et al., 1996). The ORAC value of the Andean
25.7  1.0 blackberry (18.7) is in total agreement with the ORAC values (20.3–
24.6) from other cultivars of blackberries obtained by Wang and
1.25  0.05 Lin (2000). For the CE extracts, none extraction was performed.
1.7  0.06 Free hydrosoluble compounds, such as organic acids, vitamin C,
15.8  0.1 some phenolic compounds, hydrosoluble, carotenoids, proteins,
minerals, etc, were present. The antioxidant activities of these CE
resulted from the interaction of all these compounds having
antioxidant or pro-oxidant activities.
The ORAC values of the AEs were higher compared to those of CE
(from 8.1 to 42.6). 70% acetone enables the extraction of phenolic
compounds bounded to insoluble cell wall material, therefore not
386 C. Mertz et al. / Journal of Food Composition and Analysis 22 (2009) 381–387

Table 4
ORAC values expressed as mM TE/g FW of different extracts of naranjilla, tree tomatoes and Andean blackberry.

Fruit CEa AEb WAEc XAD-7Ed HEe

Yellow tree tomato 6.5f  0.3g Ah 8.1  0.4 B 7.3  0.3 C 4.5  0.4 D 0.14  0.02
Red tree tomato 10.0  0.3 A 14.8  0.5 B 14.7  0.7 B 12.1  0.1 C 0.18  0.06
Naranjilla 11.6  0.4 A 16.4  1.3 B 15.0  1.2 B 11.8  1.6 A 0.15  0.02
Andean blackberry 18.7  0.9 A 42.6  1.0 B 43.6  1.4 B 38.6  2.2 C 0.12  0.01
a
Crude extract (CE).
b
Acetone extract (AE).
c
Washed acetone extract (WAE).
d
XAD-7 extract (XAD-7E).
e
Hexane extract (HE).
f
Mean of four repetitions.
g
Standard deviation.
h
Values with the same letter do not present significant difference (P = 0.05).

Fig. 3. Preparation of crude, acetone, washed acetone, XAD-7 and hexane extracts for ORAC assays.

present in the crude centrifuged extract. It is particularly true for
the Andean blackberry extract. The acetone 70% solvent improved
considerably the extraction of anthocyanins. Thus, for the Andean
blackberry, the antioxidant activity of AE was more than twice
higher than the antioxidant activity of CE. Concerning the other
fruits, the ORAC values increased from 25% to 48% between CE and
AE for the yellow and red tree tomato, respectively. Although the
antioxidant activity was mainly due to the phenolic compounds, it
may be also due to the more hydrophilic carotenoids (i.e.
xanthophylls), organic acids, vitamin C and reducing sugars, also
extracted with this solvent mixture.
The further step consisted in removing carotenoids by two
consecutive liquid–liquid extractions with hexane (WAE). No
significant difference appeared for the naranjilla, red tree tomato
and Andean blackberry. However, there was a significant
difference between the AE and WAE for the yellow tree tomato,
even if values were closed from each other. The combination of
more hydrophilic carotenoids with other antioxidant compounds
do not seem having neither a synergic nor an antagonist effect. The
ORAC value of the WAE of Andean blackberry (43.6) is in
agreement with the values obtained by Moyer et al. (2002). These
authors reported ORAC values between 41.6 and 78.8 mM TE/g FW
for different species of blackberry.
ORAC values of the XAD-7E ranging from 4.5 to 38.6 were all
lower than the ORAC values of WAEs. The losses of antioxidant
activity, after purification on XAD-7, represented 11–38% of the
WAEs. The higher the WAEs values, the lower were the losses of
antioxidant activity. We can note that the fruits were ranked in the
same order regarding their total phenolic content (expressed as mg
GAE per 100 g of fresh weight) but no linear correlation can be
established between them (data not shown). The purification of
WAEs on XAD-7 enabled to remove all the hydrophilic compounds
that were not phenolic compounds, such as vitamin C, organic
acids, minerals, etc. Thus, for all the studied fruits, phenolic
compounds were the main contributors of the hydrophilic
antioxidant power.
Finally the ORAC values of the HEs that represented the extract
of carotenoids were very low compared to the ORAC values of the
XAD-7E. They ranged from 0.12 to 0.18 mM TE/g FW for the Andean
blackberry and the red tree tomato, respectively. These results are
similar to those obtained by Wu et al. (2004), with ORAC values
being 0.11 for the honeydew, 0.24 for the tomato and the kiwi, and
0.35 for the grapefruit.
4. Conclusions
The analysis of polyphenols and carotenoids was achieved in the
Andean blackberry, the tree tomato and the naranjilla. Ellagitannins
and anthocyanins were major phenolic compounds in blackberry. To
our knowledge, anthocyanins and hydroxycinnamic acids were
 C. Mertz et al. / Journal of Food Composition and Analysis 22 (2009) 381–387
identified and quantified in tree tomato and naranjilla for the first
time. Carotenol fatty acid esters were abundant in tree tomato with
b-cryptoxanthin esters being the major one. b-Carotene was the
major hydrocarbon carotenoid. The carotenoid composition was
similar in both varieties of tree tomato. The three fruits studied have
a higher antioxidant potential than that of most of fruits, making
them nutritionally interesting.
Acknowledgements
Tropical fruits were supplied by the Centro Nacional de
Investigación en Tecnologia de Alimentos (CITA, Costa-Rica) and
the Escuela Politecnica Nacional (EPN, Ecuador). We express our
gratitude to Emmanuelle Meudec (INRA, UMR 1083, plateforme
Polyphénols) for assistance with LC–MS analysis; Gilles Morel
(CIRAD) and Hélène Fulcrand (Institut National de la Recherche
Agronomique) for help and advices in this study. This work was
financially supported by the European Union (PAVUC project, INCO
no. 015279).
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