HPLC–DAD–ESI–MS Analysis of Phenolic

Compounds During Ripening in Exocarp and

Mesocarp of Tomato Fruit
Armando Carrillo-López and Elhadi Yahia

C: Food Chemistry
Abstract: Identification of phenolic compounds was done by means of liquid chromatography (HPLC) coupled to mass
spectrometry (MS) using the electrospray ionization interface (ESI). Quantification of phenolic compounds was carried
out by using HPLC with diode array detector (DAD) in exocarp and mesocarp of tomato fruit at 6 different ripeness
stages (mature-green, breakers, turning, pink, light-red, and red). Several phenolic compounds were identified including
chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, and rutin and some combined phenolic acids were tentatively
identified, mainly glycosides, such as caffeoyl hexose I, caffeoyl hexose II, caffeoylquinic acid isomer, dicaffeoylquinic
acid, p-coumaroyl hexose I, p-coumaroyl hexose II, feruloyl hexose I, feruloyl hexose II, siringyl hexose, and caffeoyl
deoxyhexose hexose. Fruit exocarp had higher quantities of total soluble phenolics (TSP) compared to mesocarp. During
ripening, TSP increased in both exocarp and mesocarp, mainly in exocarp. While rutin increased, chlorogenic acid
decreased in both tissues: exocarp and mesocarp.

Keywords: caffeic acid, chlorogenic acid, Licopersicon esculentum, rutin, TOF-MS

Practical Application:Since exocarp showed higher TSP content than mesocarp, it is recommended to food processors
that tomato exocarp should not be discarded but processed along with tomato mesocarp.

Introduction nolic compounds besides flavonoids in tomatoes. Concerning to

It has been suggested that the consumption of fruit and veg-
etables is able to reduce the risk of some chronic diseases, such
as cancer and cardiovascular diseases (Willett 1994; Temple 2000).
For example, the consumption of fresh tomatoes and tomato prod-
ucts has been inversely related to the development of some types
of cancer (Giovannucci 1999). Phenolic compounds have been
suggested to play an important role in human health due to the
properties related to their antioxidant capacity, by which they
may provide antioxidant protection against oxidative processes and
achieve the prevention of some diseases (Le Marchand and oth-
ers 2000; Manach and others 2004). Phenolic compounds are
important components in many fruits and vegetables. They are
water-soluble substances that tend to accumulate in the dermal
tissues of plant body because of their potential role in protection
against ultraviolet radiation (Strack 1997), and have been related
to growth, development, and defense activities of the plant against
pathogens, parasites, and predators, in addition of imparting color
to some fruits and vegetables (Robards and Antolovich 1997).
In tomato, Minoggio and others (2003) reported that the main
phenolic acids were chlorogenic and caffeic acid, among others
such as ferulic acid, rutin, and a chlorogenic acid derivative that
were also present. Slimestad and Verheulm (2009) also reported
that chlorogenic acids and related compounds are the main phe-
chlorogenic acid health properties, Farah and Donangelo (2006)
reported that this phenolic compound has a number of benefi-
cial health properties related to their potent antioxidant activity
as well as hepatoprotective, hypoglycemic, and antiviral activity.
Analyses of phenolic compounds usually start with acid or alkali
hydrolysis to release phenolic compounds from their glycosides
and ester conjugates. However, in plants the free forms of phenolic
acids are rarely present, whereas the natural occurrence of phe-
nolic compounds is mainly as conjugated forms (Robbins 2003).
The exocarp of several fruit and vegetables has been shown to
be richer in nutrients/phytochemicals (including phenolic com-
pounds) compared to the mesocarp (Arima and Rodriguez-Amaya
1988; Chang and others 2000; Tomás-Barberán and others 2001;
Silva and others 2002). This work investigated the qualitative and
quantitative changes of phenolic compounds in mesocarp and ex-
ocarp of “Caiman” tomato fruit during ripening, looking for
differences between peel and pulp, among fruit ripeness stages and
between hydrolyzed and nonhydrolyzed extracts since the reports
of phenolic composition are mostly concerned with the free phe-
nolic acids (those derived from the hydrolyzed extracts plus the
naturally occurring free phenolic acids) rather than conjugated
phenolics (those derived from the nonhydrolyzed extracts as nat-
urally occurring conjugated compounds)

Materials and Methods

MS 20130317 Submitted 3/6/2013, Accepted 9/19/2013. Author Armando
Carrillo-López is with Maestrı́a en Ciencia y Tecnologı́a de Alimentos, Facultad de Sources of standards and solvents
Ciencias Quı́mico-Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Standards of p-coumaric acid, chlorogenic acid, caffeic acid,
80000, México. Author Elhadi M. Yahia is with Facultad de Ciencias Naturales,
Universidad Autónoma de Querétaro, Juriquilla, 76230, Querétaro, México. Direct and ferulic acid were purchased from Sigma-Aldrich (St. Louis,
inquiries to author Carrillo-López (E-mail: acarrill@uas.edu.mx). Mo., U.S.A.). Methanol (HPLC grade), acetonitrile (HPLC grade)
and formic acid (99% purity) were obtained from JT baker



C 2013 Institute of Food Technologists
R

doi: 10.1111/1750-3841.12295 Vol. 78, Nr. 12, 2013 r Journal of Food Science C1839
Further reproduction without permission is prohibited
 Phenolics changes during tomato ripening . . .

(Baker Mallinckrodt, México). HPLC-grade water was used for Ciocalteu reagent (dilution 1:10) and 120 μL of 7.5% of Na2 CO3
identification analyses. All other chemical reagents used were of were added. The plates were incubated for 2 h in darkness, and ab-
analytical grade. sorbance was measured at 630 nm using a Dynex MRX microplate
reader (Dynex Technology Chantilly, Va., U.S.A.). Results were
Plant Material and Sampling expressed as milligrams of gallic acid equivalents (GAE)/100 g
Tomato (Lycopersicon esculentum Cv Caiman) was grown in fresh weight (fw).
greenhouses from Acámbaro, Guanajuato, México. It was har-
C: Food Chemistry

vested at its mature-green stage of ripeness and transported to the

Phytochemicals & Nutrition Laboratory at the Faculty of Natu- HPLC–DAD–ESI–MS analytical conditions
ral Sciences of the Autonomous Univ. in Queretaro. Fruits were Samples (50 μL) from the different extracts were injected into
ripened in air at 25 ◦ C, 80% to 85% RH, and sampled at 6 dif- a HP 1100 series HPLC–DAD system (Hewlett-Packard GmbH,
ferent ripeness stages (mature green, breakers, turning, pink, light Waldbronn, Germany) coupled to a 6210 time of flight (TOF)
red, and red) for evaluation. The ripeness assessment was done mass spectrometer (Agilent, Palo Alto, Calif., U.S.A.). The phe-
using the color classification requirements in tomatoes from the nolic compounds were separated using a 5-μm X-Terra (4.6 × 250
United States Standards for Grades of Fresh Tomatoes. Exocarp mm) column (Waters Co., Milford, Mass., U.S.A.) operating at 25
(thickness ∼ 1 mm) was separated from mesocarp using a potato ◦ C. The mobile phase was composed of 1% formic acid in water
peeler. Exocarp was freeze-dried, and both exocarp and mesocarp (A) and acetonitrile (B) following a linear gradient from 100% A
were kept at –80 ◦ C until analysis. Seeds were discarded. and 0% B to 0% A and 100% B in 60 min using a flow rate of 0.5
mL/min. The 1% formic acid in water was to promote molecular
Phenolic compounds extraction ionization. Mass spectrometer system was equipped with an elec-
Five grams of fresh mesocarp or 0.4 g of freeze-dried exocarp trospray interface operating in the negative ionization mode. A
were each mixed with 10 mL of extraction solution (80% methanol mass hunter manager software (A.02.01) was used. Nitrogen was
in water, containing 1% formic acid) and thoroughly ground in utilized as drying gas at 350 ◦ C, at a flow rate of 11.5 L/min and
mortar and pestle. The homogenate was sonicated during 15 min at a pressure of 45 . Fragmentor and capillary conditions were
at room temperature and filtered through Whatman 3 filter paper. 100 and 4000 V, respectively.
This was named the nonhydrolyzed extract. Two milliliters of this
extract were mixed with 2 mL of 2.4 N HCl for hydrolysis in
water bath at 80 ◦ C for 2 h and then the test tubes were cooled Statistical analysis
with water at room temperature. This latter extract was named the Data are means of 3 determinations. Analysis of variance
hydrolyzed extract. Both extracts were filtered through 0.45-μm (ANOVA) was conducted and the significance of difference be-
Millipore membrane (Millipore Corp., Bedford, Mass., U.S.A.) tween means was determined by Tukey method at a 5% signifi-
before injection to the HPLC–DAD–ESI–MS system. cance level. Limit of detection (LOD) and limit of quantitation
(LOQ) were obtained from the calibration curves for chlorogenic,
Quantification of TSP caffeic, ferulic, and p-coumaric acids, using the slope value (S)
Aliquots from exocarp or mesocarp nonhydrolyzed extracts of the respective regression line and the standard deviation of the
were diluted 1:10 with HPLC-grade water and 30 μL of diluted y-intercepts (σ ). The used formulas were LOD = 3.3σ /S and
sample per hole was placed in 96-hole plates, and 150 μL of Folin– LOQ = 10σ /S.

40 Figure 1–Total soluble phenolics content in

exocarp and mesocarp of “Caiman” tomato at
Total soluble phenolics, mg GAE/100 g fw

different stages of ripeness. MG, mature green; B,

Exocarp breakers; T, turning; P, pink: LR, light-red; R, red.
Mesocarp
30

20

10

0
MG B T P LR R

Tomato ripeness stage

C1840 Journal of Food Science r Vol. 78, Nr. 12, 2013

Phenolics changes during tomato ripening . . .

Table 1–Fragment-ion identification of nonhydrolyzed extracts from exocarp and mesocarp of tomato.

Peak(Tr, min) Fragments of negative ions, (m/z) Tentative fragment ion identification Tentative compound
2 (17.1) 325 [326-H ]− p-Coumaroyl hexose p-Coumaroyl hexose I
163 [326-H-162] − p-Coumaroyl ion
119 [326-H-162-44] − Decarboxilated p-coumaroyl
3 (17.3) 341 [342-H] − Caffeoyl-hexose Caffeoyl hexose I
179 [341-H-162] − Caffeoyl-ion

C: Food Chemistry
135 [341-H-162-44] − Decarboxilated caffeoyl
4 (17.9) 515 [516-H ] − Dicaffeoylquinic acid ion Dicaffeoylquinic acid
353 [516-H-162] − CQ acid
5 (18.2) 355 [356-H] − Feruloyl hexose Feruloyl hexose I
193 [356-H-162] − Feruloyl ion
6 (18.5) 341 [342-H] − Caffeoyl hexose p-Caffeoyl hexose II
179 [342-H-162] − Caffeoyl ion
135 [342-H-162-44] − Decarboxilated caffeoyl
7 (18.8) 353 [354-H] − CQ CQ isomer
173 [354-H-180] − CQ ion minus dehydrated quinic moiety
179 [354-H-174] − Caffeic acid ion
191 [354-H-162 ] − CQ ion minus caffeoyl moiety
8 (19.2) 325 [326-H ] − p-Coumaroyl hexose p-Coumaroyl hexose II
163 [326-H-162] − p-Coumaroyl ion
Chlo (19.5) 353 [354-H] −
173 [354-H-162-18] − Chlo acid ion minus dehydrated quinic acid
179 [354-H-174] − Caffeic acid ion
191 [354-H-162] − Chlo ion minus caffeoyl moiety
10 (20.8) 355 [356-H] − Feruloyl hexose Feruloyl hexose II
193 [356-H-162] − Feruloyl ion
11 (21.6) 359 [359-H] − Syringyl hexose Siringyl hexose
197 [359-H-162] − Syringic ion
153 [359-H-162-44] − Decarboxylated siringic ion
Caf (22.7) 179 [360-H-179] −
135 [180-H-44] − Decarboxylated caffeoyl ion
14 (23.4) 487 [488-H] − Deoxyhexose hexose caffeoyl Caffeoyl deoxyhexose hexose
179 [488-H-162-146] − Caffeoyl ion
Rut (24.2) 609 [610-H] −
Cou (25.6) 163 [164-H] − Coumaroyl ion
119 [164-H-44] − Decarboxilated coumaroyl
Peaks 1, 9, 12, 13, 16, 17, 18, 19, 20 were not identified. Tr, retention time; Chlo, Chlorogenic acid; Caf, Caffeic acid; Rut, Rutin; Cou, p-Coumaric acid; CQ, caffeoylquinic acid.

Results and Discussion
Total soluble phenolics
TSP were more than twice higher in the exocarp than in the
mesocarp during all stages of ripening (Figure 1). TSP increased
significantly (P < 0.05) in both tissues during ripening, more pro-
nouncedly in exocarp. The TSP levels observed in the mesocarp
(12.7 to 16.8 mg/100 g fw) were similar to those observed by
Minoggio and others (2003) in tomato, but higher than those
observed by Kacjan-Maršić and others (2011) for 11 cultivars
from which the cultivar cherry showed the highest content (10.39
mg/100 g fw). The higher TSP content in the exocarp is in agree-
ment with the fact that phenolic compounds tend to accumulate
mainly in the dermal tissues of plant body because of their poten-
tial role in protection against ultraviolet radiation (Strack 1997),
in addition to the defense activities of the plant against pathogens,
parasites, and predators (Robards and Antolovich 1997).
UV and mass spectra by comparison with commercial standards,
and a tentatively identification of naturally occurring combined
phenolic compounds was based on their fragmentation patterns of
the mass spectra. The quantification of the identified compounds
was done by HPLC–DAD comparing the UV-peaks areas with
calibration curves produced with commercially obtained standards.
A total of 15 phenolic compounds were identified or tentatively
identified, taking into account exocarp, mesocarp and if the ex-
tracts were hydrolyzed or nonhydrolyzed.
Naturally occurring aglycones
From the nonhydrolyzed extracts, 3 naturally occurring agly-
cones of phenolic acids were identified; chlorogenic acid in
both exocarp and mesocarp, caffeic acid only in exocarp, and
p-coumaric acid only in mesocarp.

Identification and quantification of phenolic compounds in

the exocarp and mesocarp
HPLC–DAD–ESI–MS detection operating in the negative ion
mode was used to identify phenolic compounds in exocarp and
mesocarp of tomato fruit at 6 different ripeness stages. The com-
pounds were identified as aglycones obtained after acid hydrolysis
(hydrolyzed extracts) and as naturally occurring aglycones or com-
bined phenolic compounds obtained in the nonhydrolyzed ex-
tracts. Identification of aglycones was based on retention times, and
Naturally occurring combined phenolic acids
Ten naturally occurring combined phenolic acids were tenta-
tively identified in mesocarp and/or exocarp (Table 1, Figure 2).
p-Coumaroyl hexose I, caffeoyl hexose II, p-coumaroyl hexose
II, caffeoyl deoxyhexose hexose, and rutin were shown in both,
mesocarp and exocarp, whereas caffeoyl hexose I, feruloyl hexose
I, feruloyl hexose II, and syringyl hexose were observed only in
mesocarp and dicaffeoylquinic acid only in exocarp).

Vol. 78, Nr. 12, 2013 r Journal of Food Science C1841

 Phenolics changes during tomato ripening . . .
Aglycones derived from acid hydrolysis
Four aglycones were identified in the hydrolyzed extracts
(chlorogenic acid and caffeic acid in both mesocarp and exo-
carp, ferulic acid only in mesocarp and p-coumaric acid only in
exocarp).
The naturally occurring aglycone phenolic acids were iden-
tified in agreement with the retention times observed for their
C: Food Chemistry
respective commercial standards being 19.5 min for chlorogenic
acid, 22.7 min for caffeic acid, and 25.6 min for p-coumaric acid,
respectively. Furthermore, the identities were corroborated with
their respective fragmentation patterns derived from their mass
spectra (Table 1). Chlorogenic acid showed its profile of m/z 353,
173, 179, and 191, where 353 ([354-H]− ) corresponds to the [M-
H]− deprotonated molecule of 3-caffeoylquinic acid, 173 [354-H-
180]− corresponds to the ion derived from the loss of the caffeoyl
moiety from the deprotonated caffeoylquinic acid molecule, 179
Figure 2–HPLC–DAD chromatograms for phenolic compounds at 320 nm
of nonhydrolyzed extracts from exocarp and mesocarp of “Caiman” tomato
fruit. A, ripe mesocarp; B, mature-green mesocarp; C, ripe exocarp; D, pattern whose m/z of 163 ([164-H] ) and 119 ([164-H-44] )
mature-green exocarp.
C1842 Journal of Food Science r Vol. 78, Nr. 12, 2013
[354-H-173]− corresponds to the caffeoyl ion derived from the
loss of the dehydrated quinic moiety from the deprotonated caf-
feoylquinic acid, and 191 [354-H-162]− corresponds to the ion
derived from the loss of the dehydrated caffeoyl moiety. Simi-
lar results have been reported by Sun and others (2007) and by
Sánchez-Rabaneda and others (2003). Caffeic acid showed its [M-
H]− deprotonated molecule (m/z 179) and the [M-H-44]− frag-
ment ion (m/z 135) corresponding to the fragment derived from
the loss of the carbon dioxide moiety of caffeic acid (Table 1).
The identification of the naturally occurring glycoside-combined
phenolic compounds (Figure 2, Table 1) was based on the frag-
mentation pattern derived from the mass spectra obtained from
the different chromatographic peaks observed (Figure 2). Peaks 2
and 8 presented different retention times, 17.1 and 19.2 min, but
similar fragmentation patterns whose m/z of 325 ([326-H]− ), 163
[326-H-162]− , and 119 ([164-H-44]− ) correspond to the depro-
tonated molecule of p-coumaroyl hexose, the p-coumaroyl ion
derived from the deglycosilation of the molecule of coumaroyl
hexose, and the decarboxilated coumaroyl ion, respectively. Thus,
peaks 2 and 8 were tentatively identified as isomers of glycosilated
molecule of p-coumaroyl and were assigned as p-coumaroyl hex-
ose I and p-coumaroyl hexose II, respectively. Peaks 3 and 6 also
presented different retention times, 17.3 and 18.5 min, but the
same fragmentation pattern whose m/z of 341 ([342-H]− ), 179
([342-H-162]− ), and 135 ([179-H-44]− ) correspond to the depro-
tonated molecule of caffeoyl hexose, the deglycosilated molecule
of caffeoyl hexose previously dehydrated (caffeoyl ion), and the
decarboxilated caffeoyl ion, respectively. Thus, peaks 3 and 6 were
tentatively identified as glycosilated isomers of caffeic acid and was
assigned as caffeoyl hexose I and caffeoyl hexose II, respectively.
Peaks 4, 7, and Chlo, presented the retention times 17.9, 18.8,
and 19.5 min, respectively, and showed a fragmentation pattern
that exhibited in common a molecular ion m/z 353 correspond-
ing to the caffeoylquinic acid ion. Peak 4 showed a fragmentation
pattern whose m/z 515 ([516-H]− ) and 353 ([516-H-162]− ) is
believed to correspond to the deprotonated molecule of dicaf-
feoylquinic acid, in agreement with Moco and others (2006).
Both, peak 7 and peak chlo presented the same fragmentation
pattern that corresponds to the caffeolquinic acid molecule. Thus,
peak 7 corresponds to an isomerization form of the chlorogenic
acid, whereas peak chlo was identified as chlorogenic acid (3-
caffeoylquinic acid) based on the retention time and mass spectra
from the standard. Peak 10 presented a retention time of 20.8 min,
and a fragmentation pattern whose m/z of 355 ([356-H]− ) and
193 ([356-H-162]− ) correspond to the deprotonated molecule
of feruloyl hexose and the feruloyl ion (deglycosilated molecule
of feruloyl hexose), respectively. Peak 11 presented a retention
time of 21.6 min, and a fragmentation pattern whose m/z of
359 ([360-H]− ), 197 ([360-H-162]− ), and 153 ([198-H-44]− ),
correspond to the deprotonated molecule of syringyl hexose, the
syringyl ion (the deglycosilated molecule of siringyl hexose), and
the decarboxilated siringyl ion, respectively. Peak 14 presented a
retention time of 23.4 min, and a fragmentation pattern whose
m/z of 487 ([488-H]− ) and 179 ([488-H-162–146]− ) correspond
to the deprotonated molecule of deoxyhexose hexose caffeic acid
and the caffeic ion derived from the loss of the deoxyhexose hex-
ose moiety, respectively. Peak Rut presented a retention time of
24.2 min, and a fragmentation pattern whose m/z of 609 ([610-
H]− ) correspond to the deprotonated molecule of rutin. Peak
Cou with a retention time of 25.6 min presented a fragmentation
− −
Phenolics changes during tomato ripening . . .

Table 2–Phenolic acids content (mg/100 g fw) of nonhydrolyzed and hydrolyzed extracts from mesocarp and exocarp of “Caiman”
tomato fruit.

Mesocarp Nonhydrolyzed Hydrolyzed

Ripeness stage Chlorogenic Caffeic Chlorogenic Caffeic Ferulic
Mature green 0.68 ab nd 0.46 a 0.02 a U-LOQ
Breakers 0.90 a nd 0.57 a 0.07 a 0.14 a

C: Food Chemistry
Turning 0.67 ab nd 0.49 a 0.10 a 0.21 b
Pink 0.51 b nd 0.44 a 0.08 a 0.20 b
Light red 0.56 ab nd 0.44 a 0.08 a 0.23 b
Red 0.40 b nd 0.41 a 0.16 a 0.30 b
LSD 0.344 – 0.344 0.207 0.10
Exocarp Nonhydrolyzed Hydrolyzed
Ripeness stage Chlorogenic Caffeic Chlorogenic Caffeic p-Coumaric
Mature green 4.03 a U-LOQ 1.90 a 0.54 a 0.04 a
Breakers 4.09 a U-LOQ 1.80 a 0.60 a 0.09 a
Turning 3.72 a 0.06 a 1.80 a 1.43 b 0.39 b
Pink 3.30 b 0.10 a 1.80 a 1.61 b 0.36 b
Light red 2.45 c 0.12 a 1.40 b 1.34 c 0.26 b
Red 1.32 d 0.25 a 0.97 c 1.53 c 0.31 b
LSD 0.344 0.207 0.344 0.207 0.1430
LOD 0.017 0.004 0.017 0.004 0.04
LOQ 0.053 0.012 0.053 0.012 0.13
Mean values in the same column followed by different lower-case letters are significantly different (P = 0.05) using the Least Significant Difference (LSD) test. LOD, Limit of
Detection; LOQ, Limit of Quantitation; U-LOQ, Under LOQ; nd, not detected (under LOD).

correspond to the deprotonated molecule of p-coumaric acid and
the decarboxilated p-coumaric ion, respectively. Based on frag-
mentation patterns of naturally occurring phenolic compounds in
tomato, Moco and others (2006) tentatively identified 3 isomers
of chlorogenic acid (3-caffeoylquinic acid, 4-caffeoylquinic, and
5-caffeoylquinic acid), 6 glycosylated forms of caffeic acid (caffeic
acid hexose I to VI, 2 glycosilated forms of coumaric acid named
coumaric acid hexose I and II, respectively), and 2 glycosilated
forms of ferulic acid (ferulic acid hexose I and ferulic acid hex-
ose II). In our work there were tentatively identified 2 isomers of
chlorogenic acid, the dicaffeoylquinic acid, 3 glycosilated forms of
caffeic acid (caffeoyl hexose I, caffeoyl hexose II, and deoxyhexose
hexose caffeic acid), 2 glycosilated forms of p-coumaric acid (p-
coumaroyl hexose I and p-coumaroyl hexose II), and 2 forms of
ferulic acid (feruloyl hexose I and feruloyl hexose II). It seems to
be clear that the different isoforms for a given glycosilated pheno-
lic compound affect the retention time in the HPLC system. This
can be due to different molecular conformation among isomers
and not necessarily due to changes in molecular polarity.
The extraction of nonhydrolized extracts from exocarp and
mesocarp made possible the estimation of quantitative changes
of the naturally occurring combined-phenolic during ripening,
based on their mAU × s values given by the HPLC–DAD system
(Figure 2). All the combined phenolic acids showed an increasing
tendency during ripening; about 2- to 5-fold in both tissues. When
the phenolic compound extracts were nonhydrolyzed, chlorogenic
acid was the main naturally occurring phenolic acid observed in
tomato fruit as aglycone in both exocarp and mesocap tissues.
Caffeic acid was observed to appear as aglycone at the “turning”
stage of ripeness only in exocarp tissue and tended to increase
about 4-fold during ripening (Table 2). In mature-green toma-
toes, chlorogenic acid content was about 6-fold higher in exocarp
than in mesocarp, and decreased during ripening about 3-fold in
exocarp and about 2-fold in mesocarp. When the extracts were
hydrolyzed, a remaining quantity of chlorogenic acid (about 0.46
mg/100 g fw) was observed in mesocarp at the different ripeness
stages, whereas in exocarp it was estimated to be 1.9 mg/100 g fw
at the mature-green stage and decreased about 50% at the end of
tomato fruit ripening. In mesocarp, chlorogenic acid content from
nonhydrolyzed extract tended to be higher than the hydrolyzed
extract, whereas in exocarp, chlorogenic acid content from non-
hydrolyzed extract was about 2-fold higher than in the hydrolyzed
extract. This result suggests that chlorogenic acid was partially
hydrolyzed when the exocarp was subjected to acidic extraction
conditions (time of exposure and acid concentration) of the hy-
drolyzed extracts. Interestingly, such effect was observed mainly
at relatively high chlorogenic acid levels, such as those shown in
exocarp at mature-green ripeness stage (about 4.03 mg/100 g fw).
This decreased chlorogenic acid content is in agreement with the
concomitantly increment in caffeic acid during ripening observed
mainly in the hydrolyzed extracts from exocarp. Taga and oth-
ers (1984) reported that chlorogenic acid is readily hydrolyzed to
caffeic and quinic acids by some acid treatments. In hydrolyzed
extracts, caffeic acid was observed to increase in exocarp during
ripening from 0.54 to 1.53 mg/100 g fw, whereas in mesocarp it
tended to increase from 0.02 to 0.16 mg/100 g fw. The caffeic
acid levels showed in hydrolyzed extracts in both, mesocarp and
exocarp, could have been derived from the breakdown of caffeoyl
hexose and from the partial hydrolysis of chlorogenic acid, in ad-
dition to that derived from the naturally occurring biosynthesis of
caffeic acid observed only in exocarp. Minoggio and others (2003)
similarly to our results, reported the presence in tomato of chloro-
genic acid as the main phenolic acid, followed by caffeic acid.
From our results, ferulic acid in mesocarp and p-coumaric acid
in exocarp appeared as free phenolic acids only when the extracts
were hydrolyzed. More advanced ripeness stages showed higher
quantities of both compounds. The approach of the present work
consisting in the comparison of hydrolyzed versus nonhydrolyzed
extracts made possible to identify the naturally occurring phe-
nolic acids present as aglycones in the exocarp (chlorogenic acid
and caffeic acid) and in mesocarp (chlorogenic acid) of tomato
Cv Caiman. Furthermore, we noticed that ferulic acid is present
naturally as conjugated forms (tentatively as feruloyl hexose I and
feruloyl hexose II) but not as free acid. In the case of p-coumaric

Vol. 78, Nr. 12, 2013 r Journal of Food Science C1843

 Phenolics changes during tomato ripening . . .

acid, this was identified as free form (Figure 2), but the level cuted the lab analysis, interpreted results and drafted the
was too low to be quantified (LOQ = 0.0022 mg/100 g fw). manuscript.
However, p-coumaric acid was quantified from the hydrolyzed
extract of exocarp, and it is believed to be derived from the References
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Elhadi M. Yahia designed the study and helped in the
interpretation of the results. Armando Carrillo-López exe-

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