Food Research International 64 (2014) 188–199

Contents lists available at ScienceDirect

Food Research International

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

Chemical composition, nutritional value and antioxidant properties of

autochthonous Prunus avium cultivars from Campania Region
Severina Pacifico a,⁎,1, Antimo Di Maro a,1, Milena Petriccione b, Silvia Galasso a, Simona Piccolella a,
Antonella M.A. Di Giuseppe a, Marco Scortichini b, Pietro Monaco a
a
Department of Environmental Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, I-81100 Caserta Italy
b
Consiglio per la Ricerca e la Sperimentazione in Agricoltura (C.R.A.) - Unità di Ricerca per la Frutticoltura Via Torrino 3, I-81100 Caserta Italy

a r t i c l e i n f o a b s t r a c t

Article history: During a screening program aimed at the evaluation of antioxidative and antiproliferative properties, as well as
Received 14 April 2014 nutritional properties of local edible plants, two endemic sweet cherry cultivars (‘Del Monte’ and ‘Della Recca’)
Accepted 15 June 2014 were of interest. Macronutrient components (proteins, carbohydrates and lipids) of both the cherry cultivars
Available online 21 June 2014
were determined as well as free and total amino acids. Pomological traits were defined. HPLC–ESI/MSn analysis,
carried out on phenolic extracts properly prepared by extractive techniques from freeze dried fruits of both the
Keywords:
Prunus avium cvs
cherry cultivars, showed that investigated cultivars differed in their colorless phenolic composition.
Nutritional value Hydroxycinnamoyl quinic acid derivatives were present in both the cherry cultivars. ‘Della Recca’ cv. was partic-
LC–MS/MS phenol profile ularly rich in 4-O-coumaroyl quinic and 5-O-caffeoylquinic acid, whereas quercetin-3-O-rutinoside was the main
Antioxidant activity phenol compound of ‘Del Monte’ cultivar.
Cytotoxicity The antiradical properties of the extracts were investigated by DPPH and ABTS methods. ‘Della Recca’ cv. cherries
exhibited a pronounced antiradical activity: at 62.5 μg/mL dose level ABTS radical cation was converted in its
reduced form by 88.7% and DPPH radical was reduced by 75.3%. The antiproliferative efficacy of ‘Della Recca’
and ‘Del Monte’ extracts were evaluated towards five cancer cell lines (HepG2, A549, HeLa, SK-B-NE(2)-C, and
SH-SY5Y) through MTT assay. ‘Della Recca’ phenol extract showed a dose-dependent inhibiting activity towards
cervical cancer HeLa cell line.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction Phenolic compounds have attracted special attention due to their

Overproduction of oxidants that overwhelm the cellular antioxidant
capacity results in oxidative stress, a deleterious and pathogenic process
that can be an important mediator of damage to cell structures, includ-
ing lipids and membranes, proteins, and DNA (Valko et al., 2007). On the
other hand, cells and organisms are constantly exposed to some oxidiz-
ing agents, some of which are necessary for life (Liu, 2003). The key fac-
tor is to maintain a balance between oxidants and antioxidants to
sustain optimal physiologic conditions in the body. In this framework,
the large intake of functional foods as source of bioactive phytochemi-
cals or nutraceutical compounds represents a valuable strategy for
counteracting oxidative stress damages (Aruoma, 2010). In the last
years epidemiological evidences have highlighted a strong correlation
between plant metabolites having bioactive properties and human
health (Pacifico et al., 2013). Edible plants, that counteract free radical
overproduction during oxidative stress, are considered an effective
tool to prevent several diseases and indeed to increase the well-being.
⁎ Corresponding author. Tel.: +39 0823 274572; fax: +39 0823 274571.
E-mail address: severina.pacifico@unina2.it (S. Pacifico).
1
These authors equally contributed to this paper.
health-promoting characteristics (Sumbul, Ahmad, & Mohd, 2011).
These natural products, with considerable diversity in their structure,
are important components of plant foods to which they contribute to
flavor, color and sensory properties such as bitterness and astringency
(Lesschaeve & Noble, 2005). Cells respond to polyphenols mainly
through direct interactions with receptors or enzymes involved in sig-
nal transduction, which may result in modification of the redox status
of the cell and may trigger a series of redox-dependent reactions
(Scalbert, Johnson, & Saltmarsh, 2005). Both antioxidant and prooxidant
effects of polyphenols have been described, with contrasting effects on
cell physiologic processes. As antioxidants, polyphenols may improve
cell survival; as prooxidants, they may induce apoptosis and prevent
tumor growth. However, the biological effects of polyphenols may ex-
tend well beyond the modulation of oxidative stress (Scalbert et al.,
2005).
Nevertheless, in Mediterranean basin, known as much bio-diversity
reserve, several cultivars continue to be consumed as traditional food
(Guarrera & Savo, 2013), considered not only a simple nutritional in-
take, but also a potential source of healthy and natural products, as re-
ported by scientific research and underlies new trends in current food
plant science (Aruoma, 2010).

http://dx.doi.org/10.1016/j.foodres.2014.06.020
0963-9969/© 2014 Elsevier Ltd. All rights reserved.
 S. Pacifico et al. / Food Research International 64 (2014) 188–199
In this framework, sweet cherry (Prunus avium L.) is known as a
large source of antioxidant phenolic compounds. Sweet cherry tree is
a species with a great economic importance, due to the nutritional, tech-
nological and commercial value of its fruits (Pérez-Sánchez, Gómez-
Sánchez, & Morales-Corts, 2010). The nutritional importance especially
depends on the chemical composition, which represents a major source
of antioxidant compounds (Beceanu, 2008; Coşofreţ, Beceanu, & Radu,
2006; Serrano, Guillén, Martnez-Romero, Castillo, & Valero, 2005;
Usenik, Fabcic, & Stampar, 2008), such as anthocyanins, phenolic acids
(hydroxycinnamic acids) and flavonoids (Jakobek, Šeruga, Voća,
Šindrak, & Dobričević, 2009). The major phenolic antioxidants in
sweet cherries are anthocyanins but sweet cherries also have significant
amounts of phenolic acids and flavonols. The major phenolic acids in
sweet cherries are hydroxycinnamic acids (Jakobek et al., 2009).
Among hydroxycinnamates, sweet cherries have neochlorogenic acid
and p-coumaroylquinic acid as the predominant compounds. Small
amounts of chlorogenic acid and ferulic acid were found as well.
Hydroxybenzoic acids were found in sweet cherries only in small
amounts (Mattila, Hellstrom, & Torronen, 2006). Consumption of
sweet cherry has been associated with beneficial health effects as arthri-
tis alleviation and gout-related pain (Wang, Nair, Straburg, Booren, &
Gray, 1999). Reduction of the proliferation of human colon cancer
cells has been specifically associated with the consumption of cherries
(González-Gómez et al., 2010). Furthermore, phenolic antioxidants
from sweet cherries have shown protective effects on neuronal cells
(Kim, Heo, Kim, Yang, & Lee, 2005).
In Campania Region (Italy) there is a long tradition of cultivating
cherries. The Vesuvian area, on the slopes of Monte Somma, is well
known for the production of the so called “ciliegia del Monte” or
“Durona del Monte” (Cherry of the Mountain), that made history of
the cherry cultivation in this area. “Ciliegia della Recca” grows in the
Flegrean area. It comes from the Camaldoli zone and ripens between
the first and second decade of June. It is considered the most precious
sweet cherry cultivar of Campania Region. For this reason, during a
screening program aimed at the evaluation of antioxidative and anti-
proliferative properties, as well as nutritional properties of local edible
plants, two autochthonous sweet cherries cultivars are of interest.
Thus, this work reports nutritional values of ‘Del Monte’ and ‘Della
Recca’ cvs., determined in terms of their macronutrient content, and
the assessment of the antioxidant and antiproliferative activities of
both lipophilic and alcoholic extracts. The extracts were tested for
their radical scavenging power by measuring their capacity to scavenge
DPPH and ABTS cation radicals. Total phenol content was determined
using the Folin–Ciocalteau method. Moreover, the antiproliferative ef-
fects towards HepG2, A549, HeLa, SK-N-BE(2) and SH-SY5Y human
cell lines were evaluated. Finally, LC–ESI/MS/MS analysis was used to
identify and quantify the main polyphenol metabolites.
2. Material and methods
2.1. Reagents and chemicals
All of the solvents, quercetin and reagents used for assessing antiox-
idant screening were purchased from Sigma-Aldrich (Buchs,
Switzerland) except for ABTS [2,2′-azinobis-(3-ethylbenzothiazolin-6-
sulfonic acid)], which was from Roche Diagnostics (Roche Diagnostics,
Mannheim, Germany). Cell culture media and reagents for cytotoxicity
testing were purchased from Invitrogen (Paisley, UK), MTT [3-(4,5-di-
methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] was from
Sigma-Aldrich (Milan, Italy). Chemicals and solvents for the Sprint
Rapid Protein Analyzer and for the automated amino acid analysis
were provided by CEM Srl (CEM, Cologno al Serio, Bergamo, Italy) and
Biochrom (Cambridge, UK), respectively. Standard monosaccharides
were purchased from Sigma-Aldrich. For chromatographic purposes,
acetonitrile (LC-MS grade) and formic acid (98%, for mass spectrome-
try), were both purchased from Sigma-Aldrich.
189
2.2. Sampling and fractionation
Sweet cherry (Prunus avium L. cvs. ‘Del Monte’ and ‘Della Recca’)
fruits were collected in 2013 from trees grown under standard commer-
cial practices and trained in the same experimental orchard located at
Pignataro Maggiore (Caserta, Southern Italy) owned by the C.R.A. Re-
search Unit on Fruit Trees. Fruits were randomly harvested at a stage
12 of ripening stage, in which they reach consumers with the maximum
organoleptic, nutritional, and functional properties (Serrano et al.,
2005). After harvest, the fruits were immediately transported to the lab-
oratory and screened for uniformity, appearance and the absence of
physical defects or decay.
Three replicate samples (100.0 g each) of each cherry cv. were se-
lected, cleaned with MilliQ water, drained, and gently blotted with a
paper towel. Cherries were pitted, and then ground in a porcelain mor-
tar and pestle chilled with liquid N2, until particles of homogeneous size
were obtained. Frozen powdered cherry samples were lyophilized using
an FTS-System Flex-DryTM instrument (SP Scientific, Stone Ridge, NY,
USA). Three aliquots of dried cherries (~2.0 g each) were extracted for
4 h through a Soxhlet apparatus, using hexane as first extracting solvent
(150.0 mL) in order to remove fat from samples. The obtained solutions
were dried under vacuum by a rotary evaporator (Heidolph Hei-VAP
Advantage, Germany) to yield crude hexane extracts: PaDM-1 (Prunus
avium Del Monte-1), and PaDR-1 (Prunus avium Della Recca-1).
Defatted cherry samples underwent Soxhlet extraction using as extract-
ant pure MeOH (150.0 mL). PaDM-2 and PaDR-2 extracts thus obtained
were afterwards fractionated through discontinuous liquid-liquid ex-
traction (H2O/EtOAc) that allowed us to obtain aqueous (PaDM-2a,
and PaDR-2a) and organic fractions (PaDM-2b, and PaDR-2b). The frac-
tionation scheme is reported in Fig. 1.
2.3. Pomological analyses
The pomological and qualitative traits were evaluated according to
the International Union for the Protection of New Varieties of Plants
(UPOV) descriptors. The physical and chemical harvest indices were es-
timated as follows: (i) the total soluble solids content (SSC, °Brix) was
determined on the flesh juice using a digital refractometer (Sinergica
Soluzioni, DBR35, Pescara, Italy); (ii) the total acid content (TA) was de-
termined by titrating 10 ml flesh juice with 0.1 N NaOH (Wills, Scriven,
& Greenfield, 1983) and the results are expressed as g malic acid/L juice;
and (iii) the fruit color L* (brightness or lightness; 0 = black, 100 =
white), a* (− a* = greenness, + a* = redness) and b* (− b* = blue-
ness, + b* = yellowness) variables were measured, in the equatorial
perimeter at two opposite fruit zones, by using the chromatometer
Minolta (Model CR-5; Minolta, Ramsey, NJ, USA).
2.4. Macronutrient content
Total protein content: total protein content was estimated by Sprint
Rapid Protein Analyzer (iTAG technology). The cherry homogenates
were directly analyzed on a Sprint Rapid Protein Analyzer (CEM, Cologno
al Serio, Bergamo, Italy) as suggested by manufacturer's instruction.
Total lipid content: lipid extraction was performed for 4 h by Soxhlet
with hexane solvent. The obtained solution was dried by a rotary evap-
orator and then, the lipid content was determined gravimetrically
(AOAC, 1997).
Total carbohydrate content: carbohydrate content was obtained
using the difference approach (FAO, 2003).
2.5. Free and total amino acid composition
Frozen powdered cherry samples (1.0 g) was further powdered in
liquid nitrogen in a pre-cooled mortar with a pestle. For the analysis
of free amino acids, three aliquots of about 200 mg of cherry samples
were precipitated with 80% cold ethanol (1.0 mL), in the presence of
190 S. Pacifico et al. / Food Research International 64 (2014) 188–199
Fig. 1. Fractionation scheme of Prunus avium cherries cvs. ‘Della Recca’ and ‘Del Monte’. SE = Soxhlet Extraction; LLE = Liquid Liquid Extraction; PaDR = Prunus avium Della
Recca; PaDM = Prunus avium Del Monte.
nor-Leu (50 nmol) as internal standard, homogenized with a Teflon
pestle and centrifuged at 14,000 ×g, at 4 °C. The supernatant was lyoph-
ilized, treated with 3% sulfosalicylic acid (500 mL) to precipitate any
protein fraction still present, and centrifuged again. The obtained super-
natant was then analyzed (Di Maro et al., 2013). For the analysis of total
amino acids, about 50 mg of frozen cherry samples were hydrolysed in
glass tubes under vacuum with 0.5 mL of 6 M HCl containing 0.02%
phenol and nor-Leu as internal standard, at 110 °C for 20 h. Following
the hydrolysis, HCl was removed under vacuum and samples were
resuspended in 0.5 mL of 0.2 M lithium citrate buffer (pH 2.2) (Dosi
et al., 2013; Tamburino et al., 2012). Aliquots of hydrolysed and non-
hydrolysed samples were directly analyzed on a Biochrom 20
amino acid analyzer (Biochrom, Cambridge, UK) as suggested by
manufacturer's instruction.
2.6. Free glucose, fructose and sucrose content
The free glucose, fructose and sucrose composition was determined
using a Bio-LC (Dionex Corp., Sunnyvale, CA, USA) equipped with a
CarboPac® PA10 column (2 × 250 mm; Dionex Milan, Italy) and a
guard column Amino Trap™ (2 × 50 mm; Dionex Milan, Italy). Protocols
employed were as suggested by the manufacturer (Application Note 82
by Dionex Corp., Sunnyvale, CA, USA).
2.7. RP-HPLC–ESI/MS/MS analyzes
Chromatographic analyses were carried out on an Agilent 1200
HPLC system (Agilent Technologies Inc., Palo Alto, USA), equipped
with a binary pump, a vacuum degasser, an autosampler, a thermostatic
column compartment and a UV–Vis detector. A Luna C18-reversed
phase column (5 μm particle size, 4.6 × 250 mm i.d., Phenomenex)
was used for chromatographic separation at 25 °C and a flow rate of
0.35 mL/min. The UV-detection of phenolic compounds was performed
at 280 nm. The mobile phase consisted of 0.1% formic acid in water (v/v;
mobile phase A) and acetonitrile (mobile phase B). Starting with 5% B, a
linear gradient was followed to 15% B in 10 min, then increasing to 35%
B in 20 min, and finally to 80% B in 10 min, continuing for 5 min, before
re-equilibration to starting conditions. The samples (10.0 mg each)
were dissolved in a MeOH:water (1:4; v:v), sonicated for 2 min and
filled in a volumetric flask on a final volume of 10.0 mL. Then, the sam-
ples were centrifuged at 19,064 × g (5 min) before use. The injection
volume of each sample was 10.0 μL. The LC system was coupled to API
QTRAP 2000 (Applied Biosystem) with an ESI ion source operating in
the negative mode with the following parameters: HV voltage:
4500 V; dry gas flow (N2): 10.00 L/min with a capillary temperature
set at 365 °C; nebulizer pressure: 35 psi. Mass spectra were recorded
between m/z 50–2000 with a compound stability and trap drive level
of 100%. To obtain further structural information, these ions were
trapped and fragmented to yield the precursor product patterns of the
analytes. MSn data were acquired in the auto MS/MS mode. Instrument
control and data processing software used were Agilent ChemStation
B.01.03 (Agilent Technologies Inc., Palo Alto, USA) as well as Analyst
1.6 (AB Sciex Instruments, Darmstadt, Germany). The constituents
were identified on the basis of their MS/MS fragmentation patterns
and retention time order compared to literature data and reference
compounds if available. Multiple reaction monitoring (MRM) mode
was chosen for quantification of identified compounds, using quercetin
as internal standard. To this aim quercetin (30.2 μg), chromatographi-
cally distinguishable from the constituents of each extract, was added
to 1.0 mL of each sample. The intraday and interday precision,
expressed as RSD, were evaluated by analysis of three replicates on
the same day and on five separate days, respectively. RSD values did
never exceed 15%.
2.8. Determination of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging capacity
In order to estimate the DPPH• scavenging capability, investigated
extracts (3.90, 7.81, 15.62, 31.25, 62.50, 125.0 and 250.0 μg/mL, final
concentration levels) were dissolved in a DPPH• methanol solution
(9.4 × 10−5 M; 1.0 mL) at room temperature. After 30 min, the absorp-
tion at 515 nm was measured by a Shimadzu UV-1700 spectrophotom-
eter in reference to a blank. The results were expressed in terms of the
percentage decrease of the initial DPPH radical absorption by the test
samples. ID50 values were calculated on the basis of dose-response
data. Tests were carried out performing three replicate measurements
for three samples (n = 3) of each extract (in total, 3 × 3 measurements).
Recorded activities were compared to a blank. Results are the mean ±
SD values. Student t-test was applied in order to determine statistical
significance (significance level was set at P b 0.05).
2.9. Determination of ABTS [2,2′-azinobis-(3-ethylbenzothiazolin-6-sulfonic
acid)] radical cation scavenging capacity
The determination of ABTS•+ solution scavenging capacity was esti-
mated as previously reported (Di Maro et al., 2013). ABTS radical cation
 S. Pacifico et al. / Food Research International 64 (2014) 188–199
was generated by the reaction between ABTS (7.0 mM) and potassium
persulfate (2.45 mM). The mixture was allowed to stand in the dark at
room temperature for 12–16 h. Thus, the ABTS•+ solution was diluted
with PBS (pH 7.4) in order to reach an absorbance of 0.70 at 734 nm.
Investigated extracts (3.90, 7.81, 15.62, 31.25, 62.50, 125.0 and
250.0 μg/mL, final concentration levels) were dissolved in 1.0 mL of di-
luted ABTS•+ solution. After 6 min of incubation, the absorption at 734
nm was measured by a Shimadzu UV-1700 spectrophotometer in refer-
ence to a blank. The results were expressed in terms of the percentage
decrease of the initial ABTS•+ absorption by the test samples. ID50 values
were calculated on the basis of dose–response data. Tests were carried
out performing three replicate measurements for three samples (n =
3) of each extract (in total, 3 × 3 measurements). Recorded activities
were compared to a blank. Results are the mean ± SD values. Student
t-test was applied in order to determine statistical significance (signifi-
cance level was set at P b 0.05).
2.10. Cell cultures
SH-SY5Y human neuroblastoma cells were purchased from ATCC
(American Type Culture Collection); SK-N-BE(2)-C human bone mar-
row neuroblastoma cells, HepG2 human hepatoblastoma cell line,
A549 lung epithelial cell line and HeLa cervical carcinoma cell line
were purchased from ICLC (Interlab Cell Line Collection) at Istituto
Nazionale per la Ricerca sul Cancro, Genoa (Italy). SH-SY5Y and A549
cell lines were grown in DMEM high glucose medium supplemented
with 10% fetal bovine serum, 50.0 U/mL penicillin, and 100.0 μg/mL
streptomycin, at 37 °C in a humidified atmosphere containing 5% CO2.
SK-N-BE(2)-C, HepG2 and HeLa cell lines were plated and grown
under the same conditions, except that RPMI 1640 was used instead
of DMEM.
2.11. MTT cell viability test
SH-SY5Y, SK-N-BE(2)-C, HepG2, A549 and HeLa cell lines were seed-
ed in 96-multiwell plates at a density of 1.0 × 104 cells/well. After 24 h of
incubation, cells were treated with cherry extracts at five dose levels
(15.62, 31.25, 62.50, 125.0 and 250.0 μg/mL, final concentration levels).
At 24, 48, and 72 h of incubation, cells were treated with 150 μL of MTT
(0.50 mg/mL), dissolved in the culture medium, for 1 h at 37 °C in a 5%
CO2 humidified atmosphere. The MTT solution was then removed and
100 μL of DMSO were added to dissolve the formazan originated. Finally,
the absorbance at 570 nm of each well was determined using a Tecan
SpectraFluor fluorescence and absorbance reader. Cell viability was
expressed as percentage of mitochondrial redox activity of the cells treat-
ed with the extracts compared to the untreated control (Pacifico et al.,
2012). Cell viability inhibition (CVI, %) was calculated using the following
formula: [(Absuntreated cells) − (Abstreated cells) / (Absuntreated cells)] × 100.
Tests were carried out performing twelve replicate (n = 12) measure-
ments for three samples of each extract (in total: 12 × 3 measurements).
Recorded activities were compared to a blank arranged in parallel to
the samples. Results are the mean ± SD values. Student t-test was
applied in order to verify statistical significance (significance level was
set at P b 0.05).
3. Results and discussion
3.1. Pomological traits
In this work both sweet cherry cultivars were located in the same
experimental orchard, under similar cultural practices. Therefore, differ-
ences in agronomical and quality attributes between cultivars that will
be described below should be attributed to genetic characteristics of
each cultivar.
‘Del Monte’ and ‘Della Recca’ are important cherry cultivars of au-
tochthonous germoplasm grown in Campania region (Southern Italy)
191
which are harvested between the end of May and the second decade
of June. Both ‘Del Monte’ and ‘Della Recca’ have an excellent taste and
texture; their quality is very good, and they could be distributed and
commercialized in local market.
‘Del Monte’ has large fruit (9.40 ± 0.6 g) with cordate shape, red-
yellow skin color (L* 22.45; a 20.37; b 7.49) and white-yellow flesh
color (L* 32.5; a 16.28; b 12.43), while ‘Della Recca’ has medium fruit
(6.8 ± 0.9 g) with depressed shape, red skin color (L* 26.42; a 25.12;
b 14.85) and white-yellow flesh color (L* 35.75; a 8.29; b 19.80). The
fruits of both cultivars have a thin stem of medium length, with values
of 34.5 ± 2.4 and 38.4 ± 2.2 mm in ‘Della Recca’ and ‘Del Monte’,
respectively.
In both the cultivars the stone is large with elongated shape and it is
not adherent to the flesh but shows a very prominent crest on it. Our
study showed that at harvest, the values of SSC and TA were similar be-
tween two cultivars. SSC values are 17.1°Brix and 16.2°Brix, while TA
values are 92 meq/L and 95 meq/L in ‘Della Recca’ and ‘Del Monte’, re-
spectively. SSC and TA, strongly influence the consumer preference for
fruit quality (Lopez, Larrigaudiere, Girona, Behboudian, & Marsal,
2011). The TA estimates the organic acids content of fleshy fruits. This
trait is an important component of fruit organoleptic quality and is
mainly due to the presence of malic and citric acids, the main organic
acids that are found in most ripe fruits (Etienne, Génard, Lobit,
Mbeguié-A-Mbéguié, & Bugaud, 2013).
3.2. Nutritional values
Nutritional values determined on both ‘Della Recca’ and ‘Del Monte’
Prunus avium cherries are reported in Tables 1 and 2. Both ‘Della Recca’
and ‘Del Monte’ Prunus avium cherries had similar moisture content
(about 83%). Protein (0.58 g/100 g) and lipid (0.032 g/100 g) contents
in ‘Della Recca’ were slightly higher (about 1.01 and 1.14 fold, respec-
tively) than those of ‘Del Monte’.
The content of both glucose and fructose was slightly higher in ‘Della
Recca’ (about 1.01-fold for both the hexoses), while sucrose was about
0.79-fold in ‘Della Recca’. The glucose/fructose ratio was 1.22 for both
‘Della Recca’ and ‘Del Monte’ cherries.
3.3. Amino acid content
The content of total and free amino acids is reported in Table 3.
Comparing the free amino acid content of ‘Del Monte’ with that of
‘Della Recca’, both qualitative and quantitative differences were found.
The free amino acid content for 100 g fresh product of ‘Del Monte’ was
about 1.18 fold that of ‘Della Recca’ (2403.24 and 2037.13 mg, respec-
tively). Asparagine was by far the most abundant among free amino
acids (about 97.65% for ‘Del Monte’ vs. 95.84% for ‘Della Recca’). The
free amount of asparagine was confirmed by the aspartic acid increase
after acid hydrolysis of the free amino acid samples (Ferrara et al.,
2011). Glutamine content was also higher in ‘Del Monte’ (about 2.90%
of the total) that in ‘Della Recca’ (about 1.76% of the total). Among
other free amino acid, in descending order, glutamic acid (Glu), proline
(Pro) and aspartic acid (Asp) were higher in ‘Del Monte’, whereas the
Table 1
Nutritional composition and content of glucose, fructose and sucrose of fruits from Prunus
avium cvs. ‘Della Recca’ and ‘Del Monte’. Values are means (±SD) of triplicate analyses
(n = 3) and are expressed in 100 g fresh weight (FW) basis.
cv. ‘Della Recca’ cv. ‘Del Monte’
Moisture content (%) 82.55 83.40
Proteins (g/100 g) 0.58 ± 0.02 0.57 ± 0.04
Lipids (g/100 g) 0.032 ± 0.01 0.028 ± 0.01
Carbohydrates (g/100 g) 16.82 ± 0.54 15.87 ± 0.49
Glucose (mg/100 g) 9.30 ± 0.39 8.44 ± 0.29
Fructose (mg/100 g) 7.62 ± 0.24 6.89 ± 0.20
Sucrose (mg/100 g) 0.74 ± 0.04 0.93 ± 0.03
192 S. Pacifico et al. / Food Research International 64 (2014) 188–199

Table 2
Free and total amino acid composition of Prunus avium cherries cvs. ‘Della Recca’ and ‘Del Monte’. Protein amino acids are reported in bold. Values are means (±SD) of triplicate analyses
(n = 3) and are expressed in 100 g fresh weight (FW) basis.

cv. ‘Della Recca’ cv. ‘Del Monte’

Amino acidsa Total amino acidsb Free amino acid content Total amino acidb Free amino acid content
(mg/100 g) (mg/100 g) (mg/100 g) (mg/100 g)

Essential amino acids

His* 3.58 ± 0.43 0.21 ± 0.01 3.90 ± 0.43 0.14 ± 0.01
Ile 13.88 ± 1.12 0.17 ± 0.01 13.07 ± 0.11 0.18 ± 0.01
Leu 29.01 ± 1.42 0.14 ± 0.02 27.77 ± 0.99 0.12 ± 0.00
Lys 36.72 ± 2.15 0.15 ± 0.01 48.53 ± 2.63 0.08 ± 0.00
Met 3.69 ± 0.23 0.01 ± 0.00 3.10 ± 0.04 0.02 ± 0.00
Phe 15.78 ± 1.44 0.02 ± 0.00 14.68 ± 0.29 0.19 ± 0.01
Thr 24.25 ± 1.21 0.79 ± 0.07 22.85 ± 0.69 0.93 ± 0.04
Trp n.d. 0.72 ± 0.05 n.d. 0.33 ± 0.03
Val 17.57 ± 1.24 1.44 ± 0.11 16.78 ± 0.09 1.82 ± 0.09
1-mhis 0.05 ± 0.01 0.06 ± 0.00
3-mhis 0.03 ± 0.00 0.02 ± 0.02
Ala 23.57 ± 1.62 0.71 ± 0.03 32.65 ± 1.99 0.48 ± 0.03
Arg 16.42 ± 0.62 0.04 ± 0.02 14.50 ± 0.07 0.10 ± 0.01
Asn 1952.32 ± 84.44 2346.88 ± 92.70
Asp 770.63 ± 97.48 2.71 ± 0.19 1131.02 ± 103.36 2.03 ± 0.02
β-ala 0.13 ± 0.02 0.16 ± 0.00
Citr 0.27 ± 0.01 0.13 ± 0.01
Cys 0.05 ± 0.01
Cysteic acid 60.99 ± 2.96 26.66 ± 1.13
Ethan 0.13 ± 0.01 0.24 ± 0.01
GABA 2.73 ± 0.28 0.88 ± 0.08
Gln 59.24 ± 2.52 42.27 ± 1.68
Glu 38.69 ± 1.62 9.04 ± 0.28 42.07 ± 3.62 2.85 ± 0.10
Gly 22.35 ± 1.39 0.16 ± 0.02 33.27 ± 13.91 0.15 ± 0.00
Homocys 0.01 ± 0.00 0.03 ± 0.00
Hylys 0.20 ± 0.02
Orn 0.05 ± 0.00 Nd
Pea 0.25 ± 0.02 0.12 ± 0.01
Phser 0.38 ± 0.04 0.48 ± 0.05
Pro 127.49 ± 10.75 2.93 ± 0.23 162.62 ± 20.28 1.43 ± 0.12
Sarc 0.17 ± 0.02 0.26 ± 0.01
Ser 29.02 ± 0.34 1.63 ± 0.14 27.23 ± 0.89 0.14 ± 0.05
Taur 0.08 ± 0.00 0.03 ± 0.00
Tyr 10.42 ± 0.82 0.32 ± 0.06 10.54 ± 0.42 0.34 ± 0.01
Urea 0.10 ± 0.03 0.10 ± 0.03
Total (mg) 1244.06 2037.13 1631.28 2403.24
(minus Asp) (473.43) (84.81) (500.22) (56.36)

n.d., not determined.

a
Free and protein amino acids. The three letter code was used: 1-mhis: 1-methyl-L-histidine; 3-mhis: 3-methyl-L-histidine; β-ala: β-alanine; Ala: L-alanine; Asn: L-asparagine; Asp: L-aspartic
acid; Arg: L-arginine; citr, citrulline; Cys: L-half cystine; Ethan: ethanolamine; GABA: γ-amino-n-butyric acid; Gln: L-glutamine; Glu: L-glutamic acid; Gly: glycine; His: L-Histidine; Hylys:
δ-hydroxylysine; Homocys, homocystein; Ile, L-isoleucine; Leu: L-leucine; Lys: L-lysine; Met: L-methionine; Orn: L-ornithine; Pea, o-phosphoethanolamine; Phe: L-phenylalanine; Phser:
o-phospho-L-serine; Pro: L-proline; Sarc: sarcosine; Ser: L-serine; Taur: taurine; Thr: L-threonine; Trp: L-tryptophan; Tyr: L-tyrosine; Val: L-valine.
b
free plus protein-derived amino acids (see text).

valine (Val) content was slightly higher in ‘Della Recca’ cherries.
Both samples showed the presence of non-protein amino acids. The
total amount of them in ‘Della Recca’ was 1.61 fold higher than in
‘Del Monte’ (4.38 compared to 2.71 mg/100 g). Moreover, γ-amino-n-
butyric acid (GABA), and citrulline (Citr) were the most abundant
in ‘Della Recca’ (2.73 and 0.27 mg/100 g compared to 0.88 and
0.13 mg/100 g, respectively) whereas o-phospho-L-serine (Phser)
was higher in ‘Del Monte’ (0.48 and 0.38 mg/100 g, respectively). The
amount of each of the other non-protein amino acids did not exceed
1 mg/100 g of fresh product in both cherries.
Table 3
Total polyphenol content expressed as mg gallic acid equivalents (GAE) per 100 g FW
fresh weight (FW) basis. Values are means (±SD) of triplicate analyses (n = 3) and are
expressed in 100 g fresh weight (FW) basis.
GAE mg per 100 g FW GAE mg per 100 g FW
Among total amino acids in both ‘Del Monte’ and ‘Della Recca’ cvs.
Prunus avium cherries, the more abundant, in decreasing order, were
proline, glutamic acid, lysine (Lys), leucine (Leu) and serine (Ser), over-
all accounting for about 20% of the total amino acid. On the other hand,
the percentage of the four amino acids exceeds 50% of the total not con-
sidering the aspartic acid content in both samples. In fact, the high
aspartic acid content derived in large part from the free deaminated
asparagine (Asn) contained in samples subjected to acid hydrolysis. Fur-
thermore, the amount of essential amino acids [His, Ile, Leu, Lys, Met,
Phe, Thr, Val; Tryptophan (Trp) is not included as it was not determined
in the total hydrolyzed samples — see Table 3] in ‘Della Recca’ was
144.48 mg/100 mg vs. 150.68 mg/100 mg in ‘Del Monte’. Moreover,
the other amino acid contents were similar in both ‘Del Monte’ and
‘Della Recca’ cherries.
3.4. Antiradical capacity assessment

Pa-DM1 20.2 ± 3.2 Pa-DR1 31.2 ± 1.3 Conventional methods for determining the antioxidant activity of
Pa-DM2 145.3 ± 1.9 Pa-DR2 241.3 ± 2.3 plant materials are the measurements of total phenol content and radi-
Pa-DM2a 73.5 ± 3.3 Pa-DR2a 62.7 ± 2.4 cal scavenging activity. Thus, Folin–Ciocalteau Reagent assay and DPPH
Pa-DM2b 189.3 ± 3.1 Pa-DR2b 362.4 ± 1.5
and ABTS methods were applied on all the crude extracts.
 S. Pacifico et al. / Food Research International 64 (2014) 188–199 193

The Folin–Ciocalteau assay has been proposed as a standardized
method for use in the routine quality control and measurement of anti-
oxidant capacity of food products and dietary supplements (Prior, Wu,
& Schaich, 2005). The alcoholic extract of both the sweet cherry culti-
vars showed a strong reducing capacity estimated, through interpola-
tion on a calibration curve of gallic acid, to be equal to 145.3 and
241.3 mg gallic acid equivalents (GAEs) per 100 g of ‘Del Monte’ and
‘Della Recca’ fresh fruits, respectively (Table 4). These values are slightly
higher than those recently reported for sweet cherry cultivars grown in
Sicily (Ballistreri et al., 2013) but are in line with data from cv. ‘Saco’ at
fully ripened stage (Gonçalves et al., 2004) and cv. ‘Burlat’ (Liu et al.,
2011). Recently it was observed that the total phenolic contents of
Coruh valley fresh wild sweet cherries per 100 g ranged from 148 GAE
in yellow skin colored fruit to 321 mg GAE in blackish skin colored
fruit (Karlidag, Ercisli, Sengul, & Tosun, 2009). Growth pedo-climatic
conditions and plant genotype strongly affect total phenol content in
sweet cherries. Extracts derived from alcoholic extract showed different
FCR reducing capability. In particular, ethyl acetate fractions (Pa-DM2b
and Pa-DR2b), obtained by liquid–liquid extraction method, were
enriched in phenolic compounds exhibiting GAE values equal to 189.3
and 362.4 mg gallic acid equivalents per 100 g of ‘Del Monte’ and
‘Della Recca’ fresh fruits, respectively whereas aqueous extracts exhibit-
ed lower GAE values than own parental alcoholic extracts. Hexane ex-
tracts (Pa-DM1 and Pa-DR1), probably poor of constituents able to
react with Folin–Ciocalteau reagent, exhibited the lowest GAE values.
The antiradical properties of the extracts were investigated by DPPH
radical and ABTS radical cation, each of which is based on a single
electron or hydrogen atom transfer reaction (Fig. 2). ‘Della Recca’ cv.
cherries were more effective than ‘Del Monte’ cherries to reduce both
the radicals target. The different antiradical capabilities of the investi-
gated sweet cherries cvs. were evident when Pa-DR2b and Pa-DM2b ex-
tracts were tested. In fact, although both the sweet cherries extracts
scavenged DPPH radical and ABTS radical cation in a dose dependent
manner, ‘Della Recca’ cv. Pa-DR2b extract exhibited a pronounced anti-
radical activity: at 62.5 μg/mL dose level ABTS radical cation was con-
verted in its reduced form by 88.7% and DPPH radical was reduced by
75.3%. Pa-DM2b showed a scavenging effect lower than Pa-DR2b vs.
DPPH radical. Calculated ID50 values underlined that Pa-DR2b reduced
by 50% ABTS radical cation at 19.3 and DPPH radical at 28.9 μg/mL;
Pa-DM2b doses equal to 12.5 and 38.8 μg/mL defined the same
percentage decrease. TEAC values (Trolox Equivalent Antioxidant Ca-
pacity) allowed us to estimate that both the extracts exerted an anti-
radical efficacy comparable to that elicited by Trolox® when its concen-
trations are equal to 8.98 μM (Pa-DR2b) and 9.95 μM (Pa-DM2b).
3.5. Antiproliferative activity assessment
The potential cell growth inhibitory effects of ‘Della Recca’ and ‘Del
Monte’ extracts were evaluated towards five cultured cancer cell lines
(HepG2, A549, HeLa, SK-B-NE(2)-C and SH-SY5Y). The level of MTT re-
duction varied greatly between the different tested cell types (Fig. 3).
Cervical cancer HeLa cell line appears more sensitive to the anti-
proliferative action of the organic Pa-DM2b and Pa-DR2b fractions
than the other cell lines. Pa-DR2b extract ID50 values vs. HeLa cell line
were equal to 86.5, and 53.4 μg/mL after 24, and 48 h, respectively.
Pa-DM1 and Pa-DR1 fractions strongly affected the viability of neuronal
SK-B-NE(2)-C and SH-SY5Ycell lines. Pa-DR1 extract was capable of de-
termining a percentage decrease in cell viability of SH-SY5Y cells by
89.0% at a dose of 250 μg/mL.
3.6. LC–MS/MS based metabolic fingerprinting of Pa-DM2b and Pa-DR2b
extracts
Pa-DM2b extract was more rich in phenolic constituents than
Pa-DR2b one. The chemical complexity of both extracts was evaluated
through LC/MS based metabolic fingerprinting techniques (Fig. 4).
Twenty-three phenolic compounds (1–23) were detected as the
main constituents of Pa-DM2b fraction (Fig. 5, Table 5).
Compounds 1 and 2 were identified on the basis of their fragmenta-
tion pattern as quinic acid and citric acid, respectively. In fact, the MS2
spectrum of the [M–H]− ion at m/z 191 showed for compound 1 frag-
ment ions at m/z 127 and 93, that allowed us to ensure the identity of
quinic acid, whereas citric acid (2) gave a characteristic fragment ion
at m/z 111 ([M–H–CO2–2H2O]−) (Oen, 1985).
The MS2 spectrum of compound 3 (m/z 295) showed the most in-
tense fragment ion at m/z 133 (− 162 Da) and two other fragment
ions at m/z 115 and 71, attributable to a malic acid moiety. The loss of
162 Da suggested the presence of a malic acid hexoside. Malic acid
was found as the main organic acid in different sweet cherry cultivars
(8.5–10 mg/g FW; Kelebek & Selli, 2011).

Table 4
LC–MS/MS data of the main phenol constituents of Pa-DM2b extract. Relative content is expressed as percentage of the total polyphenols.

Peak Tentative assignment MW tR Pseudomolecular and fragment ions [m/z] Relative content
[Da] [min] in [MS]- mode

MS MS2

1 Quinic acid 192 9.7 191 127, 93, 86 2%

2 Citric acid 192 9.8 191 111, 93, 87 1%
3 Malic acid hexoside 296 10.3 295 133, 115, 71 2%
4 Feruloyl hexoside 356 24.2 355 355, 193, 135 4%
5 3-O-caffeoyl quinic acid 354 24.4 353 191, 179, 161, 135, 93, 85 2%
6 5-O-caffeoyl quinic acid 354 27.5 353 337, 191, 179, 137, 93 14%
7 3-O-p-coumaroyl quinic acid 338 27.9 337 191, 163, 155, 119, 111 1%
8 4-O-caffeoyl quinic acid 354 28.1 353 191, 179, 173, 135, 93 3%
9 3-O-feruloyl quinic acid 368 28.7 367 193, 191, 149, 134, 89 1%
10 5-O-feruloyl quinic acid 368 29.2 367 191, 173 4%
11 4-O-feruloyl quinic acid 368 33.1 367 367, 191, 173, 161, 135 1%
12 4-O-p-coumaroyl quinic acid 338 33.5 337 191, 173, 163, 155, 119, 93 3%
13 quercetin-3-O-(6″-O-deoxyhexosyl)hexoside (rutin) 610 34.8 609 301, 300, 271, 255 24%
14 cis-5-O-feruloyl quinic acid 368 35.5 367 367, 193, 191, 173 2%
15 5-O-caffeoyl shikimic acid 336 36.5 335 335, 161, 135, 113 b1%
16 3-O-caffeoyl shikimic acid 336 36.7 335 179, 161, 135 b1%
17 kaempferol-3-O-(6″-O-deoxyhexosyl)hexoside 594 36.8 593 285, 284, 255, 227 10%
18 α-Hydroxy hydroferuloyl quercetin 496 37.1 495 301, 193 5%
19 kaempferol-3-O-hexoside 448 39.0 447 447, 284, 255, 227 3%
20 dicaffeoyl quinic acid 516 40.8 515 353, 191, 169 2%
21 quercetin-3-O-(deoxyhexosyll)hexoside 610 43.7 609 609, 463, 301, 300 3%
22 3-O-p-coumaroyl-5-O-caffeoylquinic acid 500 44.3 499 337, 191, 179, 173, 163, 135, 119 3%
23 ferulic acid 194 45.2 193 149, 133, 105 8%
194 S. Pacifico et al. / Food Research International 64 (2014) 188–199

100

75

RSC (%)
50

25

0 µg/mL

125

125
250

125
250
125
250

250
15.62
31.25

31.25
15.62

15.62
31.25

15,6
31,5
62,5
61.5

3.90

62.5

3.90
7.81

61.5
3.90
7.81

7.81

3,9
7,8
Pa DM-1 Pa DM-2 Pa DM-2a Pa DM-2b
100

75
RSC (%)

50

25

0 µg/mL
125
250

125
250

250
125

125
250
15,62
31,25
15.62
15.62
31.25

31.25

15,6
31,5

61,5
62,5

7,81
3.90
7.81

61.5

3.90
7.81

61.5

7,8
3,9

3,9

PaDR-1 PaDR-2 Pa DR-2a PaDR-2b

Fig. 2. Radical scavenging capacity (RSC, %) of extracts from Prunus avium cherries cvs. ‘Della Recca’ and ‘Del Monte’ towards DPPH radical and ABTS radical cation. Values, reported as
percentage vs. blank, are the mean ± SD of measurements carried out on 3 samples (n = 3) analyzed three times.

Deprotonated compound 4 at m/z 355 was consistent with a feruloyl
hexoside. In fact the MS2 spectrum of the ion at m/z 355 showed the
feruloyl fragment at m/z 193 (Marín, Ferreres, Tomás-Barberán, & Gil,
2004) following the loss of a 162 Da moiety.
Compounds 5, 6, and 8 displayed the same [M–H]− ion at m/z 353
and were characterized as caffeoylquinic acid isomers. In particular,
compounds 5 and 6 were identified as 3-O-caffeoylquinic acid and 5-
O-caffeoylquinic acid, respectively, based on the hierarchical key for
identification by LC/MSn of quinic acid derivatives proposed by Clifford
and co-workers (Clifford, Johnston, Knight, & Kuhnert, 2003; Clifford,
Knight, & Kuhnert, 2005) and comparison of their retention times and
fragmentation patterns with those of pure standard compounds previ-
ously isolated from Prunus cerasus (Piccolella et al., 2008). Compound
8 was distinguished from the other two isomers by its base peak at
m/z 173 [quinic acid–H–H2O]– and identified as 4-O-caffeoylquinic
acid (Clifford et al., 2003).
Compounds 7 and 12 were tentatively identified as 3-O- and 4-O-p-
coumaroylquinic acid, respectively. In fact, both the compounds showed
[M–H]– ions at m/z 337, which yield a product ion at m/z 191, due to the
deprotonated quinic acid, and a less intense fragment ion at m/z 163
corresponding to [p-coumaric acid-H]– ion. The MS2 spectrum of
compound 12 showed a base peak at m/z 173, which was coherent
with 4-O-p-coumaroylquinic acid.
The MS spectrum of metabolites 9, 10 and 11 displayed [M–H]− ions
at m/z 367 indicating the presence of feruloylquinic acids. In MS2
analysis, compound 9 generated the most intense fragment ion at m/z
193 ([ferulic acid–H]–), which was in keeping with that previously
described in literature for 3-O-feruloylquinic acid (Guimarães et al.,
2013). The [M–H]− fragmentation produced for compound 10 a main
product ion at m/z 191, according to 5-O-feruloylquinic acid, whereas
MS2 spectrum of compound 11 showed a base peak at m/z 173 that
allowed us to identify it as 4-O-feruloylquinic acid. Hydroxycinnamic
acid derivatives, consisted of neo-chlorogenic acid, chlorogenic acid,
and p-coumaric acid derivatives, were broadly reported as the main
constituents of sweet cherry fruits (Ballistreri et al., 2013; Kim et al.,
2005).
The mass spectra of compounds 13 and 21 were in accordance with
the presence of O-diglycosyl derivatives of the flavonol quercetin, differ-
ing in the position of the sugar substituent and in the interglycosidic
linkage type between the monosaccharide moieties. In particular, the
MS2 spectrum of the deprotonated ion at m/z 609 generated for com-
pound 13 the ion at m/z 301, likely the flavonol quercetin, correspond-
ing to the direct loss of a disaccharide hexose-deoxyhexose moiety
(162 + 146 Da). The ions at m/z 271 and 255 were observed in the
MS3 spectrum that glycosylation occurred at C-3 position (Cuyckens &
Claeys, 2005). MS2 analysis of [M–H]– at m/z 609 from compound 21
showed more pronounced fragmentation than the previous isomer
with a product ion mass spectrum revealing the ion at m/z 463 due to
the loss of a deoxyhexosyl moiety (Cuyckens, Rozenberg, de
Hoffmann, & Claeys, 2001). Thus, O-diglycosyl quercetin was tentatively
identified as quercetin-3-O-rutinoside (13) and quercetin-3-O-
neohesperedoside (21). Compound 14 was tentatively identified as
 S. Pacifico et al. / Food Research International 64 (2014) 188–199 195

100 100

75 75

HepG2 ICV (%)

50 50

25 25

0 0
100 100
PaDM-1 PaDM-2 PaDM-2a PaDM-2b PaDR-1 PaDR-2 PaDR-2a PaDR-2b
75 75
A549 ICV (%)

50 50

25 25

0 0
100 100

PaDM-1 PaDM-2 PaDM-2a PaDM- PaDR-1 PaDR-2 PaDR-2a PaDR-2b

75 75
HeLa ICV (%)

50 50

25 25

0 0
100 100
PaDM- PaDR-2b
SK-N-BE(2)-C ICV (%)

PaDM-1 PaDM-2 PaDM-2a PaDR-1 PaDR-2 PaDR-2a

75 75

50 50

25 25

0 0
100 100
PaDM-1 PaDM-2 PaDM-2a PaDM-2b PaDR-1 PaDR-2 PaDR-2a PaDR-2b
75
SH-SY5Y ICV (%)

75

50 50

25 25

0 0 µg/mL
250

125
250

125
125
250

125

250
15.62
31.25

15.62
31.25

15.62
31.25

15.62
31.25
62.5
62.5

62.5

62.5

125
250

125
250

125
250

125
250
31.25

15.62
31.25
15.62
31.25

15.62
31.25

15.62
62.5

62.5

62.5
62.5

Pa DM-1 Pa DM-2 Pa DM-2a Pa DM-2b Pa DR-1 Pa DR-2 Pa DR-2a Pa DR-2b

Fig. 3. Inhibition of cell viability (ICV, %) of extracts from Prunus avium cherries cvs. ‘Della Recca’ and ‘Del Monte’ towards HepG2, A549, HeLa, SK-N-BE(2) and SH-SY5Y cell lines. Values,
reported as percentage vs. an untreated control, are the mean ± SD of measurements carried out on 3 samples (n = 3) analyzed twelve times.

the cis isomer of 5-O-feruloylquinic acid; although geometrical isomer-
ism did not influence the fragmentation behavior of the positional iso-
mers, cis-5-O-feruloyl isomer was appreciably more hydrophobic than
its trans counterpart (Clifford, Kirkpatrick, Kuhnert, Roozendaal, &
Salgado, 2008).
The mass spectra of compounds 15 and 16 showed [M–H]− ions at
m/z 335. The MS2 spectrum of both the compounds showed fragment
ions at m/z 179, 161 and m/z 135, characteristic of a caffeoyl moiety
and the following neutral loss of water and CO2, respectively (Gouveia
& Castilho, 2011). Moreover, compound 15 gave a base peak at m/z
161, whereas the fragmentation of [M–H]− ion at m/z 335 for com-
pound 16 generated the base peak at m/z 179. The relative losses of
174 Da (shikimic acid) and 156 Da (dehydrated shikimic acid) were in
accordance with the presence of 3-O- and 5-O-caffeoylshikimic acids
(Jaiswal, Sovdat, Vivan, & Kuhnert, 2010).
Metabolite 17 was tentatively identified as kaempferol-3-O-(6″-O-
deoxyhexosyl)-hexoside. The MS spectrum displayed the [M–H]− ion
at m/z 593, which in turn gave a deprotonated aglycone at m/z 285,
together with the formation of the radical anion [aglycone–H]•− at m/z
284. The aglycone fragment ions at m/z 255, due to the loss of a CH2O
moiety, and at m/z 227, due to the further neutral loss of CO, were also
detectable (Gao & Mazza, 1995).
Compound 18 was putatively identified as an acylated flavonol. In
fact the MS2 spectrum of the deprotonated molecular ion at m/z 495
showed two fragment ions at m/z 301 and 193. The first one likely
corresponded to the deprotonated flavonol quercetin. The formation
of the fragment ion at m/z 193 could be due to the neutral loss of the
quercetin moiety (−302 Da) according with a charge remote fragmen-
tation mechanism (Cheng & Gross, 2000).
Compound 19 was tentatively identified as kaempferol hexoside.
The fragmentation of the [M–H]– ion relative to this metabolite (m/z
447) provided the [aglycone–H]– fragment at m/z 285 (− 162 Da) as
well as an aglycone radical at m/z 284 (−163 Da), attributable to the
flavonol kaempferol. The loss of a fragment of 162 Da suggested the
presence of a hexose sugar. The presence of fragment ions at m/z 255
and 227 suggested that glycosylation site was at the C-3 position.
196 S. Pacifico et al. / Food Research International 64 (2014) 188–199
Fig. 4. ESI MS TICs of A) PaDR-2b extract, and B) PaDM-2b extract.
Compound 20 was identified as an isomer of dicaffeoyl quinic acid.
The fragmentation of the ion [M–H]– at m/z 515 provided the fragments
[M–H–162]− and [M–H–324]− corresponding, respectively, to the loss
of one and two caffeoyl moieties. The fragment at m/z 179 was due to
the presence of caffeic acid anion (Clifford et al., 2005).
Compound 22 was identified as a caffeoyl-p-coumaroylquinic acid
isomer. The MS2 spectrum of the deprotonated molecular ion at m/z
499 showed a fragment ion at m/z 337 as base peak, indicating the
loss of 162 Da and suggesting a coumaroylquinic acid derivative. MS3
fragmentation of the ion at m/z 337 resulted in a fragment ion at m/z
163, as base peak, pointing to a 3-p-O-coumaroylquinic acid structure,
according to literature reports (Clifford et al., 2003). Since the caffeoyl
group is the first to be lost it should be linked to the 5-OH position.
Taking into account these data, compound 22 was identified as 3-p-O-
coumaroyl-5-O-caffeoylquinic acid (Gouveia & Castilho, 2013).
Compound 23 was identified as free ferulic acid previously reported
as organic acid constituent of Prunus avium fruits (Gündoğdu &
Bilge, 2012). The fragmentation of the [M–H]− ion at m/z 193 ([ferulic
acid–H]−) gave the ion at m/z 149 due to the loss of CO2 moiety.
LC/MSn analysis of Pa-DR2b extract highlighted that it showed a less
complex phenolic compounds profile than Pa-DM2b extract (Fig. 6,
Table 5) from which differs mainly for the presence of two compounds
(10′ and 13′). In fact, Pa-DR2b was equally rich in caffeoyl and feruloyl
quinic acids. Feruloyl hexose, 5-O-caffeoylshikimic acid and rutin were
also present. The [M–H]− ion at m/z 309 which produced fragment
ions at m/z 193 and 115 suggested the presence of feruloyl malate (10′).
Compound 13′ was tentatively identified as phloretin hexoside. In
fact, the [M–H]− ion at m/z 435 provided characteristic fragments
such as m/z 273 (phloretin) and 179, 167 and 123. The neutral loss of
162 Da suggested the presence of a hexose residue. The molecule was
previously reported as constituent of Prunus avium fruits (Gündoğdu
& Bilge, 2012).
3.7. LC-MS based quantitative analysis of Pa-DM2b and Pa-DR2b extracts
The LC–MS/MS quantitative analysis allowed us to estimate in both
the investigated matrices the abundance of the individual identified
constituents as percentage of the total amount of constituents.
The aim of the quantification was to attribute the biological activities
to the presence of the most abundant constituents or eventually to their
synergy. The analyses were carried out using pure quercetin as internal
standard, which did not interfere with the other metabolites in terms of
retention time and was useful to compensate for potential variations in
instrumental analyses. It has been shown that a large percentage of the
 S. Pacifico et al. / Food Research International 64 (2014) 188–199 197

Fig. 5. Main metabolites tentatively identified in Pa-DM2b extract from Prunus avium cherries cv. ‘Del Monte’.

polyphenolic content of the extract Pa-DM2b, accounting for 26% of the
total, was constituted by quercetin-3-O-rutinoside. 5-O-caffeoylquinic
acid and kaempferol-3-O-rutinoside were also particularly abundant
(Table 4). Instead, 4-O-coumaroyl quinic acid and 5-O-caffeoylquinic
acid were the main constituents of Pa-DR2b extract (Table 5). Previous-
ly, neochlorogenic acid, p-coumaroylquinic acid and chlorogenic acid
were detected and quantified in the sweet cherry cultivars by HPLC-
PDA analysis (Ballistreri et al., 2013). The same phenolic compounds
were present in each cultivar, but there were differences in the relative
levels. In almost all genotypes, neochlorogenic acid was the major
hydroxycinnamic acid derivative followed by p-coumaroylquinic acid
and chlorogenic acid (Mozetic, Trebse, & Hribar, 2002) demonstrated
that the levels of hydroxycinnamic derivatives such as neochlorogenic
acid and p-coumarylquinic acid decrease during ripening. A change in
their ratio could also be due to the hydroxylation of p-coumarylquinic
acid to neochlorogenic acid.
4. Conclusions
Investigated cherries from Campania Region (Italy) contain high
amounts of polyphenol phytochemicals whose quality and quantity
vary considerably with the cultivar, thus providing dissimilar bioactivity
responses. In particular, it was assessed that the polyphenolic extract of
cv. ‘Della Recca’ was richer in hydroxycinnamoyl quinic acids deriva-
tives, while the extract of cv. ‘Del Monte’ was characterized by a high
flavonoid content, of which the most abundant was quercetin-3-O-
rutinoside. The relative abundance of the constituents and their
synergy led to the formulation of extracts able to exert a similar

Table 5
LC-MS/MS data of the main phenol constituents of Pa-DR2b extract. Relative content is expressed as percentage of the total polyphenols.

Peak Tentative assignment MW tR Pseudomolecular and fragment ions [m/z] Relative content
[Da] [min] in [MS]- mode

MS MS2

1′ feruloyl hexose 356 24.2 355 355, 193, 135 5%

2′ 3-O-caffeoyl quinic acid 354 24.3 353 191, 179, 161, 135, 93, 85 2%
3′ 5-O-caffeoyl quinic acid 354 27.4 353 191, 179, 137, 93 35%
4′ 3-O-p-coumaroyl quinic acid 338 27.8 337 191, 163, 155, 119, 111 41%
5′ 5-O-p-coumaroyl quinic acid 338 28.0 337 191, 163, 119, 111, 93, 67 b1%
6′ 4-O-caffeoyl quinic acid 354 28.1 353 191, 179, 173, 135, 93 b1%
7′ 3-O-feruloyl quinic acid 368 28.8 367 193, 191, 149, 134, 89 3%
8′ 5-O-feruloyl quinic acid 368 29.5 367 191, 173 4%
9′ 4-O-feruloyl quinic acid 368 33.3 367 367, 191, 179, 173, 161, 135 3%
10′ feruloyl malic acid 310 33.6 309 193, 161, 135 1%
11′ quercetin-3-O-(6″-O-deoxyhexosyl)-hexoside (rutin) 610 34.8 609 301, 300, 271, 255 b1%
12′ 5-O-caffeoyl shikimic acid 336 36.5 335 161, 135, 93 2%
13′ phloretin hexoside 436 43.2 435 273, 179, 167, 123 1%
14′ ferulic acid 194 45.2 193 149, 133, 105 3%
198 S. Pacifico et al. / Food Research International 64 (2014) 188–199
Fig. 6. Main metabolites tentatively identified in Pa-DR2b extract from Prunus avium cherries cv. ‘Della Recca’.
radical scavenging activity but different antiproliferative activity.
Pa-DR2b polyphenolic extract differently modulated cell viability,
where the extract Pa-DM2b showed a weak antiproliferative activity.
Our findings suggest that the massive presence of compounds charac-
terized by hydroxycinnamic skeleton makes Pa-DR2b a potential
chemopreventive agent.
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