Inhibitory effect of pomegranate (Punica granatum L.) polyphenol extracts on the bacterial growth and survival of clinical isolates of pathogenic Staphylococcus aureus and Escherichia coli

Full text

Inhibitory effect of pomegranate (Punica granatum L.) polyphenol
extracts on the bacterial growth and survival of clinical isolates
of pathogenic Staphylococcus aureus and Escherichia coli
Caterina Pagliarulo
a
, Valentina De Vito
b
, Gianluca Picariello
b
, Roberta Colicchio
c,d
, Gabiria Pastore
a
,
Paola Salvatore
c,e
, Maria Grazia Volpe
b,
⇑
a
Dipartimento di Scienze e Tecnologie, Università degli Studi del Sannio, via Port’Arsa 11, 82100 Benevento, Italy
b
Istituto di Scienze dell’Alimentazione-CNR, Via Roma 64, 83100 Avellino, Italy
c
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli ‘‘Federico II’’, via S. Pansini 5, 80131 Napoli, Italy
d
Fondazione SDN, Via Gianturco 113, 80143 Napoli, Italy
e
CEINGE-Biotecnologie Avanzate, Via G. Salvatore 486, 80145 Napoli, Italy
article info
Article history:
Received 4 March 2015
Received in revised form 8 June 2015
Accepted 9 June 2015
Available online 10 June 2015
Keywords:
Pomegranate polyphenols
HPLC-DAD
Mass spectrometry
Antimicrobial effects
Clinical isolates of pathogenic
microorganisms
abstract
In the present study major polyphenols of pomegranate arils and peel by-products were extracted in 50%
(v/v) aqueous ethanol, characterized and used in microbiological assays in order to test antimicrobial
activity against clinically isolated human pathogenic microorganisms.
Total concentration of polyphenols and in vitro antioxidant properties were determined by the Folin–
Ciocalteu and DPPH methods, respectively. The most abundant bioactive molecules, including antho-
cyanins, catechins, tannins, gallic and ellagic acids were identified by RP-HPLC-DAD, also coupled to
off-line matrix assisted laser desorption/ionization (MALDI-TOF) mass spectrometry (MS). The inhibitory
spectrum of extracts against test microorganisms was assessed by the agar well-diffusion method. Data
herein indicated that both pomegranate aril and peel extracts have an effective antimicrobial activity, as
evidenced by the inhibitory effect on the bacterial growth of two important human pathogens, including
Staphylococcus aureus and Escherichia coli, which are often involved in foodborne illness.
Ó2015 Elsevier Ltd. All rights reserved.
1. Introduction
Punica granatum L., member of two species comprising the
Punicaceae family, is a shrub native to occidental Asia and
Mediterranean Europe, also grown in warm climate areas of the
Americas and other parts of the world, which is popularly referred
to as pomegranate (Vidal et al., 2003).
The ripe pomegranate fruit is grenade-shaped, crowned by the
pointed calyx; it can be up to ten-centimeters diameter wide and
it is characterized by a deep red, leathery bark. The fruit contains
many seeds (arils) separated by a white, membranous peel, indi-
vidually surrounded by small amounts of tart and red juice.
In addition to its ancient historical uses, pomegranate is used in
several systems of medicine and for a variety of ailments. In
Ayurvedic medicine pomegranate is considered ‘‘a pharmacy unto
itself’’ and is used as an antiparasitic agent, a ‘‘blood tonic’’ as well
as to heal aphthae, diarrhea and ulcers. The current explosion of
interest in pomegranate as a medicinal and nutritional product is
testified by hundreds of recent scientific papers pertaining to its
health effects. The potential therapeutic properties of pomegranate
are wide-ranging and include prevention and treatment of cancer,
cardiovascular diseases, dental conditions, and protection against
ultraviolet (UV) radiation (Basu & Penugonda, 2009; Di Silvestro,
Di Silvestro, & Di Silvestro, 2009; Pacheco-Palencia, Noratto,
Hingorani, Talcott, & Mertens-Talcott, 2008). Further potential
applications include infant brain ischemia, Alzheimer’s disease,
male infertility and arthritis (Bhandari, 2012; Mohammad &
Kashani, 2012; Türk et al., 2008).
According to preliminary toxicity assessments, pomegranate is
relatively non-toxic even at massive doses (Vidal et al., 2003).
Therapeutic properties have been attributed to all the fruit com-
partments, and specific studies report that bark, roots, and tree
leaves have medicinal benefit as well. Current research agrees to
indicate ellagic acid, ellagitannins (including punicalagins), punicic
acid, flavonoids, anthocyanidins, anthocyanins, and estrogenic fla-
vonols and flavones as the most beneficial constituents of
http://dx.doi.org/10.1016/j.foodchem.2015.06.028
0308-8146/Ó2015 Elsevier Ltd. All rights reserved.
⇑
Corresponding author.
E-mail address: mgvolpe@isa.cnr.it (M.G. Volpe).
Food Chemistry 190 (2016) 824–831
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

pomegranate in therapeutic terms. Moreover, the antimicrobial
activity of these constituents is well documented too (Cowan,
1999; Machado et al., 2003;Naz, Siddiqi, Ahmad, Rasool &
Sayeed, 2007). To this purpose, P. granatum peel extracts that are
particularly high in ellagitannins exert remarkable antimicrobial
activity against pathogenic Staphylococcus aureus (Machado et al.,
2003).
Although the whole complex of pomegranate constituents
appears to exhibit a bioactive potency that is greater than the indi-
vidual components, as for instance a synergistic anti-proliferative
action on cancer cells in vitro (Lansky et al., 2005), an exhaustive
assessment of the antimicrobial properties of juice and peel
extracts on specific strains of human pathogenic microorganisms
is still missing.
Phenolic compounds of pomegranate fruit (juice and peel) have
been traditionally isolated and characterized by reversed
phase-high performance liquid chromatography (RP-HPLC) cou-
pled to UV–Vis and/or diode array detection (DAD). However, the
informative level of the RP-HPLC analysis can be limited due to
possible co-elution of different classes of polyphenols. In contrast,
the strategies based on the molecular profiling of anthocyanins and
tannins, especially those relying on mass spectrometry (MS), have
been demonstrated to be more effective and reliable to compre-
hensively identify polyphenol compounds in complex extracts. In
particular, the matrix assisted laser desorption ionization-time of
light (MALDI-TOF) MS analysis, both in mixture or after isolation
of the components, delivers a series of analytical advantages such
as the speed, specificity, high resolution and sensitivity
(Picariello et al., 2012). Herein, the main bioactive molecules,
including anthocyanins, catechins, tannins, gallic and ellagic acids
of pomegranate juice and peel were identified by RP-HPLC-DAD
coupled to off-line MALDI-TOF MS.
The primary aims of this study were to extract and characterize
the pomegranates juice and peel polyphenols as well as to assay
the extracts for their antimicrobial activity. Although the antibac-
terial activity of pomegranate polyphenols has already been
demonstrated, the novelty of this study consisted in assaying the
pomegranate extracts against clinical isolates of pathogenic S. aur-
eus and E. coli. Indeed, results of microbiological tests carried out
are interesting in view of the increasing multidrug-resistance of
clinical pathogens.
2. Materials and methods
2.1. Extraction
The pomegranate fruits were collected in local farms of the
Campania Region (Southern Italy). Arils were manually separated
from peel and seeds were carefully removed. Pomegranate juice
was obtained from arils by hand pressing. The peel was air dried
a few days and subsequently pulverized. The samples were stored
at 20 °C until analyzed. Solvents were purchased from Carlo Erba
(Carlo Erba Reagents, Milan, Italy) and were HPLC-grade or better.
2.2. Extraction of polyphenols
Pomegranate juice and peel (5 g) were homogenized in 25 ml of
50% ethanol/water (v/v) for 30 min in the dark. The extracts were
paper filtered to remove particles (crude extracts). Only the peel
extracts were ten-fold diluted with deionized water (Gözlekçi,
Saraçog
˘lu, Onursal, & Özgen, 2011). Sugars and organic acids inter-
fere with the absorbance measurement in the Folin–Ciocalteu
assay (Kim & Lee, 2002). Therefore, an aliquot of the crude extracts
was purified by solid phase extraction, using pre-packed C
18
reversed phase cartridges (Maxi-CleanTM Cartridge 300 mg,
Alltech Associates Inc., Deerfield, IL, USA). C
18
resin-bound phenol
compounds were washed with 0.01 N HCl and eluted with metha-
nol containing 0.1% (v/v) HCl, according to the manufacturer’s
indications.
2.3. Determination of total phenols (TP)
The concentration of total phenols (TP) in extracts were deter-
mined by the Folin–Ciocalteu colorimetric method (Singleton &
Rossi, 1965). Samples (100) were mixed with 5 ml of the 0.2 N
Folin–Ciocalteu reagent and 4 ml of 7.5% sodium carbonate. The
mixture was allowed to stand for 2 h at room temperature in the
dark. The absorbance at 765 nm was measured by automated
UV–Vis spectrophotometer (Beckman Coulter mod DU730).
Standard gallic acid within the 100–500 mg/l concentration range
was used to construct a calibration curve. Samples were assayed
in triplicate and the absorbance values were averaged. Outcomes
are expressed as mg gallic acid equivalent/l of sample (mgGAE/l).
2.4. Antioxidant activity (DPPH assay)
The free radical scavenging capacity was determined by the
DPPH assay (Blois, 1958). An aliquot of 500
l
l of sample solution
in ethanol was mixed with 500
l
l of 0.5 mM DPPH in ethanol.
The mixture was shaken vigorously and incubated for 30 min at
room temperature in the dark. Ethanol was used as the blank.
The absorbance was measured at 517 nm. The antiradical activity
was expressed as percentage of inhibition (I%) of the sample (As)
compared to the initial concentration of DPPH

(Ac) according to
the formula: I% = (Ac As/Ac) 100.
2.5. RP-HPLC and MS analysis of pomegranate juice and peel
2.5.1. RP-HPLC
Polyphenols were separated using a modular chromatographer
HP 1100 (Agilent Technologies, Paolo Alto, CA, USA) equipped with
a diode array detector (DAD). The stationary phase was a 250
2.1 mm i.d. C18 reversed-phase column, 4
l
m particle diameter
(Jupiter Phenomenex, Torrance, CA, USA). The column temperature
was maintained at 37 °C during the HPLC analyses. Runs were per-
formed at a constant flow rate of 0.2 ml/min applying the following
gradient of the solvent B (acetonitrile/0.1%TFA): 0–4 min: 0% B; 4–
14 min 0–14% B; 14–30 min 14–28% B; 30–34 min 28% B; 34–
42 min 28–60% B; 42–45–27 min 60–80% B; 45–50 min 80–100%
B. Solvent A was 0.1% TFA in HPLC-grade water. For each analysis
20
l
l of peel and juice pomegranate extracts 100-fold and
10-fold diluted with 0.1% TFA, respectively, were injected.
The k= 520, 360, 320 and 280 nm wavelengths were used to
monitor the HPLC separations.
2.5.2. MALDI-TOF MS
Crude extracts and HPLC isolated pomegranate polyphenols
were analyzed by MALDI-TOF MS analysis using a Voyager
DE-Pro
R
(PerSeptive Biosystems, Framingham, MA, U.S.A.)
equipped with a N
2
laser (337 nm, 3n-sec pulse width). Prior to
MS analysis, ethanol extracts of pomegranate juice and peel were
purified by Zip-Tip C
18
reversed-phase pre-packed microcolumns
(Millipore, Bedford, MA, USA), washing with 0.1% TFA and eluting
with 50% acetonitrile (ACN, v/v) containing 0.1% TFA. The HPLC
peaks were analyzed without further desalting. Spectra were
acquired in both positive and negative reflector ion modes using
2,5-hydroxybenzoic acid (DHB) as the matrix (10 mg/ml of crys-
talline powders in 50% ACN, v/v). When acquiring in the positive
ion mode, the matrix solution also contained 0.1% TFA. Spectra
were acquired using the Delay Extraction technology at an acceler-
ating voltage of 20 kV. Typically, the m/z 4001500 range was
C. Pagliarulo et al. / Food Chemistry 190 (2016) 824–831 825

explored. The positive and negative mass ranges were externally
calibrated with a mixture of standard polyphenols (Sigma, Milan,
Italy). Spectra were elaborated with the Data Explorer 4.0 pur-
chased with the instrument. Components were definitely identified
combining the indication of the HPLC retention times, DAD spec-
trophotometric data and molecular mass measurements.
2.6. Antimicrobial activity of pomegranate extracts
2.6.1. Preparation of extracts for microbiological assay
Crude and filtered pomegranate extracts (juice and peel) were
dissolved in a hydro-alcoholic buffer containing ethanol 50%
(v/v), at the final 1 mg/ml concentration. The extracts were steril-
ized by passage through a membrane filter (0.45
l
m).
2.6.2. Microorganisms and growth conditions
The antimicrobial activity of the pomegranate extracts were
evaluated against clinical isolates of S. aureus and E. coli, kindly
provided from the UOC of Clinical Microbiology, AOU Federico II
of Naples. The strain of S. aureus was isolated from a pharyngeal
swab while E. coli strain comes from a sputum sample. The identi-
fication of isolates was performed by mass spectrometry using the
matrix assisted laser desorption/ionization (MALDI) mass spec-
trometer (Bruker Daltonics, MALDI Biotyper, Fremont, CA, USA), a
high-throughput proteomic technique for identification of a vari-
ety of bacterial species (Neville et al., 2011; Sogawa et al., 2011),
and by biochemical phenotyping method in an BD Phoenix™
Automated Microbiology System (Becton Dickinson, BD Franklin
Lakes, NJ, USA), according to the manufacturer’s instruction. The
profile of susceptibility to antibiotics was also evaluated using
the BD Phoenix™ system (Tables S1 and S2). The microorganisms
were cultured in broth and agar media at 37 °C. The media used
were BD Brain Heart Infusion (BHI), BD Trypticase Soy agar with
5% sheep blood (BD, Franklin Lakes, NJ, USA) and Mueller-Hinton
(Simad s.a.s., Naples, Italy). Microbial strains were maintained at
4°C on agar media. The isolates were stored frozen at 80 °Cin
BHI broth supplemented with 10% glycerol (v/v) (Carlo Erba,
Reagents, Milan, Italy) until use and the working cultures were
activated in the respective broth at 37 °C for 15–18 h.
2.6.3. In vitro antimicrobial activity assay
The agar diffusion method, similar to that of Bauer, Kirby,
Sherris, and Turck (1966), was conducted to evaluate the inhibitory
spectrum of extracts against test microorganisms. The bacteria
were grown in BHI broth to an optical density of 0.5 at 600 nm
and the bacterial suspension was spread onto surface of agar
media. Paper discs (6 mm in diameter, Oxoid s.p.a., Rodano,
Italy), impregnated with 20
l
l of extracts, were positioned on
media using a sterile forceps. Different concentrations of pomegra-
nate extracts (1, 2, 4, 8, 10 e 20 mg/disc) were used to evaluate the
antimicrobial activity. Ampicillin (80
l
g/disc) was used as the pos-
itive control. The hydro-alcoholic buffer 50% ethanol (v/v) was
used as the negative control.
Plates were incubates at 37 °C for 12–48 h. The diameter of the
inhibition areas (including disc diameter) induced by extracts
around discs was measured and expressed in mm. The experiment
was repeated three times.
2.6.4. Effect of pomegranate extracts on bacterial growth and survival
The susceptibility of S. aureus and E. coli to different concentra-
tions of P. granatum extracts was determined by the use of dilution
tube method with 1 10
5
CFU/ml as standard inoculums (Varaldo,
2002, NCCLS). The extracts were added in the series of tubes
achieving final concentrations of 0, 10, 20, 30, 40, 45, 50, 55, 60,
65, 70, 75, 80, 100, 120, 160, 200, 250, 300, and 400
l
g/
l
l, and
tubes were incubated at 37 °C for 24 h. The isolates were also
tested with ampicillin as positive control and with the extraction
buffer as the positive and negative controls, respectively. After
incubation, samples from each tube were used to determine the
optical density at 600 nm, and an aliquot was spread onto the sur-
faces of BHI-agar (BD), plates in duplicate. Plates were then incu-
bated for 24–48 h and colony counts performed. The minimum
bactericidal concentration (MBC) was defined as the minimum
extract concentration that killed 99% of bacteria in the initial
inoculums. Minimum inhibitory concentration (MIC) was assigned
to lowest concentration of pomegranate extract, which prevent
bacterial growth.
To verify the effect of pomegranate crude juice and peel extracts
on the S. aureus and E. coli fitness, growth and survival assays were
performed in presence of increasing concentrations of the extracts.
To evaluate the fitness of each strain, during the observation period
(72 h), serial dilutions were plated on BHI-agar, and incubated at
37 °C for 24–48 h. After the growth, cells viable count was carried
out. All experiments were performed in triplicate with three inde-
pendent cultures; the results obtained were analyzed and graphi-
cally reported by using ‘‘GraphPad Prism 4’’ software, validating
the statistical significance by the Student’s t-test.
3. Result and discussion
3.1. Determination of total phenols and total antioxidant activity
Several literature methods were screened to extract polyphe-
nols from pomegranate aril juice and peel. Finally, the extraction
with 50% ethanol/water (v/v) was established as the most efficient
in terms of yield of total polyphenol and minimization of the inter-
ferences. The amount of total polyphenols varied depending on the
parts of the fruit (Table 1), in particular being higher in the peel
than in the juice extracts. The literature values reported for the
total polyphenol content are variable, probably also related to
the cultivar and to the geographical area where fruits are grown.
Gözlekçi et al. (2011) found the concentration of pomegranate
juice and peel polyphenols to vary in the 784–1551 and 1775–
3547 mgGAE/l range, respectively. According to other Authors the
pomegranate juice polyphenols vary within the 1245–
2076 mgGAE/l range (Ozgen, Durgaç, Serçe, & Kay, 2008). In any
case, the in vitro antioxidant potential of pomegranate juice is
much higher that red wine or green tea (Gil, Tomás-Barberán,
Hess-Pierce, Holcroft, & Kader, 2000). Our determinations
(Table 1) are roughly in line with the previous ones, as values lay
close or within the range of concentrations already defined.
Similarly, the in vitro antioxidant activity was expected to depend
on the parts of the fruit which polyphenols are extracted from. The
range of % radical inhibition already reported for pomegranate
juice are quite contrasting, varying according to the cultivar in
the 23.8–38.0% (Eghdami & Asli, 2010) or 71–91% (Çam, Hıs
ßıl, &
Durmaz, 2009) ranges. In general, the in vitro antioxidant activity
of the peel is significantly higher than the juice, being reported
as 71.3–86.3% (Negi & Jayaprakasha, 2003) and 46.3–99.3%
(Shiban, Al-Otaibi, & Al-Zoreky, 2012) by previous investigations.
Consistently, our determination supported a higher in vitro antiox-
idant activity of peel (94.7% of radical inhibition) if compared to
the juice (75.8%).
Table 1
Total phenols and total antioxidant activity of pomegranate juice and peel extracts.
Extract Total phenols (mgGAE/l) % Yield % Phenols (I%) DPPH
Juice 2703 ± 18 76.5 ± 1.3 18.6 ± 2.1 75.83 ± 6.71
Peel 3019 ± 26 49.9 ± 3.1 20.9 ± 5.4 94.78 ± 1.53
826 C. Pagliarulo et al. / Food Chemistry 190 (2016) 824–831

Fig. 1. RP-HPLC separation of pomegranate juice polyphenols monitored at k= 520 (A); k= 280 (B) and k= 360 (C). The most abundant components were identified by DAD
and off-line MALDI-TOF MS and are assigned in the figure. Dp = delphinidin; Cy = cyanidin; DG = diglucoside; G = glucoside; RUT = rutinoside; EA = ellagic acid;
HHDP = hexahydroxydiphenoyl; hex = hexose; deoxyhex = deoxyhexose/metylpentose; pent = pentose.
C. Pagliarulo et al. / Food Chemistry 190 (2016) 824–831 827

3.2. Characterization of phenolic compounds by HPLC-DAD and MS
The most abundant pomegranate polyphenols were identified
combining retention time (t
r
), UV–Vis, spectrophotometric (DAD)
and MALDI-TOF MS data. In the case of uncertain assignment, the
identifications were confirmed by parallel analysis of opportune
standards.
A typical RP-HPLC chromatogram of pomegranate juice antho-
cyanins, recorded at 520 nm, is shown in Fig. 1A along with the
assignment of peaks. The RP-HPLC profile of pomegranate juice is
characterized by several distinctive traits which include the exclu-
sive presence of delphinidin and cyanidin as the aglycons, both
occurring in the 3,5-O-diglucoside and 3-O-monoglucoside forms.
As expected on the basis of their polarity, diglycosylated antho-
cyanins were eluted at shorter t
r
than the monoglycosylated coun-
terparts (Fischer, Carle, & Kammerer, 2011). Trace amounts of
delphinidin (m/z 611) and cyanidin (m/z 595) 3-O-rutinosides were
also detected at longer t
r
. The pomegranate anthocyanins have
been previously characterized (Gil et al., 2000, and their relative
amounts were shown to significantly fluctuate depending on the
cultivars (Algihourchi & Barzegar, 2008). Nevertheless, the ruti-
noside anthocyanins have been detected in a relatively few inves-
tigations, demonstrating that the comprehensive pigment profile
of pomegranate still remains to be completely elucidate.
Differently from other reports (Fischer et al., 2011) we did not
detect pelargonidin derivatives.
The HPLC separation of the non-anthocyanin phenolic com-
pounds from pomegranate juice, monitored at k= 280 and
360 nm exhibited a complex pattern, because of the occurrence
of heterogenous classes of compounds, including hydrolysable tan-
nins (gallotannins), ellagitannins and gallagyl esters, as well as
hydroxybenzoic and hydroxycinnamic acids. The main
non-anthocyanin phenolics were assigned in the chromatogram
(Fig. 1B and C) according to spectrophotometric (k
max
) and
off-line MALDI-TOF MS determinations. The simple measurement
of the molecular mass did not allow a more refined discrimination
between isomers. A more comprehensive characterization of the
pomegranate juice phenolics has been carried out by
HPLC-DAD-ESI-multicollisional MS (Fischer et al., 2011; Mena
et al., 2012), although many compositional traits still remain
controversial.
The major pomegranate peel polyphenols assigned in Fig. 2
were similarly identified through the convergent indications of
RP-HPLC (monitoring at k= 360), UV–Vis spectra (DAD) and
MALDI-TOF MS data and by comparison with previous structural
characterizations (Gil et al., 2000; Seeram, Lee, Hardy, & Heber,
2005).
The RP-HPLC profile of pomegranate peel was characterized by
the dominant presence of hydrolysable ellagitannins such as
HHDP-gallagyl-hex isomers (
a
-and b-punicalagin,
[MH]

= 1082). The negative ion mode MALDI-TOF mass spec-
trum of the HPLC peak at t
r
= 24.5 min (inset of Fig. 2) showed
Fig. 2. RP-HPLC separation of pomegranate peel polyphenols monitored at k= 360. The inset shows the negative ion mode MALDI-TOF mass spectrum of the peak at
t
r
= 24.5 min, corresponding to b-punicalagin ([MH]

= 1082), co-eluting with minor amounts of other components (see text). For abbreviations cfr. legend of Fig. 1.
Table 2
Antibacterial activity of different extracts of pomegranate fruit determined with the agar-diffusion method.
Inhibition zone (mm) Inhibition zone (mm)
Extracts Extracts
Crude juice (10 mg/d) Not clear zone Crude juice (10 mg/d) Not clear zone
Crude juice (20 mg/d) 13 Crude juice (20 mg/d) 13
Purified juice (4 mg/d) 10 Purified juice (4 mg/d) 8
Crude peel (8 mg/d) 20 Crude peel (8 mg/d) 30
Purified peel (2.4, e 8 mg/d) 15–20 Purified peel (2.4, e 8 mg/d) 20–30
Controls Controls
Ampicillin (80
l
g/d) 15 Ampicillin (80
l
g/d) 30
Extraction buffer (20
l
l/d) 0 Extraction buffer (20
l
l/d) 0
Staphylococcus aureus (clinical isolate) Escherichia coli (clinical isolate)
828 C. Pagliarulo et al. / Food Chemistry 190 (2016) 824–831

the presence of a co-eluting compound that has been
previously identified as di (HHDP-galloylglucose)-pentose or
digalloyl-triHHDP-diglucose at [MH]

= 1413 (Mena et al.,
2012). The low-abundance peak at t
r
= 20.0 was ascribable to
pedunculagin ([MH]

= 782).
Less abundant peaks at higher t
r
were glycosylated derivatives
of ellagic acid, including the hexose, pentose and deoxyhexose
derivatives, along with free ellagic acid.
3.3. Preliminary screening of antimicrobial activity (agar-diffusion
method)
The antimicrobial properties of pomegranate extracts were
assayed against clinical isolates strains of pathogenic S. aureus
and E. coli.Table 2 presents diameters of inhibition zones (clear
zones around discs) exerted by the various extracts (crude and
purified juice, crude and purified peel) towards challenged
microorganisms. The hydro-alcoholic buffer assayed as the nega-
tive control resulted inactive against tested microorganisms.
Ampicillin (80
l
g/disc), used as the positive control, exerted the
expected antibacterial efficacy against both S. aureus that E. coli,
with inhibition zones of 15 and 30 mm, respectively. As reported
in Table 2, the pomegranate juice extracts exhibited a clear
antibacterial activity. Particularly, the crude juice extracts, at
concentration of 20 mg/disc, induced the formation of a clear inhi-
bition zone of 13 mm, against both S. aureus and E. coli. Differently,
the juice purified extract, at contraction of 4 mg/disc, demon-
strated a lower antibacterial effect against both S. aureus that
E. coli, forming a inhibition zone of 10 mm and 8 mm, respectively.
The crude and purified peel extracts, at concentrations of 2, 4,
8 mg/disc demonstrated a higher antibacterial activity, forming a
large zone inhibition (15–30 mm) against both the tested microor-
ganisms as reported in Table 2.
Overall, these findings clearly indicate that the hydro-alcoholic
extracts of pomegranate are able, in vitro, to effectively antagonize
the growth both of gram-positive bacteria, such as S. aureus, and
even to a greater extent that of gram-negative bacteria, such as
E. coli (Voravuthikunchai & Limsuwan, 2006). These results are in
agreement with those recently obtained in several studies on the
antimicrobial activity of pomegranate fruit extracts (Al-Zoreky,
2009; Braga et al., 2005; Priya, Shilpy, Eswara, & Girish-Babu,
2012). The relative potency of antibacterial activity of pomegra-
nate extracts against gram-positive and gram-negative microor-
ganisms (determined through the MIC) emerges as a quite
controversial issue from the literature. The relative degree of
antibacterial activity depends on several parameters including
extraction method as well as cultivar, seasonality and geographical
origin of pomegranate (Zografou, Kallimanis, Akrida-Demertzi, &
Demertzis 2013). In this current case, the variable response
between cultured and clinically isolated bacterial strains should
not be considered surprising, because it is typically recorded even
with conventional antimicrobials against clinical pathogens,
though generally within a more restricted range.
According to the results of the Shan, Cai, Brooks, and Corke
(2007), the antibacterial activity of pomegranate fruit extracts is
correlated to the total polyphenol content of different extracts
(juice: 18.6%; peel: 20.1%) as reported in Table 1. Nevertheless, it
is to be highlighted that the antimicrobial activity of pomegranate
extracts is not exclusively related to the toxicity of phenolic compo-
nents against microorganisms (Machado et al., 2003), but it proba-
bly is the result of a synergistic action of the components in the
phyto-complex, as reported in other studies on anti-inflammatory
and anticancer properties of pomegranate extracts (Adams,
Zhang, Seeram, Heber, & Chen, 2010; Lansky et al., 2005).
3.4. Effects of pomegranate extracts on bacterial growth and bacterial
survival
Quantitative evaluation of the antimicrobial activity of pome-
granate crude extracts against S. aureus and E. coli was carried
out using the dilution tube method, according to the CLSI
(Clinical and Laboratory Standards Institute) guidelines (CLSI,
2012).
Growth of S. aureus isolate was inhibited by a concentration of
65
l
g/
l
l of juice extract, while the MIC against E. coli isolate was
lower (40
l
g/
l
l). The MIC values of pomegranate extracts
Fig. 3. Inhibitory effect of pomegranate crude juice extracts on growth and fitness (A) and on survival (B) of clinically isolated E. coli and on growth and fitness (C) and on
survival (D) of clinically isolated S. aureus.
C. Pagliarulo et al. / Food Chemistry 190 (2016) 824–831 829

determined in different studies significantly vary. Indeed, MIC
against S. aureus are reported to range from 0.62 to P250
l
g/
l
l
(Machado et al., 2003; McCarrell et al., 2008; Prashanth, Asha, &
Amit, 2001). This variability is not surprising, considered it is typ-
ically recorded even with conventional antimicrobials against all
clinical isolates (EUCAST data 2013, URL http://www.eucast.org/),
though generally within a more restricted range. The MBC of
pomegranate crude juice extracts was 160
l
g/
l
l against both clin-
ically isolated microorganisms tested in this study.
Differently from the juice extracts, the peel pomegranate
extracts exhibited the following antimicrobial activity: MIC
30
l
g/
l
l and MBC 70
l
g/
l
l for E. coli; MIC 20
l
g/
l
l and MBC
50
l
g/
l
l, for S. aureus. Both the isolates used in this study are sen-
sitive to ampicillin. The values obtained are MIC 5
l
g/
l
l and MBC
40
l
g/
l
l for E. coli, and MIC 10
l
g/
l
l and MBC 280
l
g/
l
l for S.
aureus.
Ultimately, the results of the microbiological tests carried out
indicate that the antimicrobial potency of the crude extracts is
greater than purified extracts and, in particular, the crude peel
extracts more efficiently antagonize the growth and survival of
the clinical isolates. The clearly higher activity of crude extracts
support the concept that possible hydrophilic components,
removed during the purifying step, can synergistically contribute
to the antibacterial properties.
The growth, survival and fitness of the E. coli and S. aureus were
monitored in presence of pomegranate crude juice and pomegra-
nate crude peel extracts. The inhibitory effect of pomegranate
crude juice extracts on E. coli growth and fitness is showed in
Fig. 3A. The bactericidal effect is showed in Fig. 3B. The inhibitory
and bactericidal effects of pomegranate crude juice extracts on S.
aureus are showed in Fig. 3C and D, respectively. In Fig. 3A and C
the spectrophotometric determination were biased by the turbid-
ity of the extracts, as clearly evident from the survival assay
(Fig. 3B and D), demonstrating that a concentration of 160
l
g/
l
l
had a total bactericidal effect on both clinical isolates. The inhibi-
tory effect of pomegranate crude peel extracts on survival of the
E. coli and S. aureus is showed in Fig. 4. Therefore, the pomegranate
crude extracts interfered with the bacterial growth, survival and
fitness in a dose dependent manner and with time-lasting effects.
The crude peel extracts in ethanol were cloudy so it was impossible
to subject them at the growth assay.
4. Conclusions
The extracts of pomegranate juice are sources of phenolic com-
pounds, in particular anthocyanins (especially delphinidin and
cyanidin glucosides), while by-product peel is high in derivatives
of the gallagic and/or ellagic acids, such as bis-HHDP-hexoside
(pedunculagin I), punicalin, ellagic acid hexose, ellagic acid pentose
and ellagic acid deoxyhexose. Our data indicate that pomegranate
phyto-complex extracts have an effective antimicrobial activity, as
evidenced by the inhibitory effect on bacterial growth of two
important human pathogens, often involved in foodborne illness.
The antibiotic properties of pomegranate fruit extracts are of
extreme interest in the light of the ongoing threat of bacterial
strains developing resistance to conventional antibiotics. These
results are also interesting in the perspective of recovering biolog-
ically active molecules from agro-industrial by-products.
Acknowledgments
This research was funded in part by PRIN 2012 [grant number
2012WJSX8K]: ‘‘Host-microbe interaction models in mucosal
infections: development of novel therapeutic strategies’’; and
POR Campania FSE 2007–2013, Project CREME.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.
06.028.
References
Adams, L. S., Zhang, Y., Seeram, N. P., Heber, D., & Chen, S. (2010). Pomegranate
ellagitannin-derived compounds exhibit antiproliferative and antiaromatase
activity in breast cancer cells in vitro.Cancer Prevention Research (Phila), 3,
108–113.
Algihourchi, H., & Barzegar, M. (2008). Anthocyanins characterization of 15 Iranian
pomegranate (Punica granatum L.) varieties and their variation after cold
storage and pasteurization. European Food Research and Technology, 227,
881–887.
Al-Zoreky, N. S. (2009). Antimicrobial activity of pomegranate (Punica granatum L.)
fruit peels. International Journal of Food Microbiology, 134, 244–248.
Basu, A., & Penugonda, K. (2009). Pomegranate juice: a heart-healthy fruit juice.
Nutrition Reviews, 67, 49–56.
Bauer, A. W., Kirby, W. M., Sherris, J. C., & Turck, M. (1966). Antibiotic susceptibility
testing by a standardized single disk method. American Journal of Clinical
Pathology, 45, 493–496.
Bhandari, P. R. (2012). Pomegranate (Punica granatum L). Ancient seeds for modern
cure? Review of potential therapeutic applications. International Journal of
Nutrition, Pharmacology, Neurological Diseases, 2, 171–184.
Blois, M. S. (1958). Antioxidant determinations by the use of a stable free radical.
Nature, 18, 1199–1200.
Braga, L., Shupp, J., Cummings, C., Jett, M., Takahashi, J., Carmo, L., et al. (2005).
Pomegranate extract inhibits Staphylococcus aureus growth and subsequent
enterotoxin production. Journal of Ethnopharmacology, 96, 335–339.
Çam, M., Hısßıl, Y., & Durmaz, G. (2009). Classification of eight pomegranate juices
based on antioxidant capacity measured by four methods. Food Chemistry, 112,
721–726.
CLSI (Clinical and Laboratory Standards Institute). (2012). Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically; Approved
Standard-Ninth Edition, M07-A9, Vol. 32 No. 2. URL http://clsi.org/.
Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical Microbiology
Reviews, 12, 564–582.
Fig. 4. Inhibitory effect of pomegranate crude peel extracts on survival of (A) E. coli
and (B) S. aureus.
830 C. Pagliarulo et al. / Food Chemistry 190 (2016) 824–831

Di Silvestro, R. A., Di Silvestro, D. J., & Di Silvestro, D. J. (2009). Pomegranate extract
mouth rinsing effects on saliva measures relevant to gingivitis risk.
Phytotherapy Research, 23, 1123–1127.
Eghdami, A., & Asli, D. E. (2010). Determination of antioxidant capacity of
pomegranate juice by using 2,2-diphenyl-1-picrylhydrazyl. The Journal of
Organic Chemistry, 1, 30–33.
Fischer, U. A., Carle, R., & Kammerer, D. R. (2011). Identification and quantification
of phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp,
aril and differently produced juices by HPLC-DAD–ESI/MS
n
.Food Chemistry, 127,
807–821.
Gil, M., Tomás-Barberán, F., Hess-Pierce, B., Holcroft, D., & Kader, A. (2000).
Antioxidant activity of pomegranate juice and its relationship with phenolic
composition and processing. Journal of Agricultural and Food Chemistry, 48,
4581–4589.
Gözlekçi, S., Saraçog
˘lu, O., Onursal, E., & Özgen, M. (2011). Total phenolic
distribution of juice, peel, and seed extracts of four pomegranate cultivars.
Pharmacognosy Magazine, 7, 161–164.
Kim, D., & Lee, C. Y. (2002). In R. E. Wrolstad, T. E. Acree, H. An, E. A. Decker, M. H.
Penner, D. S. Reid, P. Sporns, S. J. Schwartz, & C. F. Shoemaker (Eds.), Current
protocols in food analytical chemistry, I.0.1–I.0.2. New York: John Wiley & Sons
Inc.
Lansky, E. P., Jiang, W., Mo, H., Bravo, L., Froom, P., Yu, W., et al. (2005). Possible
synergistic prostate cancer suppression by anatomically discrete pomegranate
fractions. Investigational New Drugs, 23, 11–20.
Machado, T., Pinto, A., Pinto, M., Leal, I., Silva, M., Amaral, A., et al. (2003). In vitro
activity of Brazilian medicinal plants, naturally occurring naphthoquinones and
their analogues, against methicillin-resistant Staphylococcus aureus.The
International Journal of Antimicrobial Agents, 21, 279–284.
McCarrell, E., Gould, S., Fielder, M., Kelly, A., El Sankary, W., & Naughton, D. (2008).
Antimicrobial activities of pomegranate rind extracts: enhancement by
addition of metal salts and vitamin C. BMC Complementary and Alternative
Medicine, 8,64.
Mena, P., Calani, L., Dall’Asta, C., Galaverna, G., García-Viguera, C., Bruni, R., et al.
(2012). Rapid and comprehensive evaluation of (poly)phenolic compounds in
pomegranate (Punica granatum L.) juice by UHPLC-MS
n
.Molecules, 17,
14821–14840.
Mohammad, S. M., & Kashani, H. H. (2012). Chemical composition of the plant
Punica granatum L. (Pomegranate) and its effect on heart and cancer. Journal of
Medicinal Plants Research, 6, 5306–5310.
Naz, S., Siddiqi, R., Ahmad, S., Rasool, S., & Sayeed, S. (2007). Antibacterial activity
directed isolation of compounds from Punica granatum.Journal of Food Science,
72, 341–345.
Negi, P. S., & Jayaprakasha, G. K. (2003). Antioxidant and antibacterial activities of
Punica granatum peel extracts. Journal of Food Science, 68, 1473–1477.
Neville, S. A., Lecordier, A., Ziochos, H., Chater, M. J., Gosbell, I. B., Maley, M. W., et al.
(2011). Utility of matrix-assisted laser desorption ionization-time of flight mass
spectrometry following introduction for routine laboratory bacterial
identification. Journal of Clinical Microbiology, 49(8), 2980–2984.
Ozgen, M., Durgaç, C., Serçe, S., & Kay, C. (2008). Chemical and antioxidant
properties of pomegranate cultivars grown in the Mediterranean region of
Turkey. Food Chemistry, 111, 703–706.
Pacheco-Palencia, L. A., Noratto, G., Hingorani, L., Talcott, S. T., & Mertens-Talcott, S.
U. (2008). Protective effects of standardized pomegranate (Punica Granatum L.)
polyphenolic extract in ultra-violet-irradiated human skin fibroblasts. Journal of
Agricultural and Food Chemistry, 56, 8434–8441.
Picariello, G., Ferranti, P., Garro, G., Manganiello, G., Chianese, L., Coppola, R., et al.
(2012). Profiling of anthocyanins for the taxonomic assessment of ancient
purebred V. vinifera red grape varieties. Food Chemistry, 146, 15–22.
Prashanth, D., Asha, M., & Amit, A. (2001). Antibacterial activity of Punica granatum.
Fitoterapia, 72, 171–173.
Priya, S., Shilpy, D., Eswara, U., & Girish-Babu, K. L. (2012). Effect of pomegranate
and aloe vera extract on Streptococcus mutans:Anin vitro study. Dental
Hypotheses, 3, 99–105.
Seeram, N., Lee, R., Hardy, M., & Heber, D. (2005). Rapid large scale purification of
ellagitannins from pomegranate husk, a by-product of the commercial juice
industry. Separation and Purification Technology, 41, 49–55.
Shan, B., Cai, Y. Z., Brooks, J., & Corke, H. (2007). The in vitro antibacterial activity of
dietary species and medicinal herb extracts. International Journal of Food
Microbiology, 117, 112–119.
Shiban, M. S., Al-Otaibi, M. M., & Al-Zoreky, N. S. (2012). Antioxidant activity of
pomegranate (Punica granatum L.) fruit peels. Food and Nutrition Sciences, 3,
991–996.
Singleton, V., & Rossi, J. Jr., (1965). Colorimetry of total phenolics with
phosphomolybdic phosphotungstic acid reagents. American Journal of Enology
and Viticulture, 16, 144–158.
Sogawa, K., Watanabe, M., Sato, K., Segawa, S., Ishii, C., Miyabe, A., et al. (2011). Use
of the MALDI BioTyper system with MALDI-TOF mass spectrometry for rapid
identification of microorganisms. Analytical and Bioanalytical Chemistry, 400(7),
1905–1911.
Türk, G., Sönmez, M., Aydin, M., Yüce, A., Gür, S., Yüksel, M., et al. (2008). Effects of
pomegranate juice consumption on sperm quality, spermatogenic cell density,
antioxidant activity and testosterone level in male rats. Clinical Nutrition, 27,
289–296.
Varaldo, P. E. (2002). Antimicrobial resistance and susceptibility testing: an
evergreen topic. Journal of Antimicrobial Chemotherapy, 50, 1–4.
Vidal, A., Fallarero, A., Peña, B. R., Medina, M. E., Gra, B., Rivera, F., et al. (2003).
Studies on the toxicity of Punica granatum L. (punicaceae) whole fruit extracts.
Journal of Ethnopharmacology, 89, 295–300.
Voravuthikunchai, S. P., & Limsuwan, S. (2006). Medicinal plant extracts as anti-
Escherichia coli O157:H7 agents and their effects on bacterial cell aggregation.
Journal of Food Protection, 69, 2336–2341.
Zografou, E. Ch., Kallimanis, A., Akrida-Demertzi, K., & Demertzis, P. G. (2013). Total
phenolics, antioxidant and antibacterial activity of commercial pomegranate
based juices from Greece. In C.-T. Ho, C. Mussinan, F. Shahidi, & E. T. Contis
(Eds.), Nutrition, functional and sensory properties of foods. London, UK: Royal
Society of Chemistry.
C. Pagliarulo et al. / Food Chemistry 190 (2016) 824–831 831