Food Chemistry 127 (2011) 18211827

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Antioxidant and antilisterial activity of olive oil, cocoa and rosemary

extract polyphenols
Marina Bubonja-Sonje a,, Jasminka Giacometti b, Maja Abram a
a
Department of Microbiology, Faculty of Medicine, University of Rijeka, Brace Branchetta 20, 51000 Rijeka, Croatia
b
Department of Biotechnology, University of Rijeka, S. Krautzeka bb, 51000 Rijeka, Croatia

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

Article history: Antibacterial and antioxidant activity of polyphenols from olive oil, cocoa, and rosemary extract was
Received 23 March 2010 tested. Antimicrobial activity against Listeria strains was assessed using broth dilution and time-kill curve
Received in revised form 20 October 2010 methods. The 2,2-diphenyl-2-picrylhydracyl hydrate (DPPH) radical scavenging method, FolinCiocalteu
Accepted 17 February 2011
method, and high performance liquid chromatography (HPLC) were used for phenolics identication and
Available online 22 February 2011
determination of antioxidants level. Antibacterial and antioxidant activity of main pure phenolic com-
pounds, such as hydroxytyrosol in olive oil, epicatechin in cocoa and carnosic acid in rosemary was each
Keywords:
compared with their extracts.
Antibacterial
Antioxidant
Rosemary extract showed strong, while olive oil and particularly cocoa phenolic extract showed lower
Cocoa antilisterial activity. The overall relative antioxidant capacities of the samples were as follows in decreas-
Listeria ing order: rosemary > cocoa > olive oil. These results indicate that rosemary, olive oil, and cocoa polyphe-
Olive oil nols could be potentially used as alternative food additives for the prevention of food spoilage, and
Polyphenols contamination with Listeria monocytogenes.
Rosemary 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Phenolic compounds of plant origin have attracted considerable
attention due to their benecial functional and nutritional effects
including antioxidant and antimicrobial activity. Oxidation is one
of the most important free radical-producing processes in food,
chemicals and even in living systems. Free radicals play an impor-
tant role in food and chemical material degradation. In addition to
extending shelf-life of foods by inhibition of lipid peroxidation, the
phenolics act in the scavenging of free radical and can protect the
human body against damage caused by them (Cicerale, Conlan,
Sinclair, & Keast, 2009). Reduction or elimination of chemically
synthesised additives is a current demand in food industry. An ap-
proach to prevent the proliferation of microorganisms or protect
food from oxidation is the use of essential oils or plant extracts
as natural food additives.
Although polyphenols are present in almost all plants, rose-
mary, olives, and cocoa beans are exceptionally rich sources of dif-
ferent phenolic compounds.
The fresh and dried leaves of rosemary, used frequently in
traditional Mediterranean cuisine contain a number of potentially
biologically active compounds. The antioxidant activity of
rosemary has been attributed mainly to the phenolic diterpenes,
Corresponding author. Tel.: +385 51 651 266; fax: +385 51 651 177.
E-mail address: mbubonja@medri.hr (M. Bubonja-Sonje).
carnosol and carnosic acid (Lo, Liang, Lin-Shiau, Ho, & Lin, 2002).
Beside strong antioxidative properties rosemary extract was
shown to inhibit the growth of different foodborne pathogens
and spoilage moulds (Del Campo, Amiot, & Nguyen-The, 2000).
Also, numerous studies have demonstrated antioxidant activity
of olive oil polyphenols (Servili et al., 2009; Visioli, Poli, & Gall,
2002). Far less research has been carried out on antibacterial prop-
erties of olive oil derived polyphenols. Secoiridoides (oleuropein
and derivatives), one of the major classes of phenolic compounds
contained in olives and olive oil, have been shown to inhibit or de-
lay the rate of growth of pathogenic bacteria (Bisignano et al.,
1999; Furneri, Piperno, Sajia, & Bisignano, 2004). Hydroxytyrosol
(derived from oleuropein by enzymatic hydrolysis and responsible
for the high stability of olive oil) was found to be the most active
compound amongst the various olive polyphenols and is powerful
antioxidants.
Cocoa been, the seed of Theobromina cacao, is known to be rich
in polyphenols, such as the avanol monomers ((+)-catechin and
()-epicatechin) and procyanidin oligomers (B-type procyanidins
that are linked by C4C8 bonds) (Wollgast & Anklam, 2000).
However, there are only few data on the antimicrobial properties
of cocoa polyphenols, for example against oral streptococci (Perci-
val, Devine, Duggal, Chartron, & Marsh, 2006; Smullen, Koutsou,
Foster, Zumbe, & Storey, 2007).
Listeria monocytogenes is a common foodborne pathogen of
increasing public health concern in many countries. Infection of

0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.02.071
1822 M. Bubonja-Sonje et al. / Food Chemistry 127 (2011) 18211827
humans predominantly occurs as a result of occasional contamina-
tion of ready-to-eat and raw food products. The ubiquitous distri-
bution of this pathogen in nature, its ability to proliferate at
refrigeration temperature and its tolerance to certain preservatives
has resulted in an extensive effort to develop processes to control
its growth in foods. Antilisterial activity of different plant polyphe-
nols is previously referred only in a few studies with incoherent re-
sults. This bacterium was found to be moderately susceptible to
cumin extract with high content of caffeic, ferulic and gallic acid
(Ani, Varadaraj, & Naidu, 2006), while another study showed only
low susceptibility to various types of puried polyphenols and
plant extracts (Taguri, Tanaka, & Kouno, 2006). Although some nat-
ural products successfully prolonged rancidity susceptible meat
products storage shelf life they were unable to reduce Listeria
counts in the product (Fernandez-Lopez, Zhi, Aleson-Carbonell,
Perez-Alvarez, & Kuri, 2005).
The main objectives of this work were to evaluate the antimi-
crobial activity of rosemary, olive oil and cocoa phenolic extracts
against Listeria spp., and to compare the antimicrobial activity
and antioxidant capacity of each plant extract and pure phenolic
compound which is an integral part of each extract. The sensitivity
of different antimicrobial assays was also assessed and compared.
2. Materials and methods
2.1. Plant extracts formulations and pure substances
The total phenolic compounds of cocoa (Theobroma cacao) and
olive (Olea europaea L.) oil, commercial rosemary (Rosmarinus of-
cinalis L.) extract, as well as pure polyphenols were used in this
study. Pure cocoa sample from Madagascar was kindly donated.
Commercial monovarietal extra virgin olive oil sample (cultivar
Ascolana tenera) was obtained from the OLEA B.B. d.o.o. (Rabac,
Croatia), and oil-soluble commercial rosemary extract V40 (Vitiva
d.d., Markovci, Slovenia) containing 40% carnosic acid was donated
by Sonja Smole Mozina (Biotechnical Faculty, University of Ljublj-
ana, Slovenia). Methanol, n-hexane, sodium carbonate (Na2CO3),
FolinCiocalteau reagent, 1,10 -diphenyl-1-picrylhydrazyl (DPPH)
and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox) were obtained from SigmaAldrich (Steinheim, Germany).
The following pure phenolics were used: hydroxytyrosol (HT) ob-
tained from Extrasynthese (Genay, France); gallic acid (GA) and
other phenolic standards for HPLC obtained from Fluka (Buchs,
Switzerland); carnosic acid (CA) and ()-epicatechin (EPI) obtained
from SigmaAldrich (Steinheim, Germany).
2.2. Preparation of plant polyphenol extracts
Olive oil polyphenol extract was characterised according to the
method described previously by Caponio, Alloggio, and Gomes
(1999). In brief, the aqueous polyphenol extract was isolated from
30 g virgin olive oil and evaporated in a rotating evaporator to dry-
ness. The cocoa polyphenol extract was prepared as described by
Adamson et al. (1999). In brief, the lipids were extracted from 2 g
of pure cocoa through exhaustive extraction with n-hexane. After
air-drying, sample was extracted threefold with 10 ml of an extrac-
tion solvent composed of acetone, water and acetic acid in a ratio
of 70:29.5:0.5 (v/v/v), respectively, using sonication. The result-
ing slurry was pelleted at 3000g, and then the supernatant was l-
tered through a 0.45-mm nylon lter. Solvent was than evaporated
under reduced pressure to dryness. Dry olive oil and cocoa extracts
residues were weight and dissolved in sterile water in concentra-
tion of 10 mg/ml for antimicrobial activity testing.
Five grams of the commercial rosemary extract formulations
V40 were extracted in 50 ml mixture of methanol:water:acetic
acid (90:9:1, v/v/v) at 24 C for 24 h. After ltration, the ltrate
was evaporated to dryness in a rotating evaporator. The residue
was weighed and dissolved in 96% ethanol at nal concentration
of 10 mg/ml.
The prepared extracts were subjected to high performance li-
quid chromatographic (HPLC) analysis, total phenolic content and
antioxidant capacity determinations. All extracts were prepared
and analysed in triplicates. TSP Spectra System with P2000 gradi-
ent binary pump HPLC, equipped UV/VIS2000 detector Spectra Sys-
tem, SN4000 controller and ChromQuest software was used for the
phenolic compounds analyses.
2.3. Total phenolic compound analysis
The total phenolic contents of extracts were determined accord-
ing to the method described by Swain and Hillis (1959), and mod-
ied against the recommendation of Ainsworth and Gillespie
(2007). Briey, 18 ll of each extract was transferred into a 96-well
microplate (Biotek, USA) and mixed with 36 ll FolinCiocalteu re-
agent (10%) and 145 ll sodium carbonate (700 mmol). The absor-
bance of the mixture was measured at 750 nm after 60 min
incubation in the dark at room temperature.
The total phenolic contents were calculated as a gallic acid
equivalent (GAE) from a calibration curve of GA standard solutions
(ranging from 25 to 800 mg/ml), and expressed as mg of GAE per
gram of dry extracts residue. All measurements were done in
triplicate.
2.4. Antioxidant activity determination (DPPH assay)
The antioxidant activity of the olive oil, rosemary and cocoa pol-
yphenol extracts was tested using a stable radical 2,2-diphenyl-1-
picrylhydrazyl (DPPH). The extracts were added to the DPPH solu-
tion at the nal concentration of 1 mg/ml. After incubation at 37 C
for 30 min, the activity was monitored by a decrease in an absor-
bance at 515 nm in a 96-well microplate. The radical scavenging
activity (RSA) was calculated as a percentage of DPPH scavenging
activity using the equation: %RSA = 100  [1  (AE/AD)], where AE
is the absorbance of the DPPH solution with an extract added,
and AD is the absorbance of the DPPH solution. The absorbance of
the resulting oxidised solution was compared with that of the cal-
ibrated Trolox standard (7.81000 lmol in 80% methanol). Results
are expressed in terms of Trolox equivalent antioxidant capacity
(TEAC) as lmol Trolox per g of dry extracts residue. The extract
concentration providing 50% inhibition (EC50) was calculated from
the graph of RSA percentage against extract concentration (TE
(lmol/l) = 5.012 + 3.3305 DPPH scavenging activity (%),
r = .99923). The higher RSA and TEAC values would imply greater
antioxidant activity of the sample, while a higher EC50 value indi-
cates a weaker capability to scavenge DPPH radicals.
2.5. Bacterial strains and preparation of the standardised bacterial
suspension
The standard laboratory strain of L. monocytogenes EGD (sero-
type 1/2a), four invasive clinical isolates of L. monocytogenes from
our culture collection and avirulent L. innocua strain were cultured
at 37 C for 24 h in MuellerHinton broth (MHB) (Difco, MD, USA).
A density of bacterial inoculum was adjusted to 0.5 MacFarland
turbidity standard and diluted in MHB 1:100. The optical density
of the bacterial suspension was additionally estimated using a
spectrophotometer at 550 nm, and number of bacterial cells was
extrapolated from a standard growth curve. The viable bacterial
count used in experiments was obtained by plating 10-fold dilu-
tions onto blood agar. After incubating plates for 24 h at 37 C,
the number of bacteria was calculated as colony forming units
 M. Bubonja-Sonje et al. / Food Chemistry 127 (2011) 18211827
(CFU)/ml. Starting inoculum for all experiments was approxi-
mately 1.5  106 CFU/ml.
2.6. Determination of antibacterial activity
Antimicrobial effect of olive oil, cocoa and rosemary polyphenol
extracts and pure phenolic compounds (HT, EPI and CA) was deter-
mined using agar-well diffusion, disc-diffusion, and broth dilution
methods. Bactericidal activity of each extract was also assessed by
time-kill curves. Each phenolic compound, as well as total poly-
phenol extracts, except V40, was dissolved in sterile distilled water
to prepare stock solutions. V40 was dissolved in 96% ethanol to
prepare stock solutions at concentration 100 mg/ml. Further, two-
fold serial dilutions in MHB were prepared from stock solutions of
each polyphenols and total polyphenol extracts to give nal con-
centrations ranging from 0.006 to 6.4 mg/ml. Measurement for
each antimicrobial method was repeated three times, and data
were expressed as mean SD.
2.6.1. Agar-well diffusion method
Suspensions of the tested microorganisms were spread onto the
surface of MuellerHinton agar (MHA) plates. The wells of 6 mm
diameter were cut from the agar and lled with 150 ll of polyphe-
nol extract or individual polyphenol (concentrations in the range of
0.2 to 6.4 mg/ml). The inoculated plates were incubated at 37 C for
24 h. Antimicrobial activity was evaluated by measuring the diam-
eter of the growth inhibition zone around the well.
2.6.2. Disk diffusion method
For the disk diffusion assay (Van der Berghe & Vlietinck, 1991),
Petri dishes containing MHA were inoculated with microbial sus-
pensions. Disks of lter paper (Whatman No. 5) of 5 mm diameter
were impregnated with 5 ll of each dilution of extracts or individ-
ual polyphenols (concentrations in the range of 0.26.4 mg/ml)
and placed on the agar surface. Discs of ampicillin (10 lg) were
used as positive controls. The plates were incubated overnight at
37 C and the diameter of any resulting zones of growth inhibition
was measured.
2.6.3. Broth microdilution method
Minimum inhibitory (MIC) and minimum bactericidal concen-
trations (MBC) of the lyophilised rosemary extract V40, olive oil
and cocoa polyphenol extracts, and pure phenolic compounds
(HT, CA and EPI) were determined using a standard microdilution
techniques in MHB according to Clinical and Laboratory Standards
Institute (CLSI) guidelines and repeated three times (CLSI, 2007).
Two control tubes were maintained for each test batch. These in-
cluded antibiotic control (tube containing phenolic and growth
medium without inoculum) and organism control (tube containing
bacterial suspension). MIC values were taken as the lowest concen-
tration of compound (highest dilution) that produced no visible
bacterial growth (no turbidity) when compared with the control
tubes after 24 h of incubation at 37 C. MBC is measured by inocu-
lating the broths used for MIC determinations onto blood agar and
incubated further for 1824 h. MBC was dened as the lowest con-
centration yielding negative subcultures on the solid medium.
Antimicrobial effect was dened as strong for MIC < 0.4 mg/ml,
moderate for 0.40.8 mg/ml, and weak for >0.8 mg/ml as proposed
earlier (Taguri et al., 2006).
2.6.4. Time-kill curve methodology
The antilisterial activity (against EGD strain) of total olive oil
phenolic content, cocoa phenolic content and V40 was studied over
a range of multiples MICs encompassing 0.5 to 8  MIC and incu-
bated at 37 C. Drug-free controls were included. At predetermined
time points (0, 3, 6, 9, 12 h) a 100 ll sample was removed from
1823
each test suspension, 10-fold serially diluted in sterile saline and
plated on blood agar plates for colony count determination. Data
from triplicate runs were averaged and plotted as log CFU/ml ver-
sus time (h) for each time point.
3. Results and discussion
3.1. Comparative analysis of antilisterial activity
L. monocytogenes is still a concern for both consumers and the
food industry despite the use of various food preservation meth-
ods. The preliminary antimicrobial activity screening of olive oil,
cocoa and V40 rosemary polyphenol extracts, and selected pure
phenolic compounds hydroxytyrosol (HT), carnosic acid (CA),
and ()-epicatechin (EPI) against all of the Listeria strains was per-
formed using a paper disk as well as agar-well diffusion methods.
The results of both methods have shown no evidence of antilisteri-
al activities, neither for total polyphenol extracts nor for phenolic
compounds (data not shown). However, the size of the inhibition
zone may be affected by the solubility of the oil, the diffusion range
in agar, the evaporation etc. Considering that the absence of inhi-
bition zones does not necessarily mean that compounds are inac-
tive, a further evaluation of antilisterial activity of different
polyphenols was made through the broth dilution technique that
is known to be a gold standard for determining the susceptibility
of microorganisms to antimicrobials. As shown in Table 1, all
tested extracts and pure compounds possess signicant dose-
dependent antilisterial activity. Whereas there was no signicant
difference in susceptibility amongst the all tested Listeria isolates
against different extracts and pure phenols the results of microdi-
lution are shown together. Total olive oil polyphenol extract
showed strong and HT moderate antilisterial activity. Other olive
oil polyphenols showed lower antilisterial activity than HT or total
polyphenol extract (data not shown). The lowest MICs and MBCs
obtained for total polyphenol extract, in comparison to its main
polyphenol HT, suggests an additive or synergistic effect between
certain phenolic compounds of olive oil. Our results are in concor-
dance with results reported by Medina, de Castro, Romero, and
Brenes (2006) who found simple polyphenol hydroxytyrosol and
complex polyphenol oleuropein to have the most potent antimi-
crobial activity amongst olive oil phenols against different harmful
bacteria of the intestinal microbiota. It has been shown recently
that virgin olive oil in mayonnaise and salad signicantly reduces
the counts of inoculated foodborne pathogens Salmonella Enteriti-
dis and L. monocytogenes (Medina, Romero, Brenes, & De Castro,
2007).
Although Park, Stankiewicz, Rayman, and Hauschild (1979) ob-
served considerable inhibitory effect of 5% cocoa powder on differ-
ent bacteria such as Staphylococcus, Micrococcus, Shigella and
Table 1
Minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of total
polyphenol extracts and pure polyphenols against Listeria spp.
Phenolsa Antibacterial activity (mg/ml)b
MIC MBC
OOPE 0.400 0.096 0.925 0.242
HT 0.600 0.218 1.025 0.371
CPE 1.200 0.438 >6.4
EPI 3.733 1.306 >6.4
V40 0.083 0.023 0.083 0.025
CA 0.166 0.051 0.400 0.309
a
Abbreviations: EPI = epicatechin; HT = hydroxytyrosol; OOPE = olive oil poly-
phenol extract; CPE = cocoa polyphenol extract; CA = carnosic acid; V40 = rosemary
extract.
b
The values represent means standard deviation for all tested Listeria strains.
1824 M. Bubonja-Sonje et al. / Food Chemistry 127 (2011) 18211827

Bacillus, our results (Table 1) show that total cocoa phenolic ex- 12
tract, as well as main cocoa polyphenol, epicatechin, have only
10
weak antilisterial activity (MBC higher than 6.4 mg/ml). However,
trace quantities of certain long-chain fatty acids have long been
8
known to inhibit microorganisms, especially gram positive bacte-

log 10 CFU
ria. Thus, we assume that other components contained in cocoa 6
powder, such as fatty acids and glycerides (monounsaturated,
diunsaturated and fully saturated forms), were likely responsible 4
for antibacterial effects reported in previous study. There are no
data in the literature that address the antilisterial properties of co- 2

coa polyphenols. Moreover, during the routine food monitoring, limit of quantification
0
Kiss, Tirczka, Szita, Bernath, and Csiko (2006) reported isolation
0 3 6 9 12
of L. monocytogenes strains from a total of 10 cocoa samples (9%
Time (h)
of all examined cocoa samples).
The rosemary extract, on the other hand, had the greatest antil- Fig. 1. Time-kill curve of total olive oil polyphenols against Listeria monocytogenes
isterial potential amongst all extracts as well as pure polyphenols EGD. The values represent means standard deviation. Symbols indicate inhibitory
tested in our study. Listeria growth inhibition was achieved at very concentrations as follows; j control (no drug); h 0.2 mg/ml (0.5  MIC); N 0.4
mg/ml (1  MIC); D 0.8 mg/ml (2  MIC); d 1.6 mg/ml (4  MIC); s 3.2 mg/ml
low concentration of V40 extract or carnosic acid (CA) its major
(8  MIC). The limit of quantication is 2 log10 CFU/ml.
bioactive phenolic (MIC at 0.083 mg/ml and 0.166 mg/ml respec-
tively). While the MIC value of V40 was at the same time bacteri-
cidal, CA showed approximately one dilution higher MBC value in
12
comparison to MIC. Since pure CA had a slightly higher MIC in
comparison to V40 we can conclude that minor components of 10
V40 rosemary extract also have a part in antilisterial activity pro-
ducing an additive or synergistic effect. The relationship between 8
log 10 CFU

antimicrobial activity and chemical composition of different rose- 6

mary extracts has been demonstrated previously. Authors showed
a strong antimicrobial activity of oil soluble rosemary extracts, 4
which contain CA as the main phenolic component, against gram
2
positive bacteria Bacillus cereus and Staphylococcus aureus (Klanc-
limit of quantification
nik, Guzej, Hadolin-Kolar, Abramovic, & Mozina, 2009). Similarly, 0
rosemary extract containing 30% of CA had the best antimicrobial 0 3 6 9 12
efcacy of all tested rosemary extracts against different bacteria Time (h)
and yeasts (Moreno, Scheyer, Romano, & Vojnov, 2006). Moreover,
Pandit and Shelef (1994) demonstrated inhibitory effect of differ- Fig. 2. Time-kill curve of total cocoa polyphenols against Listeria monocytogenes
EGD. The values represent means standard deviation. Symbols indicate inhibitory
ent rosemary extracts against L. monocytogenes in a pork liver sau- concentrations as follows; j control (no drug); N 0.8 mg/ml (0.5  MIC); D 1.6 mg/
sage. Although Fernandez-Lopez et al. (2005) proved in vitro ml (1  MIC); h 3.2 mg/ml (2  MIC); d 6.4 mg/ml (4  MIC). The limit of
antilisterial activity of different rosemary extracts; in the same quantication is 2 log10 CFU/ml.
study, antilisterial activity of extracts has not been manifested in
meat product (cooked meatballs). Authors explain this discrepancy
by the dilution of the rosemary extracts necessary for its use in 12
meat products, and possibility of reduction of the effectiveness of
the antimicrobial extract due to physical interactions with the food 10

matrix.
8
log 10 CFU

Our results of antilisterial activity of polyphenol extracts ob-

tained by dilution method were conrmed by the time-kill curves. 6
Total olive oil polyphenol extract inhibited bacterial growth at con-
4
centration of 0.4 mg/ml (1  MIC) while concentrations above
0.8 mg/ml (2  MIC) exhibited bactericidal effect after few hours 2
(Fig. 1). As shown in Fig. 2, the kinetics of inactivation monitored limit of quantification
over 12 h conrmed weak activity of cocoa polyphenol extract 0
against L. monocytogenes EGD strain. Concentration of 6.4 mg/ml 0 3 6 9 12

(4  MIC) of cocoa extract was not sufcient to achieve bactericidal
effect after 12 h. On the other hand, quick inactivation of bacteria
was achieved (within rst 3 h) using V40 at a very low concentra-
tion (Fig. 3).
Although the antimicrobial effects of various phenolic extracts
are well documented, the mechanisms of action of these products
and their components are not fully understood. Polyphenols are
thought to cause surface activity that damages the membranes of
bacterial cells, to inhibit its enzymes, or to interfere with the pro-
duction of certain amino acids necessary for the bacterial growth.
According to Bisignano et al. (1999) antibacterial activity of olive
oils phenolic compounds is due to the presence of the ortho
diphenolic system (catechol). Therefore, the position and number
of hydroxyl groups on the phenol group are thought to be related
Time (h)
Fig. 3. Time-kill curve of rosemary polyphenol extract against Listeria monocytog-
enes EGD. The values represent means standard deviation. Symbols indicate
inhibitory concentrations as follows; j control (no drug); h 0.025 mg/ml
(0.5  MIC); N0.05 mg/ml (1  MIC). The limit of quantication is 2 log10 CFU/ml.
to their relative toxicity to microorganisms, with evidence that
increasing hydroxylation results in an increase of antimicrobial
activity (Marjorie, 1999). Perez-Fons, Aranda, Guillen, Villalain
and Micol (2006) indicated that rosemary polyphenols may affect
membrane uidity. However, further studies are needed to inves-
tigate the action mechanisms of CA, and other polyphenols tested
in this study against listerial growth.
 M. Bubonja-Sonje et al. / Food Chemistry 127 (2011) 18211827
3.2. Antioxidant activity
The antioxidant potential of phenolic compounds has been the
subject of considerable interest, both because of its chemoprotec-
tive effect against degenerative diseases such as cardiovascular
and some neurological disease, and due to inhibition of lipid oxida-
tion of food. In present study phenolic extracts and their main phe-
nolic compounds were selected for further assessment of the
antioxidant potential after HPLC analysis of each sample.
Table 2 summarises the contents of total phenolics in examined
samples, expressed as GAE/g of dry extract residues. The amount of
total phenolics in extracts varied widely and ranged from 0.279 to
450 mg/g. Furthermore, considerable variation was found in phe-
nolic composition of samples. Phenolic alcohols HT and tyrosol
are found to be the predominant simple phenolic compounds in ol-
ive oil dry extract (0,0424 mg/g and 0,0408 mg/g; respectively) fol-
lowed by caffeic acid (0005 mg/g), 3,4-dihydroxyphenylacetic acid
(0.004 mg/g), and 4-hydroxyphenylacetic acid (0.002 mg/g). Major
secoiridoids in olive oil extract were demethyl-oleuropein
(0.0603 mg/g), demethyl ligstroside (0.062 mg/g), oleuropein agly-
con (0.011 mg/g) and ligstroside aglycon (0.0063 mg/g), while lign-
ans were detected in concentration of 0.035 mg/g dry extract.
EPI was predominant compound in cocoa powder (1.2 mg/g dry
extract) followed by caffeic acid derivative (0475 mg/g), ()-epigal-
locatechin (0.42 mg/g), ()-gallocatechin (0.19 mg/g), and (+)-cate-
chin (0.10 mg/g). Cocoa powder dry extract contained 0.249 mg/g
procyanidin B1 and 0068 mg/g procyanidin B2.
CA was found in commercial V40 rosemary extract in much lar-
ger quantity than others phenolics (403 mg/g per dry matter), car-
nosol was detected in traces while rosmarinic acid was not present.
HT was selected from olive oil extract, EPI from cocoa extract and
CA from rosemary extract for assessment of antioxidant activity.
Phenolic extracts and selected phenolic compounds exhibited
appreciable scavenging activity as is evident from the results ob-
tained by DPPH test. Table 2 shows the antioxidant activity of phe-
nolic extracts at a concentration of 1 mg/ml expressed as RSA
percentage, Trolox equivalent antioxidant capacity and EC50 val-
ues. Antioxidant activity of tested extracts was in decreasing order,
as follows: V40 > CPE > OOPE. Similarly, antioxidant activity of
their pure phenolics tested in equal concentration was: CA >
EPI > HT (Table 3). As can be seen, polyphenol extracts possess
stronger antioxidant activity than their respective major com-
pounds suggesting synergistic or additive effect of the multiple
compounds compared to single puried active ingredients. The ex-
tracts and pure phenolic compounds also demonstrated a signi-
cant dose dependent antioxidant activity (data not shown).
DPPH radical scavenging assay is widely used to test the ability
of compounds to act as free radical scavengers or hydrogen donors,
and to evaluate antioxidant activity of foods. Our observations are
in line with data from other studies based on the DPPH assay. Thus,
Table 2
The total phenolic content and antioxidant activity determined by DPPH method in
different samples.
Polyphenol Total Antioxidant activityb
extracta phenolic
RSAa TEa EC50a
content
(%) (lmol/g) (mg/ml)
(mg/g)
OOPE 0.279 69.66 0.27 0.08 0.0331 0.0008
CPE 32.48 78.34 0.30 0.10 0.0318 0.0008
V40 450 81.60 0.31 0.05 0.0282 0.0030
a
Abbreviations: CPE = cocoa polyphenol extract; EC50 = concentration of phenolic
in reaction mixture that causes a 50% decrease in the initial 2,20 -diphenyl-1-pic-
rylhydrazyl (DPPH) concentration; HT = hydroxytyrosol; OOPE = olive oil polyphe-
nol extract; RSA = radical-scavenging activity; TE = trolox equivalent;
V40 = rosemary extract.
b
The values represent means standard deviation.
1825
Table 3
The antioxidant activity of main olive oil, cocoa and rosemary phenols determined by
DPPH method.
Phenolsa Concentration Antioxidant activityb
(mg/ml)
RSAa (%) TEa EC50a
(lmol/g) (mg/ml)
HT 1 56.50 0.2180 0.005 0.0376 0.0092
EPI 1 69.31 0.2688 0.009 0.0336 0.0041
CA 1 77.30 0.3005 0.010 0.0273 0.0052
a
Abbreviations: CA = carnosic acid; EPI = epicatechin; EC50 = concentration of
phenolic in reaction mixture that causes a 50% decrease in the initial 2,20 -diphenyl-
1-picrylhydrazyl (DPPH) concentration; HT = hydroxytyrosol; RSA = radical-scav-
enging activity; TE = trolox equivalent.
b
The values represent means standard deviation.
Gomez-Alonso, Fregapane, Salvador, and Gordon (2003) reported
that antioxidant activity of the olive oil phenolic extract is
0.25 lmol/g (expressed in TE), while Lesage-Meessen et al.
(2001) reported values 2.615.27 mg/l (expressed in EC50). Our re-
sults showed that hydroxytyrosol, one of the major compounds
occurring in the olive oil, has appreciable antioxidant power com-
parable to olive oil phenolic extract. Using an oxygen radical anti-
oxidant capacity assay (ORAC), authors found that hydroxytyrosol
has the greatest antioxidant capacity amongst all olive oil polyphe-
nols (Zullo & Ciafardini, 2008).
Apart from DPPH assay used in this study, several methods can
be applied for determination of antioxidant effect, like the ferric
reducing antioxidant power (FRAP) assay, the 2,20 -azinobis (3-eth-
ylbenzothiazoline-6-sulphonic acid) (ABTS) radical cation assay
etc. Selection of method depends on type of samples, pH, hydro-
philic and hydrophobic properties, and application of different
methods can give different values resulting in different ranking
of the same food. Thereby, Gu, House, Wu, Ou, and Prior (2006)
using an ethanolic extraction and ORAC assay reported values of
826 103 lmol TE/g for cocoa powder, while Jona-Essien, West,
Alderson, and Tucker (2008) reported considerable less values ran-
ged from 12.4 to 45.5 lmol TE/g for traditional and hybrid cocoa
beans (methanolic extraction and the FRAP assay). Nevertheless,
using ABTS assay, researchers showed that cocoa has more pheno-
lic phytochemicals (611 mg of gallic equivalents/serving), and a
higher antioxidant capacity (1128 mg/serving) than teas and red
wine (Lee, Kim, Lee, & Lee, 2003). The results presented in this
study are in agreement with previously reported data, but they
cannot be directly compared due to differences in the applied
extraction solvent and presentation of the results. Additionally,
some literature data mentioned above are expressed per gram of
cocoa bean/powder, while our research results are given as mg
per gram of defatted cocoa extract, which also contributes to minor
discrepancies between the results. Although three groups of poly-
phenols in cocoa can be distinguished (catechins, antohocyanins
and proanthocyanidins), it has recently been proposed that antiox-
idant capacity of cocoa correlated to a high degree with the content
of EPI, its main catechin (Miller et al., 2009). The present study pro-
vides additional data in support of potent antioxidant capacity of
EPI, the major compound found in cocoa polyphenol extract.
Strong antioxidant activity of rosemary extract and its main
compounds CA, reported here is in line with previous observations.
Thus, Wellwood and Cole (2004) reported that free radical scav-
enging activity of different rosemary extracts correlated with CA
concentrations, as measured by the DPHH assay. The reducing
power of the rosemary polyphenol extracts based on this assay is
supported with the results obtained by other assays. It has been
shown, using Rancimat assay, that commercial rosemary extract
added in chicken frankfurters has higher antioxidant activity than
commercial food preservative (Riznar et al., 2006). Furthermore,
the use of rosemary extracts has proved to be effective as an
1826 M. Bubonja-Sonje et al. / Food Chemistry 127 (2011) 18211827
antioxidant in cooked swedish-style meatballs (Fernandez-Lopez
et al., 2005).
Antioxidants can bind metals, scavenge species that initiate or
perpetuate oxidation, quench high energy oxygen species prevent-
ing formation of peroxides, or decompose lipid peroxides. Phenolic
compounds can act as antioxidants in various ways, phenolic diter-
pen CA acts as potent metal chelator and free radical scavenger. In
addition to its ability to scavenge peroxyl and hydroxyl radicals,
compounds produced in sites of inammation that may damage
biological membranes, CA effectively inhibits mitochondrial and
microsomal lipid peroxidation (Aruoma, Halliwell, Aeschbach, &
Loligers, 1992). It is known that the antioxidant efciency of a phe-
nolic compound is very dependent on structural factors such as
number and position of hydroxyl groups in the molecule. The anti-
oxidant activity of CA is thought to be due to the cooperation of its
ortho phenolic group with its isopropyl groups. Masuda, Inaba, and
Takeda (2001) proposed that antioxidant mechanism of CA con-
sists of the oxidative coupling reaction with the peroxyl radical
at the 12- or 14-position, and subsequent degradation reactions.
Similarly, major dietary avan-3-ol, EPI exerts its antioxidant
actions through different mechanisms. EPI inhibits lipid peroxida-
tion and protein tyrosine nitration of low-density lipoprotein (LDL)
by myeloperoxidase/H2O2/nitrite, and by Cu2+ system. In addition,
EPI protects against cytotoxic and proapoptotic actions of oxyster-
ols toward endothelial cells (Steffen, Wiswedel, Peter, Schewe, &
Sies, 2006). The antioxidant properties of compounds with a cate-
chol group, such as HT found in olive oil, are associated with their
ability to stabilise free radicals through the formation of intramo-
lecular hydrogen bonds (Visioli et al., 2002).
4. Conclusions
Natural products from plants are nding increased use as anti-
microbials and antioxidants in foods. We determined and com-
pared antilisterial activity of selected polyphenol extracts using
different methods. Discrepancies between results obtained by agar
diffusion and broth dilution techniques for all tested extracts and
phenolics suggest that agar diffusion assays cannot be used to
determine the inhibition spectrum of polyphenols and that dilu-
tion method and time-kill curve studies give a more accurate indi-
cation for the true potential of the bioactive compounds.
Extravirgin olive oil, cocoa and rosemary possess varying degree
of antilisterial and antioxidant activities. The rosemary extract,
applicable as food additive, was the most effective antimicrobial
and antioxidative agent in this study.
We might assume that the most abundant polyphenols are
mainly responsible for shown antilisterial and antioxidant effects
of tested extracts. However, each phenolic extract contained differ-
ent types and amounts of phenolic compounds. Moreover, main
polyphenols were presented in different concentrations in their ex-
tracts; while CA constitutes substantial part of rosemary extract,
HT and EPI were found in considerably less concentration in re-
lated extracts. So we can conclude that the antibacterial as well
as antioxidant activity of tested extracts (especially cocoa and olive
oil extract) could be partly attributed to other minor constituents.
In other words, a positive additive and/or synergistic effect could
exist between other phenolic compounds present in extracts.
Acknowledgements
This research was supported by grants from Croatian Ministry
of Science, Education and Sports No. 062-0621273-1235 to MA
and No. 062-0000000-0221 to JG. The authors thank Sonja Smole
Mozina (Biotechnical Faculty, University of Ljubljana, Slovenia)
for donating VivOX40 rosemary extract used in this study.
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