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Hazel (Corylus avellana L.) leaves as source of antimicrobial
and antioxidative compounds
Ivo Oliveira
a
, Anabela Sousa
a
, Patrı
´cia Valenta
˜o
b
, Paula B. Andrade
b
,
Isabel C.F.R. Ferreira
a
, Federico Ferreres
c
, Albino Bento
a
, Rosa Seabra
b
,
Letı
´cia Estevinho
a
, Jose
´Alberto Pereira
a,*
a
CIMO/Escola Superior Agra
´ria, Instituto Polite
´cnico de Bragancßa, Campus Sta Apolo
´nia, Apartado 1 172, 5301-855 Bragancßa, Portugal
b
REQUIMTE/Servicßo de Farmacognosia, Faculdade de Farma
´cia da Universidade do Porto, Rua Anı
´bal Cunha, 164, 4099-030 Porto, Portugal
c
Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 164,
30100 Campus Univ. Espinardo, Murcia, Spain
Received 14 November 2006; received in revised form 21 February 2007; accepted 29 April 2007
Abstract
Aqueous extracts of leaves of different hazel (Corylus avellana L.) cultivars (Cv. M. Bollwiller, Fertille de Coutard and Daviana), were
analysed by reversed-phase HPLC/DAD for the definition of their phenolic composition. Antioxidant potential was assessed by the
reducing power assay, and the scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and b-carotene linoleate model sys-
tem. Their antimicrobial capacity was also tested against Gram positive (Bacillus cereus,Bacillus subtilis,Staphylococcus aureus) and
Gram negative bacteria (Pseudomonas aeruginosa,Escherichia coli,Klebsiella pneumoniae) and fungi (Candida albicans,Cryptococcus neo-
formans). Eight phenolic compounds were identified: 3-, 4- and 5-caffeoylquinic acids, caffeoyltartaric acid, p-coumaroyltartaric acid,
myricetin-rhamnoside, quercetin 3-rhamnoside and kaempferol 3-rhamnoside. A p-coumaric acid, three myricetin and one quercetin
derivatives were also detected. The hazel leaves extract presented high antioxidant activity in a concentration-dependent way, in general
with similar behaviour of all cultivars. Gram positive bacteria revealed to be very sensitive to hazel leaf extract (MIC 0.1 mg/ml for
B. cereus and S. aureus and 1 mg/ml for B. subtilis). However, Gram negative and the fungi displayed much lower sensitivity, being
P. aeruginosa and C. albicans resistant at 100 mg/ml. Cv. M. Bollwiller exhibited the most potent antimicrobial activity.
Ó2007 Elsevier Ltd. All rights reserved.
Keywords: Hazel leaves; Phenolics; Antioxidant potential; Antimicrobial activity
1. Introduction
Although a number of Corylus species are found
throughout the world, C. avellana and its hybrids, are the
most important as regards nut production. Hazel is a tree
or bush which may grow to 6 m high, exhibiting deciduous
leaves that are rounded, 6–12 cm long and across, softly
hairy on both surfaces, and with a double-serrate margin.
It grows wild in Europe, and western Asia (Vaughan &
Geissler, 1997). Despite its wide cultivation for nuts collec-
tion, hazel leaves are also largely consumed as an infusion.
They are used in folk medicine for the treatment of haem-
orrhoids, varicose veins, phlebitis and lower members’
oedema, as consequence of its astringency, vasoprotective
and anti-oedema properties (Valnet, 1992).
Nowadays there is considerable evidence that the anti-
oxidants contained in fruits, vegetables and beverages play
an important role in the maintenance of health and in the
prevention of disease. In fact, plant-derived products con-
tain a wide range of phytochemicals, namely phenolic com-
pounds, with antioxidant capacity, providing protection
against the harmful effects resultant of oxidative stress
(Pereira et al., 2006; Proestos, Chorianopoulos, Nychas,
0308-8146/$ - see front matter Ó2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2007.04.059
*
Corresponding author. Tel.: +351 273 303277; fax: +351 273 325405.
E-mail address: jpereira@ipb.pt (J.A. Pereira).
www.elsevier.com/locate/foodchem
Food Chemistry 105 (2007) 1018–1025
Food
Chemistry
& Komaitis, 2005; Seabra et al., 2006). Furthermore, there
is growing interest in using natural antimicrobial com-
pounds because of consumer pressure on the food industry
to avoid chemical preservatives and the increasing resis-
tance to antibiotics. With this regard the antimicrobial
capacity of phenolic compounds has also been reported
(Pereira et al., 2006; Proestos et al., 2005; Puupponen-
Pimia
¨et al., 2001; Rauha et al., 2000; Zhu, Zhang, & Lo,
2004).
Previous studies on hazel leaves concerned the measure-
ment of the bidirectional reflectance distribution function
(Bousquet, Lache
´rade, Jacquemoud, & Moya, 2005) and
the determination of organochlorine pesticides (Barriada-
Pereira et al., 2004) hormone contents (Andre
´s, Ferna
´ndez,
Rodrı
´guez, & Rodrı
´guez, 2002), nitrate accumulation
(Stams & Lutke Schipholt, 1990), polycyclic aromatic
hydrocarbons (Howsam, Jones, & Ineson, 2000; Howsam,
Jones, & Ineson, 2001) and free polyamines (Rey, Dı
´az-
Sala, & Rodrı
´guez, 1998). In addition, the phenolic compo-
sition has already been described by our (Amaral et al.,
2005) and another research group (Fraisse, Carnat, Carnat,
& Lamaison, 1999) but, as far as we know, nothing has
been reported about their antioxidant and antimicrobial
potential.
The aim of this work was to determine the phenolics com-
position of the leaves of three hazel cultivars (Cv. M. Bollw-
iller, Fertille de Coutard and Daviana) grown in Portugal,
and to assess their antimicrobial and antioxidant abilities.
The phenolic compounds were identified and quantified by
HPLC/DAD. The antimicrobial activity was evaluated
against different microorganisms, namely Gram positive
(Bacillus cereus,Bacillus subtilis and Staphylococcus aureus)
and Gram negative (Pseudomonas aeruginosa,Escherichia
coli and Klebsiella pneumoniae) bacteria and fungi (Candida
albicans and Cryptococcus neoformans). The antioxidant
potential was tested in three distinct assays: reducing power,
scavenging of DPPH radicals and b-carotene bleaching.
2. Materials and methods
2.1. Samples
Hazel leaves were obtained from three Corylus avellana
L. cultivars: M. Bollwiller, Fertille de Coutard and Davi-
ana, and were collected on 3rd July 2006 in Bragancßa,
northeast of Portugal (6°460W, 41°490N, 670 m a.s.l.).
The orchard has a planting density of 3.5 7 m. The trees
were ten years old, and had been pruned when necessary.
No phytosanitary treatments were applied. The leaves were
collected from the middle third of branches exposed to sun-
light, put in plastic bags and immediately frozen at 20 °C.
The plant material was then freeze dried.
2.2. Extract preparation
For each cultivar, three powdered sub samples (5g;20
mesh) were extracted with 250 ml of boiling water for
45 min and filtered through Whatman no. 4 paper. The
aqueous extract was frozen, lyophilized and redissolved
in water at concentrations of 100 mg/ml and 10 mg/ml
for antimicrobial and antioxidant activities assays,
respectively.
2.3. Phenolic compounds analysis
2.3.1. Standards and reagents
The standards used were from Sigma (St. Louis, MO,
USA) or Extrasynthe
`se (Genay, France). Methanol and
formic acid were obtained from Merck (Darmstadt, Ger-
many). The water was treated in a Milli-Q water purifica-
tion system (Millipore, Bedford, MA, USA) before use.
2.3.2. HPLC-DAD analytical conditions
Chromatographic separation was achieved as previ-
ously reported (Amaral et al., 2005) with an analytical
HPLC unit (Gilson), using a reversed-phase Spherisorb
ODS2 (250 4.6 mm, 5 lm particle size, Merck, Darms-
tadt, Germany) column. The solvent system used was a
gradient of water/formic acid (19:1) (A) and methanol
(B), starting with 15% methanol and installing a gradient
to obtain 30%B at 15 min, 45%B at 30 min, 52.5%B at
40 min and 100%B at 42 min. The flow rate was
1 ml min
1
, and the injection volume was 20 ll. Detection
was accomplished with a diode array detector (DAD) (Gil-
son), and chromatograms were recorded at 320 and
350 nm. Spectral data from all peaks were accumulated
in the 200–400 nm range. Data were processed on Uni-
point system software (Gilson Medical Electronics, Villiers
le Bel, France).
Phenolic compounds quantification was achieved by the
absorbance recorded in the chromatograms relative to
external standards, with detection at 320 nm for phenolic
acids and at 350 nm for flavonoids. 3- and 4-Caffeoylquinic
acids were quantified as 5-caffeoylquinic acid, caffeoyltar-
taric acid as caffeic acid, p-coumaric acid derivative and
p-coumaroyltartaric acid as p-coumaric acid, myricetin 3-
hexoside + myricetin derivative as myricetin-rhamnoside,
quercetin 3-hexoside + myricetin derivative as quercetin
3-rhamnoside and kaempferol 3-rhamnoside as kaempferol
3-glucoside. The other compounds were quantified as
themselves.
2.4. Antioxidant activity
2.4.1. Reagents
BHA (2-tert-butyl-4-methoxyphenol), TBHQ (tert-butyl
hydroquinone) and a-tocopherol were purchased from
Sigma (St. Louis, MO, USA). 2,2-Diphenyl-1-picryl-
hydrazyl (DPPH) was obtained from Alfa Aesar. All other
chemicals were obtained from Sigma Chemical Co. (St.
Louis, USA). Methanol was obtained from Pronalab
(Lisboa, Portugal). Water was treated in a Mili-Q
water purification system (TGI Pure Water Systems,
USA).
I. Oliveira et al. / Food Chemistry 105 (2007) 1018–1025 1019
2.4.2. Reducing power assay
The reducing power was determined according to a
described procedure (Oyaizu, 1986). Various concentra-
tions of sample extracts (2.5 ml) were mixed with 2.5 ml
of 200 mmol/l sodium phosphate buffer (pH 6.6) and
2.5 ml of 1% potassium ferricyanide. The mixture was incu-
bated at 50 °C for 20 min. After incubation 2.5 ml of 10%
tricloroacetic acid (w/v) were added and then the mixture
was centrifuged at 1000 rpm in a refrigerated centrifuge
(Centorion K24OR-2003), for 8 min. The upper layer
(5 ml) was mixed with 5 ml of deionised water and 1 ml
of 0.1% of ferric chloride, and the absorbance was mea-
sured spectrophotometrically at 700 nm. The extract con-
centration providing 0.5 of absorbance (EC
50
) was
calculated from the graph of absorbance registered at
700 nm against the correspondent extract concentration.
BHA and a-tocopherol were used as reference compounds.
2.4.3. Scavenging effect assay
The capacity to scavenge the 2,2-diphenyl-1-picryl-
hydrazyl (DPPH) free radical was monitored according
to a method reported before (Hatano, Kagawa, Yasuhara,
& Okuda, 1988). Various concentrations of sample extracts
(0.3 ml) were mixed with 2.7 ml of methanolic solution
containing DPPH radicals (6 10
5
mol/l). The mixture
was shaken vigorously and left to stand in the dark until
stable absorption values were obtained. The reduction of
the DPPH radical was measured by monitoring continu-
ously the decrease of absorption at 517 nm. DPPH scav-
enging effect was calculated as percentage of DPPH
discolouration using the equation: % scavenging effect =
[(A
DPPH
A
S
)/A
DPPH
]100, where A
S
is the absorbance
of the solution when the sample extract has been added
at a particular level and A
DPPH
is the absorbance of the
DPPH solution. The extract concentration providing 50%
inhibition (EC
50
) was calculated from the graph of scav-
enging effect percentage against extract concentration.
BHA and a-tocopherol were used as reference compounds.
2.4.4. b-Carotene linoleate model system
The antioxidant activity of hazel leaf extracts was eval-
uated according to a described procedure (Mi-Yae, Tae-
Hun, & Nak-Ju, 2003). b-Carotene solution was prepared
by dissolving 2 mg of b-carotene in 10 ml of chloroform.
Two millilitres of this solution were placed in a 100 ml
round-bottom flask. After chloroform removal, at 40 °C
under vacuum, 40 mg of linoleic acid, 400 mg of Tween
80 emulsifier, and 100 ml of distilled water were added to
the flask under vigorous shaking. Aliquots (4.8 ml) of this
emulsion were transferred into different test tubes contain-
ing 0.2 ml of different concentrations of hazel leaf extracts.
The tubes were shaken and incubated at 50 °C in a water
bath. As soon as the emulsion was added to each tube,
the zero time absorbance at 470 nm was measured. Absor-
bance readings were then recorded until the control sample
had changed colour. A blank assay, devoid of b-carotene,
was prepared for background subtraction. Antioxidant
activity was calculated using the following equation: Anti-
oxidant activity = (b-carotene content after 2 h of assay/
initial b-carotene content) 100. The assays were carried
out in triplicate and the results were expressed as mean val-
ues ± standard deviations. The extract concentration pro-
viding 50% antioxidant activity (EC
50
) was calculated
from the graph of antioxidant percentage against extract
concentration. TBHQ was used as reference compound.
2.5. Antimicrobial activity
2.5.1. Reagents
Ampicillin and cycloheximide were of the highest avail-
able quality, and purchased from Merck (Darmstadt, Ger-
many). Water was treated in a Milli-Q water purification
system (TGI Pure Water Systems, USA).
2.5.2. Microorganisms and culture conditions
Microorganisms CECT were obtained from the Spanish
type culture collection (CECT) of Valencia University,
while microorganisms ESA were clinically isolated strains
identified in Microbiology Laboratory of Escola Superior
Agra
´ria de Bragancßa. Gram + (B. cereus CECT 148, B.
subtilis CECT 498 and S. aureus ESA 7 isolated from
pus) and Gram – (E. coli CECT 101, P. aeruginosa CECT
108 and K. pneumoniae ESA 8 isolated from urine) bacte-
ria, and fungi (C. albicans CECT 1394 and C. neofor-
mansESA 3 isolated from vaginal fluid) were used to
screen antimicrobial activity of the three hazel leaves culti-
vars. Microorganisms were cultured aerobically at 37 °C
(Scientific 222 oven model, 2003) in nutrient agar medium
for bacteria, and at 30 °C (Scientific 222 oven model, 2003)
in sabouraud dextrose agar medium for fungi.
2.5.3. Test assays for antimicrobial activity
The screening of antibacterial activities against Gram +
and Gram-bacteria and fungi and the determination of the
minimal inhibitory concentration (MIC) were achieved by
an adaptation of the agar streak dilution method based
on radial diffusion (Hawkey & Lewis, 1994; Ferreira,
Calhelha, Estevinho, & Queiroz, 2004; Sousa et al.,
2006). Suspensions of the microorganism were prepared
to contain approximately 10
8
cfu/ml, and the plates con-
taining agar medium were inoculated (100 ll; spread on
the surface). Each sample (50 ll) was placed in a hole
(3 mm depth, 4 mm diameter) made in the centre of the
agar. Under the same conditions, different solutions of
ampicillin (antibacterial) and cycloheximide (antifungal)
were used as standards. The assays with the standards were
carried out using DMSO solutions, which was chosen as
the best solvent. After comparative toxicity assays this sol-
vent was shown to be non-toxic. The MIC was considered
to be the lowest concentration of the tested sample able to
inhibit the growth of bacteria or fungi, after 24 h. The
diameters of the inhibition zones corresponding to the
MICs were measured using a ruler, with an accuracy of
0.5 mm. Each inhibition zone diameter was measured three
1020 I. Oliveira et al. / Food Chemistry 105 (2007) 1018–1025
times (three different plates) and the average was consid-
ered. A control using only inoculation was also carried out.
3. Results and discussion
3.1. Phenolic compounds in hazel leaves
The HPLC-DAD analysis of hazel leaves aqueous
extracts (Fig. 1) revealed the presence of several hydroxy-
cinnamic acid and flavonoid derivatives. The three
analysed cultivars exhibited a common qualitative compo-
sition, in which eight phenolic compounds were identified:
3-, 4- and 5-caffeoylquinic acids, caffeoyltartaric acid,
p-coumaroyltartaric acid, myricetin-rhamnoside, quercetin
3-rhamnoside and kaempferol 3-rhamnoside (Fig. 2). In
addition, another p-coumaric acid, three myricetin and
one quercetin derivative were also detected (Fig. 1). All
these compounds were previously reported to occur in
hazel leaves (Amaral et al., 2005), with the exceptions of
4-caffeoylquinic acid and of the unidentified p-coumaric
acid derivative.
The quantification of the phenolics present in the differ-
ent aqueous extracts revealed a high amount of these com-
pounds, ranging from ca. 38 to 44 g/kg, dry basis (Table 1),
which are considerably higher than the values found before
for methanolic extracts of the same and other hazel culti-
vars (Amaral et al., 2005). As observed before, flavonoids
were always the major compounds, varying between 71%
and 80% of total phenolics (Table 1). Cv. M. Bollwiller
showed the highest content of phenolic compounds (Table
1).
All samples exhibited a similar phenolic profile, in which
myricetin-rhamnoside was the major compound, represent-
ing ca. 62.2% of total phenolics (Fig. 3). However, in what
concerns phenolic acids, some quantitative differences were
noticed: in Fertille Coutard leaves the main phenolic acid is
3-caffeoylquinic acid, while in M. Bollwiller and Daviana
cultivars 5-caffeoylquinic and caffeoyltartaric acids are the
major phenolic acid, respectively. Thus, it seems that the
nature of the cultivar influences the phenolic acid composi-
tion. The pair quercetin 3-hexoside plus myricetin deriva-
tive presented the smallest content in all cultivars,
corresponding to ca. 0.9% of total phenolics (Fig. 3).
When comparing the results with those previously
obtained for Fertille Coutard and M. Bollwiller cultivars
(Amaral et al., 2005) it could be noticed that the increase
R
1
R
2
8OH OH
10 H OH
11 H H
O
OH
OH
OH
CH
3
O
O
O
OH
OH
R
OH
R
1
2
R
4OH
6H
OH
RO
O
OH
O
O
OH
OH
O
O
OH
OH
OH
OH COOH
OH
O
O
OH
OH
OH
OH COOH
OH
OH OH
O
OH COOH
O
OH
OH
13
2
Fig. 1. Chemical structures of identified phenolic compounds from hazel leaves. (1) 3-Caffeoylquinic acid; (2) 4-caffeoylquinic acid; (3) 5-caffeoylquinic
acid; (4) caffeoyltartaric acid; (6) p-coumaroyltartaric acid; (8) myricetin 3-rhamnoside; (10) quercetin 3-rhamnoside; (11) kaempferol 3-rhamnoside.
I. Oliveira et al. / Food Chemistry 105 (2007) 1018–1025 1021
of the total phenolics content is mainly due to an increase
of phenolic acid derivatives contents. This could be attrib-
uted to the environmental factors that allowed an increased
production of these compounds, since samples’ geographi-
cal origin is different from that of the previous work. Nev-
ertheless, this rise could also be partially explained by the
drying process of the samples: in this work the leaves were
dried by lyophilisation, which is faster and less drastic than
the use of a ventilated stove at 30 °C reported before (Ama-
ral et al., 2005), that allows enzymatic reactions, with
possible alteration and loss of compounds. In addition,
3-caffeoylquinic and caffeoyltartaric acids have now been
detected in Fertille Coutard and M. Bollwiller cultivars,
respectively.
3.2. Antioxidant activity
Although several publications dealing with antioxidant
activity of hazelnut kernel (Alasalvar, Karamaca
¨, Amar-
owicz, & Shahidi, 2006; Duraka et al., 1999; Krings & Ber-
ger, 2001; Moure et al., 2001; Sivakumar & Bacchetta,
2005) and green leafy cover (Alasalvar et al., 2006) have
appeared, no such information is available about antioxi-
dant properties of their leaves. Recently, Sivakumar and
Bacchetta (2005) reported the determination of natural
vitamin E, a potent antioxidant, from Italian hazel leaves
but did not present antioxidant activity studies. In the pres-
ent study, the antioxidant potential of hazel leaves samples
was measured by different biochemical assays: reducing
power, scavenging activity on DPPH radicals and lipid per-
oxidation inhibition by the b-carotene linoleate system.
From the analysis of Fig. 4, we can conclude that the
reducing power of the extracts increased with increasing
concentration and were excellent, presenting high reducing
powers at very low concentrations (<1 mg/ml), and being
even more potent than BHA (A
700
= 0.12 at 3.6 mg/ml)
and a-tocopherol (A
700
= 0.13 at 8.6 mg/ml) standards.
The reducing power of the different cultivars was very sim-
ilar and followed the order Fertille Coutard > Daviana
M. Bollwiller.
The scavenging effect of hazel leaves extracts on DPPH
radicals also increased with concentration, specially for
Table 1
Phenolic compounds in hazel leaf samples (mg/kg, dry basis)
a
Sample Compound
b
Total
12345678 91011
M. Bollwiller 1241.0 1396.8 3466.1 2939.7 443.5 1551.9 1105.3 26655.9 356.9 3967.0 867.8 43991.9
(10.1) (5.4) (50.6) (41.7) (0.5) (14.5) (23.5) (76.4) (9.6) (27.6) (17.1)
Fertille Coutard 2438.7 1825.3 1442.6 2031.4 741.0 2355.3 642.7 22629.9 303.4 2760.3 545.0 37715.6
(12.5) (13.3) (0.7) (21.8) (0.1) (30.4) (25.8) (400.9) (9.2) (63.8) (7.9)
Daviana 632.7 708.0 1471.0 2480.1 544.9 1802.7 1560.1 25771.1 436.8 2979.5 606.5 38993.4
(7.0) (19.2) (14.6) (1.5) (2.7) (17.8) (12.8) (348.2) (7.3) (27.1) (7.6)
a
Results are expressed as mean (standard deviation) of three determinations.
b
(1) 3-Caffeoylquinic acid; (2) 4-caffeoylquinic acid; (3) 5-caffeoylquinic acid; (4) caffeoyltartaric acid; (5) p-coumaric acid derivative; (6) p-couma-
royltartaric acid; (7) myricetin 3-hexoside + myricetin derivative; (8) myricetin 3-rhamnoside; (9) quercetin 3-hexoside + myricetin derivative; (10)
quercetin 3-rhamnoside; (11) kaempferol 3-rhamnoside.
Phenolic compound
%
1234567891011
0
25
50
75
Fig. 3. Phenolic compounds profile of hazel leaves. (1) 3-Caffeoylquinic
acid; (2) 4-caffeoylquinic acid; (3) 5-caffeoylquinic acid; (4) caffeoyltartaric
acid; (5) p-coumaric acid derivative; (6) p-coumaroyltartaric acid; (7)
myricetin 3-hexoside + myricetin derivative; (8) myricetin 3-rhamnoside;
(9) quercetin 3-hexoside + myricetin derivative; (10) quercetin 3-rhamno-
side; (11) kaempferol 3-rhamnoside.
0.0
1.0
AU
02040
Minutes
1
2
3
4
5
6
7
8
9
10
11
Fig. 2. HPLC-DAD of phenolic compounds in hazel leaves (Cv. Fertille
de Coutard). Detection at 320 nm. Peaks: (1) 3-Caffeoylquinic acid; (2)
4-caffeoylquinic acid; (3) 5-caffeoylquinic acid; (4) caffeoyltartaric acid; (5)
p-coumaric acid derivative; (6) p-coumaroyltartaric acid; (7) myricetin 3-
hexoside + myricetin derivative; (8) myricetin 3-rhamnoside; (9) quercetin
3-hexoside + myricetin derivative; (10) quercetin 3-rhamnoside; (11)
kaempferol 3-rhamnoside.
1022 I. Oliveira et al. / Food Chemistry 105 (2007) 1018–1025
concentrations below 0.5 mg/ml (Fig. 5). Fertille Coutard
and M. Bollwiller cultivars showed the highest and the low-
est activities, respectively, being the obtained results much
better than those obtained for BHA (96% at 3.6 mg/ml)
and a-tocopherol (95% at 8.6 mg/ml). For all the tested
hazel cultivars DPPH scavenging activity values were
higher than 93.1% at 0.5 mg/ml. These results are much
better than DPPH radical scavenging effects described in
the literature (Moure et al., 2001) for methanol extracts
from hazel fruits (14.2% at 2 mg/mL). Nevertheless, etha-
nol extract from hazelnut was reported as possessing higher
radical scavenging effects than other roasted foods such as
almond (Krings & Berger, 2001).
The antioxidant activity of hazel leaves extracts mea-
sured by the bleaching of b-carotene is shown in Fig. 6.
The results obtained indicated a concentration-dependent
antioxidant capacity, following the order Fertille Cou-
tard > Daviana > M. Bollwiller which presented values at
2 mg/ml of 61.3%, 51.3% and 50.3%, respectively. It is
probable that the antioxidative components in the extracts
can reduce the extent of b-carotene destruction by neutral-
izing the linoleate free radical and other free radicals
formed in the system (Mi-Yae et al., 2003). The protection
of b-carotene bleaching provided by TBHQ standard
reached 82.2% at 2 mg/ml and was slightly more efficient
than the samples. Nevertheless, this and other synthetic
antioxidants applied in fat and oily foods to prevent oxida-
tive deterioration were found to be anticarcinogenic as well
as carcinogenic in experimental animals (Loliger, 1991).
For an overview of the results, at Table 2 there are pre-
sented the EC
50
values for the antioxidant activity assays
obtained from each hazel leaves sample. Fertille Coutard
cultivar revealed better antioxidant properties (lower
EC
50
values) than the other samples, for all the biochemical
assays used in the antioxidant activity screening. The EC
50
values obtained for reducing power and scavenging effects
on DPPH radicals (<0.3 mg/ml) were better than for
b-carotene bleaching inhibition (>1.2 mg/ml).
3.3. Antimicrobial activity
The antimicrobial capacity of phenolic compounds, in a
general way, is well-known (Pereira et al., 2006; Proestos
et al., 2005; Puupponen-Pimia
¨et al., 2001; Rauha et al.,
2000; Zhu et al., 2004) and we propose, for the first time,
the use of hazel leaves extracts as a source of antimicrobi-
als. As previously described, individual phenolic com-
pounds present in the hazel leaves extracts were identified
and quantified, but we choose to submit the entire extracts
to antimicrobial activity studies. In fact, food extracts may
be more beneficial than isolated constituents, since a bioac-
tive individual component can change its properties in the
presence of other compounds present in the extracts (Bor-
chers, Keen, & Gerstiwin, 2004). The minimal inhibitory
concentration (MIC) values for bacteria (B. cereus,B. sub-
tilis,S. aureus,E. coli,P. aeruginosa, K. Pneumoniae) and
fungi (C. albicans and C. neoformans) were determined as
an evaluation of the antimicrobial activity of the hazel sam-
ples and were presented in Table 3.
The tested samples revealed antimicrobial activity
against all the microorganisms apart from P. aeruginosa
0
0.5
1
1.5
2
2.5
00.2
0.4 0.6 0.8 1
Concentration (mg/ml)
Abs at 700 nm
Bollwiller Fertille de Coutard Daviana
Fig. 4. Reducing power values of different cultivars of hazel leaf extracts.
Each value is expressed as mean ± standard deviation.
0
25
50
75
100
0 0.2 0.4 0.6 0.8 1
Concentration (mg/ml)
Scavenging Effect (%)
Bollwiller Fertille de Coutard Daviana
Fig. 5. Scavenging effect on DPPH of different cultivars of hazel leaf
extracts. Each value is expressed as mean ± standard deviation.
0
15
30
45
60
75
012345
Concentration (mg/ml)
Antioxidant activity (%)
Bollwiller Fertille de Coutard Daviana
Fig. 6. Antioxidant activity (%) by b-carotene bleaching method of
different cultivars of hazel leaf extracts. Each value is expressed as
mean ± standard deviation.
Table 2
EC
50
values (mg/ml) of hazel leaf samples
Samples Reducing
power (EC
50
)
DPPH (EC
50
)b-Carotene
bleaching (EC
50
)
M. Bollwiller 0.224 0.203 1.981
Fertille Coutard 0.199 0.164 1.243
Daviana 0.223 0.188 1.861
I. Oliveira et al. / Food Chemistry 105 (2007) 1018–1025 1023
and C. albicans, which were resistant to the extracts at a
concentration of 100 mg/ml. M. Bollwiller cultivar proved
to be the most promising hazel cultivar to inhibit microbial
growth, presenting lower MICs and higher growth inhibi-
tion zones. Gram positive bacteria were more susceptible
than either Gram negative bacteria or fungi, presenting
MICs of 0.1 mg/ml for B. cereus and S. aureus, and
1 mg/ml for B. subtilis. These results are of a great impor-
tance particularly in the case of S. aureus which is well-
known for being resistant to a number of phytochemicals
and for the production of several types of enterotoxins that
cause gastroenteritis (Halpin-Dohnalek & Marth, 1989).
The antifungicide activity of the hazel leaves was weak,
being C. albicans resistant to all the samples and C. neofor-
mans susceptible only to samples from M. Bollwiller culti-
var but in a high concentration (100 mg/ml). Nevertheless,
the MIC values obtained for these extracts were even better
than the results obtained by us in previous works with table
olives (Pereira et al., 2006) and ‘‘alcaparras”(Sousa et al.,
2006).
In conclusion, the results obtained in this study demon-
strate that hazel leaves may be a good candidate for
employment as antimicrobial agent against bacteria
responsible for human gastrointestinal and respiratory
tract infections. It may also constitute a good source of
healthy compounds, namely phenolic compounds, suggest-
ing that it could be useful in the prevention of diseases in
which free radicals are implicated.
Acknowledgements
The authors are grateful to INTERREG III A Program,
Project PIREFI for financial support of this work.
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