Fe(III) reductive and free radical-scavenging properties of summer savory (Satureja hortensis L.) extract and subfractions


Dorman, H.J.D.; Hiltunen, R.

Food Chemistry 88(2): 193-199

2004


Satureja hortensis L. (summer savory) is an annual herb belonging to the family Lamiaceae. It is used as a condiment and as a plant in traditional folk medicine to treat infectious diseases and disorders. A number of studies have suggested that the activity of Satureja hortensis may relate to the antioxidant properties of its secondary metabolites. Therefore, this study attempts to characterise the antioxidant properties of an acidified aqueous methanol extract from commercially available material using Fe(III) reductive and DPPH, ABTS+ and hydroxyl free radical-scavenging assays. The crude extract demonstrated promising in vitro activity and thus was further fractionated, by liquid-liquid partitioning against water, to determine which fraction possessed the most potent activity. The ethyl acetate-soluble fraction was the most effective fraction, with reductive activity of 2648+/-41.4 micromol ascorbic acid/g extract, and DPPH and hydroxyl radical-scavenging IC50 concentrations of 138+/-6.0 and 45.0+/-7.0 microgram/ml, respectively. The TEAC value for this fraction was 2.59+/-0.06 mM Trolox. The experimental results are discussed and potential applications are explored.

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ELSEVIER
Food
Chemistry
88
(2004)
193-199
www.elsevier.com/locate/foodchem
Fe(III)
reductive
and
free
radical-scavenging
properties
of
summer
savory
(Satureja
hortensis
L.)
extract
and
subfractions
H.J.D.
Dorman
*,
R.
Hiltunen
Department
of
Pharmacy,
Division
of
Pharmacognosy,
University
of
Helsinki,
P.O.
Box
56
(Viikinkaari
5E),
FIN-00014
Helsinki,
Finland
Received
27
October
2003;
received
in
revised
form
20
December
2003;
accepted
20
December
2003
Abstract
Satureja
hortensis
L.
(summer
savory)
is
an
annual
herb
belonging
to
the
family
Lamiaceae.
It
is
used
as
a
condiment
and
as
a
plant
in
traditional
folk
medicine
to
treat
infectious
diseases
and
disorders.
A
number
of
studies
have
suggested
that
the
activity
of
Satureja
hortensis
may
relate
to
the
antioxidant
properties
of
its
secondary
metabolites.
Therefore,
this
study
attempts
to
cha-
racterise
the
antioxidant
properties
of
an
acidified
aqueous
methanol
extract
from
commercially
available
material
using
Fe(III)
reductive
and
DPPI-I
.
,
ABTS•+
and
hydroxyl
free
radical-scavenging
assays.
The
crude
extract
demonstrated
promising
in
vitro
activity
and
thus
was
further
fractionated,
by
liquid—liquid
partitioning
against
water,
to
determine
which
fraction
possessed
the
most
potent
activity.
The
ethyl
acetate-soluble
fraction
was
the
most
effective
fraction,
with
reductive
activity
of
2648
±
41.4
µmol
ascorbic
acid/g
extract,
and
DPPH•
and
hydroxyl
radical-scavenging
IC
5
o
concentrations
of
138
±
6.0
and
45.0
±
7.0
µg/ml,
re-
spectively.
The
TEAC
value
for
this
fraction
was
2.59
±
0.06
mM
Trolox.
The
experimental
results
are
discussed
and
potential
applications
are
explored.
©
2004
Elsevier
Ltd.
All
rights
reserved.
Keywords.•
Satureja
hortensis;
Antioxidant
activity;
Free
radicals;
Fe(III)
reduction;
Summer
savory;
Herbs
1.
Introduction
Interest
in
natural
antioxidants
has
increased
dra-
matically
in
recent
times
due
to:
(1)
concerns
regarding
the
safety
of
the
chronic
consumption
of
synthetic
an-
tioxidants
(butylated
hydroxyltoluene
and
butylated
hydroxylanisole),
(2)
the
antioxidative
efficacy
of
a
va-
riety
of
phytochemicals,
(3)
the
consensus
that
foods
rich
in
certain
phytochemicals
can
affect
the
aetiology
and
pathology
of
chronic
diseases
and
the
ageing
pro-
cess
and
(4)
the
public's
conceived
belief
that
natural
compounds
are
innately
safer
than
synthetic
compounds
and
are
thus
more
commercially
acceptable.
As
part
of
our
ongoing
in
vitro
screening
programme
to
identify
antioxidant-enriched
extracts
from
edible
herbs,
spices
and
agricultural
products
for
industrial
applications,
we
have
investigated
plant
extracts
from
different
genera
Corresponding
author.
Tel.:
+358-9-191-59181;
fax.:
+358-9-191-
59138.
E-mail
address.•
(H.J.D.
Dorman).
0308-8146/$
-
see
front
matter
©
2004
Elsevier
Ltd.
All
rights
reserved.
doi:10.1016/j.foodchem.2003.12.039
belonging
to
the
family
Lamiaceae
(Labiatae),
a
family
composed
of
species
with
exploitable
antioxidant
activ-
ity
(Jayasinhe,
Gotoh,
Aoki,
&
Wada,
2003;
Jose
del
Bano
et
al.,
2003;
Marinova
&
Yanishlieva,
1997).
Satureja
hortensis
L.
is
an
aromatic
and
medicinal
plant
belonging
to
the
family
Lamiaceae.
The
aerial
material
has
a
distinctive
taste
and
can
be
added
to
stuffing,
meat
pies
and
sausages
as
a
seasoning.
Fresh
sprigs
can
be
boiled
with
pulses,
such
as
peas,
beans
or
lentils,
for
flavouring
or,
alternatively,
they
can
be
used
instead
of
parsley
and
chervil
as
a
garnish.
The
leaves,
flowers
and
stems
are
used
for
herbal
tea
and,
in
tradi-
tional
medicine,
to
treat
various
ailments,
such
as
cramps,
muscle
pains,
nausea,
indigestion,
diarrhoea
and
infectious
diseases
(Giilliice
et
al.,
2003).
Extracts
from
Satureja
hortensis
have
demonstrated
a
variety
of
activities
including
antibacterial,
antifungal,
antioxi-
dant,
antispasmodic,
antidiarrhoeal
and
sedative
prop-
erties
amongst
others
(Deans
&
Svoboda,
1989;
Giilliice
et
al.,
2003;
Hajhashemi,
Sadraei,
Ghannadi,
&
Moh-
seni,
2000;
Madsen,
Andersen,
Christiansen,
Brockhoff,
194
H.J.D.
Dorman,
It
Hiltunen
/
Food
Chemistry
88
(2004)
193-199
&
Bertelsen,
1996).
In
this
study,
we
have
characterised
the
antioxidant
[Fe(III)
reductive
capacity
and
DPPH
.
,
ABTS.±
and
hydroxyl
radical-scavenging]
properties
of
a
crude
acidified
aqueous
methanol
extract
and
four
subfractions,
based
upon
solubility,
from
commercially
available
Satureja
hortensis
aerial
material.
Further-
more,
the
total
phenolic
content
of
each
sample
was
determined
by
the
Folin-Ciocalteu
method.
2.
Materials
and
methods
2.1.
Materials
Air-dried
aerial
parts
of
Satureja
hortensis
L.
were
obtained
from
Pimenta
Oy
(Finland).
Pycnogenol
was
obtained
from
Biolandes
Aromes
(France).
Ultra-pure
water
(HPLC
grade)
was
prepared,
using
a
Millipore
Milli-RO
12
plus
system
(Millipore
Corp.,
USA).
All
reagents
used
in
this
study
were
of
the
highest
purity
commercially
available
and
were
obtained
from
the
usual
sources.
2.2.
Extraction
and
fractionation
Plant
material
(40
g)
was
macerated
with
500
ml
(80:20:1,
v/v/v)
methanol:
water:
acetic
acid
at
room
temperature
for
12
h
with
constant
stirring.
The
pro-
cess
was
repeated
twice
with
fresh
extractant.
The
re-
sulting
extracts
were
combined,
filtered,
concentrated
in
vacuo
and
lyophilized.
The
dried
crude
extract
was
suspended
in
500
ml
ultra-pure
water
and
sequentially
extracted
with
250
ml
hexane,
300
ml
ethyl
acetate
(EtOAc)
and
350
ml
n-butanol
(n-BuOH).
The
re-
maining
aqueous
phase
was
filtered,
concentrated
in
vacuo
and
lyophilized.
The
organic
phases
were
filtered
through
anhydrous
magnesium
sulphate
and
the
sol-
vent
was
removed
in
vacuo.
A
schematic
of
the
ex-
traction
and
fractionation
of
the
plant
material
is
provided
in
Fig.
1.
2.3.
Total
phenol
content
Total
phenols
were
estimated
as
gallic
acid
equiva-
lents
(Singleton,
Orthofer,
&
Lamuela-Raventos,
1999).
To
ca.
6.0
ml
ultra-pure
water,
a
100
µI
sample
in
80%
aqueous
Me0H
was
transferred
to
a
10.0
ml
volumetric
flask,
to
which
was
subsequently
added
500
µI
undiluted
Folin-Ciocalteu
reagent.
After
1
min,
1.5
ml
20%
(w/v)
Na
2
CO
3
was
added
and
the
volume
was
made
up
to
10.0
ml
with
ultra-pure
water.
After
a
30
min
incubation
at
25
°C,
the
absorbance
was
measured
at
760
nm
and
compared
to
a
gallic
acid
calibration
curve.
Satureja
hortensis
Maceration
(RT,
24
h,
x
3)
500
ml
MeOH:H20:HOAc
(80:20:1
v/v/v)
Vol.
red.
in
vacuo,
lyophilised.
Crude
Extract
Partitioned
H
2
0/Hexane
I
Dried
with
MgSO
4
Sol.
removed
in
vacuo
Hexane
Fraction
Aqueous
Phase
Partitioned
EtOAc
1Dried
with
MgSO
4
Sol.
removed
in
vacuo
EtOAc
Fraction
Aqueous
Phase
1
Dried
with
MgSO
4
Sol.
removed
in
vacuo
n-BuOH
Fraction
Aqueous
Phase
I
Filtered
Vol.
red.
in
vacuo
Lyophilised
H
2
O
Fraction
Dried
at
RT
Insoluble
Fraction
Fig.
1.
Fractionation
scheme
for
the
production
of
the
S.
hortensis
crude
extract
and
subfractions.
Scheme
key:
RT,
room
temperature;
vol.,
volume;
red.,
reduced;
sol.,
solvent.
I
Partitioned
n-BuOH
Percentage
inhibition
=
[
(Abseontroi
-
Abssampie)
Ab
Snontrol
H.J.D.
Dorman,
R
Hiltunen
1
Food
Chemistry
88
(2004)
193-199
195
2.4.
Fe(III)
to
Fe(II)
reductive
capacity
The
Fe(III)
reductive
capacity
of
the
extracts
was
assessed
spectrophotometrically
(Oyaizu,
1986).
One
ml
of
each
extract
in
80%
aqueous
methanol
was
mixed
with
2.5
ml
phosphate
buffer
(0.2
M,
pH
6.6)
and
2.5
ml
of
a
1%
(w/v)
potassium
hexacyanoferrate
[K
3
Fe(CN)
6
]
solution.
After
30
min
at
50
°C,
2.5
ml
(10%,
w/v)
tri-
chloroacetic
acid
(TCA)
were
added
and
the
mixture
was
centrifuged
for
10
min
(2000
rpm).
Finally,
a
2.5
ml
aliquot
was
mixed
with
2.5
ml
ultra-pure
water
and
0.5
ml
(0.1%,
w/v)
FeC1
3
and
the
absorbance
was
recorded
at
700
nm.
Values
are
presented
as
ascorbic
acid
equivalents,
AscAE
(µmol
ascorbic
acid/g
extract).
2.5.
Dipheny1-2-picrylhydrazyl
free
radical-scavenging
activity
The
ability
of
the
extracts
to
scavenge
1,1-diphenyl-
2-picrylhydrazyl
radicals
was
assessed
spectrophoto-
metrically
(Gyamfi,
Yonamine,
&
Aniya,
1999).
A
50
µI
aliquot
of
each
extract,
in
80%
aqueous
methanol,
was
mixed
with
450
µI
Tris-HC1
buffer
(50
mM,
pH
7.4)
and
1.0
ml
1,1-dipheny1-2-picrylhydrazyl
(0.1
mM,
in
methanol).
After
a
30
min
reaction
period,
the
re-
sultant
absorbance
was
recorded
at
517
nm.
The
percentage
inhibition
was
calculated
using
equation
1
and
IC
50
s
were
estimated
using
a
non-linear
regression
algorithm.
2.7.
Ascorbate-Fe(III)-catalysed
phospholipid
peroxida-
tion
inhibition
The
ability
of
the
extracts
to
scavenge
hydroxyl
rad-
icals
was
determined
by
the
method
of
Aruoma
et
al.
(1997).
Bovine
brain
extract
(Folch
type
VII)
was
mixed
with
10
mM
PBS
(pH
7.4)
and
sonicated
in
an
ice
bath
until
an
opalescent
suspension
was
obtained
containing
5
mg/ml
phospholipid
liposomes.
The
liposomes
(0.2
ml)
were
combined
with
0.5
ml
of
PBS
buffer,
0.1
ml
of
1
mM
FeC1
3
,
and
0.1
ml
of
extract
in
80%
aqueous
Me0H.
The
peroxidation
was
initiated
by
adding
0.1
ml
of
1
mM
ascorbate.
The
mixture
was
incubated
at
37
°C
for
60
min.
After
incubation,
50
µI
of
2%
(w/v)
butylated
hydroxytoluene
in
EtOH
were
added
to
each
tube,
fol-
lowed
by
1
ml
of
2.8%
(w/v)
trichloroacetic
acid
and
1
ml
of
1%
(w/v)
2-thiobarbituric
acid
(TBA)
in
0.05
M
NaOH.
The
solutions
were
heated
in
a
water
bath
at
100
°C
for
20
min.
The
resulting
(TBA)
2
-MDA
chromogen
was
extracted
into
2
ml
of
n-butanol
and
the
extent
of
peroxidation
was
determined
in
the
organic
layer
at
532
nm.
The
percentage
inhibition
was
calculated
using
equation
1
and
IC
5
0
values
were
estimated
using
a
non-
linear
regression
algorithm.
2.8.
Statistical
analysis
Data
are
presented
as
mean
values
±95%
confidence
limits.
Analysis
of
variance
was
performed
using
AN-
OVA
procedures.
Significant
differences
between
means
were
determined
by
Tukey's
pairwise
comparison
test
at
a
level
of
P
<
0.05.
IC
50
values
were
estimated
using
a
non-linear
regression
algorithm.
x
100.
(
1
)
2.6.
ABTS
free
radical-scavenging
activity
The
determination
of
ABTS
.
+
radical-scavenging
was
carried
out
as
described
by
Re
et
al.
(1998).
The
ABTS
.
+
radical
was
generated
by
reacting
an
(7
mM)
ABTS
aqueous
solution
with
K
2
S
2
0
8
(2.45
mM,
final
concentration)
in
the
dark
for
12-16
h
and
adjusting
the
Abs734
nm
to
0.700
(±0.030)
at
ambient
temperature.
Extracts
were
diluted
such
that
a
15
µI
sample,
when
added
to
1.485
ml
ABTS
solution,
resulted
in
a
20%-
80%
inhibition
of
the
absorbance.
After
15
µI
extract/
Trolox/standards
were
added
to
1.485
ml
ABTS
.
±,
the
absorbance
at
734
nm
was
recorded
at
1
min
after
initial
mixing
and
subsequently
at
1
min
intervals
(15
min
in
toto).
The
percentage
inhibition
was
plotted
as
a
func-
tion
of
concentration
and
the
Trolox
equivalent
anti-
oxidant
capacity
(TEAC)
was
calculated
against
a
Trolox
calibration
curve.
3.
Results
3.1.
Extract
yield
and
total
phenol
content
The
yield
of
the
crude
Satureja
hortensis
extract
was
369
mg
extract/g
plant
material
and
consisted
of
approxi-
mately
6.5%
non-polar
(hexane-soluble)
components,
5.1%
moderately
polar
(EtOAc-soluble)
components,
56.8%
polar
(n-BuOH-soluble)
components,
28.7%
highly
polar
(H
2
0-soluble)
components
and
2.9%
insoluble
components.
The
total
extractable
phenolic
contents
of
the
crude
extract
and
hexane,
ethyl
acetate,
n-butanol
and
water
fractions
were
estimated
by
the
Folin-Ciocalteu
method
as
166
±
2.7,
37.1
±
1.1,
500
±
47.3,
27.0
±
9.1
and
67.2
±
25.2
mg
gallic
acid/g
extract,
respectively.
3.2.
Fe(III)
to
Fe(II)
reductive
capacity
As
can
be
seen
from
the
AscAE
values
presented
in
Fig.
2
and
the
associated
regression
parameters
(Table
1),
H.J.D.
Dorman,
It
Hiltunen
/
Food
Chemistry
88
(2004)
193-199
196
extract.
This
was
significantly
better
than
the
activities
calculated
for
the
positive
controls
(P
<
0.05).
7000
e
e
0
4
,
&
c•
ic
c
a
t
''°\
g
.6'
z
'
o
41
'
A
S'
0+
p
ct
+
iP
'SP'
't
4
P
4
r\
G
O'
•c
,
Sample
Fig.
2.
AscAE
values
calculated
for
the
positive
controls
and
S.
hort-
ensis
samples.
Values
are
presented
as
mean
values
±95%
confidence
interval.
Bars
with
the
same
lower
case
letter
(a-f)
are
not
significantly
(P
>
0.05)
different.
Table
1
Fe(III)
to
Fe(II)
reductive
capacity
linear
regression
parameters
for
the
test
samples
Sample
Slope
Intercept
(x10
-2
)
r
2
Ascorbic
acid
12.192
±
0.316
1.2
±
0.6
0.999
BHA
4.925
±
0.173
10.5
±
1.8
0.994
Pycnogenol
3.886
±
0.263
7.0
±
1.6
0.998
Trolox
3.800
±
0.029
2.3
±
0.5
0.998
Crude
extract
1.682
±
0.015
1.9
±
0.5
1.000
Hexane
Fr.
0.373
±
0.005
0.7
±
0.3
0.997
EtOAc
Fr.
5.658
±
0.091
2.7
±
0.3
0.997
n-BuOH
Fr.
0.115
±
0.002
7.7
±
1.1
0.996
H
2
O
Fr.
0.340
±
0.007
6.5
±
1.9
0.998
Values
are
presented
as
means
±95%
confidence
limits.
all
the
extracts
and
fractions
were
capable
of
catalysing
the
reduction
of
Fe(III)
and
did
so
in
a
linear
dose-de-
pendent
fashion.
The
crude
extract
possessed
an
AscAE
(ascorbic
acid
equivalent)
of
792
±
6.0
µmol
ascorbic
acid/g
extract
which
was
much
lower
than
the
value
observed
for
the
reference
substance
ascorbic
acid
or
for
the
positive
controls.
This
suggests
that
this
extract
is
not
as
powerful
a
reducing
agent
as
these
substances.
The
non-polar
hexane-soluble
fraction
possessed
a
very
low
AscAE
value
of
37.1
±
1.3
µmol
ascorbic
acid/g
extract
as
did
the
n-BuOH
fraction
(27.0
±
9.1
µmol
ascorbic
acid/g
extract),
with
the
H
2
O
fraction
con-
taining
a
better
reducing
potency
(67.2
±
25.2
µmol
ascorbic
acid/g
extract).
The
EtOAc
fraction,
on
the
other
hand,
possessed
very
potent
reductive
power,
with
an
AscAE
equal
to
2648
±
41.4
µmol
ascorbic
acid/g
3.3.
Dipheny1-2-picrylhydrazyl
radical-scavenging
activity
The
extract
and
fractions
were
capable
of
scavenging
DPPH•
free
radicals
in
a
dose-dependent
manor
via
electron-/hydrogen-donation
converting
it
to
the
non-
radical
hydrazine
form.
The
concentrations
of
crude
extract
and
fractions
required
to
scavenge
50%
of
the
DPPH•
radicals,
the
IC
5
0
values,
are
presented
in
Fig.
3.
The
hierarchy
of
ability
to
scavenging
DPPH•
radicals
was
determined
as
the
EtOAc
fraction
>
crude
extract
>
H
2
O
fraction
>
n-BuOH
fraction
>
hexane
fraction.
The
hexane-soluble
fraction
required
7.12
±
0.050
mg/ml
in
order
to
scavenge
50%
of
the
DPPH•
radicals,
which
suggests
that
the
components
within
this
fraction
are
extremely
weak
radical-scavenging
components.
Simi-
larly,
the
components
within
both
the
n-BuOH
and
H
2
O
fractions
were
not
efficient
free
radical
scavengers,
with
IC
50
values
of
3.43
±
0.082
and
2.16
±
0.040
mg/ml,
respectively.
3.4.
ABTS+
free
radical-scavenging
activity
The
extract
and
fractions
were
capable
of
scavenging
the
ABTS
.
+
free
radical
and
could
be
ranked
according
to
their
calculated
TEAC
values
at
1,
5,
10
and
15
min
time
points
(Table
2).
The
EtOAc-soluble
fraction
was
8.0
7.5
-
7.0
-
6.5
-
4.0/
1 1
1 1
45',
4,
4J
4
<s.c,
6'c>
zs
z,
cP
c
'
'`r\
Sample
Fig.
3.
The
effect
of
the
positive
controls
and
S.
hortensis
samples
upon
1,1-diphenyl-2-picrylhydrazyl
scavenging.
Values
are
presented
as
mean
values
±95%
confidence
intervals.
Bars
with
the
same
lower
case
letter
(a-h)
are
not
significantly
(1
3
>
0.05)
different.
6000
to
5000
4000
0
3000
-
g_
2000
-
1000
-
DPPI
-1
*
ra
dica
l
scaveng
ing
IC
5
0
[mg
/m
1]
3.0
2.0
1.0
0.0
a
b
c
c
e
H.J.D.
Dorman,
R
Hiltunen
1
Food
Chemistry
88
(2004)
193-199
Table
2
Radical-scavenging
activities
of
the
test
samples
ABTS
.
+
radicals
197
Sample
ABTS•+
(TEAC,
mM
Trolox)
1
min
5
min
10
min
15
min
Ascorbic
acid
5.60
±
0.26a,A
5.60
±
0.24a,A
5.59
±
0.25a,A
5.59
±
0.24a,A
BHA
6.39
±
0.34b,A
6.53
±
0.29b,A
6.56
±
0.28b,A
6.56
±
0.26b,A
Pycnogenol
3.34
±
0.19c,A
3.77
±
0.20c,A
4.00
±
0.21c,A
4.08
±
0.20c,A
Trolox
4.01
±
0.12d,A
4.00
±
0.15c,A
4.00
±
0.15c,A
4.00
±
0.15c,A
Crude
extract
0.77
±
0.04e,A
0.91
±
0.04d,B
1.00
±
0.05d,B
1.05
±
0.05d,B
Hexane
Fr.
0.15
±
0.01f,A
0.20±
0.01e,B
0.23
±
0.01e,C
0.24
±
0.02e,C
EtOAc
Fr.
2.22
±
0.05g,A
2.50±
0.07f,B
2.59
±
0.06f,B
2.64
±
0.05f,B
n-BuOH
Fr.
0.31
±
0.08h,A
0.48
±
0.08g,B
0.61
±
0.08g,C
0.70
±
0.08g,C
H
2
O
Fr.
0.33
±
0.02h,A
0.37
±
0.02g,B
0.39
±
0.03h,B
0.41
±
0.02h,B
Values
are
presented
as
means
±95%
confidence
limits.
Trolox
equivalent
antioxidant
capacity
(TEAC)
is
defined
as
the
concentration
of
Trolox
having
the
ABTS
.
±
scavenging
activity
equal
to
a
1.0
mg/ml
sample
solution.
Analysis
of
variance
was
performed
by
ANOVA
procedures,
with
significant
differences
between
means
determined
using
Tukey's
pairwise
comparisons.
Values
with
the
same
lowercase
letter
within
each
column
are
not
significantly
(P
>
0.05)
different.
Values
with
the
same
uppercase
letter
within
each
row
are
not
significantly
(1
3
>
0.05)
different.
the
most
efficient
scavenger
over
the
reaction
time
fol-
lowed
by
the
crude
extract.
Over
time,
however,
the
potencies
of
the
n-butanol
and
H
2
O
fractions
changed;
at
1
and
5
min
these
fractions
did
not
differ
significantly
(P
>
0.05)
in
their
ABTS
.
+
radical-scavenging
activity
but,
after
10
min,
the
n-BuOH
fraction
was
a
signifi-
cantly
(P
<
0.05)
better
scavenger
than
H
2
O
fraction.
The
hexane
fraction
was
the
least
active,
with
TEAC
values
ranging
fron
0.33
±
0.02
mM
Trolox
at
t=
1
min
to
0.41
±
0.02
mM
Trolox
t=
15
min.
There
was
no
significant
(P
>
0.05)
change
in
the
ability
of
the
crude
extract
or
the
EtOAc
and
H
2
O
fraction
to
reduce
the
ABTS
.
+
free
radical
over
time
after
the
5
min
point,
which
suggests
that
the
components
within
these
sam-
ples
have
fairly
rapid
reaction
kinetics.
In
the
case
of
the
hexane
and
n-BuOH
fractions,
the
reaction
kinetics
appeared
to
be
a
little
slower,
with
no
significant
(P
>
0.05)
increase
in
activity
observed
after
the
10
min
reaction
point.
12.0
Hy
droxy
l
ra
dica
l
scav
eng
ing
I
C50
[mg
/m
il
10.0
-
8.0
-
6.0/
0.8/
0.6
-
0.4
-
0.2
-
0.0
b b
a
r
-T-
1
I
T
I
I
I
I
,
s's°
6
6
1
\-
9
c•5v.
4
0
)
bc
'
e
Samples
3.5.
Ascorbate-Fe(III)-catalysed
phospholipid
peroxida-
tion
inhibition
The
crude
extract
and
fractions
were
capable
of
preventing
the
formation
of
the
malondialdehyde-thio-
barbituric
acid
MDA-(TBA)
2
chromogen
in
a
concen-
tration-dependent
fashion
and,
therefore,
hydroxyl
radical-mediated
phospholipid
peroxidation.
The
esti-
mated
IC
50
values
for
the
samples
are
presented
in
Fig.
4.
In
this
assay,
the
EtOAc
fraction
was
a
considerably
more
active
inhibitor
of
ascorbate-Fe(III)-catalysed
phospho-
lipid
peroxidation
than
the
crude
extract
or
the
n-BuOH
and
H
2
O
fractions.
The
EtOAc-soluble
fraction
was
as
effective
as
the
positive
control
substances
Pycnogenol
and
Trolox
but
not
as
effective
as
BHA.
Ascorbic
acid
was
not
used
as
a
positive
control
in
this
assay
system
as
it
is
used
as
a
catalyst
for
the
production
of
hydroxyl
radicals
in
this
assay
system.
The
polar
and
highly
polar
fractions
were
only
poorly
active,
however,
with
IC
5
0
values
of
Fig.
4.
The
effect
of
the
positive
controls
and
S.
hortensis
samples
upon
hydroxyl
radical-scavenging.
Values
are
presented
as
mean
values
±95%
confidence
intervals.
Bars
with
the
same
lower
case
letter
(a-f)
are
not
significantly
(1
3
>
0.05)
different.
5.49
±
0.892
mg/ml
and
8.82
±
1.23
mg/ml,
respectively.
It
was
not
possible
to
obtain
an
IC
50
value
for
the
hexane
fraction
as
the
biphasic
dose-response
curve
prevented
a
reliable
estimation
by
the
non-linear
regression
algorithm
used
in
this
study.
4.
Discussion
The
ability
of
isolated
compounds
or
plant
extracts
to
reduce
Fe(III)
is
often
used
as
an
indicator
of
electron
donating
activity,
which
is
an
important
mechanism
of
phenolic
antioxidant
action
(Dorman,
Peltoketo,
Hilt-
unen,
&
Tikkanen,
2003a;
Yildirim
et
al.,
2000).
The
data
198
H.J.D.
Dorman,
It
Hiltunen
/
Food
Chemistry
88
(2004)
193-199
presented
in
this
study
suggests
that
components
in
Satureja
hortensis
which
are
soluble
in
the
acidic
aqueous
methanol
extractant
are
capable
of
electron
donation
and
therefore
should
be
able
to
donate
electrons
to
un-
stable
free
radicals,
converting
them
into
more
stable
non-reactive
species,
with
the
EtOAc-soluble
compo-
nents
being
the
most
effective
electron
donators.
There
was
no
apparent
association
between
extraction
yields
and
Fe(III)
reduction
(r
2
=
0.074;
P
=
0.658).
There
was,
however,
a
very
strong
association
between
the
Folin-Ciocalteu-reactive
(total
phenol)
content
and
re-
ductive
capacity
(r
2
=
0.997;
P
<
0.001).
It
is
known
that
chemicals
which
readily
undergo
redox
reactions
are
capable
of
producing
a
high
level
of
activity
in
the
Folin-
Ciocalteu
method
(Singleton
et
al.,
1999),
which
may
explain
the
high
correlation
between
these
two
indices.
The
cardinal
mode
of
action
of
natural
antioxidants
is
their
ability
to
scavenge
free
radicals
before
they
can
initiate
free
radical
chain
reactions
in
cellular
mem-
branes
or
lipid-rich
matrices
in
foodstuffs,
cosmetics
or
pharmaceutical
preparations.
In
this
study,
three
dif-
ferent
free
radicals,
were
used
to
assess
the
potential
free
radical-scavenging
activities
of
the
Satureja
hortensis
extract
and
subfractions,
namely
the
DPPH•
and
ABTS•
+
synthetic
free
radicals
and
the
hydroxyl
radical.
The
stable
nitrogen-centered
free
radicals,
DPPH•
and
ABTS•
+are
frequently
used
for
the
estimation
of
free
radical-scavenging
ability
(Dorman
et
al.,
2003a;
Re
et
al.,
1998).
The
ABTS•+
free
radical
is
commonly
used
when
issues
of
solubility
or
interference
arise
and
the
use
of
DPPH•
based
assays
become
inappropriate
(Arnao,
2000).
However,
it
has
been
argued
that
these
techniques
are
only
capable
of
indicating
potential
antioxidant
ac-
tivity
as
they
do
not
utilise
a
food/biologically
relevant
oxidizable
substrate
nor
a
relevant
reactive
species
and,
therefore,
no
direct
information
on
protective
perfor-
mance
can
be
determined
(Dorman,
Kolar,Kahlos,
Holm,
&
Hiltunen,
2003b).
Therefore,
the
ability
of
the
samples
to
inhibit
ascorbate-Fe(III)-generated
hydroxyl
radical-mediated
peroxidation
of
a
heterogeneous
phospholipid-aqueous
phosphate
buffered
system
was
determined,
as
phospholipids
are
believed
to
play
a
principal
role
in
oxidative
deterioration
and
off-flavour
development
in
foodstuffs
(Wu
&
Sheldon,
1988)
and
the
hydroxyl
radical
is
a
highly
reactive
radical,
capable
of
being
generated
in
vivo.
Recently,
a
number
of
re-
views
detailing
the
range
of
antioxidant
methodologies
currently
available
and
describing
their
advantages
and
disadvantages
have
been
published
(Antolovich,
Pren-
zler,
Patsalides,
McDonald,
&
Robards,
2002;
Frankel
&
Meyer,
2000;
Niki
&
Naguchi,
2000).
The
free
radical-scavenging
data
presented
in
Figs.
2-
4
and
Table
2
suggests
that
the
crude
extract
and
subfractions
are
capable
of
scavenging
reactive
free
radical
species
via
the
mechanism
of
electron-/hydrogen-
donation.
As
in
the
case
of
the
samples'
performance
in
the
Fe(III)
reduction
assay,
the
EtOAc-soluble
compo-
nents
were
the
most
active,
followed
by
the
crude
extract
in
the
three
in
vitro
scavenging
assays
used.
There
was
no
association
between
the
extraction
yields
and
the
reciprocal
IC
50
DPPH•
scavenging
values
(r
2
=
0.110;
P
=
0.586).
There
was,
however,
a
robust
association
between
the
reciprocal
IC
50
values
and
the
Folin-Cio-
calteu
reagent-reactive
content
(r
2
=
0.990;
P
<
0.001)
and
Fe(III)
reductive
index
(r
2
=
0.992;
P
<
0.001).
There
was
a
strong
correlation
between
the
calculated
TEAC
values
at
t
=
10
min
and
the
Folin-Ciocalteu
reagent
reactive
content
(r
2
=
0.968;
P
=
0.003).
The
association
was
stronger
between
the
Folin-Ciocalteu
reactive
content
and
the
calculated
area
under
the
curve,
an
index
used
to
assess
the
overall
antioxidant
perfor-
mance
across
the
total
reaction
time
in
comparison
to
that
of
Trolox
(Re
et
al.,
1998)
(r
2
=
0.999;
P
<
0.001).
There
was
no
association
between
the
extraction
yields
and
the
reciprocal
IC
50
hydroxyl
radical-scavenging
values
(r
2
=
0.434;
P
=
0.341)
but
there
was
a
robust
association
between
the
Folin-Ciocalteu
reagent
reactive
content
(r
2
=
0.950;
P
=
0.025)
and
the
Fe(III)
reductive
(r
2
=
0.955;
P
=
0.023)
index
and
the
reciprocal
ICso
values.
The
crude
extract
and
subfractions
of
Satureja
hortensis
may
be
able
to
protect
susceptible
components
such
as
amino
acids,
DNA,
lipoproteins,
polyunsatu-
rated
fatty
acids,
sugars
and
proteins
in
biological
and
food
matrices
from
free
radical-mediated
degradation
(Halliwell,
Aeschbach,
Loliger,
&
Aruoma,
1995).
The
hexane-soluble
fraction
could
not
be
reliably
assessed
in
the
hydroxyl
radical-phospholipid
assay,
thus
no
conclusions
can
be
draw
about
its
performance.
5.
Conclusions
Satureja
hortensis
is
an
annual
herb
belonging
to
the
family
Lamiaceae
which
is
used
as
a
condiment
and
as
a
traditional
folk
medicine
especially
in
Mediterranean
re-
gions.
For
example,
in
the
Eastern
Anatolia
region
of
Turkey,
it
is
used
to
treat
infectious
diseases.
Aerial
ma-
terial
from
commercially
available
plant
material
was
macerated
with
acidified
aqueous
methanol
to
produce
a
crude
extract.
An
aliquot
of
this
extract
was
subfractioned
by
liquid—liquid
partitioning
against
water
with
hexane,
EtOAc
and
n-BuOH.
These
samples
were
screened
to
characterise
their
antioxidant
properties,
using
a
Fe(III)
reduction
assay
and
DPPH
.
,
ABTS•
+
and
hydroxyl
free
radical-scavenging
assays.
The
crude
extract
and
the
EtOAc
fraction
were
the
most
effective
samples,
with
the
remaining
subfractions
demonstrating
considerably
weaker
activities.
The
activity
of
the
EtOAc-soluble
components
of
the
crude
extract
showed
particularly
strong
properties
and
may
find
potential
application
as
preservatives
to
retard
free
radical-mediated
degradation
of
susceptible
components.
Furthermore,
due
to
the
role
H.J.D.
Dorman,
R
Hiltunen
/
Food
Chemistry
88
(2004)
193-199
199
free
radicals
play
in
the
deterioration
of
human
health,
the
EtOAc
subfraction
may
potentially
have
beneficial
effects
upon
human
biology
if
incorporated
in
foodstuffs
in
ap-
propriate
quantities.
However,
before
such
claims
can
be
made
with
confidence,
further
research
on
the
qualitative-
quantitative
chemical
composition
and
in
vivo
efficacy
studies,
including
absorption
and
metabolism
studies,
should
be
carried
out.
Acknowledgements
HJDD
acknowledges
the
financial
support
of
the
Paulig
Group
Ltd.,
Finland.
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