Rosmarinic acid-rich extracts of summer savory (Satureja hortensis L.) protect Jurkat T cells against oxidative stress


Chkhikvishvili, I.; Sanikidze, T.; Gogia, N.; Mchedlishvili, T.; Enukidze, M.; Machavariani, M.; Vinokur, Y.; Rodov, V.

Oxidative Medicine and Cellular Longevity 2013: 456253-456253

2014


Summer savory (Satureja hortensis L., Lamiaceae) is used in several regions of the world as a spice and folk medicine. Anti-inflammatory and cytoprotective effects of S. hortensis and of its rosmarinic acid-rich phenolic fraction have been demonstrated in animal trials. However, previous studies of rosmarinic acid in cell models have yielded controversial results. In this study, we investigated the effects of summer savory extracts on H2O2-challenged human lymphoblastoid Jurkat T cells. LC-MS analysis confirmed the presence of rosmarinic acid and flavonoids such as hesperidin and naringin in the phenolic fraction. Adding 25 or 50 µM of H2O2 to the cell culture caused oxidative stress, manifested as generation of superoxide and peroxyl radicals, reduced cell viability, G0/G1 arrest, and enhanced apoptosis. This stress was significantly alleviated by the ethanolic and aqueous extracts of S. hortensis and by the partially purified rosmarinic acid fraction. The application of an aqueous S. hortensis extract doubled the activity of catalase and superoxide dismutase in the cells. The production of IL-2 and IL-10 interleukins was stimulated by H2O2 and was further enhanced by the addition of the S. hortensis extract or rosmarinic acid fraction. The H2O2-challenged Jurkat cells may serve as a model for investigating cellular mechanisms of cytoprotective phytonutrient effects.

Hindawi
Publishing
Corporation
Oxidative
Medicine
and
Cellular
Longevity
Volume
2013,
Article
ID
456253,
9
pages
http://dx.doi.org/10.1155/2013/456253
CS)
Hindawi
Research
Article
Rosmarinic
Acid-Rich
Extracts
of
Summer
Savory
(Satureja
hortensis
L.)
Protect
Jurkat
T
Cells
against
Oxidative
Stress
Irakli
Chkhikvishvili,'
Tamar
Sanilddze,
1
Nunu
Gogia,'
Tamar
Mchedlishvili,'
Mafia
Enukidze,'
Marine
Machavariani,'
Yakov
Vinokur,
2
and
Victor
Rodov
2
Institute
of
Medical
Biotechnology,
Tbilisi
State
Medical
University,
33
Vazha
Pshavela
Ave.
017Z
Tbilisi,
Georgia
2
Department
of
Postharvest
Science
of
Fresh
Produce,
Agricultural
Research
Organization,
The
Volcani
Center,
P.O.
Box
6,
50250
Bet
Dagan,
Israel
Correspondence
should
be
addressed
to
Victor
Rodov;
vrodov@agri.gov.il
Received
5
July
2013;
Revised
13
October
2013;
Accepted
17
October
2013
Academic
Editor:
David
Vauzour
Copyright
©
2013
Irakli
Chkhikvishvili
et
al.
This
is
an
open
access
article
distributed
under
the
Creative
Commons
Attribution
License,
which
permits
unrestricted
use,
distribution,
and
reproduction
in
any
medium,
provided
the
original
work
is
properly
cited.
Summer
savory
(Satureja
hortensis
L.,
Lamiaceae)
is
used
in
several
regions
of
the
world
as
a
spice
and
folk
medicine.
Anti-
inflammatory
and
cytoprotective
effects
of
S.
hortensis
and
of
its
rosmarinic
acid-rich
phenolic
fraction
have
been
demonstrated
in
animal
trials.
However,
previous
studies
of
rosmarinic
acid
in
cell
models
have
yielded
controversial
results.
In
this
study,
we
investigated
the
effects
of
summer
savory
extracts
on
H
2
0
2
-challenged
human
lymphoblastoid
Jurkat
T
cells.
LC-MS
analysis
confirmed
the
presence
of
rosmarinic
acid
and
flavonoids
such
as
hesperidin
and
naringin
in
the
phenolic
fraction.
Adding
25
or
50µM
of
H
2
0
2
to
the
cell
culture
caused
oxidative
stress,
manifested
as
generation
of
superoxide
and
peroxyl
radicals,
reduced
cell
viability,
GO/G1
arrest,
and
enhanced
apoptosis.
This
stress
was
significantly
alleviated
by
the
ethanolic
and
aqueous
extracts
of
S.
hortensis
and
by
the
partially
purified
rosmarinic
acid
fraction.
The
application
of
an
aqueous
S.
hortensis
extract
doubled
the
activity
of
catalase
and
superoxide
dismutase
in
the
cells.
The
production
of
IL-2
and
IL-10
interleukins
was
stimulated
by
H
2
0
2
and
was
further
enhanced
by
the
addition
of
the
S.
hortensis
extract
or
rosmarinic
acid
fraction.
The
H
2
0
2
-challenged
Jurkat
cells
may
serve
as
a
model
for
investigating
cellular
mechanisms
of
cytoprotective
phytonutrient
effects.
1.
Introduction
Summer
savory
(Satureja
hortensis
L.)
is
an
herb
of
the
Lami-
aceae
family
that
is
used
in
cooking
and
folk
medicine
in
several
regions
of
the
world
[1].
In
Georgia,
dried
and
ground
summer
savory
(local
name
kondari)
is
one
of
the
most
pop-
ular
spices,
used
either
on
its
own
or
as
an
ingredient
in
spice
blends.
In
addition,
from
ancient
times,
it
has
been
known
locally
as
an
antimicrobial
folk
remedy
for
gastrointestinal
problems
[2].
Indigenous
landraces
of
summer
savory
are
cultivated
in
Georgia
[3].
The
leaves
of
summer
savory
are
rich
in
phenolic
com-
pounds,
particularly
rosmarinic
acid
and
flavonoids,
which
account
for
the
high
antioxidant
capacity
of
these
leaves
[4,
5].
In
our
previous
study
of
Georgian
spices,
we
found
that
kondari
had
one
of
the
highest
total
phenolic
content
levels
and
one
of
the
highest
hydrophilic
antioxidant
capacity
levels
[6].
Rosmarinic
(a-0-caffeoy1-3,4-dihydroxy-phenyl
lactic)
acid
was
found
to
be
the
major
compound
in
ethanolic
extracts
of
summer
savory
and
some
other
Lamiaceae
herbs
[4].
Rosmarinic
acid
is
a
phenylpropanoid
derivative
that
is
the
second
most
common
ester
of
caffeic
acid
in
the
plant
kingdom.
Animal
studies
have
revealed
anti-inflammatory
activity
of
S.
hortensis
extract
and
its
polyphenolic
fraction,
in
partic-
ular
[7,
8].
This
activity
might
be
associated,
at
least
partially,
with
rosmarinic
acid,
whose
antiinflammatory
and
antialler-
gic
properties
have
been
demonstrated
in
animal
and
human
trials
[9,10].
Osakabe
et
al.
[10]
suggested
that
the
antiallergic
effect
of
rosmarinic
acid
might
be
due
to
two
independent
2
Oxidative
Medicine
and
Cellular
Longevity
mechanisms:
the
scavenging
of
reactive
oxygen
species
and
the
modulation
of
the
inflammatory
response.
For example,
the
nephroprotective
effect
of
rosmarinic
acid
was
associated
with
improved
antioxidant
potency,
including
enhanced
glutathione
content
and
activity
of
antioxidant
enzymes
[11].
However,
the
cellular
mechanisms
by
which
rosmarinic
acid
exerts
its
anti-inflammatory
effects
are
not
fully
under-
stood
and
demand
further
investigation.
The
human
lym-
phoblastoid
T-cell
Jurkat
line,
a
constitutive
producer
of
the
potent
T-cell
growth
factor
interleukin
2
(IL-2),
is
a
popular
model
for
the
study
of
immune
signaling
[12].
Jurkat
cells
can
imitate
both
healthy
and
inflammatory
T-cells
in
their
response
to
oxidative
metabolites,
such
as
hydrogen
peroxide
[13].
Therefore,
investigating
the
effect
of
S.
hortensis
extract
on
the
proliferation
and
activity
of
T-cells
may
contribute
to
our
understanding
of
the
mechanism(s)
of
its
anti-
inflammatory
and
cytoprotective
effects.
Although
H
2
0
2
plays
an
important
role
in
antigen-dependent
lymphocyte
activation
[14],
excessive
production
of
H
2
0
2
induces
oxida-
tive
stress
and
impairs
T-cell
activity,
leading
to
chronic
inflammation
and
cell
death.
The
response
of
Jurkat
cells
to
H
2
0
2
is
dose-dependent.
Reversible
oxidative
changes
that
can
be
repaired
by
cellular
antioxidant
system
occur
at
a
H
2
0
2
concentration
of
20
yM,
and
the
first
signs
of
apoptosis
are
noted
at
50
yM
H
2
0
2
[15].
Relatively
high
bolus
doses
of
H
2
0
2
(150
yM)
induce
apoptosis
in
Jurkat
cells,
but
the
continuous
presence
of
a
lower
concentration
of
H
2
0
2
(2
yM)
inhibits
the
apoptotic
process
[16].
Both
apoptosis
and
necrosis
were
observed
in
the
Jurkat
cells
exposed
to
100
yM
H
2
0
2
[17],
while
necrosis
was
far
more
common
at
500µM
H
2
0
2
[18].
Despite
its
well-documented
cytoprotective
activity
in
animal
trials,
concentrations
of
up
to
150
yM
of
rosmarinic
acid
failed
to
prevent
the
H
2
0
2
-mediated
apoptosis
of
Jurkat
cells
and
showed
no
antioxidant
properties
[19].
Moreover,
even
in
the
absence
of
exogenous
hydrogen
peroxide,
rosmarinic
acid
was
reported
to
induce
the
apoptosis
of
Jurkat
cells
[19,
20].
The
discrepancy
between
the
prooxidant
behavior
of
rosmarinic
acid
toward
Jurkat
cells
that
has
been
observed
in
previous
studies
and
its
well-known
antioxidant
and
anti-inflammatory
properties
hamper
the
use
of
Jurkat
cells
as
a
model
for
investigating
the
mode
of
action
of
this
phytonutrient.
In
the
present
work,
we
reexamined
the
effects
of
summer
savory
extracts
and
thier
rosmarinic
acid-rich
phenolic
fraction
on
H
2
0
2
-challenged
Jurkat
cells.
2.
Materials
and
Methods
2.1.
Plant
Material.
Plants
of
a
local
Georgian
landrace
of
S.
hortensis
were
grown
in
an
experimental
plot
near
Tbilisi
from
seeds
purchased
from
commercial
supplier.
The
plants
were
harvested
at
their
vegetative
state
(55
days
after
seed
germination),
the
phenological
stage
characterized
by
the
highest
phenolic
compound
content,
highest
flavonoid
content,
and
greatest
antioxidant
activity
(I.
Chkhikvishvili,
unpublished
data).
The
collected
plant
material
was
air-dried
in
the
shade
at
25-30°C.
The
dried
matter
was
stored
in
a
closed
glass
container
in
a
cool,
dry
place.
2.2.
Extraction
and
Purification.
The
dried
plant
material
(1
g
samples)
was
sequentially
extracted
with
chloroform,
ethyl
acetate,
and
ethanol
at
a
1:5
w/v
ratio
of
plant
material
to
solvent;
the
duration
of
each
extraction
step
was
24
h.
The
residue
was
extracted
with
water
by
steeping
for
20
min
at
90°C
and
subsequent
gradual
cooling
down
to
room
temper-
ature.
Direct
application
of
this
water
extraction
technique
to
the
dried
plant
material
produced
a
"total
aqueous
extract:'
The
solvents
were
removed
by
evaporation
under
vacuum
at
a
temperature
below
40°C,
and
the
extracts
were
stored
at
-80°C
until
use.
For
the
purification
of
the
phenolic
fraction,
the
total
aqueous
extract
was
percolated
through
a
polyamide
column.
The
column
was
washed
with
water
and
the
purified
fraction
was
eluted
with
96%
ethanol.
2.3.
Liquid
Chromatography-Mass
Spectrometry
(LC-MS)
Analysis.
The
samples
of
purified
phenolic
fraction
were
dissolved
in
HPLC-grade
methanol
and
filtered
through
a
Millex-HV
Durapore
(PVDF)
membrane
(0.22
ym)
before
being
injected
into
the
LC-MS
instrument.
Mass
spec-
tral
analyses
were
carried
out
using
the
Ultraperformance
LC-Quadruple
Time
of
Flight
(UPLC-QTOF)
instrument
(Waters
Premier
QTOF,
Milford,
MA,
USA),
with
the
UPLC
column
connected
online
to
a
PDA
detector
(Waters
Acquity),
and
then
to
an
MS
detector
equipped
with
an
electrospray
ion
(ESI)
source
(used
in
ESI-negative
mode).
Separation
was
performed
on
a
2.1
x
50
mm
i.d.,
1.7
ym
UPLC
BEH
C18
column
(Waters
Acquity).
The
chromatographic
and
MS
parameters
were
as
follows:
the
mobile
phase
consisted
of
0.1%
formic
acid
in
water
(phase
A)
and
0.1%
formic
acid
in
acetonitrile
(phase
B).
The
linear
gradient
program
was
as
follows:
100%
to
95%
A
over
0.1
min,
95%
to
5%
A
over
9.7
min,
held
at
5%
A
over
3.2
min,
and
then
returned
to
the
initial
conditions
(95%
A)
in
4.2
min.
The
flow
rate
was
0.3
mL
min
-1
and
the
column
was
kept
at
35°C.
Masses
of
the
eluted
compounds
were
detected
with
a
QTOF
Premier
MS
instrument.
The
UPLC-MS
runs
were
carried
out
at
the
following
settings:
capillary
voltage
of
2.8
kV,
cone
voltage
of
30
eV,
and
collision
energy
of
5
eV.
Argon
was
used
as
the
collision
gas.
The
m/z
range
was
70
to
1,000
D.
The
MS
system
was
calibrated
using
sodium
formate
and
Leu-enkephalin
was
used
as
the
lock
mass.
The
MassLynx
software
version
4.1
(Waters)
was
used
to
control
the
instrument
and
calculate
accurate
masses.
2.4.
Cell
Culture
and
Experimental
Design.
The
human
T-
cell
leukemia
lymphoblastoid
Jurkat
cells
(DSMZ
ACC
282)
were
obtained
from
the
Deutsche
Sammlung
von
Mikro-
organismen
and
Zellkulturen
(DSMZ,
Braunschweig,
Ger-
many).
The
cells
were
grown
in
suspension
culture
at
37°C
under
5%
humidified
CO
2
in
bioactive
medium
RPMI
1640
(Gibco,
Grand
Island,
NY,
USA)
containing
inactivated
embryonic
bovine
serum
(Sigma,
St.
Louis,
MO,
USA),
L-glutamine
(4
mM),
penicillin
(100
U
mL
-1
),
and
strepto-
mycin
(100
U
mL
-1
).
The
experiments
were
carried
out
at
cell
densities
of
0.3
to
0.6
x
10
6
cells
mL
-1
.
In
order
to
imitate
the
oxidative
stress
conditions,
H
2
0
2
(Sigma)
was
added
to
the
Jurkat
culture
to
reach
the
concentrations
of
25
Oxidative
Medicine
and
Cellular
Longevity
3
and
50
yM,
corresponding
to
low
and
intermediate
stress
severity,
respectively
[15]
.
In
the
unstressed
control
treatment,
water
was
added
to
the
samples
instead
of
H
2
0
2
.
The
S.
hortensis
extracts
were
added
to
the
cultures
at
a
rate
of
2
mg
mL
-1
as
the
H
2
0
2
was
added.
In
a
separate
trial,
the
effect
of
cell
pretreatment
with
S.
hortensis
extract
on
their
response
to
subsequent
H
2
O
;
oxidative
stress
was
investigated.
Cell
suspensions
(2
x
10
cells
mL
-1
)
were
incubated
with
S.
hortensis
rosmarinic
acid
fraction
as
described
above.
After
the
incubation
period,
the
cells
were
harvested
by
centrifugation
at
1500
g
for
5
minutes,
washed,
resuspended
in
fresh
medium,
and
exposed
to
H
2
02.
Cellular
responses
to
oxidative
stress
were
evaluated
by
free
radicals
generation
and
cell
viability
as
described
below.
2.5.
Hydrogen
Peroxide
Scavenging
Capacity.
The
ability
of
S.
hortensis
extracts
to
scavenge
hydrogen
peroxide
in
the
absence
of
cells
was
tested
in
order
to
check
possible
contribution
of
this
abiotic
H
2
0
2
decomposition
to
exper-
imental
results.
The
H
2
0
2
-scavenging
capacity
of
extracts
was
tested
as
described
by
Ruch
et
al.
[21].
A
solution
of
hydrogen
peroxide
50
yM
was
prepared
in
phosphate
buffer
(pH
7.4).
Phenolic
extracts
(2
mg
mL
-1
)
in
distilled
water
and
50
yM
hydrogen
peroxide
solution
were
added
to
incubation
system
comprising
bioactive
medium
RPMI
1640
(GIBSO)
with
inactivated
embryonic
bovine
serum
(Sigma),
L-glutamine
(4
mM),
penicillin
(100
U
mL
-1
),
and
strepto-
mycin
(100
U
mL
-1
).
Absorbance
of
hydrogen
peroxide
at
230
nm
was
determined
10
minutes
later
against
a
blank
solution
containing
the
incubation
medium
with
hydrogen
peroxide.
The
percentage
of
hydrogen
peroxide
scavenging
by
S.
hortensis
extracts
was
calculated.
The
trial
revealed
a
17%
reduction
of
H
2
0
2
concentration
due
to
the
interaction
with
S.
hortensis
rosmarinic
acid
fraction.
2.6.
Electron
Paramagnetic
Resonance
(EPR)
Spectroscopy.
The
effect
of
S.
hortensis
extracts
on
the
generation
of
free
radicals
in
H
2
0
2
-challenged
and
unchallenged
cells
was
studied
using
the
electron
paramagnetic
resonance
(EPR)
method.
EPR
spectra
were
registered
on
a
radiospectrometer,
RE
1307
(EPSI,
Chernogolovka,
Russia).
Peroxyl
radicals
were
detected
with
spin-trap
a-phenyl-tertbutylnitrone
(PBN;
Sigma)
(50
mM
on
0.6
x
10
6
cells
in
0.5
mL
medium)
at
room
temperature
at
microwave
power
(20
mV).
Superoxide
radi-
cals
were
detected
with
a
spin-trap
5,5
dimethyl-I-pyrrolyn-
IV-oxide
(DMPO)
(Sigma)
(50
mM
on
0.6
x
10
6
cells
in
0.5
mL
medium)
at
room
temperature
at
microwave
power
(20
mV).
2.7
Cell
Viability
and
Proliferation.
The
viability
of
the
cells
was
determined
using
the
MTT
cell
proliferation
assay.
Cell
suspensions
(2
x
10
6
cells
mL
-1
)
were
incubated
with
H
2
0
2
and
S.
hortensis
preparations
as
described
above.
After
the
incubation
period,
the
cells
were
harvested
by
centrifu-
gation
at
1500
g
for
5
minutes,
washed,
and
resuspended
in
fresh
medium.
The
8
mg
mL
-1
solution
of
3-(4,5-dimethyl-
thiazol-2)-2,5-diphenyltetrazolium
bromide
(MTT)
(Sigma)
in
buffer
(140
mM
NaCl,
5
mM
HEPES,
pH
7.4)
was
added
to
the
cell
suspension
at
a
rate
of
30
yL
per
100
yL
suspension
and
the
mixture
was
incubated
for
4h
at
37
°
C
in
a
5%
CO
2
atmosphere.
After
this
incubation,
the
supernatant
was
carefully
removed
and
the
colored
formazan
crystals
produced
from
the
MTT
were
dissolved
in
100
yL
of
dimethyl
sulfoxide
(DMSO).
The
absorption
values
of
the
solutions
were
measured
at
570
nm.
The
distribution
of
the
Jurkat
cells
among
the
different
cell-cycle
phases
was
studied
using
flow
cytometry.
Mitochondrial
transmembrane
potential
(AT)
in
the
cell
culture
was
determined
by
flow
cytometry
using
the
lipophilic
cation
test
3,3'
-dihexyloxacarbocyanine
iodide
(DiOC
6
)
described
by
Zamzami
et
al.
[22].
2.8.
Antioxidant
Enzymes.
Jurkat
cell
extract
was
prepared
by
centrifuging
the
cell
suspensions
at
500
g
and
then
homogenizing
the
cellular
precipitate
in
a
lysis
buffer
(pH
7.9)
that
was
comprised
of
1.5
mM
MgCl
2
,
10
mM
KC1,
1
mM
dithiothreitol,
1
yg
mL
-1
leupeptin,
1
yg
mL
-1
aprotinin,
and
10
mM
HEPES.
The
volume
of
the
buffer
was
twice
the
volume
of
the
precipitate.
Lysis
of
the
cells
was
performed
by
passing
the
suspension
through
a
27-gauge
needle
10
times.
The
obtained
homogenate
was
centrifuged
for
20
min
at
10,000
g.
The
supernatant
was
used
to
determine
the
levels
of
enzyme
activity.
Catalase
(EC
1.11.1.6)
activity
was
measured
spectrophotometrically
as
the
decomposition
of
H
2
0
2
at
240
nm
[23].
One
unit
of
catalase
activity
was
defined
as
the
amount
of
enzyme
decomposing
1
ymol
of
H
2
0
2
per
minute.
The
superoxide
dismutase
(SOD;
EC
1.15.1.1)
was
assayed
using
NADPH
and
phenazine
methosulfate
(PMS)
reagents
for
the
reduction
of
nitroblue
tetrazolium
salt
(NBT)
into
blue-colored
formazon
measured
spectrophotometrically
at
560
nm
[24].
One
unit
of
SOD
activity
was
defined
as
the
amount
of
enzyme
oxidizing
1
nmol
NADPH
per
minute.
The
activity
of
both
enzymes
was
expressed
in
terms
of
units
per
mg
of
protein.
A
total
protein
micro
Lowry
kit
(Sigma)
was
used
to
determine
the
protein
content.
2.9.
Interleukin
Analysis.
Jurkat
cells
were
prestimulated
by
incubation
with
50
yg/mL
phytohemagglutinin
(PHA)
at
37°C
for
5
min
and
cultured
for
24
h
with
nonstimulated
Jurkat
cells
(40%
stimulated
and
60%
non-stimulated
cells).
The
pro-
and
anti-inflammatory
cytokines
IL-2
and
IL-10
were
assayed
using
ELISA
kits
(Bender
Medsystems,
Vienna,
Austria)
and
the
Multiscan
microplate
reader
(LabSystem,
Helsinki,
Finland).
2.10.
Statistics.
The
trials
were
performed
in
five
replications.
The
statistical
analysis
of
the
obtained
results,
including
calculation
of
means
and
standard
deviations,
was
conducted
using
the
IBM
SPSS
Statistics
program.
The
statistical
sig-
nificance
of
the
differences
between
the
means
was
analyzed
by
pair-wise
comparison
of
treatment
results
with
nontreated
control
using
Student's
t-test
at
P
<
0.05.
4
Oxidative
Medicine
and
Cellular
Longevity
0.4
-
100
161.022
mi.
179
<
mi.
161
nt/z
197
I
->
O
01
OH
OH
359.78
OH
HO
135.0
179.038
0.2
-
197.050
150
200 250
300 350
2
+
3
m/z
4
0
I I I I I I I I I 1 1 1 1 1
0
5
10
(min)
FIGURE
1:
HPLC
chromatogram
of
the
S.
hortensis
rosmarinic
acid
fraction.
The
peak
5
represents
rosmarinic
acid
and
the
peak
2
+
3
partially
separated
naringin
and
hesperidin.
The
peaks
1
and
4
were
tentatively
identified
as
rutin
and
apigenin-7-glucoside,
respectively.
Insert:
mass-
spectrum
of
the
rosmarinic
acid
and
its
fragmentation
scheme.
3.
Results
3.1.
Analysis
of
S.
hortensis
Extracts.
HPLC
analysis
revealed
a
number
of
phenolic
compounds
in
the
ethanolic
extract
of
S.
hortensis,
rosmarinic
and
ferulic
acids
being
the
major
compounds.
In
addition,
a
number
of
phenolic
acids
(caffeic,
p-coumaric),
flavonoid
aglycones
(catechin,
epicatechin,
lute-
olin,
apigenin),
and
glycosides
(rutin,
hesperidin,
apigenin-7-
glucoside)
were
tentatively
identified
in
the
ethanolic
extract.
Partial
purification
of
the
rosmarinic
acid
provided
a
fraction
comprising
four
major
peaks.
The
tentative
identification
of
the
rosmarinic
acid
as
the
most
abundant
component
of
the
fraction
was
based
on
its
UV
absorption
spectrum
and
retention
time
as
compared
with
those
of
the
authentic
standard
sample.
The
identity
was
confirmed
by
LC-MS
based
on
the
presence
of
a
deprotonated
molecular
ion
[M-H]
-
at
m/z
359
and
characteristic
fragment
ions
at
m/z
123,
m/z
135,
m/z
161,
m/z
179,
and
m/z
197,
in
accordance
with
data
in
the
literature
[25,
26]
and
fragmentation
scheme
(Figure
1).
Two
flavonoid
glycosides
were
identified
by
LC-MS
through
comparisons
with
standard
samples
as
hesperidin
based
on
a
[M-H]
-
at
m/z
609,
a
characteristic
hesperetin
fragment
ion
at
m/z
301,
naringin
based
on
[M-H]
-
at
m/z
579,
and
a
characteristic
naringenin
fragment
ion
at
m/z
271.
In
addition,
two
more
flavonoid
glycosides
were
tentatively
identified
in
the
fraction
as
rutin
and
apigenin-7-glucoside.
H
2
0
2
-Induced
Oxidative
Stress
as
Affected
by
S.
hortensis
Extracts.
The
addition
of
25
or
5012M
of
hydrogen
peroxide
caused
oxidative
stress
in
the
Jurkat
cells,
which
was
mani-
fested
as
the
generation
of
superoxide
and
peroxyl
radicals
that
could
be
detected
by
EPR
spectroscopy.
The
amount
of
radicals
formed
depended
on
the
concentration
of
H202;
no
radicals
were
detected
in
the
absence
of
hydrogen
peroxide
(Table
1).
Chloroform
and
ethyl
acetate
extracts
of
S.
hortensis
had
only
limited
effects
on
the
oxidative
state
of
the
cells,
slightly
reducing
the
amount
of
radicals
detected
at
higher
hydrogen
peroxide
concentrations.
On
the
other
hand,
considerable
alleviation
of
the
oxidative
stress
and
almost
complete
elimination
of
the
radicals
were
observed
in
the
presence
of
the
ethanolic
E.
hortensis
extract.
Significant
antioxidant
effects
were
also
associated
with
the
aqueous
extract
and
with
the
partially
purified
rosmarinic
acid
fraction,
although
the
efficacy
of
the
latter
preparation
was
markedly
lower
than
that
of
the
crude
ethanolic
extract.
In
line
with
these
findings,
the
total
aqueous
extract
of
S.
hortensis
doubled
the
activity
of
the
antioxidant
enzymes
catalase
and
superoxide
dismutase
in
the
Jurkat
cells,
even
in
the
absence
of
exogenous
hydrogen
peroxide
(Figure
2).
3.2.
Effects
on
Jurkat
Cell
Viability.
In
the
absence
of
any
exo-
genous
H
2
0
2
challenge,
adding
ethanolic
S.
hortensis
extract
or
the
purified
phenolic
fraction
to
Jurkat
cells
slightly
improved
their
viability.
Other
S.
hortensis
extracts
had
no
significant
effects
on
the
viability
of
unstressed
Jurkat
cells,
as
measured
by
the
MTT
test
(Table
2).
Hydrogen
peroxide-induced
oxidative
stress
reduced
the
viability
of
Jurkat
cells
in
a
dose-dependent
manner.
This
hydrogen
peroxide
effect
was alleviated
by
the
application
of
ethanolic
and
aqueous
extracts
of
S.
hortensis
and
by
the
phenolic
fraction.
The
aqueous
S.
hortensis
extract
was
the
most
effective
for
restoring
cell
viability
to
the
level
observed
in
the
unstressed
control
culture
(Table
2).
Oxidative
Medicine
and
Cellular
Longevity
5
TABLE
1:
Effects
of
S.
hortensis
extracts
and
of
the
partially
purified
rosmarinic-acid
fraction
on
the
generation
of
superoxide
(0,
-
)
and
peroxyl
(L00
.
)
radicals
in
Jurkat
cells
subjected
to
hydrogen
peroxide-induced
oxidative
stress.
0
0,
-
LOO*
Hydrogen
peroxide
concentration,
/4M
25
EPR
signal
intensity,
arbitrary
units
0,
-
LOU
50
0,
-
LOO*
No
additives
(control)
0
0
2.4
±
0.2
3.0
±
0.2
3.1
±
0.1
3.8
±
0.3
Chloroform
extract
0
0
2.0
±
0.2
3.2
±
0.2
2.1
±
0.2*
2.8
±
0.3*
Ethyl
acetate
extract
0
0
2.0
±
0.2
3.2
±
0.2
2.1
±
0.2*
2.8
±
0.3*
Ethanolic
extract
0
0
0*
0*
0.1
±
0.1*
0*
Aqueous
extract
0
0
0*
0.3
±
0.1*
0.1
±
0.1*
0.5
±
0.1*
Rosmarinic
acid
fraction
0
0
1.0
±
0.2*
2.2
±
0.2*
1.1
±
0.2*
1.8
±
0.3*
Values
represent
averages
of
five
replications
±
standard
deviations.
Values
marked
with
the
asterisk
are
significantly different
from
the
control
in
the
same
column
at
P
<
0.05,
according
to
Student's
t-test.
120
-
0
g
80
-
A.
0
P.
2
4
0
I
SOD
Catalase
0
No
additives
S.
hortensis
FIGURE
2:
Effect
of
the
total
aqueous
S.
hortensis
extract
on
the
activities
of
superoxide
dismutase
(SOD)
and
catalase
in
Jurkat
cells.
Error
bars
represent
standard
deviations
of
five
replications.
Bars
marked
with
an
asterisk
are
significantly
different
from
the
control
at
P
<
0.05,
according
to
Student's
t-test.
3.3.
Effect
of
Pretreatment
of
Jurkat
Cells
with
S.
hortensis
Extract
on
Subsequent
Cellular
Sensitivity
to
Oxidative
Stress.
The
data
presented
in
Table
3
demonstrate
that
pretreatment
of
Jurkat
cells
with
the
rosmarinic
acid
fraction
significantly
alleviated
the
oxidative
stress
incurred
to
cells
by
subsequent
exposure
to
hydrogen
peroxide,
as
expressed
by
free
radical
generation
and
decline
in
cell
viability.
This
alleviation
could
not
be
attributed
to
the
peroxide-scavenging
activity
of
the
extracts
because
no
direct
contact
of
the
extracts
with
the
peroxide
took
place
in
that
case.
In
addition,
the
direct
peroxide-scavenging
capacity
of
the
rosmarinic
acid
fraction
did
not
exceed
17%,
so
that
its
contribution
to
the
cell
protection
was
rather
limited.
3.4.
Effects
on
the
Cell
Cycle.
Oxidative
stress
changed
the
cell-cycle
phase
distribution
of
the
Jurkat
cells,
restricting
cell
proliferation
and
increasing
the
relative
proportions
of
G0/Gl
cells
(the
G0/Gl
arrest)
and
apoptotic
cells
among
the
total
cell
population.
These
trends
were
alleviated
by
the
addition
of
the
ethanolic
S.
hortensis
extract,
so
that
the
amount
of
apoptotic
cells
in
that
treatment
was
not
significantly
different
from
that
observed
in
the
unstressed
control
(Table
4).
Adding
the
S.
hortensis
extract
alone,
without
hydrogen
peroxide,
had
no
significant
effect
on
the
cell-cycle
phase
distribution
of
the
Jurkat
cells
(data
not
shown).
The
alleviation
of
H
2
0
2
-induced
apoptosis
by
the
ethanolic
S.
hortensis
extract
and
by
the
partially
purified
rosmarinic
acid
fraction
was
also
evident
from
the
index
of
mitochondrial
transmembrane
potential
determined
by
flow
cytometry
(Table
5).
3.5.
Interleukin
Production.
The
production
of
both
IL-2
and
IL-10
interleukins
by
Jurkat
cells
was
stimulated
by
hydrogen
peroxide
and
further
enhanced
by
the
addition
of
the
S.
hortensis
extract
and
its
phenolic
fraction
(Table
6).
4.
Discussion
Our
study
has
confirmed
that
rosmarinic
acid
is
an
abundant
phenylpropanoid
compound
in
summer
savory.
To
the
best
of
our
knowledge,
hesperidin
and
naringin
have
not
been
previously
reported
in
S.
hortensis,
but
they
have
been
found
in
other
Satureja
species
[27]
and
in
other
genera
of
this
family,
such
as
Mentha
[25].
The
present
research
has
demonstrated
for
the
first
time
that
S.
hortensis
and
its
rosmarinic
acid-rich
fraction
can
protect
Jurkat
cells
from
oxidative
stress
caused
by
hydrogen
peroxide.
These
findings
are
in
line
with
the
antioxidant,
cyto-
protective,
and
anti-inflammatory
activities
of
S.
hortensis
[7]
and
rosmarinic
acid
[9,
10]
that
have
been
observed
in
vivo
in
animals
and
humans.
Similar
protective
antioxidant
proper-
ties
were
exhibited
by
S.
hortensis
extracts
when
applied
to
H
2
0
2
-stressed
lymphocytes
isolated
from
blood
taken
from
healthy
rats
[28].
In
cell
cultures,
rosmarinic
acid
protected
6
Oxidative
Medicine
and
Cellular
Longevity
TABLE
2:
Effects
of
S.
hortensis
extracts
on
the
viability
of
Jurkat
cells
in
the
presence
or
absence
of
hydrogen
peroxide.
0
Hydrogen
peroxide
concentration,
/4M
25
50
MTT
test
results,
A
570
No
additives
(control)
0.69
±
0.02
0.36
±
0.01
0.22
±
0.01
Chloroform
extract
0.63
±
0.02
0.37
±
0.01
0.24
±
0.03
Ethyl
acetate
extract
0.58
±
0.05
0.47
±
0.03
0.42
±
0.03*
Ethanolic
extract
0.74
±
0.03*
0.56
±
0.05*
0.46
±
0.03*
Aqueous
extract
0.61
±
0.01
0.68
±
0.04*
0.67
±
0.02*
Rosmarinic
acid
fraction
0.75
±
0.04*
0.62
±
0.03*
0.42
±
0.04*
Values
represent
averages
of
five
replications
±
standard
deviations.
Values
marked
with
the
asterisk
are
significantly different
from
the
control
in
the
same
column
at
P
<
0.05,
according
to
Student's
t-test.
TABLE
3:
Effect
of
pretreatment
of
the
Jurkat
cells
with
partially
purified
S.
hortensis
rosmarinic
acid
fraction
on
the
cellular
response
to
subsequent
hydrogen
peroxide-induced
oxidative
stress.
0
Hydrogen
peroxide
concentration,
/4M
25
50
Nontreated
control
Rosmarinic
acid
fraction
Peroxyl
radicals
generation,
EPR
signal
intensity
(arbitrary
units)
0
3.0
±
0.2
0
1.9
±
0.2*
3.8
±
0.3
2.1
±
03*
Nontreated
control
Rosmarinic
acid
fraction
0.69
±
0.02
0.75
±
0.04*
Cell
viability
(MTT
test
results,
A570)
0.36
±
0.01
0.59
±
0.04*
0.22
±
0.01
0.37
±
0.03*
Values
represent
averages
of
five
replications
±
standard
deviations.
Values
marked
with
the
asterisk
are
significantly different
from
the
control
in
the
same
column
at
P
<
0.05,
according
to
Student's
t-test.
TABLE
4:
Effects
of
hydrogen
peroxide
and
of
the
ethanolic
S.
hortensis
extract
on
the
cell-cycle
phase
distribution
of
Jurkat
cells.
Cell-cycle
phases,
%
GO/G1
S
G2/M
GO/Apoptosis
No
additives
(control)
23.8
±
3.4
54.5
±
3.3
19.0
±
2.9
2.7
±
3.6
H
2
0
2
25µM
42.3
±
3.3*
36.7
±
3A*
12.5
±
1.7*
8.5
±
1.9*
H
2
0
2
25µM
+
S.
hortensis
(ethanolic
extract)
37.5
±
2.5*
43.0
±
3.3*
16.0
±
3.4
3.5
±
1.3
Values
represent
averages
of
five
replications
±
standard
deviations.
Values
marked
with
the
asterisk
are
significantly different
from
the
control
in
the
same
column
at
P
<
0.05,
according
to
Student's
t-test.
human
neuronal
cells
against
hydrogen
peroxide-induced
apoptosis
[29]
and
inhibited
in
a
dose-dependent
manner
the
formation
of
reactive
oxygen
and
nitrogen
species
in
RAW264.7
macrophages
stimulated
with
lipopolysaccharide
or
phorbol
12-myristate
13-acetate
[30].
On
the
other
hand,
in
a
previous
study,
rosmarinic
acid
failed
to
protect
Jurkat
cells
from
H
2
0
2
-mediated
oxidative
damage
and
actually
induced
their
apoptosis
[19,
20].
Such
prooxidant
cytotoxic
reactions
in
cell
cultures
are
associated
with
the
generation
of
H
2
0
2
through
the
interaction
of
phenolic
compounds
with
culture
media
ingredients
(e.g.,
transient
metals)
and
can,
therefore,
be
considered
artifacts
[31,
32].
Inclusion
of
catalase
or
metmyoglobin
in
the
growth
medium
negates
these
reactions
and
allows
the
realization
of
the
cytoprotective
antioxidant
potential
of
phenolic
com-
pounds
[31].
One
possible
explanation
for
the
apparent
discrepancy
between
our
results
and
those
of
Kolettas
et
al.
[19]
might
be
that
the
high
dose
of
antioxidant
materials
used
in
our
study
could
overcome
the
influence
of
H202,
either
added
exogenously
or
generated
in
cell
cultures
with
participation
of
transient
metals.
Indeed,
in
a
metal-catalyst
system,
most
phenolic
compounds
exhibited
pro-oxidant
effects
at
low
doses
and
shifted
to
antioxidant
activity
at
higher
concen-
trations
[33].
Furthermore,
it
was
shown
recently
that
high
doses
(2-3
mM)
of
caffeic
acid
and
other
phenylpropanoids
protected
Jurkat
cells
from
H
2
0
2
-induced
DNA
damage
by
chelating
intracellular
labile
iron
[34].
The
presence
of
the
potent
flavonoid
antioxidants
in
the
phenolic
fraction,
in
addition
to
rosmarinic
acid,
might
further
strengthen
its
antioxidant
capacity.
Enhancement
of
the
activity
of
the
antioxidant
enzymes
by
S.
hortensis
(Figure
2)
might
Oxidative
Medicine
and
Cellular
Longevity
7
TABLE
5:
Effects
of
ethanolic
S.
hortensis
extract
and
of
the
partially
purified
rosmarinic-acid
fraction
on
the
incidence
of
apoptosis
in
Jurkat
cells
in
the
presence
of
hydrogen
peroxide.
Cell
counts
Healthy
Apoptotic
K
ratio*
No
additives
(control)
212
8
26.5
H
2
0
2
25µM
H
2
0
2
25µM
+
S.
hortensis
(ethanolic
extract)
H
2
0
2
25µM
+
rosmarinic
acid
fraction
268
2090
1211
3519
539
108
0.08
3.9
11.2
*K-ratio
of
healthy
to
apoptotic
Jurkat
cells.
TABLE
6:
Effects
of
the
ethanolic
S.
hortensis
extract
and
of
the
partially
purified
rosmarinic-acid
fraction
on
the
production
of
interleukins
by
Jurkat
cells
in
the
presence
of
hydrogen
peroxide.
normal
antioxidant
enzyme
activity,
inhibited
lipid
peroxi-
dation,
and
increased
the
HDL
levels
in
the
treated
animals,
resulting
in
the
alleviation
of
disorders
and
enhanced
immu-
nity.
Rosmarinic
acid
increased
the
secretion
of
IL-10
in
a
lipopolysaccharide-stimulated
macrophage
model
[41].
Addition
of
the
S.
hortensis
extract
or
its
phenolic
fraction
restored
the
viability
and
proliferation
of
H
2
0
2
-challenged
Jurkat
cells,
alleviated
the
GO/G1
arrest,
and
controlled
the
apoptosis
of
these
cells.
Altogether,
these
phenomena
were
in
line
with
the
general
scheme
of
cellular
response
to
oxidative
stress,
implying
that
low
doses
of
reactive
oxygen
species
pro-
mote
cell
proliferation,
intermediate
doses
result
in
growth
arrest,
and
severe
oxidative
stress
ultimately
causes
cell
death
via
apoptotic
or
necrotic
mechanisms
[42].
Apparently,
the
addition
of
S.
hortensis
extracts
alleviated
the
oxidative
stress
exerted
on
the
cells
by
hydrogen
peroxide.
These
effects
may
be
attributed
to
the
direct
radical-scavenging
activity
of
rosmarinic
acid
and
other
phenolic
compounds,
as
well
as
to
indirect
mechanisms
such
as
the
enhancement
of
antioxidant
enzymes
and
the
release
of
anti-inflammatory
signaling
molecules,
such
as
IL-10.
IL-2,
pg
IL-10,
pg
No
additives
(control)
0.90
±
0.05
3.21
±
0.04
H
2
0
2
25µM
H
2
0
2
25µM
+
S.
2.61
±
0.04*
6.80
±
0.05*
hortensis
(ethanolic
15.30
±
0.04*
extract)
H
2
0
2
25µM
+
S.
hortensis
rosmarinic
20.80
±
0.07*
acid
fraction
Values
represent
averages
of
five
replications
±
standard
deviations.
Values
marked
with
the
asterisk
are
significantly
different
from
the
control
in
the
same
column
at
P
<
0.05,
according
to
Student's
t-test.
also
contribute
to
the
neutralization
of
hydrogen
peroxide.
Catalase
and
SOD
play
important
roles
in
the
control
of
oxidative
stress
and
apoptosis
in
Jurkat
cells
[35].
Similar
to
our
findings,
an
aqueous
extract
of
another
rosmarinic
acid-
containing
Lamiaceae
herb,
Perilla
frutescens,
was
shown
to
upregulate
the
mRNA
and
protein
expression
of
these
antioxidant
enzymes
in
cultured
human
vein
endothelial
cells
[36].
Another
noteworthy
phenomenon
observed
in
this
work
was
a
parallel
increase
in
the
levels
of
the
IL-2
and
IL-10
interleukins.
Robust
production
of
IL-2
is
the
major
trait
of
the
Jurkat
cell
line
[12].
There
is
a
synergistic
interaction
between
these
two
interleukins
during
the
immune
response
[37].
Anti-inflammatory
factors
such
as
IL-10
may
be
released
in
order
to
balance
the
dramatic
increase
in
proinflammatory
cytokines
in
stressful
situations,
and
thereby
control
the
magnitude
and
duration
of
the
inflammatory
response
[38].
Interestingly,
adding
antioxidant-rich
plant
materials
to
the
diets
of
animals
enduring
proinflammatory
conditions
has
been
shown
to
increase
the
level
of
IL-10
[39]
or
the
levels
of
both
IL-2
and
IL-10
[40]
in
parallel
with
a
decrease
in
the
levels
of
pro-inflammatory
factors,
such
as
IL-6,
TNF-a,
and
IL-43.
In
addition,
these
dietary
interventions
preserved
5.
Conclusions
The
present
research
has
demonstrated
that
rosmarinic
acid-
rich
extract
of
S.
hortensis
can
protect
Jurkat
cells
from
oxidative
stress
caused
by
hydrogen
peroxide.
These
findings
are
in
line
with
the
antioxidant,
cytoprotective,
and
anti-
inflammatory
activities
of
rosmarinic
acid
that
have
been
observed
in
animals
and
humans.
Therefore,
the
H
2
0
2
-
challenged
Jurkat
cells
may
serve
a
model
for
investigating
cellular
mechanisms
of
cytoprotective
effects
of
phytonutri-
ents.
It
should
be
kept
in
mind,
however,
that
these
results
were
achieved
with
a
rather
high
concentration
of
rosmarinic
acid
that
supposedly
could
overcome
the
culture-associated
artifacts.
Further
research
is
needed,
in
order
to
optimize
the
experimental
system.
Acknowledgments
The
authors
are
grateful
to
Dr.
Mira
Weissberg,
Institute
of
Plant
Sciences,
The
Volcani
Center,
ARO,
for
performing
the
LC-MS
analysis.
The
Israeli
partners
acknowledge
the
finan-
cial
support
by
the
Center
of
Absorption
in
Science,
Ministry
of
Immigration
Absorption,
Israel
(KAMEA
Program).
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