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Chemical
composition,
biological
and
cytotoxic
activities
of
Cistus
salviifolius
flower
buds
and
leaves
extracts
Salma
Kammoun
El
Euch
,
Jalloul
Bouajila
,
Nabiha
Bouzouita
a
Ecole
Supérieure
des
Industries
Alimentaires,
58,
Avenue
Alain
Savary,
1003,
Cité
El-Khadra,
Tunis,
Tunisie
b
Laboratoire
de
Chimie
Organique
Structurale:
Synthèse
et
Etude
Physicochimique-Faculté
des
sciences
de
Tunis,
Tunisie
c
Université
de
Toulouse,
Université
Paul-Sabatier,
Faculté
de
pharmacie
de
Toulouse,
Laboratoire
des
IMRCP
UMR
CNRS
5623,
118
route
de
Narbonne,
F-31062
Toulouse,
France
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
11
May
2015
Received
in
revised
form
22
July
2015
Accepted
15
August
2015
Available
online
27
August
2015
Keywords:
Cistus
salviifolius
Leaves
Flower
buds
Antioxidant
activity
Biological
activities
Cytotoxicity
a
b
s
t
r
a
c
t
The
chemical
composition,
antioxidant
activity
(DPPH
•
,
ABTS
•+
and
FRAP
assays),
anti-xanthine
oxydase
(XOD),
anti-superoxide
dismutase
(SOD),
anti-acetylcholinesterase
(AChE),
anti-inflammatory
(anti-5-
lipoxygenase
(5-LOX))
and
cytotoxic
(OVCAR
and
MCF-7)
activities
comparison
between
the
leaves
and
the
flower
buds
(FB)
of
Cistus
salviifolius
separately
extracted
with
methanol
(MeOH)
80%
was
inves-
tigated.
The
highest
phenolics
content
(305.30
±4.68
mg
gallic
acid
equivalent
(E)/g
dry
weight
(dw))
and
flavonoids
(76.21
±
1.26
mg
quercetin
E/g
dw)
were
obtained
in
FB
extract,
however,
the
leaves
has
nine
times
higher
amount
of
tannins
(56.36
±
0.67
mg
catechin
E/g
dw)
and
more
important
concentra-
tion
of
anthocyanins
(0.31
±
0.02
mg
cyaniding-3-glucoside
E/g
dw).
It
was
found
that
FB
methanolic
extract
exhibited
better
antioxidant
(IC
50
=
5.11
±
0.53,
4.82
±
0.08
and
59.27
±
0.13
mg/L
assessed
by
DPPH
•
,
ABTS
•+
and
FRAP
tests,
respectively)
and
anti-SOD
(IP
(%)
=
58.39
±
11.67)
activities
than
leaves
extract.
Moreover,
FB
possessed
more
evident
cytotoxic
activity
against
OVCAR
and
MCF-7
cells
(IP
(%)
=
36.85
±
5.66
and
35.16
±
4.67%,
respectively)
in
comparison
to
leaves
which
were
inactive
at
a
con-
centration
of
50
mg/L.
However,
leaves
showed
more
powerful
inhibitory
activities
towards
the
enzymes
AChE
(IC
50
=
18
±
2.71
mg/L),
5-LOX
(IC
50
=
13.38
±
0.20
mg/L)
and
XOD
(IC
50
=
19.48
±
0.21
mg/L).
Results
showed
that
the
organ
factor
influenced
considerably
the
chemical
composition
content
and
the
biological
activities
of
C.
salviifolius.
©
2015
Elsevier
B.V.
All
rights
reserved.
1.
Introduction
The
Cistaceae
family,
known
as
rock-rose,
is
a
large
family
of
perennial
shrubs
commonly
distributed
in
the
Mediterranean
semi-arid
ecosystem
(
Most
species
of
this
family,
characterized
with
fragrant
and
sweet
smelling,
are
much
appreciated
in
the
perfume
industry
Abbreviations:
DPPH,
1,1-diphenyl-2-picrylhydrazyl;
ABTS,
2,2’-azinobis-3-
ethylbenzothiazoline-6-sulphonic
acid;
FRAP,
Ferric
reducing
antioxidant
power;
XOD,
xanthine
oxidase;
SOD,
superoxide
dismutase;
AChE,
acetylcholinesterase;
5-LOX,
5-lipoxygenase;
MeOH,
methanol;
FB,
flower
buds;
dw,
dry
weight;
TPC,
total
phenolic
content;
FC,
folin
Ciocalteu;
TFC,
total
flavonoid
content;
CTC,
con-
densed
tannin
content;
TAC,
total
anthocyanin
content;
GAE,
gallic
acid
equivalent;
QE,
quercetin
equivalent;
CE,
catechin
equivalent;
C-3GE,
cyanidin-3-glucoside
equivalent;
NDGA,
nordihydroguaiaretic
acid;
MTT,
3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium
bromide;
PBS,
phosphate
buffered
saline.
∗ Corresponding
author.
Fax:
+33
5622
56826.
∗∗ Corresponding
author.
Fax:
+21
6711
71192.
addresses:
jalloul.bouajila@univ-tlse3.fr
(J.
Bouajila),
(N.
Bouzouita).
and
for
ornamen-
tal
purposes
Cistus
species
have
been
used
since
ancient
times
in
traditional
folk
medicine
Catalán
as
anti-inflammatory
anti-ulcerogenic,
wound
healing,
anti-microbial
(
antifungal
antiviral,
anti-tumor
cytotoxic
(
anti-nociceptive
Among
the
Cistaceae
family,
Cistus
salviifolius
L.,
a
shrub
belongs
to
the
Leucocistus
subgenus,
is
traditionally
used
as
an
astringent
and
cicatrizing
agent
in
some
countries
of
the
Mediterranean
area
in
addition
to
being
used
as
a
tea
substitute
In
Jor-
dan,
it
has
been
utilized
for
the
treatment
of
gout
(
which
develops
because
of
the
deposition
of
uric
acid
in
the
form
of
urate
monohydrate
crystals
in
the
synovial
joints
during
purine
catabolism
by
xanthine
oxidase
(XOD)
which
catalyses
the
conversion
of
hypoxanthine
to
xanthine
and
xanthine
to
uric
acid
with
concomitant
production
of
hydrogen
per-
oxides
and
superoxides
anions
as
byproducts
Constituents
from
C.
salvifolius
(Cistaceae)
activate
also
peroxisome
http://dx.doi.org/10.1016/j.indcrop.2015.08.033
0926-6690/©
2015
Elsevier
B.V.
All
rights
reserved.
S.K.
El
Euch
et
al.
/
Industrial
Crops
and
Products
76
(2015)
1100–1105
1101
Table
1
Extraction
yields
determination.
Reference
Extraction
yields
(%)
FB
30.20
L
21.80
FB:
Flower
buds;
L:
Leaves.
proliferator-activated
receptor-y
and
stimulate
glucose
uptake
by
adipocytes
Research
in
Turkey
showed
that
C.
salviifolius
possess
great
efficacy
towards
ulcers
As
it
has
been
demonstrated,
in
Marocco,
that
it
exhibited
anti-mycobacterial
activity
against
Mycobacterium
aurum
A+
and
Mycobacterium
smegmatis
MC2
155
(
Cyclohexane
carboxylic
acids
(quinic
and
shikimic
acids),
flavanols
(catechins),
betuloside
(rhododendrin),
ellagitannins
(punicalagins)
and
glycosylated
flavonols
(myricetin
and
quercetin
glycosides)
were
the
most
abundant
compounds
in
C.
salviifolius
growing
in
Spain
have
reported
that
the
simultaneous
presence
of
ellagitannins,
and
flavonoid
glycosides,
may
preserve
sensitive
targets
in
C.
salvi-
ifolius
leaves
from
photochemical
damage
and
as
a
consequence,
this
plant
not
only
serve
a
key
ecological
function
in
the
stabiliza-
tion
of
highly-disturbed
ecosystems,
but
may
be
a
very
interesting,
and
still
not
fully
explored,
source
of
metabolites
with
potential
use
in
human
health
care.
The
aim
of
the
presented
study
was
to
evaluate
and
com-
pare
leaves
and
flower
buds
of
C.
salviifolius
separately
extracted
with
MeOH
80%
in
terms
of
(1)
their
total
content
of
phe-
nolics,
flavonoids,
tannins,
and
anthocyanins
and
(2)
their
antioxidant
(DPPH
•
,
ABTS
•+
,
FRAP),
anti-inflammatory,
anti-
acetylcholinesterase,
anti-superoxide
dismutase,
anti-xanthine
oxidase,
and
cytotoxic
activities
(see
also
To
our
knowledge,
no
studies
have
been
performed
on
the
flower
buds
of
C.
salviifolius.
2.
Materials
and
methods
2.1.
Chemicals
used
All
the
chemicals
used
were
of
the
analytical
reagent
grade.
All
reagents
were
purchased
from
Sigma–Aldrich–Fluka
(Saint-
Quentin,
France).
2.2.
Plant
material
collection
C.
salviifolius
leaves
and
flower
buds
(FB)
were
collected
in
March
2013
from
Nahli
mount
located
in
Ariana
governorship
in
Tunisia.
The
material
was
authenticated
by
Dr
Nadia
Ben
Brahim
(botanical
laboratory
and
ornamental
plants,
National
Institute
for
Agricul-
ture
Research
in
Tunis)
and
voucher
specimens
were
deposited
in
laboratory
of
valorisation
of
natural
substances
in
High
School
of
Food
Industries
in
Tunis.
2.3.
Methanolic
extract
preparation
Leaves
and
FB
were
separately
subjected
to
extraction
with
MeOH
80%.
25
g
of
dried
and
pulverised
plant
material
were
three
times
macerated
(for
24
h
each)
under
orbital
shaking
with
250
mL
of
solvent.
Extracts
were
combined,
filtered
and
evaporated
to
dry-
ness
under
vacuum
at
40
◦
C.
The
residue
amount
was
weighed
and
expressed
in
follows:
EY(%)
=
M
1
−
M
0
m
×
100
where
EY
is
the
yield,
in
percentage,
of
the
dry
residue
amount
extraction;
M
1
is
the
weight,
in
grams,
of
the
residue
and
the
round
bottomed
flask
after
vacuum
evaporation;
M
0
is
the
weight,
in
grams,
of
the
empty
round
bottomed
flask;
and
m
is
the
weight,
in
grams,
of
the
test
sample.
Extracts
were
kept
in
amber
vials
and
stored
at
−18
◦
C
for
further
analysis.
2.4.
Total
phenolic
content
determination
The
total
phenolic
content
(TPC)
of
the
different
extracts
was
assessed
by
spectrometry
using
the
“Folin
Ciocalteu”
(FC)
reagent
assay
20
L
of
the
diluted
extract
solution
was
mixed
with
100
L
of
FC
reagent
(0.2N).
The
mixture,
prepared
in
96-well
plate,
was
agitated
for
30
s
and
rested
in
the
darkness
for
5
min.
80
L
of
Na
2
CO
3
(75
g/L)
was
added.
After
agitation
and
incubation
during
15
min,
the
absorbance
was
measured
at
765
nm.
Gallic
acid
(GA)
(0–30
mg/L)
was
used
as
a
standard
for
the
calibra-
tion
curve.
The
total
phenolic
content
was
expressed
as
milligram
of
GA
equivalent
per
gram
of
dry
weight
(mg
GAE/g
dw).
2.5.
Total
flavonoid
content
determination
Total
flavonoid
content
(TFC)
was
measured
according
to
Each
extract
solution
(100
L)
was
mixed
with
an
equal
volume
of
2%
AlCl
3
methanolic
solution.
After
incu-
bation
for
15
min,
the
absorption
was
measured
at
415
nm.
The
amount
of
total
flavonoids
in
different
extracts
is
expressed
as
mil-
ligram
of
quercetin
equivalent
per
gram
of
dry
weight
(mg
QE/g
dw)
and
was
determined
from
a
standard
curve
of
quercetin
ranged
from
2
to
10
mg/L.
2.6.
Total
condensed
tannin
content
determination
To
evaluate
the
condensed
tannin
content
(CTC),
the
vanillin
method
was
used
(
50
L
of
extract
solution
was
added
to
100
L
of
freshly
prepared
vanillin
solution
(1%
in
7
M
H
2
SO
4
)
and
then
incubation
in
darkness
during
15
min.
The
absorbance
was
measured
at
500
nm.
The
concentration
was
cal-
culated
as
milligram
catechin
equivalent
per
gram
of
dry
weight
(mg
CE/g
dw)
from
a
calibration
curve.
2.7.
Total
anthocyanin
content
determination
Total
anthocyanin
content
(TAC)
was
quantified
with
the
pH
dif-
ferential
absorbance
method
(
The
absorbance
of
each
extract
was
measured
at
510
and
700
nm
in
two
buffers
at
pH
1.0
(hydrochloric
acid-potassium
chloride,
0.2
M)
and
4.5
(acetic
acid-sodium
acetate,
0.2
M)
after
15
min
incubation
in
darkness
of
equal
volume
of
extract
solution
and
buffer
(100
L).
The
real
absorbance
was
calculated
as
follows:
A
=
(
A
510
−
A
700
)
pH1
− (A
510
−
A
700
)
pH4
.5
The
TAC
was
measured
using
the
molar
extinction
coef-
ficient
of
the
cyanidin-3-glucoside
(C3G)
used
as
a
standard
(
=
26900
L
mol
−1
cm
−1
)
and
its
molecular
weight
and
the
well’s
diameter.
Results
were
expressed
as
mg
C3GE/g
dw.
2.8.
DPPH
•
radical
scavenging
assay
The
hydrogen
atom
donation
ability
of
chemical
compounds
in
FB
and
leaves
was
measured
on
the
basis
to
scavenge
the
sta-
ble
free
radical
1,1-diphenyl-2-picrylhydrazyl
(
In
a
96-well
plates,
20
L
of
each
extract
solution,
at
a
con-
centration
ranging
from
1
to
80
mg/L,
were
added
to
180
L
of
a
freshly
prepared
80%
aqueous
methanolic
DPPH
•
solution
1102
S.K.
El
Euch
et
al.
/
Industrial
Crops
and
Products
76
(2015)
1100–1105
(0.2
mM).
The
mixture
was
incubated
in
the
dark
for
25
min
and
the
absorbance
was
measured
against
a
blank
at
524
nm.
The
free
radical-scavenging
activity
was
calculated
as
percent
inhibition
using
the
following
formula:
PI (%)
=
A
blank
−
A
sample
A
blank
×
100
where
A
blank
is
the
absorbance
of
the
control
negative
reaction
without
extract.
A
sample
is
the
absorbance
of
the
test
sample.
Extract
concentration
providing
50%
inhibition
(IC
50
)
of
the
initial
DPPH
•
concentration
was
calculated
using
the
linear
relation
between
the
compound
concentration
and
the
probability
of
the
percentage
of
DPPH
•
inhibition.
The
ascorbic
acid
was
used
as
a
positive
control.
2.9.
ABTS
•+
radical
scavenging
assay
The
2,2
-azinobis-3-ethylbenzothiazoline-6-sulphonic
acid
(ABTS)
radical
cation
was
generated
by
the
oxidation
of
ABTS
with
potassium
persulfate,
and
its
reduction
in
the
presence
of
hydrogen-donating
antioxidants
is
measured
spectrophotomet-
rically
at
734
nm
Briefly,
mix
7
mM
solution
of
ABTS
at
pH
7.4
(5
mM
NaH
2
PO
4
,
5
mM
Na
2
HPO
4
and
154
mM
NaCl)
with
2.45
mM
potassium
persulfate
(final
concentrations)
followed
by
storage
in
the
dark
at
4
◦
C
for
16
h
before
use.
The
mixture
was
diluted
with
distilled
water
to
give
an
absorbance
ranging
between
0.70
and
0.9.
For
each
sample,
20
L
of
different
dilutions
(1–80
mg/L)
were
allowed
to
react
with
180
L
ABTS
•+
solution.
The
absorbance
was
measured
6
min
after
initial
mixing.
Free
radical
scavenging
ability
was
expressed
by
IC
50
(mg/L)
values
determined
as
described
previously
in
DPPH
•
assay.
Ascorbic
acid
was
used
as
a
standard.
2.10.
Ferric
reducing
antioxidant
power
assay
(FRAP)
The
reducing
power
of
the
extract
was
assessed
according
to
the
method
of
2
mL
of
various
concentra-
tions
(1–200
mg/L)
were
mixed
with
phosphate
buffer
(2.5
mL,
0.2
M)
and
2.5
mL
of
1%
potassium
ferricyanide
water
solution
K
3
[Fe(CN)
6
].
The
mixture
was
incubated
at
50
◦
C
during
30
min.
2.5
mL
of
trichloroacetic
acid
(10%
aqueous
solution)
were
added
to
the
mixture
which
was
subsequently
centrifuged
at
3000
rpm
for
10
min.
The
supernatant
(2.5
mL)
was
mixed
with
an
equal
volume
of
distilled
water
and
0.5
mL
of
a
freshly
prepared
FeCl
3
solution
(0.1%).
The
absorbance
was
measured
after
10
min
incubation
in
darkness,
at
700
nm.
IC
50
value
(mg/L)
is
the
effective
concentra-
tion
the
absorbance
was
0.5
for
reducing
antioxidant
power
and
was
obtained
by
interpolation
from
linear
regression
analysis.
The
ascorbic
acid
was
used
as
a
positive
control.
2.11.
Anti-inflammatory
activity
The
anti-inflammatory
activity
was
assessed
using
the
spec-
trophotometric
measurement
of
a
conjugated
diene
the
result
of
linoleic
acid
oxidation
by
the
enzyme
5-LOX
Briefly,
150
L
of
buffer
(pH
=
7.4)
(Na
2
HPO
4
,
2H
2
O;
KH
2
PO
4
;
NaCl)
were
mixed
with
20
L
of
extract
(50
mg/L
in
well),
60
L
of
linoleic
acid
and
20
L
of
5-LOX
enzyme
solution.
The
mixture
was
homog-
enized
and
incubated
for
10
min
at
25
◦
C.
The
absorbance
was
determined
at
234
nm
against
a
blank.
Nordihydroguaiaretic
acid
(NDGA)
was
used
as
a
standard,
IC
50
value
is
the
concentration
of
the
extract
that
caused
50%
enzyme
inhibition.
2.12.
Anti-acetylcholinesterase
activity
evaluation
Ellman’s
method
(
was
the
simple
tech-
nique
to
evaluate
the
activity
of
the
acetyl-cholinesterase
enzyme
(AChE).
Briefly,
50
L
of
buffer
A
(Na
2
HPO
4
,
12H
2
O
(0.1
M;
pH
=
8))
were
mixed
with
25
L
of
extract
solution
(50
mg/L),
125
L
of
5,5’-dithio-bis(2-nitrobenzoic
acid)
(DTNB)
solution
(3
mM,
pH
=
7)
prepared
in
Buffer
C
(Na
2
HPO
4
,
12H
2
O
(0.1
M,
pH
=
7))
and
25
L
of
the
enzyme
AChE
solution
(1.4
U/mL)
dissolved
in
buffer
B
(Na
2
HPO
4
,
12H
2
O
(0.02
M,
pH
=
7))
already
placed
in
an
ice
bath.
The
96-well
plate
was
placed
inside
the
plate
reader,
incubated
at
25
◦
C
with
agitation
during
15
min.
The
plate
was
recuperated
to
add
25
L
of
acetylthiocholine
iodidesolution
(15
mM,
in
Buffer
A)
and
left
to
incubate
for
10
min.
The
absorbance
was
read
at
412
nm
against
a
blank.
Galanthamine
was
used
as
a
positive
control
and
the
IC
50
value
is
the
concentration
of
the
extract
causing
50%
of
AChE
inhibition.
2.13.
Xanthine
oxidase
inhibition
(XOD)
assay
XOD
inhibition
was
determined
by
measuring
at
295
nm,
the
formation
of
uric
acid
from
xanthine
(
In
96-well
plate,
60
L
of
buffer
(Na
2
HPO
4
,
70
mM,
pH
=
7.5)
were
mixed
with
50
L
of
extracts
(50
mg/L
in
well)
and
30
L
of
the
XOD
solution
(0.1
U/mL).
After
incubation
and
agitation
during
15
min
at
25
◦
C,
60
L
of
xanthine
solution
(150
M)
were
added.
The
mixture
were
subsequently
homogenized
and
incubated
for
5
min.
The
absorbance
was
measured
against
a
blank
without
the
extract
test
and
the
inhibition
percentage
of
XOD
activity
was
calculated
according
to
the
formula:
PI (%)
=
A
blank
−
A
sample
A
blank
×
10
Allopurinol
was
used
as
a
reference
and
the
IC
50
value
is
the
extract
concentration
causing
50%
of
XOD
inhibition.
2.14.
Superoxide
dismutase
(SOD)
inhibition
test
SOD
activity
assay
was
performed
by
pyrogallol
autoxidation
method
50
L
of
extracts
solutions
(50
mg/L
in
well)
were
mixed
with
120
L
of
SOD
solution
(0.2
mg/mL)
pre-
pared
in
buffer
(tris
(50
mM)/DTPA
(1
mM),
pH
=
7.4).
The
mixture
was
incubated
at
25
◦
C
for
4
min
and
30
L
of
pyrogallol
solution
(30
mM)
were
added.
After
incubation
for
7
min
at
37
◦
C,
the
first
absorbance
was
measured
at
325
nm.
The
following
photometries
were
recorded
every
minute
during
4
min.
The
inhibition
percent-
age
of
SOD
activity
was
calculated
according
to
the
formula
in
case
(
OD/min)
=
Abs
ti+1
−
Abs
ti
=
0.04
±
0.01for
the
control
and
blank
control:
PI (%)
=
A
sample
−
A
blank
sample
A
C
−
A
blank
sample
×
100
where
A
blanksample
is
the
absorbance
measured
without
the
extract;
A
C
is
the
absorbance
of
the
control
measured
without
the
enzyme
SOD.
If
(
OD
control
/min)
>
0.05,
the
extract
absorbs
and
(A
C
–
A
BC
)
must
be
subtracted
from
A
sample
.
If
(
OD
control
/min)
<
0.03,
the
extract
inhibits
the
pyrogallol
and
(A
C
–
A
BC
)
must
be
added
to
A
sample
.
A
BC
is
the
absorbance
measured
without
both
the
extract
and
the
enzyme.
2.15.
Cytotoxicity
evaluation
Cytotoxicity
of
extracts
against
human
breast
cancer
cells
(MCF-7)
and
ovarian
cancer
cells
(OVCAR)
was
estimated
by
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bro-
mide
(MTT)
assay
of
100
L
of
cells
were
distributed
in
96-well
plates
at
a
concentration
of
10
4
cells/wells
and
incubated
at
37
◦
C
during
24
h.
100
L
of
cells
in
exponential
growth
phase
were
incubated
at
37
◦
C
for
48
h
with
the
presence
of
S.K.
El
Euch
et
al.
/
Industrial
Crops
and
Products
76
(2015)
1100–1105
1103
Table
2
Chemical
composition
of
the
methanolic
Cistus
salviifolius
flower
buds
and
leaves
extracts.
Reference
Phenols
Flavonoids
(QE)
Tannins
(CE)
Anthocyanins
FB
305.30
±
4.68
76.21
±
1.26
6.10
±
0.26
0.24
±
0.02
L
286.99
±
2.96
65.58
±
0.86
56.36
±
0.67
0.31
±
0.02
GAE:
Gallic
Acid
Equivalent.
QE:
Quercetin
Equivalent.
CE:
Catechin
Equivalent.
CGE:
Cyanidin
3-Glucoside
Equivalent.
Values
are
expressed
as
means
±
Standard
Deviation
(S.D),
n
=
3.
a
mg/g
dw
extract.
100
L
of
the
culture
medium
containing
extracts
at
a
concentra-
tion
of
50
mg/L.
The
medium
was
removed
and
cells
were
treated
with
MTT
solution
50
L,
1
mg/mL
in
phosphate
buffered
saline
(PBS)
and
incubated
at
37
◦
C
for
40
min.
MTT
solution
was
then
discarded
and
DMSO
(50
L)
was
added
to
dissolve
insoluble
blue
crystals.
Optical
density
was
measured
at
605
nm.
Tamoxifen
was
used
as
positive
control.
All
data
were
expressed
as
means
±
standard
deviations
of
trip-
licate
measurements.
Standard
deviations
(S.D.)
did
not
exceed
5%
for
the
majority
of
the
obtained
values.
3.
Results
and
discussion
3.1.
Extraction
yields
Extraction
yields
of
C.
salviifolius
were
given
in
The
FB
provided
higher
yield
(30.20%)
than
the
leaves
(21.75%).
Vari-
ation
in
the
yields
of
both
extracts
was
attributed
to
the
organ
factor.
These
results
were
much
better
than
that
reported
by
they
found
12.50
and
1.275%
of
yields
obtained
from
successive
extraction
of
acetone-water
and
ethyl
acetate
of
C.
salviifolius
aerial
part,
respectively;
also,
more
important
than
that
determined
by
with
aqueous
and
hydroalcoholic
macerations
and
which
were
ranging
from
6.20
to
8.50%
and
10.00
to
12.10%,
respectively.
We
did
not
find
any
data
concerning
C.
salviifolius
flower
buds
extract.
3.2.
Total
phenolics,
flavonoids,
tannins
and
anthocyanins
content
TPC,
TFC,
CTC
and
TAC
in
the
FB
and
leaves
extracts
of
C.
salvi-
ifolius
were
cited
in
chemical
composition
of
leaves
and
FB
extracts
showed
that
this
species
is
very
rich
in
phenolic
com-
pounds.
FB
contained
higher
amounts
of
TPC
and
TFC
than
leaves
(305.30
±
4.68
against
286.99
±
2.96
mg
GAE/g
dw
and
76.21
±
1.26
against
65.58
±
0.86
g
QE/g,
respectively).
However
the
CTC
were
nine
times
more
abundant
in
leaves
than
FB
(56.36
±
0.67
against
6.10
±
0.26
mg
CE/g
dw).
TAC
were
weakly
represented
in
both
leaves
and
FB
extracts
compared
to
the
other
families
and
they
were
more
abundant
in
leaves
than
FB
(0.31
±
0.02
against
0.24
±
0.02
mg
C3GE/g
dw).
C.
salviifolius
possessed
a
considerable
concentration
of
phenolics
and
flavonoids
when
compared
to
results
found
in
the
literature.
In
fact,
TPC
obtained
in
FB
extracts
(305.30
±
4.68
mg
GAE/g
dw)
and
in
leaves
extract
(286.99
±
2.96
mg
GAE/g
dw)
were
higher
than
that
reported
by
Spain
sample
(273.30
±
0.20
and
243.80
±
0.95
mg
GAE/g
ethano-
lic
and
aqueous
leaves
dw,
respectively).
Moreover,
TFC
in
the
both
extracts
were
three
to
five
times
more
important
than
that
determined
by
±
0.04
and
10.50
±
0.06
mg
QE/g
ethanolic
and
aqueous
leaves
dw,
respec-
tively).
In
addition,
in
comparison
with
other
Spanish
Cistus
species,
C.
salviifolius
TPC
and
TFC,
in
leaves
and
FB
extracts,
were
more
interesting
than
that
found
in
C.
ladanifer
leaves
(229.30
±
1.08
mg
GAE/g
dw
and
30.40
±
0.49
mg
QE/g
dw,
respectively)
and
obtained
with
aqueous
extraction.
We
note
also
higher
concentration
of
flavonoids
in
our
extracts
than
that
assessed
in
C.
populifolius
leaves
Table
3
Antioxidant
activity
assessed
by
means
DPPH
•
,
ABTS
•+
and
FRAP
assays.
Reference
IC
50
(mg/L)
DPPH
•
ABTS
•+
FRAP
FB
5.11
±
0.53
4.82
±
0.08
59.27
±
0.13
L
6.48
±
0.19
7.87
±
0.28
77.35
±
0.32
Vitamin
C
4.68
±
0.12
4.07
±
0.20
18.00
±
0.05
Values
are
expressed
as
means
±
S.D,
n
=
3.
aqueous
extract
(59.50
±
1.08
mg
QE/g
dw)
(
3.3.
Antioxidant
capacity
Leaves
and
FB
extracts
were
individually
assessed
for
antiox-
idant
activity
using
three
different
spectrophotometric
tests
and
were
compared
to
the
positive
control,
ascorbic
acid.
The
results
were
summarized
in
Our
findings
revealed
that
both
extracts
have
very
notable
antioxidant
activity
which
was
com-
parable
to
the
positive
control
due
to
their
richness
of
TPC
and
TFC.
We
observed
also
that
FB
exhibited
more
potent
antioxidant
activity
than
the
leaves
justified
by
the
DPPH
•
,
ABTS
•+
and
FRAP
tests
(IC
50
=
5.11
±0.53
against
6.48
±
0.19
mg/L;
4.82
±
0.08
against
7.87
±
0.28
mg/L
and
59.27
±
0.13
against
77.35
±
0.32
mg/L,
respectively).
This
result
may
be
explained
by
its
higher
content
of
phenols
and
flavonoids
when
compared
to
leaves.
No
antioxidant
activity
of
C.
salviifolius
extract
has
been
previously
reported.
We
should
notice
that
the
antioxidant
activity
of
leaves
and
flower
buds
extracts,
as
complex
mixtures,
have
exerted
very
strong
antioxidant
activities
which
were
highly
comparable
to
the
pure
compound
vitamin
C.
These
results
prove
the
presence
in
extracts
interesting
and
more
potent
antioxidants
when
compared
to
vitamin
C.
3.4.
Anti-xanthine
oxidase
activity
From
the
observed
that
both
extracts,
at
a
concen-
tration
of
50
mg/L,
possessed
notable
inhibition
potency
against
XOD.
The
leaves
extract
exhibited
more
powerful
activity
(IP
(%)
=
63.51
±
4.76)
than
FB
extract
(IP
(%)
=
54.73
±
2.09).
This
result
may
be
explained
by
the
presence
of
greatly
higher
CTC
in
leaves
than
FB
(56.36
±
0.67
against
6.10
±
0.26
mg
CE/g
dw),
also
more
important
concentration
of
anthocyanins
(0.31
±
0.02
against
0.24
±
0.02
mg
C3GE/g
dw).
Flavonoids
and
proantho-
cyanidins
are
reported
to
be
antioxidants
inhibitors
of
xanthine
oxidase
(
Moreover,
we
observed,
that
the
complex
mixture
of
leaves
extract
had
an
important
IC
50
value
(19.48
±
0.21)
which
was
near
to
that
of
the
pure
compound,
allop-
urinol
(IC
50
=
1.13
±
0.78
mg/L).
This
obtained
result
could
highlight
the
idea
of
pure
active
compounds
isolation
and
identification
as
a
possible
candidate.
No
investigations
have
been
conducted
before
on
C.
salviifolius
extract
inhibitory
activity
against
XOD.
The
inhibition
percent-
age
obtained
in
this
study
by
C.
salviifolius
leaves
extract
at
1104
S.K.
El
Euch
et
al.
/
Industrial
Crops
and
Products
76
(2015)
1100–1105
Table
4
Anti-xanthine
oxidase,
anti-superoxide
dismutase,
anti-acetylcholinesterase
and
anti-inflammatory
activities
of
Cistus
salviifolius
extracts.
Samples
Anti-XOD
activity
Anti-AChE
activity
Anti-SOD
activity
Anti-5-LOX
activity
IP
(%)
IC
50
IP
(%)
IC
50
IP
IP
IC
50
FB
54.73
±
2.09
–
34.72
±
2.02
–
58.39
±
11.67
21.03
±
0.23
–
L
63.51
±
4.76
19.48
±
0.21
87.18
±
0.74
18.00
±
2.71
37.70
±
5.36
82.27
±
3.06
13.38
±
0.20
Allopurinol
94.50
±
2.22
1.13
±
0.78
–
–
–
–
–
Galanthamine
–
–
–
1.17
±
0.06
–
–
–
NDGA
–
–
–
–
–
–
1.81
±
0.05
a
Inhibition
percentage
at
a
concentration
of
50
mg/L.
b
mg/L;
NDGA:
nordihydroguaiaretic
acid.
Values
are
expressed
as
means
±
S.D,
n
=
3.
a
concentration
of
50
mg/L
was
higher
than
those
obtained
by
they
reported
62.42
±
1.8%
of
inhibition
percentage
at
a
concentration
of
50
mg/L
of
Tephrosia
purprea
Linn.
root
methanolic
extract
and
47.21
±
0.01%
at
higher
concentration
of
1000
mg/L
of
methanolic
Swietenia
mahagoni
seed
extract,
respectively.
3.5.
Anti-acetylcholinesterase
activity
Anti-AChE
activity
of
C.
salviifolius
leaves
and
FB
extracts
was
tested
(
at
a
concentration
of
50
mg/L.
We
notice
the
absence
of
anti-AChE
activity
evaluation
for
this
plant
in
the
liter-
ature.
From
the
observed
that
leaves
extract
exhibited
more
than
two
times
interesting
inhibitory
activity
towards
the
enzyme
AChE
(IP
(%)
=
87.18
±
0.74)
in
comparison
to
FB
extract
(34.72
±
0.02%).
This
result
may
be
explained
by
the
considerably
higher
amount
of
condensed
tannins
and
anthocyanins
in
leaves
extract
than
FB.
C.
salviifolius
leaves
extract
(at
a
concentration
of
50
mg/L)
exhibited
considerably
better
AChE
inhibitory
activity
(87.18
±
0.74%)
compared
to
a
variety
of
reported
plants
(e.g.:
65.16
±
8.13%
obtained
from
methanolic
stems
extracts
of
Piper
interruptum;
58.02
±
3.83%
got
from
methanolic
seed
extracts
of
Piper
nigrum
at
a
concentration
of
100
mg/L
and
68.2
±
15.6
obtained
from
95%
ethanolic
extracts
of
whole
Salvia
officinalis
at
a
concentration
of
2500
mg/L).
Due
to
its
interesting
IC
50
values
(IC
50
=
18
±
2.71
mg/L),
bioactive
pure
compounds
existing
in
C.
salviifolius
leaves
extracts
could
be
good
candidates
to
replace
galantamine
while
it
has
been
reported
to
have
adverse
effects
including
gastrointestinal
disturbances
and
problems
associated
with
bioavailability
3.6.
Anti-superoxide
dismutase
activity
SOD,
a
natural
metallo-enzyme
present
in
various
plant
extracts,
is
produced
endogenously
in
all
aerobic
cells.
It
catalyses
the
dis-
mutase
of
the
superoxide
anion
(O
2
2
−
)
into
oxygen
and
hydrogen
peroxide.
This
antioxidant
enzyme
is
unfortunately
involved
in
chemo-resistant
ovarian
cancer
cells
processes.
showed
that,
at
a
concentration
of
50
mg/L,
both
extracts
exhibited
good
anti-SOD
activity.
The
best
activity
was
observed
in
flower
buds
extract
(IP
(%)
=
58.39
±
11.67)
due
to
its
higher
content
of
phenols
and
flavonoids.
To
our
knowledge,
no
previously
study
on
the
anti-SOD
activ-
ity
of
any
plant
extract
has
been
reported
and
until
now,
there
is
no
known
inhibitor
used
for
this
enzyme.
So
obtained
values
are
encouraging
enough
to
prompt
us
to
try
to
identify
the
molecules
responsible
for
this
activity.
Table
5
Cytotoxic
activities
of
methanolic
extracts
assessed
by
means
OVCAR
and
MCF-7
tests.
Reference
Cytotoxic
activities
MCF-7
FB
36.85
±
5.66
35.16
±
4.67
L
NA
NA
Tamoxifen
(0.2
mg/L)
61.76
±
4.21
47.17
±
4.31
NA:
not
active.
Values
are
expressed
as
average
±
S.D,
n
=
3.
a
Inhibition
percentage
at
a
concentration
of
50
mg/L.
3.7.
Anti-inflammatory
activity
The
5-LOX
inhibition
by
leaves
and
FB
extracts
of
C.
salviifolius
was
reported
for
the
first
time
in
the
literature
in
this
study.
Leaves
extract
exhibited
four
times
stronger
anti-inflammatory
activity
(IP
=
82.27
±
3.06%)
than
FB
extracts
(IP
=
21.03
±
0.23%).
This
result
might
be
related
to
its
considerable
richness
in
condensed
tannins
and
anthocyanins
in
comparison
with
leaves.
NDGA,
known
as
a
potent
antioxidant
was
used
as
a
standard.
This
pure
compound
has
seven
times
stronger
anti-inflammatory
activity
(IC
50
=
1.81
±
0.05
mg/L)
when
compared
to
the
complex
mixture
of
leaves
extract
(IC
50
=
13.38
±
0.20
mg/L).
So,
we
should
notice
that
this
extract
fractionation
could
take
out
pure
active
compounds
against
5-LOX
enzyme,
more
active
than
NDGA.
More-
over,
we
deduce
that
this
result
is
much
better
than
some
varieties
of
Punica
granatum
reported
by
they
obtained
IC
50
=
21.80
±
0.50
and
14.30
±
1.50
mg/L
for
the
Garsi
and
Gabsi
varieties,
respectively.
We
should
notice
that
the
most
prominent
anti-inflammatory,
anti-acetylcholinesterase
and
anti-XOD
activities
were
found
in
the
same
most
active
extract:
leaves
extract.
This
result
may
be
explained
by
the
possible
presence
of
a
correlation
between
these
activities
such
as
a
radical
mechanism
link.
3.8.
Cytotoxic
activity
Cytotoxic
activity
evaluation
of
leaves
and
FB
extracts,
at
a
con-
centration
of
50
mg/L,
against
the
cancer
cell
lines,
OVCAR
and
MCF-7,
using
the
MTT
assay,
was
showed
for
the
first
time
in
the
lit-
erature
for
this
plant
(
The
anti-cancer
activity
of
FB
extract
was
moderate
(IP
=
36.85
±
5.66
and
35.16
±
4.67%
towards
OVCAR
and
MCF-7
respectively),
but
more
interesting
than
leaves
extract
found
to
be
inactive
against
the
both
cells
lines.
This
result
can
be
explained
by
the
presence
of
higher
concentrations
of
phenols
and
flavonoids
in
FB
than
leaves.
The
absence
of
cytotoxic
property
could
highlight
the
idea
of
C.
salviifolius
leaves
extract
use
in
food
preservation,
essentially
to
fight
or
delay
the
oxidation
process
as
it
exhibited
powerful
antioxidant
activity.
S.K.
El
Euch
et
al.
/
Industrial
Crops
and
Products
76
(2015)
1100–1105
1105
4.
Conclusions
Our
work
highlighted
the
significant
difference
in
chemical
composition
between
the
two
organs,
leaves
and
flower
buds,
and
its
significant
influence
on
the
biological
activities.
The
concentra-
tions
of
phenols
and
flavonoids
were
determined
to
be
highest
in
flower
buds,
however
the
tannins
and
the
anthocyanins
were
much
more
abundant
in
leaves.
Both
extracts
are
endowed
with
very
potent
antioxidant
activity,
especially
FB
extract,
proved
by
three
different
spectrophotometric
tests.
These
findings
lead
us
to
con-
clude
that
C.
salviifolius
extracts
could
be
considered
as
potential
alternatives
for
synthetic
antioxidants
used
in
the
food
indus-
try.
Furthermore,
SOD
enzyme
was
more
sensible
to
FB
extract
compounds.
However,
C.
salviifolius
leaves
extract
exerted
more
interesting
inhibitory
activities
towards
XOD,
AChE
and
5-LOX
enzymes.
Obtained
results
showed
that
condensed
tannins
and
anthocyanins
seem
to
have
a
major
effect
on
those
enzymes
inhibi-
tion.
Further
work
is
in
progress
to
identify
any
specific
molecules
which
may
be
responsible
for
the
observed
biological
activities
and
these
results
open
so
a
new
horizon
about
C.
salviifolius
possible
utilizations
in
several
fields
such
as
food
industries,
medicine
and
pharmaceutical.
Acknowledgements
We
gratefully
acknowledge
the
Tunisian
Ministry
of
High
Edu-
cation
for
supporting
this
work
and
Dr.
Nadia
Ben
Brahim
for
plant
material
authentication.
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Document Outline
- Chemical composition, biological and cytotoxic activities of Cistus salviifolius flower buds and leaves extracts
- 1 Introduction
- 2 Materials and methods
- 2.1 Chemicals used
- 2.2 Plant material collection
- 2.3 Methanolic extract preparation
- 2.4 Total phenolic content determination
- 2.5 Total flavonoid content determination
- 2.6 Total condensed tannin content determination
- 2.7 Total anthocyanin content determination
- 2.8 DPPH radical scavenging assay
- 2.9 ABTS+ radical scavenging assay
- 2.10 Ferric reducing antioxidant power assay (FRAP)
- 2.11 Anti-inflammatory activity
- 2.12 Anti-acetylcholinesterase activity evaluation
- 2.13 Xanthine oxidase inhibition (XOD) assay
- 2.14 Superoxide dismutase (SOD) inhibition test
- 2.15 Cytotoxicity evaluation
- 3 Results and discussion
- 4 Conclusions
- Acknowledgements
- References
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