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Aqueous extract of Baccharis trimera improves redox status and decreases the severity of alcoholic hepatotoxicity

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The metabolism of ethanol occurs mainly in the liver, promoting increase of reactive oxygen species and nitrogen, leading to redox imbalance. Therefore, antioxidants can be seen as an alternative to reestablish the oxidizing/reducing equilibrium. The aim of this study was to evaluate the antioxidant and hepatoprotective effect of aqueous extract of Baccharis trimera (Less.) DC., Asteraceae, in a model of hepatotoxicity induced by ethanol. The extract was characterized and in vitro tests were conducted in HepG2 cells. It was evaluated the cells viability exposed to aqueous extract for 24h, ability to scavenging the radical DPPH, besides the production of reactive oxygen species and nitric oxide, and the influence on the transcriptional activity of transcription factor Nrf2 (12 and 24h) after exposure to 200mM ethanol. The results showed that aqueous extract was non-cytotoxic in any concentration tested; moreover, it was observed a decrease in ROS and NO production, also promoting the transcriptional activity of Nrf2. In vivo, we pretreatment male rats Fisher with 600mg/kg of aqueous extract and 1h later 5ml/kg of absolute ethanol was administrated. After two days of treatment, the animals were euthanized and lipid profile, hepatic and renal functions, antioxidant status and oxidative damage were evaluated. The treatment with extract improved liver function and lipid profile, reflecting the reduction of lipid microvesicules in the liver. It also promoted an increase of glutathione peroxidase activity, decrease of oxidative damage and MMP-2 activity. These results, analyzed together, suggest the hepatoprotective effect of B. trimera aqueous extract.
Content may be subject to copyright.
Revista
Brasileira
de
Farmacognosia
27
(2017)
729–738
ww
w.elsevier.com/locate/bjp
Original
Article
Aqueous
extract
of
Baccharis
trimera
improves
redox
status
and
decreases
the
severity
of
alcoholic
hepatotoxicity
Ana
Carolina
S.
Rabeloa,
Glaucy
R.
de
Araújoa,
Karine
de
P.
Lúcioa,
Carolina
M.
Araújoa,
Pedro
H.
de
A.
Mirandaa,
Breno
de
M.
Silvab,
Ana
Claudia
A.
Carneirob,
Érica
M.
de
C.
Ribeirob,
Wanderson
G.
de
Limac,
Gustavo
H.
B.
de
Souzad,
Geraldo
C.
Brandãod,
Daniela
C.
Costaa,
aLaboratório
de
Bioquímica
Metabólica,
Departamento
de
Ciências
Biológicas,
Programa
de
Pós-graduac¸
ão
em
Ciências
Biológicas,
Universidade
Federal
de
Ouro
Preto,
Ouro
Preto,
MG,
Brazil
bLaboratório
de
Biologia
e
Biotecnologia
de
Micro-organismos,
Departamento
de
Ciências
Biológicas,
Programa
de
Pós-graduac¸
ão
em
Ciências
Biológicas,
Universidade
Federal
de
Ouro
Preto,
Ouro
Preto,
MG,
Brazil
cLaboratório
de
Morfopatologia,
Departamento
de
Ciências
Biológicas,
Programa
de
Pós-graduac¸
ão
em
Ciências
Biológicas,
Universidade
Federal
de
Ouro
Preto,
Ouro
Preto,
MG,
Brazil
dLaboratório
de
Farmacognosia,
Escola
de
Farmácia,
Programa
de
Pós-graduac¸
ão
em
Ciências
Farmacêuticas,
Universidade
Federal
de
Ouro
Preto,
Ouro
Preto,
MG,
Brazil
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
23
June
2017
Accepted
13
September
2017
Available
online
4
November
2017
Keywords:
Ethanol
Redox
imbalance
Baccharis
trimera
Aqueous
extract
Hepatotoxicity
a
b
s
t
r
a
c
t
The
metabolism
of
ethanol
occurs
mainly
in
the
liver,
promoting
increase
of
reactive
oxygen
species
and
nitrogen,
leading
to
redox
imbalance.
Therefore,
antioxidants
can
be
seen
as
an
alternative
to
reestablish
the
oxidizing/reducing
equilibrium.
The
aim
of
this
study
was
to
evaluate
the
antioxidant
and
hepatopro-
tective
effect
of
aqueous
extract
of
Baccharis
trimera
(Less.)
DC.,
Asteraceae,
in
a
model
of
hepatotoxicity
induced
by
ethanol.
The
extract
was
characterized
and
in
vitro
tests
were
conducted
in
HepG2
cells.
It
was
evaluated
the
cells
viability
exposed
to
aqueous
extract
for
24
h,
ability
to
scavenging
the
radical
DPPH,
besides
the
production
of
reactive
oxygen
species
and
nitric
oxide,
and
the
influence
on
the
transcrip-
tional
activity
of
transcription
factor
Nrf2
(12
and
24
h)
after
exposure
to
200
mM
ethanol.
The
results
showed
that
aqueous
extract
was
non-cytotoxic
in
any
concentration
tested;
moreover,
it
was
observed
a
decrease
in
ROS
and
NO
production,
also
promoting
the
transcriptional
activity
of
Nrf2.
In
vivo,
we
pre-
treatment
male
rats
Fisher
with
600
mg/kg
of
aqueous
extract
and
1
h
later
5
ml/kg
of
absolute
ethanol
was
administrated.
After
two
days
of
treatment,
the
animals
were
euthanized
and
lipid
profile,
hepatic
and
renal
functions,
antioxidant
status
and
oxidative
damage
were
evaluated.
The
treatment
with
extract
improved
liver
function
and
lipid
profile,
reflecting
the
reduction
of
lipid
microvesicules
in
the
liver.
It
also
promoted
an
increase
of
glutathione
peroxidase
activity,
decrease
of
oxidative
damage
and
MMP-
2
activity.
These
results,
analyzed
together,
suggest
the
hepatoprotective
effect
of
B.
trimera
aqueous
extract.
©
2017
Sociedade
Brasileira
de
Farmacognosia.
Published
by
Elsevier
Editora
Ltda.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Ethanol
is
the
most
used
alcohol
in
alcoholic
beverages
and
its
abusive
consumption
is
associated
with
various
health
problems
worldwide
(Lívero
and
Acco,
2016).
The
metabolization
of
ethanol
occurs
mainly
in
the
liver,
where
it
is
converted
to
acetaldehyde
by
alcohol
dehydrogenase
and
subsequently
oxidized
to
acetate
by
acetaldehyde
dehydrogenase
(Cederbaum,
2012).
These
enzymes
use
NAD+as
cofactor
and
generate
NADH,
thus
decreasing
the
Corresponding
author.
E-mail:
daniela.costa@iceb.ufop.br
(D.C.
Costa).
NAD+/NADH
ratio,
affecting
several
metabolic
pathways
(Smith
et
al.,
2007),
besides
promoting
the
increase
of
acetaldehyde
adducts
and
reactive
oxygen
species
(ROS),
leading
to
oxidative
stress
(Lu
et
al.,
2012;
Han
et
al.,
2016).
In
some
circumstances,
the
microsomal
oxidation
system
of
ethanol
(CYP2E1)
can
be
acti-
vated,
which
contributes
even
more
to
the
ROS
formation
(Smith
et
al.,
2007;
Ceni
et
al.,
2014;
Hernández
et
al.,
2015).
Alcoholic
fatty
liver
disease
(AFLD)
is
the
first
response
of
the
liver
to
ethanol
use
and
is
characterized
by
accumulation
of
lipids
in
hepatocytes
(Ceni
et
al.,
2014).
An
optimal
pharmacological
treat-
ment
for
AFLD
would
reduce
inflammatory
parameters,
oxidative
stress
and
lipid
accumulation,
and
avoid
fibrotic
events.
However,
the
development
of
a
drug
that
is
capable
of
acting
on
so
many
different
pathways
is
extremely
difficult.
For
this
reason,
a
single
https://doi.org/10.1016/j.bjp.2017.09.003
0102-695X/©
2017
Sociedade
Brasileira
de
Farmacognosia.
Published
by
Elsevier
Editora
Ltda.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
730
A.C.
Rabelo
et
al.
/
Revista
Brasileira
de
Farmacognosia
27
(2017)
729–738
drug
therapy
has
not
been
developed,
but
combined
therapies
in
an
attempt
to
reverse
hepatocyte
injury
(Lívero
and
Acco,
2016).
Due
to
the
great
importance
of
oxidative
stress
in
the
pathogen-
esis
of
AFLD,
several
studies
have
focused
on
the
use
of
antioxidants
to
prevent
oxidative
damage
and
improve
liver
function
(Ceni
et
al.,
2014;
Hernández
et
al.,
2015;
Lívero
and
Acco,
2016).
ROS
can
be
neutralized
by
enzymatic
antioxidants,
such
as
superox-
ide
dismutase
(SOD),
catalase,
glutathione
peroxidase
(GPx)
and
glutathione
reductase
(GR)
and
nonenzymatic
as
glutathione,
vita-
mins
and
dietary
antioxidants
(Chen
et
al.,
2015;
Han
et
al.,
2016).
The
antioxidant
enzymes
are
mainly
regulated
by
the
factor-2
nuclear-erythroid
factor
(Nrf-2)
(Lushchak,
2014;
Chen
et
al.,
2015;
González
et
al.,
2015).
This
factor
is
usually
found
in
the
cell
cytoplasm
associated
with
the
ECH-associated
calcite-like
protein
(Keap-1),
which
allows
its
labeling
and
degradation
Nrf2
via
pro-
teasome
(Chen
et
al.,
2015).
However,
in
the
presence
of
oxidative
stress
the
factor
migrates
to
the
nucleus,
where
it
binds
to
the
antioxidant
response
element
(ARE),
promoting
the
antioxidant
enzymes
expression
(Lushchak,
2014;
Chen
et
al.,
2015;
González
et
al.,
2015;
Kim
and
Keum,
2016).
In
addition
to
the
enzymatic
antioxidants,
the
inclusion
of
antioxidants
in
the
diet
is
of
great
importance
and
the
consump-
tion
is
related
to
the
reduction
of
the
risk
of
the
development
of
diseases
associated
with
the
accumulation
of
free
radicals,
since
in
these
compounds
substances
that
act
in
synergism
in
the
pro-
tection
of
cells
and
tissues
can
be
found
(Bianchi
and
Antunes,
1999).
In
fact,
some
studies
have
demonstrated
the
beneficial
effect
of
natural
dietary
antioxidants
(Al-Sayed
et
al.,
2014;
Al-
Sayed
et
al.,
2015;
Pádua
et
al.,
2010;
Lívero
et
al.,
2016a,b;
Fahmy
et
al.,
2017).
Thereby,
medicinal
plants
have
attracted
the
atten-
tion
of
researchers
as
potential
agents
against
alcoholic
liver
injury
because
of
their
antioxidant
potential
and
the
few
side
effects
(Ding
et
al.,
2012).
However,
most
plant
species
are
only
empirically
used,
and
there
are
few
studies
that
prove
their
therapeutic
efficacy
(Foglio
et
al.,
2006).
In
this
sense,
Baccharis
trimera
(Less.)
DC.,
Asteraceae,
is
a
medic-
inal
plant
used
in
popular
culture
and
widely
distributed
in
South
America
(Bona
et
al.,
2005).
In
Brazil,
this
plant
is
popularly
known
as
carqueja
and
used
as
gastric
protector
(Lívero
et
al.,
2016a,b),
hypoglycemic
(Oliveira
et
al.,
2011),
anti-inflammatory
(Karam
et
al.,
2013)
and
antioxidant
(Pádua
et
al.,
2013;
de
Araújo
et
al.,
2016).
Some
studies
are
focused
in
the
use
of
medicinal
plants
in
ethanol-induced
intoxication,
however
most
studies
use
only
alcoholic/hydroethanolic
extracts
and
it
is
known
that
the
solvent
used
for
the
extraction
of
the
secondary
metabolites
is
involved
in
the
biological
activity
of
these
plants
(Rates,
2001).
Based
on
these
evidences
and
on
the
fact
that
there
are
no
studies
with
aqueous
extract
of
B.
trimera
in
ethanol-induced
hepatotoxicity
available,
our
goal
was
to
characterize
this
extract
and
verify
its
effect
on
pro-
tection
against
alcoholic
hepatotoxicity
in
vitro
and
in
vivo
model.
Materials
and
methods
Plant
material
The
aerial
parts
of
Baccharis
trimera
(Less.)
DC.,
Asteraceae,
were
collected
in
Ouro
Preto
city,
in
Minas
Gerais
state,
Brazil.
The
spec-
imens
were
authenticated
and
deposited
at
the
Herbarium
José
Badini
(UFOP),
OUPR
22.127.
After
identification,
the
aerial
parts
were
dried
in
a
ventilated
oven
(30 C),
pulverized
and
stored
in
plastic
bottles.
To
obtain
the
aqueous
extract,
approximately
100
g
of
the
plant
was
extracted
with
1l
of
water
for
24
h,
by
maceration.
The
solids
were
removed
by
vacuum
filtration
and
the
solvent
was
removed
by
a
rotary
evaporator
at
40 C
(Pádua
et
al.,
2010).
RP-UPLC-DAD-ESI-MS
analyses
The
aqueous
extract
was
analyzed
in
the
Ultra
Performance
Liquid
Chromatography
coupled
to
diode
arrangement
detector
and
mass
spectrometry.
In
this
assay,
20
mg
of
the
sample
was
applied
and
diluted
with
4
ml
of
MeOH/H2O
(9:1).
The
eluate
was
dried
and
resuspended
in
a
solution
of
methanol
and
then
filtered
on
Chromafil®PDVF
syringe
filters
(polyvinylidene
dilfluoride,
0.20
mm)
in
a
volume
sufficient
to
obtain
2
mg/ml
concentration
of
the
sample.
For
the
analysis
in
HPLC-DAD-EM,
20
l
of
the
sample
were
injected
into
the
liquid
chromatograph,
in
the
same
condi-
tions
described
by
de
Araújo
et
al.
(2016).
In
vitro
tests
DPPH
radical-scavenging
activity
The
percentage
of
antioxidant
activity
of
each
substance
was
assessed
by
DPPH
free
radical
assay,
according
to
Araújo
et
al.
(2015).
In
brief,
aqueous
extract
was
diluted
in
methanol
80%
and
dilutions
were
performed
to
obtain
the
concentrations
(25–500
g/ml).
The
standard
curve
was
performed
with
the
ref-
erence
antioxidant
Trolox
(6-hydroxy-2,5,7,8-tetramethylchrome-
2-carboxylic
acid).
As
blank
methanol
(80%)
was
used
and
the
antioxidant
activity
was
determined
by
the
decrease
in
the
DPPH
absorbance
and
the
percent
inhibition
was
calculated
using
the
following
equation:
%
antioxidant
activity
=
(1
ASample
515/AControl515)
×
100.
Cell
culture
Hepatocellular
carcinoma
cell
line
(HepG2)
was
acquired
from
the
Cell
Bank
from
the
Federal
University
of
Rio
de
Janeiro.
The
cells
were
placed
in
sterile
75
cm2growth
vials
containing
the
DMEM
culture
medium
and
supplemented
with
antibiotic
(Penicillin-
Streptomycin)
and
10%
(v/v)
fetal
bovine
serum.
The
bottles
were
incubated
in
an
oven
at
37 C
humidified
with
5%
carbon
diox-
ide
(CO2).
Cells
were
used
for
assays
when
the
confluence
reached
about
80%,
so
the
medium
was
aspirated
and
the
monolayer
washed
with
buffered
saline
(PBS).
After
this,
2
ml
of
trypsin
and
EDTA
solu-
tion
(0.20%
and
0.02%,
respectively)
were
used.
Subsequently,
the
cells
were
centrifuged
and
the
supernatant
was
discarded
and
the
cell
pellet
was
resuspended
in
1
ml
of
DMEM
medium.
The
cells
were
then
counted
with
Trypan
Blue
0.3%
in
the
Neubauer
chamber.
For
each
experiment
triplicate
was
used,
with
biological
duplicate
(totaling
n
=
6
per
group).
Cell
viability
assay
Cell
viability
was
determined
using
colorimetric
MTT
(3-[4,5-
dimethyl-thiazol-2-yl]-2,5-diphenyl
tetrazolium
bromide)
assay
as
described
previously
(Fotakis
and
Timbrell,
2005).
In
brief,
HepG2
cells
(1
×
105)
were
cultured
in
96
well-plates
with
or
without
different
aqueous
extract
concentrations
of
B.
trimera
(5–600
g/ml),
which
was
diluted
in
DMEM
medium,
and
ethanol
(5–800
mM)
for
24
h.
After
incubation,
medium
was
removed
and
MTT
solution
(5
mg/ml)
was
added
and
incubated
for
fur-
ther
1
h
at
37C.
Subsequently,
dimethyl
sulfoxide
was
added
to
dissolve
formazan
crystals
and
the
absorbance
was
measured
at
570
nm.
The
cell
viability
percentage
was
calculated
based
in
the
formula
below,
where
the
control
was
assigned
100%
viability.
%
of
cell
viability
=absorbance
of
treated
cells
absorbance
control ×
100
ROS
and
NO
production
For
the
determination
of
reactive
oxygen
species
and
nitric
oxide
2.5
×
104cells
were
cultured
in
white
96
well-plates
with
A.C.
Rabelo
et
al.
/
Revista
Brasileira
de
Farmacognosia
27
(2017)
729–738
731
two
different
aqueous
extract
of
B.
trimera
concentrations
(10
and
50
g/ml),
diluted
in
DMEM
medium.
After
an
incubation
of
3
h,
the
medium
was
removed
and
200
mM
of
ethanol
was
added
with
50
M
of
carboxi-H2DCFDA
(for
ROS
production)
or
10
M
of
DAF-
FM
(for
NO
production).
The
plate
was
incubated
for
24
h
and,
then,
HANKS
was
added.
The
reading
was
obtained
in
microplate
reader,
using
485
nm
for
excitation
and
535
nm
for
emission
microwave.
Luciferase
reporter
assay
To
evaluate
the
effect
of
aqueous
extract
of
B.
trimera
on
transcriptional
activity
of
Nrf2,
Luciferase
assay
System
was
used,
according
to
Silva
et
al.
(2011),
with
some
modifications.
For
this,
a
kit
Dual
Luciferase
assay
System
was
used.
Briefly,
1.5
×
105HepG2
cells
were
plated
in
24-well
plates
and
incubated
for
24
h.
Then,
medium
without
SFB
was
added
and
incubated
for
further
24
h.
After
this
time,
transfection
was
performed
with
100
l
per
well
of
mix
(500
ng
of
lipofectamine,
100
ng
of
pRL-TK,
400
ng
of
pPGL37
and
medium
HG
to
complete
the
volume).
Six-hour
incubation
was
carried
out
and,
then,
10
and
50
g/ml
of
aqueous
extract
were
added.
After
3-hour
incubation,
200
mM
of
ethanol
was
added
and
a
new
incubation
was
performed
for
12
or
24
h.
After
this,
100
l
of
lysis
buffer
(provided
by
the
kit)
was
added
and
centrifuged
for
4
min
at
10,000
rpm.
Then,
15
l
of
supernatant
and
35
l
of
Luciferase
II
reagent
(LAR-II)
were
read
in
luminometer
(580
nm),
providing
the
“Net
A”
value.
After,
50
l
of
Stop
and
Glo®were
added,
providing
the
“Net
B”
value.
For
calculations
the
NetA/NetB
ratio
was
used.
In
vivo
tests
Animals
Male
Fisher
rats
(220–250
g),
obtained
from
the
Laboratory
of
Experimental
Nutrition
from
the
Federal
University
of
Ouro
Preto,
were
kept
on
collective
cages,
in
a
12
h
light/dark
cycle
at
room
temperature
and
were
fasted
12
h
with
water
ad
libitum
before
the
experiment.
All
animals
were
used
according
to
the
Committee
guidelines
on
Care
and
Use
of
Animal
from
Federal
University
of
Ouro
Preto,
Brazil
(No.
2016/01).
The
experimental
protocol
The
animals
were
divided
into
three
groups:
-
Control
group
(C)
(n
=
7):
received
1
ml
of
water;
-
Ethanol
group
(E)
(n
=
5):
received
1
ml
of
water
and
1
h
later
5
ml/kg
of
absolute
ethanol
(El-Naga,
2015).
-
Aqueous
extract
of
B.
trimera
(Aq)
(n
=
7):
received
600
mg/kg
of
extract
and
1
h
later
5
ml/kg
of
absolute
ethanol
(Pádua
et
al.,
2010,
2013,
2014).
All
treatments
were
administrated
by
gavage,
totaling
a
volume
of
1
ml
of
solution.
The
animals
were
treated
for
two
consecutive
days
and
24
h
after
the
last
ethanol
dose,
they
were
euthanized
by
deep
anesthesia
induced
by
isoflurane.
They
were
maintained
on
a
12
h
fasting.
Analysis
of
biochemical
serum
parameters
The
serum
was
used
for
determining
the
urea,
creatinine,
ALT,
AST,
protein
total,
glucose,
total
cholesterol,
HDL,
fraction
non-HDL,
triacylglycerides.
All
measurements
were
performed
by
commer-
cial
laboratories
kits
Labtest®(Lagoa
Santa,
MG,
Brazil)
and
Bioclin®
(Belo
Horizonte,
MG,
Brazil).
Determination
of
antioxidant
system
The
antioxidant
system
was
evaluated
by
SOD,
catalase,
glu-
tathione
peroxidase
and
reductase
activity,
beyond
glutathione
total
(oxidized
and
reduced).
The
assay
for
determination
of
indirect
SOD-activity
is
based
on
SOD
competition
with
super-
oxide
radical,
formed
by
self-oxidation
of
pyrogallol,
which
is
responsible
for
MTT
reduction
and
formation
of
formazan
crystals
(Marklund
and
Marklund,
1974).
Catalase
activity
was
determined
based
on
its
ability
to
convert
hydrogen
peroxide
(H2O2)
into
water
and
molecular
oxygen
(Aebi,
1984).
Glutathione
system
was
determined
by
kit
(Sigma–Aldrich,
St.
Louis,
MO,
USA).
To
determine
catalase
and
superoxide
dismutase
activity,
100
mg
of
liver
tissue
was
homogenized
in
phosphate
buffer
(pH
7.4).
Total
glutathione
and
reduced/oxidize
glutathione
concentra-
tions
were
determined
by
the
homogenization
of
100
mg
of
tissue
in
5%
sulfosalicylic
buffer.
For
correction
of
the
dosages,
the
protein
was
measured
by
the
Lowry
method
(Lowry
et
al.,
1951).
After
homogenization,
the
samples
were
centrifuged
at
10,000
×
g
for
10
min,
at
4C.
The
supernatant
was
collected
and
used
as
the
sample
and
all
dosages
were
according
to
Bandeira
et
al.
(2017).
Determination
of
oxidative
stress
markers
In
order
to
evaluate
oxidative
damage
thiobarbituric
acid
reac-
tive
substances
(TBARS)
and
carbonyl
protein
such
as
markers
were
used.
The
TBARS
concentration
was
determined
based
on
thiobar-
bituric
acid
(TBA)
binding
to
oxidized
lipids,
according
to
Buege
and
Aust
(1978).
In
the
method
for
determining
carbonylated
protein,
it
was
used
2,4-dinitrophenylhydrazine
(DNPH),
which
reacts
with
carbonyl
groups
to
generate
the
corresponding
hydrazone
that
can
be
analyzed
spectrophotometrically,
as
described
by
Levine
et
al.
(1994).
For
correction
of
the
dosages,
the
protein
was
measured
by
the
Lowry
method
(Lowry
et
al.,
1951).
Gelatin
zymography
MMP-2
activity
was
detected
using
gelatin
zymography.
Briefly,
50
mg
of
tissue
were
homogenized
in
200
of
RIPA
buffer
(150
mM
NaCl,
50
mM
Tris,
1%
IGEPAL,
0.5%
sodium
deoxycholate,
0.1%
SDS,
1
l/ml
protease
inhibitor
at
pH
8.0).
After
homogenization,
the
samples
were
centrifuged
at
10,000
×
g
for
10
min,
at
4C
and
the
supernatant
was
collected
and
used
as
the
sample.
The
activity
was
measured
according
to
Araújo
et
al.
(2015).
Histological
evaluation
For
microscopic
analysis,
a
portion
of
the
liver
from
each
animal
of
experimental
groups
was
fixed
in
10%
formalin
and
immersed
in
paraffin.
Sections
of
4
m
were
obtained
and
the
slides
were
stained
with
hematoxylin
and
eosin
(H&E).
The
photomi-
crographs
were
obtained
at
40×
magnification
(Leica
Application
Suite,
Germany).
Liver
histology
was
examined
using
eleven
images
obtained
at
random
from
the
tissue
and
classified
for
the
degree
of
microvesicular
steatosis.
The
images
were
examined
semi
quan-
titatively,
considering
that
the
degree
of
lipid
infiltration
was
graded
reflecting
the
percentage
of
hepatocytes
containing
lipid
droplets.
It
was
given
the
values
0–3
according
to
the
steatosis,
where
0:
none;
1:
1–33%;
2:
33–66%;
3:
>66%,
as
described
by
Brunt
et
al.
(1999).
Statistical
analysis
The
data
were
analyzed
by
Kolmogorov–Smirnov
test
for
normality,
and
all
data
showed
a
normal
distribution.
All
values
are
expressed
as
the
mean
±
standard
error
of
the
mean
(SEM).
Statistical
analysis
was
performed
using
one-
way
analysis
of
variance
(ANOVA),
with
Bonferroni
posttest.
Prism
5.0
(GraphPad,
La
Jolla,
CA,
USA)
was
used
to
per-
form
the
analysis.
Differences
were
considered
significant
when
p
<
0.05.
732
A.C.
Rabelo
et
al.
/
Revista
Brasileira
de
Farmacognosia
27
(2017)
729–738
20.0
15.0
10.0
5.0
0.0
1,2,3
4,5
7
6
11
10
9
8
121314
Time
AU
1.60
1.80
2.00
2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
Fig.
1.
RP-UPLC-DAD
fingerprint
section
of
aqueous
extract
of
Baccharis
trimera.
1:
3-O-feruloylquinic
acid;
2:
4-O-caffeinoylquinic
acid;
3:
5-O-caffeinoylquinic
acid;
4:
3-O-
caffeoyylquinic
acid;
5:
4-O-feruloylquinic
acid;
6:
5-O-feruloylquinic
acid;
7:
apigenin-6,8-di-C-glucopyranosidium;
8:
3-O-isoferuloylquinic
acid;
9:
5-O-isoferuloylquinic
acid;
10:
6(8)-C-furanosyl-8(6)-C-hexosyl
flavone;
11:
6(8)-C-hexosyl-8(6)-C-furanosyl
flavone;
12:
3,4-di-O-caffeinylquinic
acid;
13:
3,5-di-O-caffeoyylquinic
acid;
14:
4,5-di-O-caffeinoylquinic
acid;
15:
4-O-isoferuloylquinic
acid.
Table
1
Substances
identified
in
the
aqueous
extract
of
Baccharis
trimera
by
LC-DAD-ESI-MS.
Peak
Compound
RT
(min)
UV
(nm)
LC–MS
[MH](m/z)
(fragmentation
m/m)
1
3-O-feruloylquinic
acid 1.73 321.1
367.29
(193.0;
155.2;
148.9;
134.0)
2
4-O-caffeinoylquinic
acid
1.74
320.1
353.38
(191.0;
178.9;
135.1)
3
5-O-caffeinoylquinic
acid
1.75
323.1
353.38
(191.2;
179.1;
135.1)
4
3-O-caffeoyylquinic
acid
1.86
323.1
353.38
(190.8;
179.0;
137.0)
5
4-O-feruloylquinic
acid
1.88
323.1
367.36
(193.2;
172.8;
149.1;
134.0)
6
5-O-feruloylquinic
acid
1.96
320.1
367.38
(192.7;
172.7;
149.2;
134.2)
7
Apigenin-6,8-di-C-glucopyranosidium 2.12
269.1;
321.0
593.42
(547.1;
503.0;
472.9;
431.1)
8
3-O-isoferuloylquinic
acid
2.25
318.1
367.59
(193.1;
172.8;
149.1;
133.8)
9
5-O-isoferuloylquinic
acid
2.47
323.1
367.17
(191.2;
172.8;
149.1;
134.1)
10
6(8)-C-furanosyl-8(6)-C-hexosyl
flavone
2.36
271.1;
331.1
563.48
(515.1;
473.1;
443.0;
383.1)
11
6(8)-C-hexosyl-8(6)-C-furanosyl
flavone
2.50
271.3;
333.2
563.20
(515.1;
472.9;
442.9;
383.2)
12
3,4-di-O-caffeinylquinic
acid
2.81
282.1;
320.5
515.20
(190.0;
162.9;
134.7)
13
3,5-di-O-caffeoyylquinic
acid
2.93
283.1;
321.2
515.17
(191.0;
163.0;
135.0)
14
4,5-di-O-caffeinoylquinic
acid
2.95
286.1;
320.3
515.40
(190.7;
163.0;
143.2;
127.0)
15
4-O-isoferuloylquinic
acid
3.46
322.1
367.22
(190.6;
163.0;
148.0)
Results
RP-UPLC-DAD-ESI-MS
analysis
of
aqueous
extract
Twelve
phenolic
acids
and
three
flavonoids
were
identified
in
the
aqueous
extract
of
B.
trimera,
by
RP-UPLC-DAD-ESI-MS.
The
RP-
UPLC-DAD
fingerprint
is
shown
in
Fig.
1
(Table
1).
Antioxidant
activity
in
vitro
of
Baccharis
trimera
To
evaluate
the
ability
of
B.
trimera
to
neutralize
the
DPPH
rad-
ical,
five
concentrations
of
extract
were
used.
It
was
observed
that
the
highest
concentration
of
extract
(500
g/ml)
was
able
to
inhibit
DPPH
by
approximately
68%,
similar
to
the
150,174–125,145
g/ml
of
trolox
standard
concentration.
This
means
that
the
extract
at
a
A.C.
Rabelo
et
al.
/
Revista
Brasileira
de
Farmacognosia
27
(2017)
729–738
733
Table
2
Evaluation
of
Baccharis
trimera
ability
to
scavenging
DPPH
radical.
The
high-
est
concentration
of
aqueous
extract
of
Baccharis
trimera
was
able
to
inhibit
DPPH
by
approximately
68%,
while
the
reference
antioxidant
(Trolox)
required
a
concentration
of
300
times
greater
to
inhibit
the
same
percentage.
DPPH:
2,2-diphenyl-1picrylhydrazyl;
Trolox:
6-hydroxy-2,5,7,8-tetra-trhylchroman-2-
carboxylic
acid.
The
values
are
expressed
as
the
mean
±
SD.
DPPH
radical
scavenging
activity
Inhibition
%
Aqueous
extract
of
Baccharis
trimera
(g/ml)
500
68.1
±
11.1
250
30.5
±
1.38
100
8.10
±
0.90
50
3.30
±
0.88
25
0.59
±
0.27
Trolox
(g/ml)
200232
95.9
±
0.19
175203
83.3
±
3.05
150174
72.0
±
2.82
125145
60.4
±
4.79
100116
47.3
±
1.24
75087
32.9
±
1.53
50058
22.0
±
0.77
25029
10.5
±
1.06
concentration
of
approximately
300
times
less
than
trolox
is
able
to
inhibit
the
same
percentage
of
DPPH
(Table
2).
Baccharis
trimera
aqueous
extract
does
not
show
cytotoxicity
in
HepG2
cells
The
results
of
Fig.
2
(panel
A)
showed
that
there
was
no
sig-
nificant
difference
in
the
HepG2
cells
viability
at
concentrations
of
5–25
g/ml
of
aqueous
extract
of
B.
trimera,
with
viability
main-
tained
above
85%.
In
the
concentrations
of
50–600
g/ml
there
was
a
significant
reduction
in
viability
in
relation
to
the
control,
but
maintained
above
75%.
In
panel
B
a
significant
reduction
was
observed
in
cell
viability
from
the
concentration
of
200
M
of
ethanol.
Baccharis
trimera
aqueous
extract
decreased
a
reactive
species
production
in
HepG2
cells
incubated
with
ethanol
It
can
be
observed
in
Fig.
2
that
ethanol
promoted
the
increase
of
ROS
(panel
C)
and
NO
(panel
D)
in
HepG2
cells.
There
was
a
reduction
in
these
parameters
when
the
cells
were
pretreated
with
aqueous
extract
in
both
concentrations
(10
and
50
g/ml),
reaching
similar
levels
to
negative
control
(cells
not
incubated
with
ethanol).
Baccharis
trimera
aqueous
extract
modulates
Nrf2
transcriptional
activity
in
HepG2
cells
It
can
be
observed
in
Fig.
2
that
the
ethanol
is
able
to
induce
the
Nrf2
transcriptional
activity
in
12
h
(panel
E)
and
24
h
(panel
F)
of
incubation,
when
compared
to
the
control.
The
Nrf2
transcriptional
activity
was
increased
when
cells
were
pretreated
with
10
g/ml
of
aqueous
extract
of
B.
trimera,
in
12
h,
when
compared
to
the
ethanol
group
without
treatment.
Whereas
regarding
the
24
h,
the
concentration
of
50
g/ml
of
aqueous
extract
was
able
to
induce
the
increase
the
Nrf2
transcriptional
activity.
Evaluation
of
the
effect
of
Baccharis
trimera
on
biochemical
parameters
in
alcoholic
hepatotoxicity
Renal
function
was
evaluated
based
on
the
parameters
creati-
nine
and
urea.
In
order
to
evaluate
liver
function,
the
parameters
ALT,
AST
and
total
protein
were
used.
Total
cholesterol,
HDL,
frac-
tion
non-HDL
and
triacylglycerol
levels
were
determined
for
the
lipid
profile.
Table
3
showed
the
increase
in
creatinine
levels,
total
Table
3
Biochemical
marker
levels
in
serum
and
plasma
of
rats.
Animals
received
absolute
ethanol
(E);
pretreatment
with
aqueous
extract
(Aq)
and
1
h
after
received
absolute
ethanol.
Control
(C)
received
water.
Different
letters
(a,
b,
c,
d)
indicate
significant
difference
from
each
other
at
p
<
0.05,
while
same
letters
indicate
no
significant
difference
(p
>
0.05).
Biochemical
parameters
Treated
groups
C
E
Aq
Urea
(mg/dl)
59.6
±
1.7
71.81
±
5.5
70.92
±
6.1
Creatinine
(mg/dl)
0.375
±
0.09b0.7361
±
0.087a0.57
±
0.079a
ALT
18.99
±
1.4a21.83
±
2.02a12.0
±
0.65b
AST
37.02
±
3.8
37.76
±
3.5
27.5
±
2.69
Total
protein
(mg/dl) 5.97
±
0.65a4.12
±
0.17b4.62
±
0.4b
Glucose
(mg/dl) 100.6
±
5.8 108.5
±
6.7
103.1
±
10.4
Total
cholesterol
(mg/dl)
97.15
±
2.6c230.0
±
28.24a144.0
±
19.11b
HDL
(mg/dl)
43.8
±
4.05
35.82
±
0.31
42.92
±
1.18
Fraction
non
HDL
(mg/dl)
58.8
±
10.61c189.1
±
20.95a99.98
±
25.82b
Triacylglycerides
(mg/dl)
63.95
±
12.72
53.86
±
6.49
53.22
±
12.78
cholesterol
and
non-HDL
fraction,
besides
a
decrease
in
total
pro-
tein
in
the
group
of
animals
that
received
ethanol.
Pretreatment
with
aqueous
extract
of
B.
trimera
promoted
a
decrease
in
ALT
activ-
ity,
total
cholesterol
and
non-HDL
fraction
and
an
increase
in
total
protein,
compared
with
ethanol
group.
It
was
not
observed
sig-
nificant
differences
in
urea
levels,
AST
activity,
glucose,
HDL
and
triacylglycerides
levels
in
any
of
the
experimental
groups.
Effect
of
Baccharis
trimera
on
the
antioxidant
system
in
alcoholic
hepatotoxicity
It
could
be
observed
in
Fig.
3
that
the
ethanol
consumption
did
not
alter
the
activities
of
SOD
(panel
A)
and
catalase
(panel
B)
enzymes,
compared
with
control
group.
The
aqueous
extract
did
not
alter
these
parameters
either.
Regarding
glutathione
system,
the
results
showed
a
significant
decrease
in
glutathione
total
(panel
C)
and
glutathione
peroxidase
(GPx)
activity
(panel
E),
together
with
an
increase
in
reduced/oxidize
glutathione
ratio
(panel
D)
in
ethanol
group
when
compared
with
control
group.
B.
trimera
aque-
ous
extract
promotes
an
increase
in
GPx
activity
and
a
decrease
in
reduced/oxidize
glutathione
ratio.
Glutathione
reductase
activity
(panel
F)
did
not
alter
in
any
of
the
experimental
groups.
Evaluation
of
the
effect
of
Baccharis
trimera
on
markers
of
oxidative
stress
in
alcoholic
hepatotoxicity
In
ethanol
group
there
was
an
increase
in
TBARS
(Fig.
4
panel
A)
and
carbonylated
protein
(panel
B)
levels,
compared
to
control
group.
The
extract
of
B.
trimera
was
able
to
reduce
only
the
levels
of
TBARS,
no
alterations
were
observed
in
the
levels
of
carbonylated
protein.
Baccharis
trimera
decreases
the
MMP-2
activity
in
alcoholic
hepatotoxicity
To
evaluate
the
MMP-2
activity
the
zymography
technic
was
used.
Fig.
5A
represents
qualitative
images
of
gel
and
5B
repre-
sents
the
quantitative
activity
(band
density).
Then,
it
was
observed
that
ethanol
promoted
an
increase
in
MMP-2
activity,
when
com-
pared
to
the
control.
The
treatment
with
B.
trimera
promotes
the
reduction
of
MMP-2
activity,
in
relation
of
ethanol
group.
Baccharis
trimera
aqueous
extract
reduces
micro-steatosis
in
alcoholic
hepatotoxicity
It
could
be
observed
that
the
animals
from
ethanol
group
show
microvesicular
steatosis,
mainly
grade
3
and
1
(Fig.
6),
whereas
animals
from
control
group
exhibit
mainly
grade
0
and
1.
Treatment
734
A.C.
Rabelo
et
al.
/
Revista
Brasileira
de
Farmacognosia
27
(2017)
729–738
Fig.
2.
Cell
viability
of
Baccharis
trimera
aqueous
extract
(5–600
g/ml)
(A)
and
ethanol
(5–800
mM)
(B)
for
24
h
measured
by
MTT
assay.
Effect
of
Baccharis
trimera
aqueous
extract
on
ethanol-induced
ROS
(C)
and
NO
(D)
production,
expression
MFI
(Medium
intensity
of
fluorescence).
We
also
evaluated
the
effect
of
Baccharis
trimera
on
ethanol-
induced
Nuclear
factor
E2-related
factor
2
(Nrf2),
via
luciferase
assay,
for
12
h
(E)
and
24
h
(F).
The
results
were
expressed
as
mean
±
S.D
(n
=
6).
Different
letters
(a,
b,
c)
indicate
significant
difference
from
each
other
at
p
<
0.05,
while
same
letters
indicate
no
significant
difference
(p
>
0.05).
with
B.
trimera
was
able
to
reduce
the
degree
of
severity
of
the
microvesicular
steatosis,
mainly
grade
1.
Discussion
The
present
study
investigated
the
potential
protective
effects
of
B.
trimera
aqueous
extract
against
hepatotoxicity
induced
by
ethanol,
as
well
the
compounds
present
in
this
extract.
In
vitro,
abil-
ity
of
the
extract
to
scavenge
free
radicals
and
the
effect
of
B.
trimera
on
ethanol
mediated
ROS,
NO
and
transcriptional
activity
of
Nrf2
in
HepG2
cells
were
examined.
It
was
provided
for
the
first
time
that
B.
trimera
aqueous
extract
stimulates
the
transcriptional
activity
of
Nrf2.
In
vivo,
using
a
rat
model
of
acute
intoxication
by
ethanol,
it
was
demonstrated
that
B.
trimera
alleviated
the
oxidative
dam-
ages,
improving
the
antioxidant
defense
and
attenuating
hepatic
steatosis
(Graphic
abstract).
This
encourages
the
advancement
of
research
indicating
B.
trimera
as
therapeutic
agent
for
hepatopro-
tection.
Flavonoids,
caffeic
acid
derivatives
and
diterpenes
have
been
isolated
from
different
extracts
of
B.
trimera
(Abad
and
Bermejo,
2007;
Verdi
et
al.,
2005;
Lívero
et
al.,
2016a,b).
In
our
study,
using
LC-DAD-ESI-MS
three
flavonoids
were
detected
in
aqueous
extract
(apigenin-6,8-di-C-glucopyranosidium
(7);
6(8)-C-furanosyl-8(6)-C-hexosyl
flavone
(10);
6(8)-C-hexosyl-
8(6)-C-furanosyl
flavone
(11))
and
twelve
phenolic
acids
(3-
O-feruloylquinic
acid
(1);
4-O-caffeinoylquinic
acid
(2);
5-
O-caffeinoylquinic
acid
(3);
3-O-caffeoyylquinic
acid
(4);
4-
O-feruloylquinic
acid
(5);
5-O-feruloylquinic
acid
(6);
3-O-
isoferuloylquinic
acid
(8);
5-O-isoferuloylquinic
acid
(9);
3,4-di-
O-caffeinylquinic
acid
(12);
3,5-di-O-caffeoyylquinic
acid
(13);
4,5-di-O-caffeinoylquinic
acid
(14);
4-O-isoferuloylquinic
acid
(15)).
The
ability
of
aqueous
extract
of
B.
trimera
to
sequester
radicals
was
evaluated
and
the
results
showed
that
all
tested
concentrations
showed
an
antioxidant
capacity
in
a
dose-dependent
manner.
For
the
purpose
of
determining
the
concentrations
that
would
be
used
A.C.
Rabelo
et
al.
/
Revista
Brasileira
de
Farmacognosia
27
(2017)
729–738
735
Fig.
3.
Effect
of
Baccharis
trimera
aqueous
extract
on
the
level
of
SOD
(A),
catalase
(B),
glutathione
total
(C),
reduced
and
oxidized
glutathione
ratio
(D),
activity
of
glutathione
peroxidase
(E)
and
glutathione
reductase
(F),
in
the
livers
of
rats.
Control
(C)
received
water.
Animals
received
absolute
ethanol
(E);
pretreatment
with
aqueous
extract
(Aq)
and
1
h
after
received
absolute
ethanol.
Different
letters
(a,
b)
indicate
significant
difference
from
each
other
at
p
<
0.05,
while
same
letters
indicate
no
significant
difference
(p
<
0.05).
in
the
other
in
vitro
assays,
HepG2
cells
were
incubated
with
differ-
ent
concentrations
of
the
aqueous
extract
and
ethanol.
The
results
showed
that
B.
trimera
aqueous
extract
was
not
cytotoxic
at
any
concentration
evaluated.
Rodrigues
et
al.
(2009)
demonstrated
that
the
aqueous
B.
trimera
extract
was
not
cytotoxic
to
bone
marrow
cells
at
any
of
the
concentrations
tested
(500–2000
g/ml).
How-
ever,
Nogueira
et
al.
(2011)
demonstrated
the
aqueous
extract
was
cytotoxic
in
500
g/ml
in
HTC
and
HEK
cells
(rat
hepatoma
cells
and
human
embryo
kidney
epithelial
cells,
respectively)
indicat-
ing
that
the
toxicity
may
be
tissue-specific.
In
relation
to
ethanol,
there
was
a
reduction
in
viability
from
200
mM.
Kumar
et
al.
(2012)
observed
a
significant
decrease
in
HepG2
cells
viability
exposed
to
ethanol
from
100
mM.
Based
on
this,
non-cytotoxic
concentrations
of
B.
trimera
were
selected
(10–50
g/ml)
and
the
ethanol
cytotoxic
concentration
(200
mM)
were
used
in
the
subsequent
experiments.
When
HepG2
cells
were
incubated
with
ethanol
the
produc-
tion
of
ROS
and
NO
was
increased.
Haorah
et
al.
(2011)
also
found
increased
ROS
and
NO
in
endothelial
cells
treated
with
ethanol.
This
increase
can
be
explained
by
the
fact
that
the
main
metabolite
of
ethanol,
acetaldehyde,
activates
NADPH
oxidase
and
inducible
nitric
oxide
synthase
(iNOS),
which
leads
to
an
increase
in
the
pro-
duction
of
ERO
and
nitric
oxide
(NO),
causing
oxidative
damages
(Haorah
et
al.,
2008;
Rump
et
al.,
2010;
Alikunju
et
al.,
2011).
Gong
and
Cederbaum
(2006)
observed
that
in
hepatocytes
isolated
from
rats
there
was
an
increase
of
Nrf2,
probably
due
to
the
induction
of
CYP2E1
promoted
by
ethanol,
which
leads
to
the
increase
in
736
A.C.
Rabelo
et
al.
/
Revista
Brasileira
de
Farmacognosia
27
(2017)
729–738
Fig.
4.
Effect
of
Baccharis
trimera
aqueous
extract
on
the
level
of
TBARS
(A)
and
carbonylated
protein
(B)
in
the
livers
of
rats.
Control
(C)
received
water.
Animals
received
absolute
ethanol
(E);
pretreatment
with
aqueous
extract
(Aq)
and
1
h
after
received
absolute
ethanol.
Different
letters
(a,
b)
indicate
significant
difference
from
each
other
at
p
<
0.05,
while
same
letters
indicate
no
significant
difference
(p
<
0.05).
Fig.
5.
Effect
of
Baccharis
trimera
aqueous
extract
on
the
MMP-2
activity,
via
gelatin
zymography,
in
the
livers
of
rats.
A:
Gel
bands;
B:
band
density.
HT1080
fibrosarcoma
cells
were
used
as
positive
control.
Control
(C)
received
water.
Animals
received
absolute
ethanol
(E);
pretreatment
with
aqueous
extract
(Aq)
and
1
h
after
received
absolute
ethanol.
Different
letters
(a,
b)
indicate
significant
difference
from
each
other
at
p
<
0.05,
while
same
letters
indicate
no
significant
difference
(p
<
0.05).
ROS
production,
with
consequent
activation
of
Nrf2.
These
findings
are
in
agreement
with
our
results
that
found
an
increase
in
Nrf2
transcriptional
activity
in
cells
that
received
only
ethanol.
Ethanol
induced
Nrf2
transcriptional
activity
has
also
been
demonstrated
by
others
authors
(Dong
et
al.,
2011;
Lu
et
al.,
2012).
The
pretreatment
with
B.
trimera
promoted
the
reduction
of
ROS
and
NO,
returning
to
values
similar
to
the
control.
Antioxidants
can
act
directly
through
the
elimination
of
ERO
and
ERN,
or
indirectly,
through
the
modulation
of
signaling
pathways
(Paiva
et
al.,
2015).
Thus,
this
study
and
others
showed
that
B.
trimera
has
the
ability
to
sequester
radicals,
inferring
that
the
decrease
in
these
species
can
be
attributed,
at
least,
to
the
plant’s
direct
action
de
Oliveira
et
al.,
2012;
Pádua
et
al.,
2013).
In
addition,
it
was
also
inferred
that
this
decrease
can
be
attributed
to
the
indirect
action,
since
B.
trimera
aqueous
extract
promoted
an
increase
in
Nrf2
transcrip-
tional
activity,
promoting
antioxidant
protection
against
the
stress
by
the
ethanol.
Lívero
et
al.
(2016a,b)
have
already
demonstrated
that
the
hydroethanolic
extract
of
B.
trimera
promotes
an
increase
expression
of
Nrf2,
but
no
other
study
has
shown
the
effect
of
B.
trimera
over
the
Nrf2
activity.
Thus,
these
data
in
agreement
with
the
decrease
of
ROS
and
NO
show
that
B.
trimera
may
be
effective
in
preventing
stress
induced
by
ethanol.
Fig.
6.
Representative
hematoxylin
and
eosin-stained
histological
sections
of
livers
from
rats
(A).
Semi-quantification
was
demonstrated
in
B.
Severe
microvesicular
steatosis
was
observed
in
the
ethanol
group
(E),
but
not
in
the
control
group
(C).
Baccharis
trimera
aqueous
extract
(Aq)
attenuated
fat
accumulation
in
hepatocytes.
The
images
were
photographed
at
400x
magnification.
Scale
bar
=
50
m.
Asterisk
(*)
indicates
significant
difference
between
two
groups.
To
confirm
the
effects
an
in
vivo
experiment
was
carried
out,
where
the
animals
received
only
absolute
ethanol
or
were
pre-
treated
with
B.
trimera
aqueous
extract.
The
results
showed
a
worsening
kidney
function,
decrease
in
total
protein,
but
ALT
and
AST
transaminases
did
not
change
in
the
ethanol
group.
There
are
several
situations
in
which
there
is
loss
of
correla-
tion
between
serum
levels
of
liver
enzymes
and
a
tissue
injury,
so
that
an
increase
of
serum
activities
of
liver
enzyme
markers
does
not
necessarily
reflect
on
liver
cell
death
(Contreras-Zentella
and
Hernández-Mu˜
noz,
2016).
However,
acetaldehyde
formed
dur-
ing
the
metabolism
of
ethanol
may
form
adducts
with
amino
acids,
reflecting
the
general
decrease
in
protein
synthesis
and
the
decrease
of
plasma
protein
secretion
(Smith
et
al.,
2007).
Pretreat-
ment
with
B.
trimera
significantly
decreased
ALT
activity.
Lívero
et
al.
(2016a,b)
also
found
a
decreased
ALT
activity
when
mice
received
hydroethanolic
extract
of
B.
trimera.
The
acute
admin-
istration
of
alcohol
may
lead
to
a
reduction
or
no
change
in
A.C.
Rabelo
et
al.
/
Revista
Brasileira
de
Farmacognosia
27
(2017)
729–738
737
glucose
concentration,
this
difference
can
be
explained
by
the
nutri-
tional
status
at
the
time
alcohol
is
administered
(Steiner
et
al.,
2015).
Probable
because
our
animals
received
balanced
commer-
cial
feed
no
changes
were
found
in
glucose
in
any
experimental
group.
The
results
also
showed
an
increase
in
total
cholesterol,
non-
HDL
fraction,
beyond
hepatic
micro-steatosis,
but
TAG
did
not
change
in
ethanol
group.
After
acute
consumption
of
high
doses
of
ethanol
the
serum
levels
of
TAG
may
increase,
decrease
or
remain
normal,
however,
the
total
flow
that
is
absorbed
by
the
liver
is
increased
due
to
the
stimulatory
effects
of
ethanol
on
liver
blood
flow
(Baraona
and
Lieber,
1979),
promoting
the
accumulation
of
micro
and/or
macrovesicles
lipids
in
hepatocytes
(Baraona
and
Lieber,
1979;
Lívero
and
Acco,
2016).
B.
trimera
improved
the
lipid
profile
and
decreased
hepatic
micro-steatosis,
protecting
against
ethanol
damage.
Lívero
et
al.
(2016a,b)
showed
that
hydroethano-
lic
extract
of
B.
trimera
decreases
the
expression
of
the
Scd1
gene,
which
is
responsible
for
encoding
the
stearoyl-CoA
desaturase-1,
an
important
enzyme
in
the
biosynthesis
of
the
main
fatty
acids
found
in
TAG.
Maybe
part
of
this
mechanism
contributes
to
the
protection
mechanism
by
the
extract.
The
ability
of
ethanol
to
induce
oxidative
stress
and
antioxidant
depletion,
such
as
glutathione,
is
well
recognized
(Lu
and
Ceder-
baum,
2008;
Han
et
al.,
2016).
Our
results
showed
that
ethanol
alterations
in
glutathione
metabolism
were
more
significant
than
alterations
in
SOD
and
CAT
activities,
since
ethanol-treated
rats
exhibited
a
decrease
in
GPx
activity
and
decrease
in
oxidized
glu-
tathione,
reflecting
an
increase
in
the
GSH/GSSG
glutathione
ratio.
The
decrease
in
GPx
activity
in
ethanol-induced
intoxication
has
also
been
demonstrated
in
other
studies
(Park
et
al.,
2013;
Li
et
al.,
2014;
Yan
et
al.,
2014).
GPx
is
one
of
the
responsible
for
the
reduction
of
H2O2to
water,
since
a
decrease
in
the
activity
of
this
enzyme
was
observed,
it
is
possible
to
infer
that
in
these
animals
there
was
probably
accumulation
of
H2O2,
contributing
to
oxidative
stress.
This
inefficiency
in
the
antioxidant
response
may
justify
the
increase
in
the
TBARS
and
carbonylated
protein
levels
observed
in
our
study.
In
carbon
tetrachloride-in