Poster presented on the 11th international ISSX Conference

1. Preclinical Pharmacokinetic Profile
of Four Traditional Medicinal Plants
André Luís Dias Araujo Mazzari1,Francisco de Assis Rocha Neves2,
Dâmaris Silveira2, Jose M. Prieto1
1. Houghton
P,
Fang
R,
Techatanawat
I,
Steventon
G,
Hylands
PJ,
Lee
CC.
The
sulphorhodamine
(SRB)
assay
and
other
approaches
to
tes9ng
plant
extracts
and
derived
compounds
for
ac9vi9es
related
to
reputed
an9cancer
ac9vity.
Methods
2007;42:
377-­‐387
2. RepeAo
G,
del
Peso
A,
Zurita
JL.
Neutral
red
uptake
assay
for
the
es9ma9on
of
cell
viability/cytotoxicity.
Nat
Protoc
2008;
3:
1125-­‐1131
3. Chieli
E,
Santoni
Rugiu
E,
Cervelli
F,
Paolicchi
A,
Tongiani
R.
Assessment
of
P-­‐glycoprotein-­‐dependent
drug
transport
in
isolated
rat
hepatocytes
using
rhodamine
123.
Cell
Biol
Toxicol.
1993;
9:
235-­‐241
4. Allen
S,
Shea
JM,
Felmet
T,
Gadra
J,
Dehn
PF.
A
kine9c
microassay
for
glutathione
in
cells
plated
on
96-­‐well
micro9ter
plates.
Methods
Cell
Sci
2000;22:
305-­‐312
5. Rebbeor,
J.F.,
Wang,
W.,
CliXon,
D.,
and
Ballatori,
N.
(1998).
Glutathione
S-­‐conjugate
forma9on
and
metabolism
in
HepG2
cells:
a
cell
model
of
mercapturic
acid
biosynthesis.
J
Toxicol
Environ
Health
A
53,
651-­‐663.
6. Rahman,
I.,
Kode,
A.,
and
Biswas,
S.K.
(2006).
"Assay
for
quan9ta9ve
determina9on
of
glutathione
and
glutathione
disulfide
levels
using
enzyma9c
recycling
method,"
in
Nat
Protoc.
(England),
3159-­‐3165.
Cell
viability
At
a
glance
Acknowledgements
P-­‐gp
Rhodamine123
GSH
CYP
3A4
P-gp
Efflux Drugs
Intracellular
Glutathione Levels
mRNA extraction
cDNA synthesis
GGT
Molecular
Biology
Studies
References
The
method
used
in
the
intracellular
determina9on
of
glutathione
levels
was
adapted
from
those
described
by
Rahman
and
coworkers
(Rahman
et
al.,
2006)
and
Allen
and
coworkers
(Allen
et
al.,
2000)
with
slight
modifica9ons.
AXer
24h
incuba9on
with
BSO
(10μM)
or
plant
extracts
(100μg/mL),
HepG2
cells
(4
×
104
cells/well)
were
washed
with
PBS
and
60µL
of
0.1%
Triton-­‐X
was
added
to
each
well
of
the
plates
to
lyse
the
cells.
25µL
of
5%
sulfosalicylic
acid
was
added
to
the
cell
lysates
and
plates
were
shaken
for
2min.
25µL
of
glutathione
reac9on
buffer
containing
NADPH
(2.39mM),
DTNB
(0.01M)
and
glutathione
reductase
(500UI)
in
sodium
phosphate
buffer
(143mM)
containing
EDTA
(6.3mM)
was
added
to
the
cell
lysates.
Absorbance
was
read
in
a
kine9c
cycle
in
the
plate
reader
every
30s
for
5min
at
405nm
(11
readings).
Absorbances
were
converted
into
absolute
amounts
by
means
of
the
i-­‐
slopes
method
using
known
concentra9ons
of
L-­‐
glutathione
reduced.
Rhodamine
uptake/efflux
assays
were
conducted
as
described
by
Chieli
and
coworkers
with
minor
modifica9ons
(Chieli
et
al.,
1993).
AXer
five
passages
in
media
containing
vincris9ne
(50µM),
Caco-­‐2
VCR
cells
(1
×
104
cells/well)
were
incubated
for
2h
with
the
P-­‐gp
inhibitor
verapamil
(20µM)
or
plant
extracts
(100µg/mL)
in
serum-­‐free
media
containing
rhodamine
123
(5µg/mL).
AXer
incuba9on,
cells
were
washed
with
verapamil
(20µM)
in
PBS.
Cells
were
lysed
with
100µL
of
0.1%
Triton
X-­‐100
in
PBS
and
the
plates
were
placed
in
the
incubator
for
15min.
The
fluorescence
intensity
of
cell
lysates
was
measured
in
the
plate
reader
(Exc-­‐485nm,
Em-­‐525nm).
The
cellular
accumula9on
of
rhodamine
123
for
each
of
the
extracts
was
expressed
as
the
percentage
of
the
accumula9on
measured
for
rhodamine
123
under
control
condi9ons.
GGT
ac9vity
assay
was
conducted
according
Rebbeor
and
coworkers
(Rebbeor
et
al.,
1998).
Briefly,
aXer
24h
incuba9on
of
HepG2
cells
(1
×
106
cells/well)
with
the
GGT
inhibitor
acivicin
(5µM)
or
plant
extracts
(100µg/mL),
media
was
aspirated
and
cells
were
washed
with
PBS.
4mL
of
pre-­‐warmed
glycylglicine
buffer
(115mM
Tris,
138mM
glycylglycine)
and
400µL
of
the
substrate
γ-­‐Glutamyl-­‐p-­‐nitroanilide
(29.6mg/mL
of
HCl
0.5mmol/L)
were
added
to
the
wells
and
plates
were
incubated
for
10min.
Then,
500µL
of
the
content
of
each
well
were
transferred
to
24-­‐well
plates
and
absorbance
was
measured
in
the
plate
reader
(405nm).
Absorbances
were
converted
into
absolute
amounts
by
means
of
a
calibra9on
line
using
4-­‐nitroaniline.
GGT
activity
Rh123
accumula9on
GSH
level
folds
of
induc9on
p-­‐nitroaniline
produc9on
1Department of Pharmaceutical & Biological Chemistry,
UCL School of Pharmacy 29-39 Brunswick Square, WC1N 1AX, London, UK
2Faculty of Health Sciences, University of Brasília, Brasília-DF, Brazil
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GSH
G
S
H
H2N CH C
CH2
OH
O
SH
Cysteine
GSH
breakdown
cDNA
mRNA
copies
of
the
target
gene
RT-­‐qPCR
amplificaLon
of
CYP3A4
gene
For
the
SRB
assay
(Houghton
et
al.,
2007),
aXer
24h
incuba9on
of
HepG2
cells
(2
×
104
cells/well)
with
the
plant
extracts
(100µg/mL),
media
containing
the
samples
was
removed
and
cells
were
fixed
with
100µL
of
cold
40%
w/v
TCA
solu9on
in
deionized
water.
The
plates
were
incubated
at
4°C
for
1h
and
then
immersed
five
9mes
in
tap
water.
The
TCA-­‐fixed
cells
were
stained
by
adding
100µL
of
SRB
solu9on
(0.4
%
SRB
in
0.1%
glacial
ace9c
acid)
and
leX
at
room
temperature
for
1h.
AXerwards,
the
plates
were
quickly
rinsed
four
9mes
with
1%
ace9c
acid
and
flicked
to
remove
unbound
dye
and
then
leX
to
air-­‐dry
overnight.
AXer
drying
completely,
the
protein
bound
SRB
was
solubilized
by
adding
100µL
of
Tris
base
buffer
solu9on
(10mM)
to
each
well.
The
plates
were
agitated
in
an
orbital
shaker
for
30min.
The
op9cal
density
was
measured
at
492nm
by
using
a
microplate
plate
reader
Infinite
M200
(Tecan
Trading
AG,
Switzerland).
Cell Viability Assays
Sulphorhodamine B (SRB)
and Neutral Red
For
Neutral
Red
(Repero
et
al.,
2008),
aXer
24h
incuba9on
of
Caco-­‐2
VCR
cells
(1
×
104
cells/well)
with
the
plant
extracts
(100µg/mL),
media
was
removed
and
100µL
of
NR
solu9on
(40µg/ml)
pre-­‐
warmed
at
37°C
was
added
to
each
well
and
all
plates
were
placed
in
the
incubator
at
37°C
for
2h.
The
cells
were
rinsed
with
150µL
of
PBS
and
the
washing
solu9on
removed
by
decan9ng
or
gently
tapping
the
plate.
150µL
of
neutral
red
destain
solu9on
(96%
ethanol,
deionised
water,
glacial
ace9c
acid;
50%:49%:1%)
was
added
to
each
well
of
the
plate
aXer
the
washing
step.
The
plate
was
immediately
shaken
on
a
microplate
shaker
(IKA
MS3,
Germany)
for
at
least
10min
un9l
the
neutral
red
had
been
completely
extracted
from
the
cells
and
formed
a
homogenous
solu9on.
The
absorbance
of
the
neutral
red
extract
was
read
out
in
the
plate
reader
at
540nm.
SH
Glutathione
(GSH)
AXer
exposing
HepG2
cells
(5
×
105
cells/
well)
to
plant
extracts
or
the
CYP3A4
inducer
Rifampicin
(50mM)
or
the
CYP
3A4
inhibitor
DMSO
1%
for
96h,
total
RNA
was
extracted
from
using
TRIzol®
Reagent
according
to
the
manufacturer’s
protocol.
Samples
were
treated
with
DNase
I
(1U/μL)
to
avoid
genomic
contamina9on.
The
quan9ty
and
quality
of
RNA
was
determined
by
differen9al
readings
at
260
and
280nm
in
a
Nanodrop
2000
(Thermo
Scien9fic).
Integrity
of
total
RNA
from
HepG2
cells
was
assessed
by
visual
inspec9on
of
the
two
rRNAs
28s
and
18s
on
agarose
gels.
cDNA
was
synthesized
from
1μg
of
total
RNA
with
the
Moloney
rine
Leukemia
Virus
Reverse
Transcriptase
(M-­‐MLV
RT)
(200U/μL)
and
oligo(dT)
12-­‐18
primer
(0.5μg/μL),
according
to
the
manufacturer’s
instruc9on
in
a
final
volume
of
21μL.
RT-qPCR conditions and analysis
CYP3A4
sense
strand
primer
sequence
was
5’-­‐
CAAGGACAACATAGATCGTTACATATACACACCCTT
TGGAAG-­‐3’
and
the
an9sense
strand
primer
was
5’-­‐
AGCTCAATGCATGTACAGAATCCCCGGTTA-­‐3’.
The
β-­‐ac9n
gene
was
used
to
control
for
varia9ons
in
RNA
loading
within
the
experimental
condi9ons.
The
sense
strand
p r i m e r
s e q u e n c e
w a s
5 ’ -­‐
CGTACCACTGGCATCGTGAT-­‐3’and
the
an9sense
s t r a n d
p r i m e r
w a s
5 ’ -­‐
GTGTTGGCGTACAGGTCTTTG-­‐3’.
The
RT-­‐qPCR
was
carried
out
in
96-­‐well
plates
using
a
Pikoreal™
Real-­‐Time
PCR
detec9on
system
(Thermo
Scien9fic).
Each
well
contained
a
final
reac9on
volume
of
10μL
(5.0μL
MasterMix
with
SYBR
Green,
2.0μL
cDNA
template
diluted
appropriately,
0.5μL
of
each
primer
at
a
final
concentra9on
0.3mM
and
2.0μL
of
RNAse/DNAse
free
dis9lled
water).
PCR
reac9on
was
performed
under
following
schema:
ini9al
denatura9on
at
95°C
for
2min,
then
40
cycles
of
denatura9on
at
95°C
for
15s,
annealing
at
55°C
(β-­‐ac9n)
or
60°C
(CYP3A4)
for
30s,
and
extension
at
72°C
for
30s.
At
the
end
of
the
run,
a
mel9ng
curve
was
generated
by
hea9ng
the
amplicon
from
60
to
95◦C
in
order
to
confirm
the
specificity
of
the
amplifica9on
for
each
primer
pair.
All
RT-­‐qPCR
were
run
in
quadruplicates.
Standard
curves
were
produced
to
check
the
PCR
efficiency
using
a
five-­‐fold
dilu9on
series
of
cDNA.
Efficiency
(E)
of
primer
pairs
was
obtained
from
the
slope
of
the
calibra9on
curve
generated.
The
rela9ve
expression
was
calculated
on
the
basis
of
‘delta
delta
Ct’
(ΔΔCt)
values.
Normaliza9on
of
target
gene
was
achieved
by
using
β-­‐ac9n
as
a
reference
gene.
RP
-­‐
Rhamnus
purshiana
DC.
(Rhamnaceae)
SA
-­‐
Salix
alba
L.
(Salicaceae)
AF
-­‐
Apuleia
ferrea
(Mart.)
Baill.
(Leguminosae)
VP
-­‐
Vernonia
polyanthes
Less.
(Compositae)
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Caco-­‐2VCR
HepG2
Herbal
Medicines
in
Brazil:
pharmacokine9c
profile
and
poten9al
herb-­‐drug
interac9ons
Pharmacovigilance
in
Brazil:
the
monitoring
of
herb-­‐drug
interac9ons
using
pharmacokine9c
data
Plant
extracts
(Conc.
100μg/mL)
GSH
GSH
GSH
GSH
GSH
GSH
GSH
GSH
GSH
Substrate
GGT
Rh
123
Rh
123
Rh
123
Rh
123
Rh
123
Rh
123
Rh
123
Rh
123
Rh
123
Substrate
Rh
123
Rh
123
Rh
123
Rh
123
Rh
123
Apuleia
ferrea
(AF),
Salix
alba
(SA),
Rhamnus
purshiana
(RP)
and
Vernonia
polyanthes
(VP)
are
medicinal
plants
shortlisted
by
the
Brazilian
Na9onal
Health
System
for
future
clinical
use.
However,
nothing
is
known
about
their
effects
on
metabolic
and
transporter
proteins
responsible
for
the
pharmacokine9c
profile
of
drugs.
• ALL
four
medicinal
plants
increased
CYP3A4
mRNA
gene
expression.
• ALL
four
medicinal
plants
significant
decreased
glutathione
(GSH)
levels,
this
effect
could
be
explained
by
the
inhibi9on
of
Gamma-­‐Glutamyl
Transferase
(GGT)
for
SA
but
not
for
the
other
medicinal
plants.
• SA
and
AF
significantly
impaired
Rh123
efflux
which
depends
on
P-­‐glycoprotein
(P-­‐gp)
ac9vity.
This
data
warrant
more
research
on
how
consump9on
of
these
medicinal
plants
may
pose
a
risk
of
herb-­‐drug
interac9ons.
INTRODUCTION
RESULTS
UPSHOT
%
Viability