Process
Biochemistry
46
(2011)
2210–2214
Contents
lists
available
at
SciVerse
ScienceDirect
Process
Biochemistry
jo
u
rn
al
hom
epa
ge:
www
.elsevier.com/locate/procbio
Short
communication
A
straightforward
method
to
determine
the
cytocidal
and
cytopathic
effects
of
the
functional
groups
of
gallic
acid
Dan
Li,
Zuojia
Liu,
Wenjing
Zhao,
Yanli
Xi,
Fenglan
Niu
∗
School
of
Public
Health,
Jilin
University,
Changchun,
130021,
PR
China
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
7
December
2010
Received
in
revised
form
16
August
2011
Accepted
19
August
2011
Available
online
26
August
2011
Keywords:
Gallic
acid
Gold
nanoparticles
Cytotoxicity
Phenolic
hydroxyl
a
b
s
t
r
a
c
t
Gallic
acid
(GA)
has
well-known
antioxidative,
antimutagenic,
antiviral
and
anti-inflammatory
proper-
ties.
GA
has
been
used
to
label
gold
nanoparticles
for
bio-analytical
applications,
but
the
effects
of
gold
nanoparticles
coated
with
GA
on
human
cell
lines
have
not
been
studied.
We
synthesized
GA-coated
gold
nanoparticles
(GA-AuNPs)
and
compared
the
biochemical
effects
of
the
particles
to
that
of
free
GA
on
the
human
hepatocyte
cell
line
HL-7702.
Cell
proliferation
measurements
and
apoptosis
assays
indicated
that
the
presence
of
GA-AuNPs
was
less
cytotoxic
and
was
less
likely
to
induce
apoptosis.
Synthesis
of
the
labeled
nanoparticles
resulted
in
the
oxidation
of
the
two
phenolic
hydroxyls
of
GA
to
their
quinine
forms.
Because
this
was
the
only
chemical
change
to
the
molecule,
the
differences
in
toxicity
between
free
GA
and
GA-AuNPs
suggested
that
these
phenolic
hydroxyls
were
necessary
for
the
bioactivity
of
GA
molecules.
This
study
might
also
reveal
a
new
and
easier
way
to
illuminate
the
importance
of
the
phenolic
hydroxyls
of
GA
in
the
cytocidal
and
cytopathic
effects
on
cells.
©
2011
Elsevier
Ltd.
All
rights
reserved.
1.
Introduction
Gallic
acid
(GA),
or
3,4,5-trihydroxybenzoic
acid,
is
found
in
fruits
and
plants,
such
as
grapes,
strawberries,
pineapples,
bananas,
lemons,
gallnuts,
tea
leaves
and
oak
bark
[1,2].
It
is
well
known
for
its
natural
and
strong
antioxidative,
antiallergic,
antimutagenic,
anticarcinogenic,
antiviral,
anti-bacterial
and
anti-inflammatory
abilities
[3–8].
It
has
also
been
found
to
induce
apoptosis
in
some
tumor
cell
lines
[9–11]
and
plays
an
important
role
in
preventing
malignant
transformation
and
cancer
development
in
vivo
[8].
Gold
nanoparticles
and
GA-coated
gold
nanoparticles
(GA-
AuNPs)
have
attracted
the
interest
of
many
researchers
in
recent
years
for
their
physical
and
chemical
properties
in
bio-analytical
applications.
Advancements
in
the
synthesis
of
GA-coated
gold
nanoparticles
have
led
to
better
techniques
for
the
detection
of
lead
by
reducing
variations
in
the
size
distribution
of
GA-AuNPs
and
minimizing
electrostatic
repulsion
between
each
nanoparti-
cle
[12,13].
These
modifications
have
decreased
the
detection
limit
for
Pb
2+
to
10
nM.
Gold
nanoparticles
have
also
been
synthesized
by
reducing
HAuCl
4
with
GA
in
the
presence
of
poly-(N-vinyl-2-
pyrrolidone)
(PVP).
GA
functions
as
the
reducing
agent,
and
PVP
stabilizes
the
construct.
The
PVP
protected
nanoparticles
were
then
used
as
nucleic
acid
probes
after
further
modification
[14].
Thomas
and
co-workers
studied
the
mechanism
of
GA-AuNPs
formation
by
conducting
several
control
experiments
using
a
∗
Corresponding
author.
Tel.:
+86
431
85619133.
E-mail
address:
niufl@jlu.edu.cn
(F.
Niu).
variety
of
model
compounds
[12].
They
demonstrated
that
during
the
formation
of
GA-AuNPs,
two
adjacent
dihydroxyl
groups
of
GA
undergo
two-electron
oxidation
to
the
corresponding
quinone
forms,
and
the
nanoparticles
that
created
are
stabilized
by
the
interaction
of
the
carboxylic
acid
group
of
GA
[12].
To
the
best
of
our
knowledge,
a
study
analyzing
the
cytocidal
or
cytopathic
effect
of
GA-AuNPs
on
any
cell
lines
has
not
been
per-
formed,
and
the
possible
medical
applications
of
GA-coated
gold
nanoparticles
make
the
cytotoxicity
of
these
structures
relevant.
Therefore,
we
synthesized
gold
nanoparticles
that
were
reduced
and
stabilized
by
GA
and
then
measured
the
biochemical
effects
on
human
hepatocyte
HL-7702
cells
compared
to
free
GA.
Dif-
ferences
were
detected
by
MTT
assay,
Hoechst
33342
staining,
Annexin
V
assays
and
mitochondrial
membrane
potential
(MMP)
measurements.
Under
these
conditions,
GA-AuNPs
were
less
toxic,
produced
a
lower
incidence
of
cell
death
and
triggered
apoptosis
less
often
than
free
GA.
Combined
with
the
mechanism
of
synthe-
sis
of
GA-AuNPs,
the
data
directly
shows
that
the
phenolic
hydroxyl
groups
of
GA
are
responsible
for
its
cytotoxicity
and
the
induction
of
apoptosis.
Thus,
this
study
provided
a
new
and
easy
method
to
ascertain
the
biochemical
effects
of
the
functional
groups
of
GA
on
a
human
cell
line.
2.
Materials
and
methods
2.1.
Materials
HAuCl
4
,
NaBH
4
,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bro-
mide
(MTT),
Hoechst
33342
and
rhodamine
123
(R123)
were
obtained
from
Sigma–Aldrich
(USA).
RPMI
1640
and
fetal
bovine
serum
(FBS)
were
purchased
from
1359-5113/$
–
see
front
matter
©
2011
Elsevier
Ltd.
All
rights
reserved.
doi:10.1016/j.procbio.2011.08.011