Effect of biologically synthesized gold nanoparticles on alloxan-induced diabetic rats-An in vivo approach

Abstract

Development of novel antidiabetic agents using various organic compounds and biomolecules has been in practice for a long time. Recently, nanomaterials are also being used in antidiabetic studies for their unique properties such as small size, biocompatibility and ability to penetrate cell membrane for carrying drugs. Herein, in vivo antidiabetic activity of gold nanoparticles (AuNPs) synthesized using the antidiabetic potent plant Gymnema sylvestre R. Br on wistar albino rats has been evaluated. The formation of AuNPs and their morphology were confirmed using spectroscopic and microscopic analyses, respectively. The treatment of AuNPs has shown significant reduction in blood glucose level on diabetic rats. AuNPs were also tested for its anti-inflammatory effect by estimating the serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and high-sensitive C-reactive protein (CRP).

Full text

Colloids
and
Surfaces
B:
Biointerfaces
122
(2014)
505–511
Contents
lists
available
at
ScienceDirect
Colloids
and
Surfaces
B:
Biointerfaces
j
o
ur
nal
ho
me
pa
ge:
www.elsevier.com/locate/colsurfb
Effect
of
biologically
synthesized
gold
nanoparticles
on
alloxan-induced
diabetic
rats—An
in
vivo
approach
V.
Karthicka,
V.
Ganesh
Kumara,∗,
T.
Stalin
Dhasa,
G.
Singaraveluc,
A.
Mohamed
Sadiqb,
K.
Govindarajua
aNanoscience
Division,
Centre
for
Ocean
Research,
Sathyabama
University,
Chennai
600119,
India
bDepartment
of
Biochemistry,
Adhiparasakthi
College
of
Arts
and
Science,
Kalavai
632506,
India
cNanoscience
Division,
Department
of
Zoology,
Thiruvalluvar
University,
Vellore
632115,
India
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
3
June
2014
Received
in
revised
form
9
July
2014
Accepted
14
July
2014
Available
online
21
July
2014
Keywords:
Diabetes
Gold
nanoparticles
Gymnema
sylvestre
Electron
microscopy
Hypoglycemia
a
b
s
t
r
a
c
t
Development
of
novel
antidiabetic
agents
using
various
organic
compounds
and
biomolecules
has
been
in
practice
for
a
long
time.
Recently,
nanomaterials
are
also
being
used
in
antidiabetic
studies
for
their
unique
properties
such
as
small
size,
biocompatibility
and
ability
to
penetrate
cell
membrane
for
carrying
drugs.
Herein,
in
vivo
antidiabetic
activity
of
gold
nanoparticles
(AuNPs)
synthesized
using
the
antidiabetic
potent
plant
Gymnema
sylvestre
R.
Br
on
wistar
albino
rats
has
been
evaluated.
The
formation
of
AuNPs
and
their
morphology
were
confirmed
using
spectroscopic
and
microscopic
analyses,
respectively.
The
treatment
of
AuNPs
has
shown
significant
reduction
in
blood
glucose
level
on
diabetic
rats.
AuNPs
were
also
tested
for
its
anti-inflammatory
effect
by
estimating
the
serum
levels
of
tumor
necrosis
factor-alpha
(TNF-␣),
interleukin-6
(IL-6)
and
high-sensitive
C-reactive
protein
(CRP).
©
2014
Elsevier
B.V.
All
rights
reserved.
1.
Introduction
With
the
ever
changing
life
style
of
humans,
the
chances
of
vulnerability
towards
disease
increases
and
one
among
the
most
common
complication
is
diabetes,
which
is
characterized
by
high
blood
glucose
levels
(BGL)
often
related
to
eating
habits,
age
and
heredity.
US
Department
of
Health
and
Human
Services
reported
that
the
risk
of
complications
like
cardiovascular
problems,
kid-
ney
disease,
neurological
disorders,
and
hypertension
is
greater
in
patients
having
diabetes
[1].
Diabetes
is
mainly
classified
into
type
I
(T1D)—diabetes
mellitus
and
type
II
(T2D)
and
gestational
diabetes
in
the
case
of
pregnant
women.
Among
prominent
classi-
fications,
T2D
receives
the
main
focus
as
it
affects
large
proportion
of
populace
throughout
the
world
by
increased
levels
of
blood
glu-
cose
owing
to
insulin
resistance
in
adipose
tissue,
muscle
and
liver
due
to
impaired
insulin
secretion
from
pancreatic
␤-cells
[2].
The
use
of
nanomedicine
for
treatment
of
diabetes
has
been
attempted
recently
[3].
Also
researchers
are
developing
various
antidiabetic
agents
for
controlling
diabetes
using
in
vitro
and
in
vivo
research
∗Corresponding
author.
Tel.:
+91
44
24500646;
fax:
+91
44
24500646.
E-mail
address:
ganeshkumar@sathyabamauniversity.ac.in
(V.G.
Kumar).
methods
[4]
but
some
are
limited
due
to
their
poor
pharmacoki-
netic
properties
[5,6].
Nanomaterials
have
innumerable
applications
in
variety
of
industries
[7].
In
general,
noble
metal
nanoparticles
are
utilized
for
various
biomedical
applications
and
significant
results
have
been
obtained
in
the
recent
past
[8].
Particularly,
gold
nanoparti-
cles
(AuNPs)
are
employed
for
the
treatment
of
various
diseases
[9]
due
to
their
unique
optical,
chemical
and
biological
properties
[10].
Though
variety
of
protocols
is
in
practice
[11],
preparation
of
AuNPs
by
biosynthesis
process
has
been
the
most
preferred
methodol-
ogy
for
its
facile
and
environment-friendly
approach.
Gymnema
sylvestre
R.
Br
is
a
woody
climber
belonging
to
the
family
of
Ascle-
piadaceae
that
has
profound
antidiabetic
potential
[12].
It
has
been
extensively
studied
for
its
antihyperglycemic
effect
[13]
as
it
contains
several
active
compounds
useful
for
reducing
the
blood
glucose
levels
in
the
case
of
diabetes
mellitus
[14].
Gymnemic
triac-
etate
isolated
from
G.
sylvestre
was
tested
for
its
antidiabetic
activity
on
streptozotocin-induced
diabetic
model
[15].
Similarly,
exten-
sive
studies
have
been
carried
out
for
antidiabetes
and
its
related
complications
using
various
medicinal
plants
[16].
In
the
present
investigation,
biosynthesis
of
AuNPs
using
antidiabetic
potent
G.
sylvestre
R.
Br.
and
its
effect
on
alloxan-induced
diabetic
model
is
discussed.
http://dx.doi.org/10.1016/j.colsurfb.2014.07.022
0927-7765/©
2014
Elsevier
B.V.
All
rights
reserved.

506
V.
Karthick
et
al.
/
Colloids
and
Surfaces
B:
Biointerfaces
122
(2014)
505–511
2.
Materials
and
methods
2.1.
Materials
Chloroauric
acid
(HAuCl4)
was
obtained
from
Loba
Chemie,
India.
All
other
chemicals
used
in
the
reaction
were
of
analytical
grade
and
used
as
received.
Fresh
leaves
of
G.
sylvestre
R.
Br.
were
collected
from
Veeranam
(12◦11N;
78◦5E),
Tiruvannamalai
dis-
trict,
Tamilnadu,
India,
in
the
month
of
September
2012.
It
was
cleaned
with
double
distilled
water
to
remove
the
dust
and
shade
dried
for
a
week
at
room
temperature.
The
leaves
were
ground
to
powder
and
stored
for
further
studies.
2.2.
Synthesis
of
AuNPs
using
G.
sylvestre
leaf
extract
The
plant
extract
was
prepared
by
taking
4
g
of
dry
G.
sylvestre
leaf
powder
along
with
40
mL
of
distilled
water
in
a
conical
flask
and
kept
in
an
orbital
shaker
for
24
h
followed
by
filtering
of
extract
using
No.
1
Whatman
filter
paper.
The
filtered
extract
was
stored
at
4–8 ◦C
for
further
use.
The
volume
of
plant
extract
required
for
the
synthesis
was
optimized
using
trial-and-error
method
and
found
to
be
0.6:10
(plant
extract:chloroauric
acid
solution).
2.4
mL
of
extract
was
added
to
40
mL
of
1
mM
HAuCl4solution
and
kept
in
shaker
for
the
reduction
of
Au3+ to
Au0.
Change
in
color
from
pale
yellow
to
ruby
red
within
5
min
indicates
that
the
reaction
was
complete
and
there
was
no
further
color
change.
2.3.
Characterization
of
AuNPs
Ultraviolet–visible
(UV–vis)
spectra
of
the
reaction
mixture
(plant
extract
and
chloroauric
acid
solution)
were
recorded
in
the
range
of
400–700
nm
using
a
Shimadzu
UV-1800
UV–vis
spec-
trophotometer.
The
functional
groups
involved
in
the
reduction
of
chloroauric
acid
were
detected
by
recording
an
infrared
spectrum
employing
potassium
bromide
(KBr)
pellet
technique
using
Perkin
Elmer
model-983/G
detector
double
beam
FT-IR
spectrophotome-
ter
in
the
range
of
400–450
cm−1.
The
high-resolution
transmission
electron
microscopy
(HRTEM)
analysis
was
carried
out
using
high-
resolution
transmission
electron
microscopy
JEOL
3010.
For
atomic
force
microscopic
(AFM)
analysis,
NTEGRA
PRIMA-NTMDT
instru-
ment
was
used
and
a
thin
film
of
the
sample
was
prepared
by
dropping
20
␮L
of
the
sample
onto
the
cover
slip.
Zeta
potential
of
the
synthesized
AuNPs
was
evaluated
with
the
help
of
Malvern
Zeta-sizer
Nano
ZS
(Malvern
Instrument,
Malvern,
Worcestershire,
UK).
2.4.
Experimental
animals
Healthy
male
Wistar
albino
rats
weighing
180
±
20
g
were
used
in
the
experimentation.
All
animals
were
maintained
under
a
con-
stant
12-h-light/12-h-dark
exposure
cycle
at
temperature
around
22 ◦C
and
were
fed
with
standard
diet
pellet
(Hindustan
Lever
Ltd.,
India)
and
water
ad
libitum.
Animals
were
maintained
as
per
the
guidelines
of
National
Institute
of
Nutrition,
Indian
Council
of
Med-
ical
Research,
Hyderabad,
and
the
experimental
part
was
approved
by
the
Institutional
Animal
Ethical
Committee
(IAEC),
Adhiparasak-
thi
College
of
Arts
and
Science,
Kalavai.
2.5.
Diabetes
induction
Prior
to
the
experiment,
rats
were
treated
with
alloxan
mono-
hydrate
in
saline
(100
mg/kg
body
weight)
and
was
optimized
since
the
test
subjects
were
unstable
when
given
with
higher
dosage
of
alloxan
[16].
After
48
h,
rats
were
tested
for
hyperglycemic
condi-
tion
(considering
the
glucose
level
of
around
250
mg/dL)
and
were
taken
for
treatment.
The
concentration
of
AuNPs
was
fixed
using
previously
established
work
for
conducting
the
study
[3].
2.6.
Experimental
design
The
rats
were
randomly
divided
into
five
groups
each
containing
six
animals
as
follows
and
were
treated
daily
for
28
days.
Group
I:
Normal
treated
with
saline
Group
II:
Diabetic
control
treated
with
alloxan
(100
mg/kg
body
weight)
Group
III:
Diabetic
rats
treated
with
glibenclamide
(0.5
mg/kg
body
weight)
Group
IV:
Diabetic
rats
treated
with
AuNPs
(0.5
mg/kg
body
weight)
Group
V:
Normal
rats
treated
with
AuNPs
(0.5
mg/kg
body
weight)
The
AuNPs
(mixed
with
saline)
and
standard
drug
were
given
orally
using
gavage.
At
the
end
of
the
experimental
period,
the
rats
were
fasted
overnight
and
sacrificed
by
decapitation
to
collect
blood
and
the
serum
was
separated
immediately
for
biochemical
analysis
[17].
The
pancreas
were
collected
and
washed
with
ice-
cold
saline
to
remove
the
blood
cells
for
histopathological
analysis.
The
degree
of
glycosylated
hemoglobin
was
evaluated
in
the
whole
blood
for
all
groups
using
chromatographic
technique
by
HbA1ckit
(BD,
Biosciences,
USA)
following
the
manufacture’s
pro-
tocol.
The
serum
levels
of
cholesterol,
high-density
lipoprotein
cholesterol
(HDL-c),
low-density
lipoprotein
cholesterol
(LDL-c)
and
triglycerides
were
measured
using
respective
kits
from
BD,
Biosciences,
USA.
The
serum
levels
of
insulin,
tumor
necrosis
factor-␣
(TNF-
␣),
interleukin-6
(IL-6)
and
high-sensitive
C-reactive
protein
(CRP)
were
assayed
using
respective
enzyme-linked
immunosor-
bent
assay
(ELISA)
kits
obtained
from
R&D
Systems,
USA,
using
Microplate
reader,
Thermo
Scientific
Pvt.
Ltd.,
India.
The
pancreas
was
fixed
in
buffered
neutral
10%
formalin
solution
and
then
the
tis-
sues
were
embedded
in
paraffin.
The
tissues
were
sectioned
at
5
␮m
thicknesses
for
hematoxylin
and
eosin
dye
staining.
The
tissues
were
further
examined
using
optical
microscope
Olympus
BX51,
Japan.
2.7.
Statistical
analysis
All
data
were
expressed
as
means
±
SD
(n
=
6)
of
the
three
repli-
cates
of
each
experiment.
Statistical
significance
of
differences
was
assessed
by
one-way
ANOVA
followed
by
Bonferroni’s
multiple
comparisons
test
where
appropriate.
All
analyses
were
performed
using
GraphPad
Prism,
version
5.01.
3.
Results
and
discussion
3.1.
UV–visible
(UV–vis)
spectroscopic
analysis
The
UV–vis
spectrum
was
recorded
for
various
concentrations
of
plant
extract
mixed
with
chloroauric
acid
solution
(Fig.
1A)
and
as
a
function
of
time
(Supplementary
Fig.
1).
The
formation
of
AuNPs
resulted
from
the
reduction
of
chloroauric
acid
by
G.
sylvestre
is
evident
as
a
ruby
red
color
appeared
4
min
after
the
start
of
the
reaction.
The
sample
F
in
the
spectrum
(0.6
mL
extract
and
10
mL
chloroauric
acid
solution)
has
been
taken
for
further
studies
as
the
spectrum
gave
a
sharp
intense
peak
compared
to
the
other
con-
centrations
(Fig.
1A).
UV–vis
spectra
obtained
gave
a
characteristic
absorption
maximum
at
535
nm,
thus
indicating
the
surface
plas-
mon
resonance
(SPR)
of
AuNPs
caused
by
the
collective
oscillation
of
free
electrons
[18,19].
However,
smaller
nanoparticles
(<1–2
nm)

V.
Karthick
et
al.
/
Colloids
and
Surfaces
B:
Biointerfaces
122
(2014)
505–511
507
Fig.
1.
(A)
UV–vis
spectrum
obtained
for
gold
nanoparticles
synthesized
using
various
concentrations
of
plant
extract
(D–200
␮L),
(E–300
␮L),
(C–400
␮L)
and
(F–600
␮L),
(B,
C)
HRTEM
micrograph
of
G.
sylvestre
reduced
AuNPs
at
various
magnifications.
do
not
possess
such
properties
as
their
electrons
exist
in
discrete
energy
levels,
whereas
bulk
gold
with
continuous
absorption
spec-
trum
gives
a
single
peak
in
the
UV–visible
region
[20].
It
is
evident
from
the
micrographs
that
the
obtained
particles
are
well
defined
with
no
aggregation
seen
as
particles
with
isotropic
nature
will
be
useful
for
drug
delivery
studies
[21].
3.2.
Electron
microscopic,
atomic
force
microscopic
and
zeta
()
potential
analysis
Generally,
electron
micrographs
are
used
to
predict
the
size
and
shape
of
the
nanoparticles.
HRTEM
results
suggest
that
the
parti-
cles
were
polydisperse
[9,22]
in
nature
with
the
spherical-shaped
particles
being
more
dominant
(Fig.
1B
and
C).
Fig.
2A
and
B
shows
the
AFM
2D
topography,
where
randomly
arranged
particles
with
some
aggregations
were
seen
[23].
The
aggregation
seen
could
have
been
caused
during
the
analysis
by
tip
convolution
effects
with
sur-
face
as
the
particles
appear
to
have
a
larger
size
as
compared
to
that
of
HRTEM
(Fig.
2C)
[24].
Zeta
potential
is
used
to
find
out
col-
loidal
stability
and
to
study
the
strength
of
electrostatic
potential
at
electrical
double
layer
[25].
Fig.
3A
shows
the
particle
size
analysis
which
indicates
that
the
average
size
of
the
synthesized
AuNPs
is
around
50
nm.
Zeta
potential
of
the
synthesized
AuNPs
is
illustrated
in
Fig.
3B
which
indicates
negative
zeta
potential
of
−17.5
mV
with
high
colloidal
stability.
From
the
literature,
it
is
evident
that
parti-
cles
having
negative
zeta
potential
will
be
stable
for
a
long
period
[26].
3.3.
Effect
of
AuNPs
on
alloxan-induced
diabetic
rats
Alloxan
causes
nucleic
acid
damage
by
reducing
the
cellular
nicotinamide
adenine
dinucleotide
(NAD)
content
in
pancreatic
islet
cells,
thereby
disturbing
the
synthesis
of
proinsulin
[27].
So,
alloxan
is
used
as
a
common
diabetogenic
agent
for
studying
the
antidiabetic
effects
of
plant
compounds/antidiabetic
agents.
Plants
are
rich
in
compounds
like
flavanoids,
alakaloids,
terepenoids
and
peptides,
which
are
primarily
used
in
drug
formulations.
G.
sylvestre
has
profound
effect
in
controlling
diabetes
by
regulating
blood
glu-
cose
level
as
indicated
by
Shanmugasundaram
and
co-workers
in
their
extensive
research
on
analyzing
the
antidiabetic
effects
of
G.
sylvestre
on
rats
[28]
and
rabbits
[29].
In
continuation
with
the
pre-
vious
reports,
we
have
evaluated
the
effect
of
G.
sylvestre-mediated

508
V.
Karthick
et
al.
/
Colloids
and
Surfaces
B:
Biointerfaces
122
(2014)
505–511
Fig.
2.
(A,
B)
AFM
image
obtained
for
AuNPs
shows
the
surface
morphology
of
the
particles.
(C)
Histogram
analysis.
Fig.
3.
(A)
Particle
size
analysis
and
(B)
Zeta
potential
measurements
obtained
for
G.
sylvestre-mediated
AuNPs.

V.
Karthick
et
al.
/
Colloids
and
Surfaces
B:
Biointerfaces
122
(2014)
505–511
509
Table
1
Changes
in
body
weight
blood
glucose,
plasma
insulin,
total
hemoglobin
and
glycosylated
hemoglobin
levels
at
the
end
of
the
study.
Group
Body
weight
(g)
Fasting
blood
glucose
(mg/dL)
Plasma
Insulin
(␮U/mL)
Hemoglobin
(g/dL)
Glycosylated
hemoglobin
(mg/gHb)
Initial
Final
Group
I
182
±
7.5
192.3
±
7.2c
76.03
±
7.6
10.02
±
0.82
11.84
±
0.54
0.28
±
0.03
Group
II 186
±
6.2
ns 181.1
±
5.8
ns,
c 217
±
6.1###
6.2
±
0.5###
6.6
±
0.5###
0.87
±
0.05###
Group
III
183
±
5.6
ns
187.4
±
5.74
ns,
ns
105.34
±
6.2***
8.32
±
0.42***
10.1
±
0.65***
0.38
±
0.015***
Group
IV
184.4
±
7.3
ns
189.7
±
7.8
ns,
ns
109.6
±
5.2***
7.367
±
0.55*
9.64
±
0.6***
0.42
±
0.045***
Group
V
181.7
±
7.04
ns
190.4
±
4.7c,
ns
72.98
±
3.2
ns
9.7
±
0.43
ns
11.58
±
0.38
ns
0.3
±
0.04
ns
ns,
not
significant
(between
initial
and
final
of
body
weight);
(a)
P
<
0.0001,
(b)
P
<
0.001,
(c)
P
<
0.01;
###P
<
0.001
compared
with
corresponding
values
of
normal
control
group
and
diabetic
control
group;
***P
<
0.001
compared
with
corresponding
values
for
diabetic
control
group
with
STD
and
treatment.
Ns,
not
significant
compared
with
normal
control
group
and
normal
treated
group.
AuNPs
on
alloxan-induced
diabetic
rats.
Table
1
shows
the
body
weight,
BGL,
insulin,
hemoglobin
and
glycosylated
hemoglobin.
The
control
rats
exhibited
normal
blood
glucose,
whereas
alloxan-
induced
diabetic
rats
showed
significantly
higher
concentration
than
those
of
control
rats.
The
existence
of
hyperglycemic
condition
has
contributed
to
weight
loss
and
the
rats
experienced
uncontrolled
glucose
regu-
lation
caused
by
alteration
in
cellular
metabolism
[30]
in
diabetic
rats.
After
treatment,
diabetic
rats
showed
normal
glucose
level
indicating
the
hypoglycemic
effect
of
AuNPs
and
seemed
normal.
In
contrast,
diabetic
rats
suffered
from
hair
loss,
dull
fur
and
showed
inactivity
[31].
Significant
elevation
of
blood
glucose
levels
and
gly-
cosylated
hemoglobin
levels
were
observed
in
diabetic
controls,
while
final
body
weight,
insulin
and
hemoglobin
were
significantly
decreased
compared
to
that
of
normal
rats.
It
should
be
noted
that
diabetic
rats
treated
with
AuNPs
significantly
(p
<
0.001)
brought
the
parameters
to
normal
level,
proving
the
positive
effect
of
AuNPs.
The
presence
of
various
compounds
in
the
extract
would
have
caused
the
antihyperglycemic
effect
as
G.
sylvestre
is
well
known
for
its
antidiabetic
activity
by
reducing
the
blood
glucose
level
[32].
HbA1c,
a
component
seen
in
red
cells,
refers
to
the
attach-
ment
of
glucose
to
the
N-terminus
of
the
␤-chain
by
a
ketoamine
linkage
which
is
found
elevated
during
diabetic
conditions
[33].
It
can
be
used
as
a
measure
of
hyperglycemic
conditions
in
diabetic
subjects
[34].
Elevated
glucose
level
leads
to
glycosylation
of
pro-
teins
and
other
molecules
like
lipoproteins,
apolipoproteins
and
also
clotting
factors
[35].
It
also
increases
the
risk
of
complications
like
reduced
bone
mineral
density
[36],
obesity
and
cardiovascular
diseases
[37].
It
should
be
noted
that
the
rats
treated
with
AuNPs
showed
normal
levels
of
glycosylated
hemoglobin
compared
with
diabetic
controls.
Lipid
profiling
is
an
important
parameter
that
needs
to
be
evaluated
in
T2D
as
increased
lipid
levels
lead
to
cardiovascu-
lar
diseases
and
is
often
seen
in
uncontrolled
diabetes.
In
the
case
of
diabetes,
the
cholesterol
absorption
will
be
elevated
due
to
decreased
cholesterol
biosynthesis
[38].
It
also
leads
to
dis-
turbance
of
lipid
metabolism
by
increased
mobilization
of
fatty
acids
from
adipose
tissue
and
secondary
elevation
of
free
fatty
acid
in
the
blood
[39].
Administration
of
alloxan
causes
hyperlipi-
demia
in
healthy
rats
[40]
as
a
marked
increase
in
lipid
content
were
found
in
alloxan-induced
diabetic
rats.
Fig.
4A
depicts
the
effect
of
AuNPs
on
serum
cholesterol,
triglycerides,
HDL-c
and
LDL-c
levels.
Alloxan-induced
rats
showed
significant
increase
in
cholesterol,
triglycerides
levels
and
reduced
HDL-c
levels
indicates
upregulation.
The
changes
in
serum
lipid
levels
are
probably
due
to
uncontrolled
biosynthesis
of
lipids
by
increased
cyclic
adenosine
monophosphate
[41].
Rats
treated
with
AuNPs
showed
signifi-
cant
reduction
in
cholesterol,
triglycerides
and
LDL-c
levels
and
showed
increase
in
HDL-c
levels
(P
<
0.001)
compared
with
dia-
betic
group.
The
possible
mechanism
for
the
antihyperlipidemic
effect
of
AuNPs
could
be
by
insulin-stimulating
effect
on
pancreatic
beta
cells
or
insulin-mimetic
effects
by
the
active
compounds
from
G.
sylvestre
[42].
Since
there
was
no
considerable
improvement
in
insulin
levels
after
treatment,
it
is
suggested
that
the
positive
effect
could
be
by
insulin-stimulating
effect
rather
than
insulin-mimetic
effect.
TNF-␣
is
known
to
play
dual
role
in
autoimmune
diabetes
depending
upon
precision
timings
where
the
autoimmune
process
Fig.
4.
(A)
Role
of
AuNPs
on
lipid
profile
in
the
plasma
of
control
and
induced
rats
at
the
end
of
the
study
and
(B)
the
effect
of
AuNPs
on
inflammatory
marker
levels
in
serum
of
control
and
treated
rats.
(a)
P
<
0.0001,
(b)
P
<
0.001,
and
(c)
P
<
0.01.
###P
<
0.001
compared
with
corresponding
values
of
normal
control
group
and
diabetic
control
group;
***P
<
0.001
compared
with
corresponding
values
for
diabetic
control
group
with
standard
drug
and
treatment.
ns
=
not
significant
compared
with
normal
control
group
and
normal
treated
group.

510
V.
Karthick
et
al.
/
Colloids
and
Surfaces
B:
Biointerfaces
122
(2014)
505–511
in
type
1
diabetes
is
driven
by
inflammatory
mediators
[43].
Literature
suggest
that
increased
serum
levels
of
proinflammatory
cytokines
like
IL-6
and
TNF-␣
represent
the
state
of
T2D
and
insulin
resistance
[44].
Since
it
is
well
known
that
the
risk
of
getting
a
cardiac
disease
in
diabetic
subjects
is
quite
high,
we
have
analyzed
the
circulating
inflammatory
marker.
Fig.
4B
shows
the
serum
levels
of
TNF-␣,
IL-6
and
hsCRP.
Alloxan
induced
diabetic
control
rats
showed
increased
TNF-␣,
IL-6
and
hsCRP
levels
showing
inflammation.
After
treatment,
it
should
be
noted
that
the
serum
levels
of
TNF-␣,
IL-6
and
hsCRP
were
brought
down
significantly
compared
to
the
diabetic
group.
hsCRP
is
an
acute
protein
and
is
usually
expressed
under
the
stimulation
of
TNF-␣
and
IL-6
[45].
Elevated
levels
of
hsCRP
are
often
associated
with
impaired
insulin
sensitivity,
myocardial
infarction
and
stroke
[37].
hsCRP
is
also
used
as
a
promising
marker
for
the
identification
of
coronary
heart
disease
(CHD)
as
it
is
considered
as
the
predictor
of
cardiovascular
disease
[46].
After
treatment
with
AuNPs,
the
serum
levels
of
TNF-␣,
IL-6
and
hsCRP
were
significantly
brought
down
to
normal
levels
as
compared
to
that
of
the
diabetic
group
and
standard
drug
(glibenclamide),
indicating
the
effect
of
AuNPs
on
suppressing
the
inflammation.
Diabetic
control
rats
usually
show
reduced
pancreatic
islet
cells
due
to
the
destruction
caused
by
diabetic
agent
[47]
as
seen
in
Sup-
plementary
Fig.
3.
After
treatment,
slight
changes
have
been
noted
with
islets
cells
in
terms
of
regeneration,
vacuolation
and
invasion
of
connective
tissues
as
compared
to
that
of
the
diabetic
control
rats.
This
indicates
the
protective
effect
of
G.
sylvestre-mediated
AuNPs
on
islet
cells
and
its
restoration
of
damaged
islet
cells.
G.
sylvestre
was
also
studied
for
its
islet
cell
regeneration
in
diabetes-
induced
rats
by
establishing
glucose
homeostasis
through
serum
insulin
levels,
repairing
endocrine
pancreas
[32].
We
know
that
the
effect
of
oral
drugs
depend
on
the
extent
of
drug
absorption
from
gut
lumen,
metabolism
of
drug
in
the
liver
and
its
excretion
into
bile
and
urine
[48].
The
action
of
drug
begins
inside
the
cell
and
is
transported
across
cell
membrane
by
transport
proteins
[49].
It
is
believed
that
the
AuNPs
are
carried
across
the
cell
membrane
by
similar
process
as
they
are
very
small
in
size
and
have
prolonged
effect
in
biosystems
[50,51].
With
the
above
discussion,
it
is
viable
to
use
an
antidiabetic
potent
plant
for
preparation
of
colloidal
drugs
may
greatly
enhance
the
nature
of
nanomaterials
for
antidiabetic
applications.
4.
Conclusion
Biologically
synthesized
AuNPs
and
its
effect
on
regulation
of
induced
hyperglycemia
and
inflammation
using
alloxan-induced
diabetic
rats
were
studied.
AuNPs
synthesized
biologically
using
the
antidiabetic
potent
G.
sylvestre
plant
showed
positive
effect
on
alloxan-induced
diabetic
model.
They
were
effective
in
reducing
the
BGL
to
normal
level
and
have
good
control
over
the
lipid
levels
of
the
treated
diabetic
rats.
AuNPs
also
have
an
anti-inflammatory
effect
on
diabetic
rats
assessed
using
inflammatory
markers
TNF-␣,
IL-6
and
hsCRP.
Based
on
the
results,
it
is
suggested
that
colloidal
nanoparticles
could
be
used
as
a
nanomedicine
for
treatment
of
diabetes.
Acknowledgements
We
thank
DST-Nanomission,
Government
of
India,
for
its
financial
support
for
the
project
(SR/NM/NS-06/2009)
and
the
man-
agement
of
Sathyabama
University,
Chennai,
for
its
stanch
support
in
research
activities.
We
gratefully
acknowledge
SAIF,
IIT
Madras,
and
Centre
for
Nanoscience
and
Nanotechnology,
Sathyabama
Uni-
versity,
for
instrumental
facilities.
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.colsurfb.
2014.07.022.
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