Pain Modulation by Lignans (Phyllanthin and Hypophyllanthin) and Tannin (Corilagin) Rich Extracts of Phyllanthus amarus in Carrageenan-induced Thermal and Mechanical Chronic Muscle Hyperalgesia


Chopade, A.R.; Sayyad, F.J.

Phytotherapy Research 29(8): 1202-1210

2015


The current study was aimed at evaluating the antihyperalgesic effects of lignans (phyllanthin and hypophyllanthin) and tannin (corilagin) rich three standardized extracts of Phyllanthus amarus in a model of chronic musculoskeletal inflammatory pain. Three percent carrageenan injected in the gastrocnemius muscle produced hyperalgesia to mechanical and heat stimuli ipsilaterally, which spreads to the contralateral side within 7 to 9 days. To investigate the effects on chronic thermal and mechanical hypersensitivity, three extracts of P. amarus in three doses (100, 200, and 400 mg/kg) were administered to animals intraperitoneally from 14th day to 22nd day after intramuscular injection of carrageenan. It was observed that intraperitoneal administrations of Phyllanthus extracts showed antihyperalgesic activity, as they elevated thermal and mechanical threshold, which was supported by histopathological observations along with reduction in prostaglandin E2 (PGE2) concentration. In conclusion, we strongly suggest that the observed antihyperalgesic and antiinflammatory effects of P. amarus in current pain model are mediated via spinal or supraspinal neuronal mechanisms, mainly by inhibition of PGE2. Modulation of chronic muscular inflammation may be due to presence of phytoconstituents like phyllanthin, hypophyllanthin, and corilagin, which offers a promising means for treatment of chronic muscle pain.

PHYTOTHERAPY
RESEARCH
Phytother.
Res.
29:
1202-1210
(2015)
Published
online
14
May
2015
in
Wiley
Online
Library
(wileyonlinelibrary.com
)
DOI:
10.1002/ptr.5366
Pain
Modulation
by
Lignans
(Phyllanthin
and
Hypophyllanthin)
and
Tannin
(Corilagin)
Rich
Extracts
of
Phyllanthus
amarus
in
Carrageenan-
induced
Thermal
and
Mechanical
Chronic
Muscle
Hyperalgesia
Atul
R.
Chopade
l
'
2
*
and
F.
J.
Sayyad
2
1
Department
of
Pharmacology,
Rajarambapu
College
of
Pharmacy,
Kasegaon
415404,
Maharashtra,
India
2
Department
of
Pharmacognosy
and
Pharmacology,
Government
College
of
Pharmacy,
Karad
41512,
Maharashtra,
India
The
current
study
was
aimed
at
evaluating
the
antihyperalgesic
effects
of
lignans
(phyllanthin
and
hypophyllanthin)
and
tannin
(corilagin)
rich
three
standardized
extracts
of
Phyllanthus
amarus
in
a
model
of
chronic
musculoskel-
etal
inflammatory
pain.
Three
percent
carrageenan
injected
in
the
gastrocnemius
muscle
produced
hyperalgesia
to
mechanical
and
heat
stimuli
ipsilaterally,
which
spreads
to
the
contralateral
side
within
7
to
9
days.
To
investi-
gate
the
effects
on
chronic
thermal
and
mec
h
anical
hypersensitivity,
three
extracts
of
P
amarus
in
three
doses
(100,
200,
and
400
mg/kg)
were
administered
to
animals
intraperitoneally
from
14th
day
to
22nd
day
after
intra-
muscular
injection
of
carrageenan.
It
was
observed
that
intraperitoneal
administrations
of
Phyllanthus
extracts
showed
antihyperalgesic
activity,
as
they
elevated
thermal
and
mechanical
threshold,
which
was
supported
by
his-
topathological
observations
along
with
reduction
in
prostaglandin
E2
(PGE2)
concentration.
In
conclusion,
we
strongly
suggest
that
the
observed
antihyperalgesic
and
antiinflaumnatory
effects
of
P
amarus
in
current
pain
model
are
mediated
via
spinal
or
supraspinal
neuronal
mechanisms,
mainly
by
inhibition
of
PGE2.
Modulation
of
chronic
muscular
inflammation
may
be
due
to
presence
of
phytoconstituents
like
phyllanthin,
hypophyllanthin,
and
corilagin,
which
offers
a
promising
means
for
treatment
of
chronic
muscle
pain.
Copyright
©
2015
John
Wiley
&
Sons,
Ltd.
Keywords:
Phyllanthus
amarus;
carrageenan;
chronic;
muscle
and
hyperalgesia.
INTRODUCTION
Pain
of
muscle
origin
is
the
major
presenting
symptom
of
many
clinically
defined
conditions
encountered
glob-
ally
(Chopade
and
Mulla,
2010).
To
study
the
underly-
ing
pathological
mechanisms
of
chronic
pain,
a
number
of
experimental
animal
models
have
been
developed
over
the
years
(Chopade
et
al.,
2008).
The
study
by
Radhakrishnan
et
al.
provides
an
animal
model
for
eval-
uating
test
compounds
on
chronic
hyperalgesia
induced
by
muscle
inflammation,
which
is
maintained
by
spinal
or
supraspinal
neuronal
mechanisms,
and
has
been
sug-
gested
to
have
greater
face
validity
to
pain
of
musculo-
skeletal
origin
in
humans
(Radhakrishnan
et
al.,
2003).
Natural
agents
derived
from
plants
can
modulate
the
expression
of
inflammatory
signals
in
chronic
pain
disor-
ders
(Khanna
et
al.,
2007).
As
number
of
medicinal
plants
from
the
ayurvedic
and
traditional
medicine
have
been
investigated
through
experimental
and
clinical
studies
on
their
possible
efficacy
in
chronic
pain
diseases
such
as
Commiphora
wightii,
Curcuma
longa,
Zingiber
officinale,
Tinospora
cordifolia,
and
Withania
somnifera
(Harvey,
2008;
Khanna
et
al.,
2007).
*
Correspondence
to:
Atul
R.
Chopade,
Department
of
Pharmacology,
Rajarambapu
college
of
Pharmacy,
Kasegaon
415404,
Maharashtra,
India.
E-mail:
chopadearv@gmail.com;
ramvij47@rediffmail.com
Copyright
©
2015
John
Wiley
&
Sons,
Ltd.
The
extracts
and
the
compounds
isolated
from
Phyllanthus
amarus
Schumm
and
Thonn
(Family
Euphor-
biaceae)
have
shown
a
wide
spectrum
of
pharmacological
activities
including
antiviral,
antibacterial,
antiplasmodial,
antimalarial,
antimicrobial,
anticancer,
antidiabetic,
hypo-
lipidemic,
antioxidant,
hepatoprotective,
nephroprotective,
and
diurectic
properties
(Calixto
et
aL,
1998;
Bagalkotkar
et
aL,
2006;
Joseph
and
Raj,
2011;
Patel
et
aL,
2011;
Sarin
et
aL,
2014).
A
number
of
preclinical
and
clinical
studies
have
confirmed
the
medicinal
properties
of
various
Phyllanthus
species
that
have
been
mentioned
in
traditional
system
of
medicine
(Calixto
et
aL,
1998;
Bagalkotkar
et
al.,
2006;
Joseph
and
Raj,
2011;
Patel
et
aL,
2011;
Sarin
et
al.,
2014).
Previous
studies
have
reported
the
analgesic,
antiinflammatory,
and
anti-arthritic
activity
of
the
standard-
ized
extracts
of
P.
amarus
(Mali
et
aL,
2011;
Kandhare
et
aL,
2013;
Chopade
and
Sayyad,
2014a,
2014b).
However,
the
effects
of
P.
amarus
plant
on
chronic
inflammatory
muscle
hyperalgesia
are
unexplored,
which
has
encouraged
us
to
evaluate
the
effects
of
standardized
extracts
of
P.
amarus
for
their
chronic
pain
modulating
potential.
MATERIALS
AND
METHODS
Phyllanthus
amarus
samples
and
chemical
agents.
The
standardized
P.
amarus
aqueous
extract
(PAAE)
of
whole
Received
24
August
2014
Revised
04
April
2015
Accepted
10
April
2015
PAIN
MODULATION
BY
EXTRACTS
OF
Phyllanthus
amarus
1203
plant
(Reference
No.
SR/KN/CL/1/2012-L12030241)
was
procured
as
a
gift
sample
from
Chemiloids
Ltd.,
Vijayawada,
while
the
standardized
methanolic
extract
of
R
amarus
leaf
(PAME),
which
contains
more
than
2.5%
of
Phyllanthin
and
Hypophyllanthin
(Report
No.
FP1112042
PA/11LOT05),
and
the
standardized
hydromethanolic
extract
of
P
amarus
leaf
(PAHME),
which
contains
more
than
5%
of
Corilagin
(Report
No.
FP1102034—PA/11LOT/02)
were
procured
as
a
gift
sample
from
Natural
Remedies
Pvt.
Ltd.,
Bangalore.
Aceclofenac
of
Alembic
Vadodra,
Carrageenan,
Anes-
thetic
ether,
Formalin,
Potassium
hydroxide,
and
meth-
anol
of
Loba
chemicals,
Mumbai,
were
used
in
the
present
study.
Experimental
animals.
Adult
male
wistar
rats
weighing
200-340
g
were
used
in
this
study.
The
animals
were
maintained
under
standard
environmental
conditions
and
were
fed
with
standard
diet
and
water
ad
libitum.
Food
and
water
were
freely
available
throughout
the
experiments.
A
prior
approval
(Approval
number
GCOPK/2011-12/CPCSEA/616)
was
obtained
from
the
Animal
Ethics
Committee
of
Govt.
College
of
Phar-
macy,
Karad.
Govt.
College
of
Pharmacy,
Karad,
is
reg-
istered
under
Committee
for
the
Purpose
of
Control
and
Supervision
of
Experiment
on
Animals
(CPCSEA),
Government
of
India,
and
Registration
No.
209/GO/a/
2000/CPCSEA.
The
experiments
were
performed
according
to
the
ethical
guidelines
of
the
International
Association
for
the
Study
of
Pain
(Zimmermann,
1983)
and
the
CPCSEA.
Induction
of
carrageenan
chronic
inflammatory
muscle
hyperalgesia.
Chronic
inflammation
was
induced
by
injecting
100µL
of
freshly
prepared
solution
of
3%
carrageenan
in
normal
saline
to
the
left
gastrocnemius
muscle
belly
of
rats,
under
light
ether
anesthesia
(Radhakrishnan
et
al.,
2003).
After
24
h
intramuscular
injection
of
carrageenan,
spontaneous
pain
behavioral
signs
were
observed
in
animals.
Paw
withdrawal
laten-
cies
(PWLs)
to
heat
and
mechanical
stimuli
were
re-
corded
in
all
groups;
after
injection
of
carrageenan,
still
the
chronic
model
was
set
such
that
until
2
weeks,
which
was
indicative
by
decrease
in
PWLs
of
the
contra-
lateral
paw
(noncarrageenan
injected
side).
Experimental
protocol
(dosages
of
Phyllanthus
amarus
extracts
and
standard
drug).
Chronic
inflammatory
mus-
cle
pain
was
induced
to
all
the
animals
as
per
the
proce-
dure
described
earlier.
To
investigate
the
effects
of
chronic
treatment
on
mechanical
and
thermal
hypersen-
sitivity,
three
extracts
of
P
amarus
in
three
different
dosages
(100,
200,
and
400
mg/kg)
were
administered
to
animals
intraperitoneally
from
14th
day
to
22nd
day
after
induction
of
chronic
pain.
Phyllanthus
extracts
were
administered
twice
a
day
(every
12
h
apart)
to
the
rats
for
a
period
of
7
days.
Standard
drug
used
for
comparison
was
Aceclofenac
(preferential
selective
COX-2
inhibitor)
given
in
a
dose
of
10
mg/kg,
intraperi-
toneally.
The
treatment
was
interrupted
for
couple
of
days
on
the
18th
and
19th
day
and
reinitiated
on
20th
day
to
investigate
the
possible
development
of
tolerance.
The
evaluation
of
nociceptive
responses
was
performed
60-80
min
after
the
first
daily
treatment
(the
time
where
the
maximal
inhibition
was
observed
in
the
acute
treatment).
Inflammatory
control
group
(hyperalgesic
rats)
treated
with
vehicle
dimethyl
sulfoxide
0.2
mL
intraperitoneally
was
used
for
simulta-
neous
comparative
assessments
with
aceclofenac
and
Phyllanthus
extracts
treated
groups.
A
parallel
group
of
healthy
rats
(normal
control)
was
kept
to
assess
the
level
of
muscle
inflammation,
histopathological
features
and
changes
in
prostaglandin
E2
concentration.
Ani-
mals
were
tested
for
PWLs
to
heat
and
mechanical
stimuli
before
carrageenan
injection
and
consecutively
thereafter
till
end
of
the
study.
The
animals
were
sacrificed
on
22nd
day
to
study
the
muscle
histology
at
the
site
of
injection.
Behavioral
testing
for
evaluation
of
thermal/heat
hyperalgesia.
The
response
to
inflammatory
hyperalgesia
was
determined
by
measuring
PWL
of
carrageenan-
injected
paw
by
dipping
it
in
the
water
bath
maintained
at
47±1
°C
(Jain
et
al.,
2001).
Baseline
latency
to
paw
withdrawal
from
thermal
source
was
established
thrice,
5
min
apart,
and
averaged.
A
cut-off
time
of
15
s
was
imposed
to
avoid
any
injury
to
the
paw.
The
PWL
for
contralateral
paw
was
observed
in
this
chronic
model
at
60-80
min
after
drug
administration
on
subsequent
dos-
ing.
Responses
to
heat
stimuli
were
measured
before
and
post
intramuscular
injection
of
carrageenan
till
end
of
study,
that
is,
22nd
day.
Behavioral
testing
for
evaluation
of
mechanical
hyperalgesia.
For
testing
of
mechanical
hyperalgesia
or
allodynia,
the
animals
were
placed
on
an
elevated
metal
grid
allowing
stimulation
of
the
plantar
surface
of
the
paw,
and
the
animals
were
allowed
to
adapt
to
their
environment
for
15
min
(Radhakrishnan
et
al.,
2003;
Radhakrishnan
and
Sluka,
2005).
The
presence
of
mechanical
hyperalgesia
was
assessed
using
a
series
of
von
Frey
nylon
hairs
or
filament
(2-20
g),
which
were
applied
in
increasing
force
until
the
rat
withdrew
its
hind
paw.
Each
hair
was
applied
five
times,
and
the
threshold
(g)
was
taken
as
the
lowest
force
that
caused
at
least
three
withdrawals
out
of
the
five
consecutive
stimuli.
Von
Frey
nylon
hairs
were
calibrated
both
prior
to
and
throughout
the
time
course
of
the
entire
study
to
ensure
that
consistent
bending
forces
were
routinely
applied.
Responses
to
mechanical
stimuli
were
also
measured
before
and
post
intramuscular
injection
of
carrageenan
till
end
of
study,
that
is,
22nd
day.
Measurement
of
muscle
circumference.
After
14
days,
the
circumference
of
inflamed
and
the
noninflamed
gastrocnemius
muscle
was
measured
over
the
skin
using
a
measuring
tape
to
confirm
the
development
of
inflammation.
The
circumference
was
also
measured
in
subsequent
groups
after
drug
treatments
to
assess
the
correlation
between
PWL
and
inflammation.
Muscle
cir-
cumference
measurement
was
performed
by
wrapping
a
piece
of
cotton
thread
round
the
muscle
of
each
rat
and
measuring
the
muscle
circumference
with
the
help
of
a
meter
ruler
as
described
previously
(Radhakrishnan
and
Copyright
©
2015
John
Wiley
&
Sons,
Ltd.
Phytother.
Res.
29:
1202-1210
(2015)
1204
A.
R.
CHOPADE
AND
F.
J.
SAYYAD
(A)
Ipsilateral
paw
(B)
Contralateral
paw
15-
15
I
10.
PAAE
(400
mg/kg)
PAAE
(200
mglkg)
PAAE
(100
mglkg)
ACECLOFENAC
-a-
CONTROL
2
10
0
-
PAAE
(400
mg/kg)
-
PAAE
(200
mg/kg)
PAAE
(100
mglkg)
-E-
ACECLOFENAC
CONTROL
E
5-
i=
°
000°
<5.
,
<5
,
<5
,
0
,
0
,
°
l
'P
<
0.05;V<0.01
a
OA
115‘
'
;0
4'
0
4
0
4
.0"
&
0
4'
5-
)—
Days
Days
1
.
F.
<0.05;
1
1
,
0.01
15
15
7
"e
,
10.
8
PAME
(400
mg/kg)
4
-
PAME
(2oo
mg/kg)
+
PAME
(100
mg/kg)
*
ACECLOFENAC
+
CONTROL
4-
PAME
(400
mg/kg)
4-
PAME
(200
mg/kg)
PAME
(100
mg/kg)
+
ACECLOFENAC
+
CONTROL
c
10-
0
a
I-
5
I-
0
e
e
e
Otis
Oti ry
°
(5'
e
e
e
Os10se Os9
Oti
o
Otis
e
2
Is
0
a
Days
15
+
PAHME
(400
mg/kg)
+
PAHME
(200
mg/kg)
+
PAHME
(100
mg/kg)
+
ACECLOFENAC
+
CONTROL
a
E
5
e
eeeee
Days
Days
15
4
-
PAHME
(400
mg/kg)
4
-
PAHME
(200
mg/kg)
c
10.
+
PAHME
(100
mg/kg)
0
+
ACECLOFENAC
a)
+
CONTROL
e
e
cis%
eeee
O
tiry
Days
Figure
1.
Effects
of
Phyllanthus
extracts
on
paw
withdrawal
latency
to
heat
stimuli
in
chronic
inflammatory
muscle
hyperalgesia
on
the
ipsilateral
(A)
and
contralateral
side
(B).
Effects
of
Phyllanthus
amarus
extracts,
aceclofenac,
and
vehicle
(inflammatory
control)
administered
post
carrageenan
injection
on
thermal
hyperalgesia.
The
mean
thermal
withdrawal
latency
(in
seconds)
was
measured
for
both
the
paws
ipsilateral
and
contralateral
in
rats
(n
=
5
for
each
group).
Each
point
represents
mean
standard
error
of
mean
of
the
paw
withdrawal
threshold
(in
seconds)
to
stimulation
by
heat.
Data
were
analyzed
by
one-way
analysis
of
variance
using
Dunnett's
multiple
comparison
test.
p
<
0.05
was
considered
significant
as
compared
with
the
inflammatory
control.
This
figure
is
available
in
color
online
at
wileyonlinelibrary.
com/journal/ptr.
Sluka,
2005).
Muscle
circumference
was
measured
before
induction
of
chronic
inflammation
and
on
13th
and
22nd
day
after
intramuscular
injection
of
carrageenan.
Histopathological
studies.
Two
animals
of
each
group
were
sacrificed
at
2
weeks
after
the
injection
of
carra-
geenan
in
control
and
drug
treatments
(Radhakrishnan
et
aL,
2003).
Ipsilateral
knee
joints
were
dissected
and
fixed
in
10%
formalin.
The
dissected
muscle
was
embedded
in
paraffin.
Paraffin
sections
of
all
the
tissue
were
stained
with
hematoxylin
and
eosin
(H
and
E)
and
examined
by
light
microscopy.
Analysis
of
histological
findings
was
descrip-
tive
and
performed
in
a
blinded
fashion
by
a
pathologist.
Measurement
of
prostaglandin
E-2.
Half
milliliter
su-
pernatant
inflammatory
immersion
of
the
left
muscle
was
added
to
2
mL
Potassium
hydroxide-methanol
solution
(0.5
mol/L)
and
kept
in
a
water
bath
at
50
°C
for
isomerization
for
20
min,
and
then
methanol
was
added
to
a
total
capacity
of
5
mL
and
thoroughly
mixed
(Matsuda
and
Tanihata,
1992;
Charles
et
al.,
1962).
After
standing
for
5
min,
the
absorbance
was
measured
at
278
nm
using
a
ultraviolet
spectrophotometer
(Shimadzu
1800).
The
optical
density
value
of
per
milli-
liter
inflammatory
exudates
corresponds
to
indicate
the
prostaglandin
E-2
(PGE2)
content.
Statistical
analysis.
All
the
statistical
calculation
were
per-
formed
using
Graph
pad
Prism
software
version
6.01
°
(GraphPad
Software,
Inc.7825
Fay
Avenue,
Suite
230La
Jolla,
CA
92037
USA),
1992-2012.
All
the
data
are
expressed
as
mean
standard
error
of
mean
and
was
ana-
lyzed
by
using
one-way
analysis
of
variance
using
Dunnett's
multiple
comparison
test
as
post
hoc
test.
p-value
<
0.05
was
considered
significant
as
compared
with
the
inflammatory
control.
RESULTS
Effect
of
carrageenan
injection
on
the
gastrocnemius
muscle
inflammation
and
spontaneous
pain
behavior
Injection
of
3%
carrageenan
into
the
left
gastrocnemius
muscle
produced
inflammation
of
the
muscle
after
24
h
and
was
still
present
until
transforming
into
chronic
Copyright
©
2015
John
Wiley
&
Sons,
Ltd.
Phytother.
Res.
29:
1202-1210
(2015)
20
20
+
PAHME
(400
mglkg)
PAHME
(200
mg/kg)
-
a
-
PAHME
(100
mglkg)
ACECLOFENAC
+
CONTROL
715.
,15
E
E
io.
PAHME
(400
mgl(g)
PAHME
(200
mglkg)
PAHME
(100
mgikg)
*
ACECLOFENAC
*
CONTROL
210
0
LL
5
U.
5.
0
00'eee,,,
,
Yeese
Days
qokeeeeeeeese
Days
1205
PAIN
MODULATION
BY
EXTRACTS
OF
Phyllanthus
amarus
model
after
2
weeks.
The
inflammation
was
accompanied
by
a
significant
reduction
in PWL
to
heat
and
mechanical
stimuli.
The
spontaneous
pain
behavior
signs
were
ob-
served
in
animals
such
as
guarding
the
carrageenan-
injected
paw
and
weight
bearing
on
the
contra
lateral
side
during
the
first
2
days.
After
48
h,
there
were
no
signs
of
spontaneous
pain
except
that
there
was
curling
of
the
paw
ipsilaterally.
Effects
of
Phyllanthus
extracts
on
heat
hyperalgesia
All
the
P
amarus
extracts
at
the
studied
doses
(100
to
400
mg/kg)
showed
a
significant
reduction
to
heat
hyperalgesia
induced
by
carrageenan.
Basal
PWLs
for
all
the
groups
in
the
experiment
were
similar
approxi-
mately
around
8.53
±
0.15
s
(n
=
55)
prior
carrageenan
injection,
which
reduced
significantly
after
2
weeks
to
2.95
±
0.23
s.
The
intraperitoneal
administration
of
Phyllanthus
extracts
caused
a
rapid
reduction
in
hyperalgesia
returning
it
to
near
normal
within
1
h.
There
were
significant
differences
in
PWL
to
heat
for
the
groups
injected
with
Phyllanthus
extracts
and
inflammatory
control
in
the
current
study
cohort.
The
maximum
effect
was
seen
during
60-90
min
after
admin-
istration
of
the
Phyllanthus
extracts.
The
effects
of
Phyllanthus
extracts
on
thermal
hyperalgesia
in
chronic
inflammatory
muscle
hyperalgesia
are
shown
in
Fig.
1.
Maximum
decrease
in
thermal
response
latency
was
observed
in
animals
treated
with
vehicle
dimethyl
sulfoxide.
The
results
show
all
doses
of
P
amarus
extracts
tested
(100,
200,
and
400
mg/kg),
significantly
attenuated
thermal
hyperalgesia,
p
<
0.01
versus
inflammatory
control
in
both
paws.
Intraperitoneal
administration
of
P
amarus
extracts
significantly
and
dose
dependently
(100-400
mg/kg)
attenuated
thermal
hyperalgesia.
Data
revealed
that
200
and
400
mg/kg
of
P.
amarus
extracts
inhibited
thermal
hyperalgesia
(all
p
<
0.01
versus
inflammatory
control)
with
similar
efficacy
to
aceclofenac
(p
<0.01
versus
vehicle).
The
lowest
effective
dose
of
extracts
100
mg/kg
also
signifi-
cantly
attenuated
thermal
hyperalgesia
(p
<
0.05
versus
inflammatory
control).
Effect
of
Phyllanthus
extracts
on
mechanical
hyperalgesia
The
effects
of
Phyllanthus
extracts
on
mechanical
hyperalgesia
in
chronic
inflammatory
muscle
hyperalgesia
are
shown
in
Fig.
2.
A
maximum
decrease
in
mechanical
response
threshold
was
observed
in
the
ipsilateral
as
well
as
contralateral
paws
in
animals
treated
with
vehicle
alone.
The
results
show
all
doses
of
P
amarus
extracts
tested
(100,
200,
and
400
mg/kg),
significantly
attenuated
mechanical
hyperalgesia,
p
<
0.05
versus
inflammatory
control
in
both
paws.
Data
also
revealed
that
treatment
(A)
Ipsilateral paw
20
(B)
20
Contralateral
paw
PAAE
(400
mg/kg)
PAAE
(400
mg/kg)
-v-
PAAE
(200
mg/kg)
10
10
-4-
-4-
PAAE
(200
mg/kg)
PAAE
(100
mg/kg)
ACECLOFENAC
CONTROL
;7;
15.
E
510.
-v-
PAAE
(100
mg/kg)
ACECLOFENAC
CONTROL
2.
5
LL
5-
0
1
.
1.<
0.00;
',0.01
I
20
o"
:5
.:,
44
<0'
<PI"
I.P
< 0.05; "P<0.01
I
Days
Days
+
PAME
(400
mglkg)
20
-A-
PAME
(400
mg/kg)
^15
PAME
(200
mglkg)
PAME
(100
mg/kg)
,T
15.
*
PAME
(200
mglkg)
4-
ACECLOFENAC
E
PAME
(100
mg/kg)
S10
+
CONTROL
210.
*
ACECLOFENAC
CONTROL
0
U.
6
0
Days
Days
p's
0
... ...
k
t
0 0
0
0 0
<'
0
.
0
0
0
0000
e
eeeeeese
Figure
2.
Effects
of
Phyllanthus
extracts
on
paw
withdrawal
latency
to
mechanical
stimuli
in
chronic
inflammatory
muscle
hyperalgesia
on
the
ipsilateral
(A)
and
contralateral
side
(B).
Effects
of
Phyllanthus
amarus
extracts,
aceclofenac,
and
vehicle
(inflammatory
control)
admin-
istered
post
carrageenan
injection
on
mechanical
hyperalgesia.
The
mean
thermal
withdrawal
latency
(in
seconds)
was
measured
for
both
the
paws
ipsilateral
and
contralateral
in
rats
(n
=
5
per
group).
Each
point
represents
mean
standard
error
of
mean
of
the
paw
withdrawal
threshold
(in
grams)
to
mechanical
stimulation
by
von
Frey
filaments.
Data
were
analyzed
by
one-way
analysis
of
variance
using
Dunnett's
multiple
comparison
test.
p
<
0.05
was
considered
significant
as
compared
with
the
inflammatory
control.
This
figure
is
available
in
color
online
at
wileyonlinelibrary.com/journal/ptr.
Copyright
©
2015
John
Wiley
&
Sons,
Ltd.
Phytother.
Res.
29:
1202-1210
(2015)
#
1D<0.05;
"P<0.01
!:1
1206
A.
R.
CHOPADE
AND
F.
J.
SAYYAD
(A)
Effect
of
the
Phyllanthus
extracts
on
muscle
inflammation
22nd
Day
after
drug
treatment
13th
day
after
carrageenan
injection
Non-inflammed
#
#
Htt
IZ:3
CONTROL
ACECLOFENAC
PAAE
(100
mglkg)
PAAE
(200
mglkg)
PAAE
(400
mglkg)
=I
PAME
(100
mg/kg)
PA
ME
(200
mglkg)
=
PAME
(400
mg/kg)
1—
#
tt
PAHME
(100
mglkg)
PAHME
(200
mglkg)
12120
PAHME
(400
mg/kg)
1—
O
Muscle
circumference
(cms)
'P<0.05
(B)
Effect
of
the
Phyllanthus
extracts
on
PGE2
concentration
10
ye
a
1
1
'
V^
V'
1#
e
(1,
t.
,C)
e
pv
e
,
c):
,e
tt
:
F
A
#•
'"
0
0
0
qv
'
"
eV
sx.
Figure
3.
(A)
Effect
of
Phyllanthus
extracts
on
muscle
inflammation
in
chronic
inflammatory
hyperalgesia.
Effect
of
administration
of
Phyllanthus
amarus
extracts
(100,
200,
and
400
mg/kg),
aceclofenac,
or
vehicle
administered
on
muscle
edema
induced
by
carrageenan.
Muscle
diameter
was
measured
only
ipsilaterally.
Each
point
represents
the
mean
standard
error
of
mean
of
muscle
thickness/diameter
(in
centimeters)
before
carrageenan
injection
(baseline)
or
at
the
times
(13th
and
22nd
day)
after
intramuscular
injection
of
Carrageenan.
Data
were
analyzed
by
one-way
analysis
of
variance
using
Dunnett's
multiple
comparison
test.
p
<
0.05
was
considered
significant
in
com-
parison
with
inflammatory
control.
(B)
Effects
of
Phyllanthus
extracts
on
prostaglandin
E-2
(PGE2)
concentration.
Effect
of
administration
of
P
amarus
extracts
(100,
200,
and
400
mg/kg,
intraperitoneally)
on
PGE2
concentration
in
muscle
exudates
on
22nd
day
after
intramuscular
injection
of
carrageenan
in
rats.
PGE2
concentration
was
measured
only
ipsilateral
carrageeenan-injected
muscle.
Each
bar
represents
the
mean
±
standard
error
of
mean
of
the
PGE2
concentration
(in
optical
density/mL).
Data
were
analyzed
by
one-way
analysis
of
variance
using
Dunnett's
multiple
comparison
test.
p
<
0.05
was
considered
significant
in
comparison
with
inflammatory
control.
This
figure
is
available
in
color
online
at
wileyonlinelibrary.com/journal/ptr.
PGE2
(
0D/m
1)
with
200
and
400
mg/kg
of
P.
amarus
extracts
inhibited
mechanical
hyperalgesia
(p
<
0.01
versus
inflammatory
control)
with
similar
efficacy
to
aceclofenac
(p
<
0.01
versus
control).
When
treatment
of
Phyllanthus
extracts
was
interrupted
for
02
days
(on
the
18th
and
19th
day),
mechanical
hypersensitivity
was
reestablished.
On
the
20th
day
after,
the
treatment
of
Phyllanthus
extracts
was
restarted,
and
it
was
observed
that
these
extracts
significantly
reduced
mechanical
hyperalgesia,
which
excluded
the
possibility
of
the
development
of
tolerance.
Effects
of
Phyllanthus
extracts
on
muscle
inflammation
Carrageenan
produced
distinct
muscle
inflammation
in
the
inflammatory
control
group
indicating
inflammatory
response
as
compared
with
the
normal
control
animals.
While
the
consequent
dosing
of
the
Phyllanthus
extracts
showed
marked
inhibition
of
muscle
inflammation
as
there
was
a
significant
decrease
in
circumference
of
muscle
as
compared
with
the
inflammatory
control
(Fig.
3A).
Statistical
significance
in
reduction
of
muscle
circumference
was
observed
at
the
set
level
of
p
<
0.05
in
comparison
with
the
inflammatory
control.
Effects
of
Phyllanthus
extracts
on
concentration
of
prostaglandin
E-2
The
treatment
with
Phyllanthus
extracts
significantly
decreased
PGE2
level
in
the
edema
exudates
as
com-
pared
with
inflammatory
control
group.
The
inhibitory
potency
of
Phyllanthus
extract
treated
groups
was
also
better
as
compared
with
the
control
as
shown
in
Fig.
3B.
A
concentration
response
curve
was
observed
after
the
treatment
with
Phyllanthus
extracts
on
concentration
of
PGE2,
as
there
was
a
gradual
decrease
in
concentra-
tion
of
PGE2
with
increase
in
dose
of
Phyllanthus
extracts.
The
basal
concentration
of
PGE2
(normal
con-
trol
animals
without
inflammation)
was
0.186
±
0.26
as
compared
with
8.736
±
0.18
with
the
inflammatory
control
animals.
P.
amarus
extract
(400
mg/kg)
and
Copyright
©
2015
John
Wiley
&
Sons,
Ltd.
Phytother.
Res.
29:
1202-1210
(2015)
PAIN
MODULATION
BY
EXTRACTS
OF
Phyllanthus
amarus
1207
11
111
(El
'
`1.4x-re
••••
./
Figure
4.
Photomicrographs
of
muscle
pathology.
Histopathological
changes
in
gastrocnemius
muscle
in
normal,
chronic
inflammatory
control,
Aceclofenac,
and
Phyllanthus
amarus
extracts
(only
400
mg/kg)
treated
groups
are
shown.
Circles
show
foci
of
muscle
necrosis,
and
arrows
show
group
of
inflammatory
cell
infiltrates
and
macrophages
in
chronic
hyperalgesic
controls
and
subsequent
drug-treated
groups.
Micro
photographs
were
taken
at
a
magnification
of
40
x
10
times.
(A)
Histological
slide
of
gastrocnemius
muscle
of
normal
healthy
rat.
(B)
Chronic
inflammatory
response
showing
the
macrophages
and
scattered
mast
cells.
(C)
Response
in
Aceclofenac-treated
rats
(0)
Response
in
Phyllanthus
amarus
aqueous
extract
(PAAE)
treated
rats
(E)
Response
in
methanolic
extract
of
P.
amarus
leaf
(PAME)
treated
rats.
(F)
Response
in
hydromethanolic
extract
of
R
amarus
leaf
(PAHME)
treated
rats.
This
figure
is
available
in
color
online
at
wileyonlinelibrary.com/journal/ptr.
aceclofenac
significantly
(p
<
0.01)
attenuated
the
PGE2
concentration
in
muscle
exudates.
Histopathological
studies
Histopathological
examination
of
the
tissues
in
the
cur-
rent
study
shows
inhibition
of
inflammatory
changes
that
parallel
the
long
lasting
hyperalgesia
observed
in
control.
In
the
hyperalgesic
controls,
the
acute
inflammation
was
severe,
accompanied
by
myonecrosis
and
presence
of
neutrophils
in
large
numbers.
The
chronic
inflammation
was
epimysial
and
perimysial,
with
presence
of
macro-
phages
and
few
scattered
mast
cells.
Aceclofenac-treated
animals
decreased
the
chronic
macrophagic
response
as
very
few
macrophages
are
seen
with
no
fibrinous
exudates.
While
the
Phyllanthus
extracts
treated
rats
showed
decrease
in
neutrophils
and
absence
of
macro-
phages
and
mast
cells
as
compared
with
hyperalgesic
con-
trols
(for
details,
see
Fig.
4).
PAAE
treatment
inhibited
the
macrophagic
response
to
lesser
extent,
while
focal
muscle
degeneration
was
observed
with
massive
leukocyte
infiltration.
PAME
treatment
inhibited
the
macrophagic
response
to
some
extent,
while
no
fibrinous
exudates
were
observed.
PAHME
treatment
inhibited
the
macrophagic
response
to
some
extent,
while
fibrinous
exudates
were
observed
with
disruption
in
muscular
morphology
DISCUSSION
In
the
present
study,
we
have
examined
the
effects
of
systemic
administration
of
the
Phyllanthus
extracts,
on
thermal
and
mechanical
hyperalgesia
evoked
by
in-
tramuscular
injection
of
carrageenan
in
chronic
model
of
muscle
hyperalgesia.
The
standardized extract
of
P.
amarus
when
evaluated
in
Freund's
complete
adjuvant-induced
arthritis
model
has
shown
prominent
anti-arthritic
activity,
which
was
attributed
to
its
antiinflammatory
activity
(Mali
et
al.,
2011).
The
study
by
Kandhare
et
al.
have
shown
that
PAAE
exerted
a
preventive
antiinflammatory,
antioxidant,
and
anti-
apoptotic
effect
in
acetic
acid-induced
colitis
model
of
inflammatory
bowel
disease
(Kandhare
et
al.,
2013).
Interestingly,
all
the
major
reported
data
deal
with
the
preventive
aspects
of
hyperalgesia,
but
to
date,
no-
body
has
evaluated
the
effects
of
Phyllanthus
extracts
on
the
established
hyperalgesia
in
more
pronounced
spinally
mediated
muscle
hyperalgesia
in
preclinical
settings.
The
chronic
thermal
and
mechanical
hyperalgesia
produced
in
the
present
model
are
maintained
by
spi-
nal
or
supraspinal
neuronal
mechanisms,
as
a
result
from
series
of
central
and
peripheral
changes
occurring
at
the
site
of
insult
(Radhakrishnan
et
al.,
2003;
Chopade
and
Sayyad,
2014a,
2014b;
Araldi
et
al.,
2013;
Ossipov
et
al.,
2010).
It
is
clear
from
the
results
of
our
study
that
P.
amarus
extracts
can
reverse
the
al-
ready
established
chronic
hyperalgesia.
It
is
also
clear
from
the
histopathological
evidences
that
there
are
no
contralateral
signs
of
inflammation
observed
in
our
studies,
supporting
a
neuronal
role,
either
spinal
or
supraspinal,
for
the
contralateral
spread
of
hyperalgesia.
The
results
of
our
study
suggest
that
mus-
cle
mediated
inflammatory
pain
can
be
alleviated
by
Phyllanthus
extracts
and
that
once
established,
ongoing
Copyright
©
2015
John
Wiley
&
Sons,
Ltd.
Phytother.
Res.
29:
1202-1210
(2015)
PAAE
0000
000
8C0
700
100
22
100
of
Imo
IOW
AO
AO
700
6W
500
401
300
200
100
PAME
P
HP
000
020
0.02
0.60 0.80
1.60
P
HP
1208
A.
R.
CHOPADE
AND
F.
J.
SAYYAD
(A)
900
eao
PAHME
700
700
6W
5:83
600
100
9C0
2W
P
HP
200
200
100
100
0
000 020
OM
080
100
(B)
Standard
0.05
020
0.60
BO
020
OM
O.W
020
IA
501
OM
OM
OM
1.00
450
400
C
450
P
AHME
C
St11
ndard
000
250
050
aso
250
200
160
100
iii
1
I
020
OB0
100
010
010
aeo
Figure
5.
HPTLC
finger
print
profile
of
Phyllanthus
extracts.
(A)
HPTLC
chromatogram
of
Phyllanthus
extracts
for
detection
of
lignans
with
help
of
standard
markers
[phyllanthin
(P)
and
hypophyllanthin
(H)]
using
mobile
phase
composed
of
chloroform:
methanol:
water
(7:3:0.4,
v/v).
(B)
HPTLC
chromatogram
of
Phyllanthus
extracts
for
detection
of
tannins,
with
help
of
standard
marker
corilagin
(C)
using
mobile
phase
composed
of
toluene:
ethyl
acetate:
formic
acid
(6:4:0.3,
v/v).
This
figure
is
available
in
color
online
at
wileyonlinelibrary.com/journal/ptr.
PAAE
C
PAME
C
inflammation
does
not
appear
to
contribute
to
the
pro-
cess
of
inflammatory
hyperalgesia.
It
is
important
to
mention
that
the
antihyperalgesic
effects
of
Phyllanthus
extracts
were
not
susceptible
to
tolerance,
because
Phyllanthus
extracts
maintains
its
efficacy
when
adminis-
tered
repeatedly
by
intraperitoneal
route.
Spinal
COX-2
plays
an
important
role
in
the
maintenance
of
hyperalgesia
induced
by
carrageenan
(Ding
et
aL,
1998).
After
inflammation,
allodynia
and
hyperalgesia
occur,
generally
because
of
an
increase
in
PGE2
level
in
the
inflamed
tissue
and
in
the
spinal
cord
that
can
be
associated
with
induction
and
activation
of
Copyright
©
2015
John
Wiley
&
Sons,
Ltd.
Phytother.
Res.
29:
1202-1210
(2015)
PAIN
MODULATION
BY
EXTRACTS
OF
Phyllanthus
amarus
1209
COX-2
(Chopade
and
Sayyad,
2014a,
2014b).
The
COX-2
enzyme
is
the
major
source
of
PGE2
in
many
inflamma-
tory
pain
models,
and
almost
all
of
the
COX-2-selective
inhibitors
have
shown
potent
antihyperalgesic
activity
(Chopade
and
Sayyad,
2014a,
2014b).
Pulichino
et
al.
sug-
gest
that
the
inhibition
of
PGE2
synthesis
by
nonsteroidal
antiinflammatory
drugs
and
COX-2
inhibitors
contributes
to
their
efficacy
in
treating
the
signs
of
chronic
inflamma-
tory
pain
(Pulichino
et
al.,
2006).
Standardized
extracts
of
P.
amarus
inhibited
the
induction
of
endotoxin-induced
cyclooxygenase-2
and
cytokine
production
in
vitro
as
well
as
in
vivo
(Kiemer
et
aL,
2003).
While
in
the
present
study,
there
was
a
significant
decrease
in
PGE2
level
in
the
edema
exudates
for
all
the
P.
amarus
and
aceclofenac-
treated
groups
as
compared
with
the
inflammatory
con-
trol,
histopathological
examination
of
the
muscle
tissues
in
the
current
study
also
supports
the
inflammatory
changes
that
parallel
the
long
lasting
hyperalgesia.
In
the
present
study,
it
is
clear
from
the
preliminary
phy-
tochemical
studies
that
the
PAAE
extract
is
rich
in
tannins
as
well
as
lignans,
the
PAME
contains
high
amount
of
lignans,
and
PAHME
has
significant
amount
of
tannins.
Lignans
are
the
major
constituents
present
in
the
Phyllanthus
extracts
and
are
reported
to
possess
analgesic,
antioxidant,
antiinflammatory,
anti-arthritic,
and
immuno-
modulatory
activity
(Raphael
and
Kuttan,
2003;
Ofuegbe
et
aL,
2014;
Patel
et
aL,
2011;
Kandhare
et
al.,
2013;
Sarin
et
al.,
2014).
Purified
lignans
such
as
phyltetralin,
nirtetralin,
and
niranthin
isolated
from
Phyllanthus
ex-
tracts
have
shown
antiinflammatory
actions
in
vivo
and
in
vitro
experiments
(Kassuya
et
al.,
2003).
Furthermore,
lignan-rich
fraction
and
mainly
niranthin
were
found
to
be
effective
in
interfering
the
inflammatory
response
in-
duced
by
platelet
activating
factor
(Kassuya
et
aL,
2006).
While
on
the
other
side,
the
tannins
are
also
the
major
constituents
present
in
the
Phyllanthus
extracts,
the
experimental
data
have
demonstrated
that
major
tannin
corilagin
present
in
these
Phyllanthus
extracts
exhibits
antihyperalgesic
activity
(Moreira
et
al.,
2013).
The
phytochemical
finger
print
analysis
of
the
Phyllanthus
extracts
clearly
indicates
that
the
observed
antihyperalgesic
activity
in
the
studied
extracts
can
di-
rectly
be
assigned
to
presence
of
lignans
and
tannins.
High
performance
thin
layer
chromatography
(HPTLC)
analysis
of
Phyllanthus
extracts
show
significant
peaks
for
each
extract,
which
are
shown
in
Fig.
5A
and
5B.
From
the
HPTLC
chromatograms,
we
can
interpret
that
Phyllanthus
extracts
contain
considerable
amount
of
lignans
(phyllanthin
and
hypophyllanthin)
and
tannin
(corilagin).
Therefore,
the
presence
of
these
compounds
might
be
the
ultimate
cause
for
their
bioactivity.
Studies
from
our
lab
have
demonstrated
chronic
pain
modulating
potential
of
Phyllanthus
species.
The
molec-
ular
docking
analysis
of
phytocompounds
from
Phyllanthus
species
with
various
inflammatory
targets
involving
enzymes
like
COX-2
and
PGE
synthase,
cyto-
kines
like
TNF-Alpha
and
IL-1
BETA
and
with
N-
methyl-D-aspartate
receptor
(NMDA)
receptor,
sug-
gests
that
lignans
and
tannins
have
ability
to
interact
with
multiple
targets
involved
in
inflammatory
hyperalgesia
and
are
involved
in
its
modulation
(Chopade
et
al.,
2015).
Thus,
results
of
the
present
study
confirm
the
ear-
lier
findings
that
the
lignans
and
tannins
from
the
Phyllanthus
species
exhibit
significant
antihyperalgesic
activity
and
are
responsible
for
the
observed
pain
modu-
lating
potential.
The
observed
antihyperalgesic
and
Phyllanthin
Corilagin
Hypophyllanthin
Figure
6.
Phyllanthus
amarus
whole
plant.
The
diagrammatic
representation
shows
that
P
amarus
plant
is
capable
of
yielding
important
antihyperalgesic
molecules
of,
namely,
phyllanthin,
hypopyllanthin,
and
corilagin.
This
figure
is
available
in
color
online
at
wileyonlinelibrary.com/journal/ptr.
antiinflammatory
effects
of
P.
amarus
in
carrageenan-
induced
chronic
pain
model
may
be
due
to
the
presence
of
phytoconstituents
like
phyllanthin,
hypophyllanthin,
and
corilagin,
which
offers
a
promising
means
for
the
treatment
of
inflammatory
chronic
muscular
pain
and
is
represented
in
graphical
form
in
Fig.
6.
In
depth
experimental
evalua-
tion
of
the
compounds
like
phyllanthin,
hypophyllanthin,
corilagin,
phyltetralin,
nirtetralin,
niranthin,
and
so
on
by
logistic
approaches
would
lead
us
to
clinically
effective
mol-
ecules
for
treating
various
chronic
pain
disorders.
To
conclude
in
the
present
carrageenan-induced
chronic
pain
model,
we
have
evaluated
the
role
of
P.
amarus
ex-
tracts
in
the
reversal
and
inhibition
of
the
state
of
chronic
muscle
hyperalgesia.
Also,
while
considering
the
observa-
tions
of
the
present
model,
we
suggest
the
importance
of
a
spinal
and
supraspinal
mechanisms
of
Phyllanthus
species
in
the
face
of
peripheral
inflammation.
Acknowledgements
The
authors
are
thankful
to
the
University
Grants
Commission
(UGC)
for
providing
fellowship
under
RGNFS
[Grant
reference
no.
E
16-1917(SC)/2010
(SA-III)]
and
to
A.
R.
C.
for
his
doctoral
studies.
Conflict
of
Interest
Both
the
authors
declare
no
financial/commercial
conflicts
of
interest.
Copyright
©
2015
John
Wiley
&
Sons,
Ltd.
Phytother.
Res.
29:
1202
-
1210
(2015)
1210
A.
R.
CHOPADE
AND
F.
J.
SAYYAD
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Copyright
©
2015
John
Wiley
&
Sons,
Ltd.
Phytother.
Res.
29:
1202-1210
(2015)