In vitro relaxant and spasmolytic effects of constituents from Viburnum prunifolium and HPLC quantification of the bioactive isolated iridoids


Cometa, M.Francesca.; Parisi, L.; Palmery, M.; Meneguz, A.; Tomassini, L.

Journal of Ethnopharmacology 123(2): 201-207

2009


Viburnum prunifolium is a North America shrub used in ethnomedicine because of its spasmolytic, sedative, and anti-asthmatic properties. Contrasting results were reported in past literature about the active principles of this plant. Our aim was to clarify this matter by evaluating the relaxant and spasmolytic activities of the main constituents obtained from the drug. The pharmacological assays were carried out on rabbit jejunum spontaneous contractions and on guinea-pig carbachol-precontracted trachea. Cumulative concentration (1-100 microg/ml) of Viburnum prunifolium methanolic extract (MeOH extract), its purified fractions soluble in ethylacetate (EtOAc fraction) and in n-butanol (BuOH fraction), and the iridoid glucosides (2 x 10(-5) to 4 x 10(-4)M): 2'-O-acetyldihydropenstemide (1), 2'-O-trans-p-coumaroyldihydropenstemide (2), 2'-O-acetylpatrinoside (3), and patrinoside (4), isolated from EtOAc fraction (1 and 2) and BuOH fraction (3 and 4), induced both relaxant effect of rabbit jejunum spontaneous contractions and spasmolytic effect on guinea-pig carbachol (5.5 x 10(-7)M)-precontracted trachea. Propranolol (10(-6)M) antagonised all Viburnum prunifolium tested components relaxant and spasmolytic effects. At non-relaxing concentrations (0.5 microg/ml), MeOH extract and its fractions induced a potentiating effect of isoprenaline cumulative concentrations also in both isolated tissues. In both tissues, the order of potency was EtOAc fraction>BuOH fraction>MeOH extract and 1>2>3>4 suggesting that the major iridoids of EtOAc fraction may be considered among the most active compounds. HPLC analysis of the bioactive iridoids indicates that 1 and 2 are present for 7.38% and 14.90% in EtOAc fraction, and 3 and 4 for 18.47% and 8.86% in BuOH fraction. By comparing the values of EC(50) of the fractions and compounds isolated from them, we may assume that the iridoids play a significant role in the biological activity of the corresponding fractions.

Journal
of
Ethnopharmacology
123
(2009)
201-207
,rnal
of
I
TEP10-
PHARMACOLOGY
Contents
lists
available
at
ScienceDirect
Journal
of
Ethnopharmacology
ELSEVIER
journal
homepage:
www.elsevier.com/locate/jethpharm
In
vitro
relaxant
and
spasmolytic
effects
of
constituents
from
Viburnum
prunifolium
and
HPLC
quantification
of
the
bioactive
isolated
iridoids
Maria
Francesca
Cometaa'*,
Laura
Parisi
a
,
Maura
Palmeryb,
Annarita
Meneguz
a
,
Lamberto
Tomassinic
a
Department
of
Therapeutic
Research
and
Medicines
Evaluation,
Istituto
Superiore
di
Sanita,
Vie
Regina
Elena
299,
00161
Rome,
Italy
b
Department
of
Human
Physiology
and
Pharmacology
"Vittorio
Erspamer",
"Sapienza
University
of
Rome",
Pie
Aldo
Moro
5,
00185
Rome,
Italy
c
Department
of
Plant
Biology,
"Sapienza
University
of
Rome",
Pie
Aldo
Moro
5,
00185
Rome,
Italy
ARTICLE
INFO
ABSTRACT
Article
history:
Received
21
October
2008
Received
in
revised
form
28
February
2009
Accepted
15
March
2009
Available
online
26
March
2009
Keywords:
Viburnum
prunifolium
Smooth
muscle
relaxation
Spasmolytic
Rabbit
jejunum
Guinea-pig
trachea
13-Adrenoceptors
Iridoid
glucosides
Ethnopharmacological
relevance:
Viburnum
prunifolium
is
a
North
America
shrub
used
in
ethnomedicine
because
of
its
spasmolytic,
sedative,
and
anti-asthmatic
properties.
Aim
of
the
study:
Contrasting
results
were
reported
in
past
literature
about
the
active
principles
of
this
plant.
Our
aim
was
to
clarify
this
matter
by
evaluating
the
relaxant
and
spasmolytic
activities
of
the
main
constituents
obtained
from
the
drug.
Materials
and
methods:
The
pharmacological
assays
were
carried
out
on
rabbit
jejunum
spontaneous
contractions
and
on
guinea-pig
carbachol-precontracted
trachea.
Results:
Cumulative
concentration
(1-100
µg/ml)
of
Viburnum
prunifolium
methanolic
extract
(MeOH
extract),
its
purified
fractions
soluble
in
ethylacetate
(EtOAc
fraction)
and
in
n-butanol
(BuOH
fraction),
and
the
iridoid
glucosides
(2
x
10
-6
to
4
x
10
-4
M):
2'-0-acetyldihydropenstemide
(1),
2'
-0-trans-p-
coumaroyldihydropenstemide
(2),
2'-0-acetylpatrinoside
(3),
and
patrinoside
(4),
isolated
from
EtOAc
fraction
(1
and
2)
and
BuOH
fraction
(3
and
4),
induced
both
relaxant
effect
of
rabbit
jejunum
sponta-
neous
contractions
and
spasmolytic
effect
on
guinea-pig
carbachol
(5.5
x
10
-7
M)-precontracted
trachea.
Propranolol
(10
-6
M)
antagonised
all
Viburnum
prunifolium
tested
components
relaxant
and
spasmolytic
effects.
At
non-relaxing
concentrations
(0.5
pLg/m1),Me0H
extract
and
its
fractions
induced
a
potentiating
effect
of
isoprenaline
cumulative
concentrations
also
in
both
isolated
tissues.
Conclusion:
In
both
tissues,
the
order
of
potency
was
EtOAc
fraction
>
BuOH
fraction
>Me0H
extract
and
1
>2
>3
>
4
suggesting
that
the
major iridoids
of
EtOAc
fraction
may
be
considered
among
the
most
active
compounds.
HPLC
analysis
of
the
bioactive
iridoids
indicates
that
1
and
2
are
present
for
738%
and
14.90%
in
EtOAc
fraction,
and
3
and
4
for
18.47%
and
8.86%
in
BuOH
fraction.
By
comparing
the
values
of
EC
50
of
the
fractions
and
compounds
isolated
from
them,
we
may
assume
that
the
iridoids
play
a
significant
role
in
the
biological
activity
of
the
corresponding
fractions.
2009
Elsevier
Ireland
Ltd.
All
rights
reserved.
1.
Introduction
The
root
and
stem
barks
of
Viburnum
prunifolium
L
(Capri-
foliaceae),
or
Black
Haw,
a
deciduous
shrub
of
North
America,
are
well
known
in
folk
medicine
for
their
spasmolytic,
sedative,
and
anti-asthmatic
properties
(Jarboe
et
al.,
1966;
British
Herbal
Pharmacopoeia,
1983;
American Herbal
Pharmacopoeia,
2000).
Furthermore,
as
well
as
many
other
Viburnum
species,
Viburnum
Abbreviations:
BuOH
fraction,
n-butanol
fraction;
CC,
column
chromatography;
DMSO,
dimethyl
sulfoxide;
Me0H
extract,
methanolic
extract;
EtOAc
fraction,
ethy-
lacetate
fraction;
HPLC,
high
performance
liquid
chromatography;
Me0H,
methanol;
UV,
ultraviolet.
*
Corresponding
author.
Tel.:
+39
06
49902389;
fax:
+39
06
49387100.
E-mail
address:
(M.F.
Cometa).
0378-8741/$
-
see
front
matter
C
2009
Elsevier
Ireland
Ltd.
All
rights
reserved.
doi:10.1016/j.jep.2009.03.021
prunifolium
is
popularly
used
in
the
treatment
of
menstrual
cramps,
as
an
anti-abortive
agent
and
to
prevent
postpartum
bleeding
(Osol
and
Farrar,
1955;
Bernal
and
Correa,
1990;
Calle
et
al.,
1999).
An
aqueous
infusion
or
decoction
of
the
bark
of
roots
and
stems
is
generally
used
(American
Herbal
Pharmacopoeia,
2000).
Despite
the
continued
use
in
folk
medicine,
contrasting
results
on
the
pharmacological
properties
of
the
plant
have
been
reported
in
previous
literature
(Benigni
et
al.,
1962),
perhaps
due
to
a
differ-
ent
preparation
of
the
extract
(e.g.
ethanolic
extraction
or
aqueous
decoction),
various
drug
administrations
(oral
and
intravenous),
or
to
common
inert
adulterant
in
Viburnum
prunifolium
for
biological
assays
(American
Herbal
Pharmacopoeia,
2000).
Apart
from
a
few
compounds
identified
as
being
possibly
responsible
for
the
proper-
ties
of
Viburnum
prunifolium
(Mariott
and
Munch,
1946;
Grote
and
Woods,
1947;
Horhammer
et
al.,
1966,
1967;
Jarboe
et
al.,
1966;
Ojewole,
1984),
several
unidentified
constituents
showing
spas-
1
R
=
H
R'
=
acetyl
2
R
=
H
R'
=
p-coumaroyl
3
R
=
OH
R'
=
acetyl
4
R
=
OH
R'
=
H
CH
2
OH
c;71
,
:).___.-0
\
.
HO
CH2
R'0
202
M.F.
Cometa
et
al
. /
journal
of
Ethnopharmacology
123
(2009)
201-207
Herbarium
Cesati
(Herbarium
Horti
Romani—RO
2988).
All
solvents
and
reagents
were
purchased
from
Carlo
Erba
Reagenti
(Milan,
Italy).
Dried
Viburnum
prunifolium
stem
bark
(300
g)
was
exhaustively
extracted
with
methanol
at
room
temperature.
The
obtained
solu-
tion
was
concentrated
to
dryness
in
vacuo
(MeOH
extract,
39.6
g).
A
part
of
the
Me0H
extract
residue
(25.0
g)
was
partitioned
between
H
2
O
and
ethylacetate,
and
the
aqueous
layer
was
re-extracted
with
n-butanol.
The
two
organic
phases
were
evaporated
to
obtain
EtOAc
and
a
n-BuOH
dry
residues
(EtOAc
fraction,
7.2
g
and
BuOH
frac-
tion,
12.5
g).
Me0H
extract
and
the
fractions
were
stored
at
4
°C
until
analysis.
2.2.
Isolation
and
structural
analysis
of
iridoids
H
OH
2
C
0
CH3
0
C
H3
Fig.
1.
Iridoids
1-4.
molytic
activity
on
different
isolated
smooth
muscles
were
reported
(Jarboe
et
al.,
1967).
As
a
consequence
of
this
situation,
Black
Haw
is
still
largely
unrecognized
by
the
public
and
conventional
medical
professionals
and
it
is
absent
from
modern
pharmacopoeia
with
the
exception
of
British
Herbal
Pharmacopoeia
(1983)
and
American
Herbal
Pharmacopoeia
and
Therapeutic
Compendium
(2000).
In
the
last
decade,
we
have
isolated
four
iridoid
gluco-
sides
from
Viburnum
prunifolium
(Fig.
1),
all
characterized
by
Valeriana-type
skeleton:
2'-0-acetyldihydropenstemide
(1),
2'-0-
trans-p-coumaroyldihydropenstemide
(2),
2'-0-acetylpatrinoside
(3),
and
patrinoside
(4).
These
iridoids
were
more
easily
obtained
by
splitting
the
total
methanolic
extract
into
two
fractions:
in
fact,
compounds
1
and
2
predominate
in
the
less
polar
EtOAc
fraction,
and
3
and
4
in
the
fraction
of
medium
polarity,
BuOH
fraction
(Tomassini
et
al.,
1999).
In
a
study
of
the
same
period,
these
two
fractions
proved
to
be
active
on
the
non-pregnant
rat
uterus,
showing
a
concentration-
dependent
relaxant
effect
of
spontaneous
contractions
(Tomassini
et
al.,
1998).
Moreover,
some
evidence
obtained
from
the
related
species
Viburnum
tinus
suggests
that
its
iridoid
glucosides
could
account
for
the
spasmolytic
and
sedative
activities
observed
for
the
methanolic
extract
of
this
plant
(Cometa
et
al.,
1998a).
On
the
basis
of
these
considerations,
the
first
aim
of
the
present
study
was
to
investigate
in
suitable
biological
systems
the
pharma-
cological
properties
of
Viburnum
prunifolium
in
order
to
support
the
wide
use
of
this
plant
in
ethnomedicine.
Considering
the
well-known
biological
activities
of
iridoids
and
our
preliminary
experience
with
Viburnum
species
on
different
muscle
isolated
preparations,
the
second
aim
was
to
characterize
relaxant
and
spasmolytic
activities
of
these
compounds
through
the
study
of
the
mechanism
of
action
and
the
relative
contribution
to
Viburnum
prunifolium
total
extract
activity.
In
the
light
of
the
ethnobotanical
use
of
this
plant,
we
tested
the
relaxant
and
spasmolytic
activities
of
the
methanolic
extract
from
the
stem
bark
of
Viburnum
prunifolium,
its
EtOAc
and
n-BuOH
purified
fractions,
and
their
major
iridoidic
compounds
on
isolated
rabbit
jejunum
and
guinea-pig
trachea.
Furthermore,
in
order
to
obtain
clear
information
regarding
the
iridoidic
contribution
to
the
extract
activity,
an
HPLC
analysis
of
bioactive
iridoids
has
been
included.
2.
Materials
and
methods
2.1.
Plant
material,
extraction
and
preparation
of
fractions
The
stem
bark
of
cultivated
Viburnum
prunifolium
was
sup-
plied
by
the
company
A.
Minardi
(Bagnacavallo,
Italy),
which
vouches
for
the
plant
identity.
The
plant
material
was
also
identi-
fied
by
the
authors
in
the
Department
of
Plant
Biology
of
Sapienza
University
of
Rome",
where
a
sample
specimen
is
deposited
in
An
amount
of
3.6
g
of
EtOAc
fraction
was
subjected
to
CC
on
sil-
ica
gel
and
eluted
with
methanol-chloroform
(Me0H-CHC1
3
,
1:9),
affording
pure
2'-0-acetyldihydropenstemide
(1)
(85
mg)
and
2'-0-
trans-p-coumaroyldihydropenstemide
(2)
(180
mg).
6.5
g
of
BuOH
fraction,
on
CC
on
silica
gel
with
Me0H-CHC1
3
(1:4),
gave
pure
2'-0-
acetylpatrinoside
(3)
(82
mg)
and
patrinoside
(4)
(108
mg).
All
the
structures
were
elucidated
by
spectroscopic
means
and
the
identity
of
the
single
compounds
confirmed
by
comparison
with
authen-
tic
samples.
The
preparation
of
extracts
and
the
isolation
of
tested
compounds
were
performed
in
accordance
with
Tomassini
et
al.
(1999).
2.3.
HPLC
analysis
2.3.1.
HPLC
equipment
HPLC
analysis
was
performed
using
a
Dionex
(Sunnyvale,
California)
Model
Ultimate
3000
liquid
chromatography
system,
equipped
with
a
quaternary
pump
(LPG-3400
Low
Pressure
Gradi-
ent
Pump
Analytical),
an
Autosampler
(WPS-3000
TSL
Analytical),
a
Photodiode
Array
Detector
(PDA
3000),
and
Thermostated
Column
Compartment
(TCC-3000).
A
Pinnacle
ODS
amine
C-18
(250
mm
x
4.6
mm,
5µm)
(Restek
Corporation),
protected
by
a
ODS
amine
C-18
guard
column
(10
mm
x
4
mm,
5µm)
(Restek
Corpora-
tion)
was
employed.
CHROMELEON
Chromatography
Data
System
(Version
6.8,
Dionex)
was
used
to
control
the
system
and
to
analyse
the
data.
2.3.2.
Sample
preparation
and
standard
solution
Viburnum
prunifolium
EtOAc
and
BuOH
fractions
were
each
dis-
solved
in
Me0H
(J.T.
Baker
HPLC
Analyzed,
Deventer,
Holland)
to
give
two
solutions
with
the
concentration
of
1900
µg/ml.
Each
solu-
tion
was
filtered
through
a
0.22
pm
PTFE
syringe
filter
before
the
HPLC
analysis.
The
four
standard
solutions
consisting
of
the
isolated
iridoids
1-4,
were
each
dissolved
in
Me0H
to
give
the
stock
solution
of
0.5
mg/ml.
2.3.3.
Chromatographic
conditions
Separations
of
iridoidic
compounds
in
the
respective
fractions
were
achieved
on
linear
gradient
of
water
(Water
Plus
for
HPLC;
Carlo
Erba
Reagents,
Milano,
Italy)
(A)
and
acetonitrile
(B)
(J.T.
Baker
HPLC
Analyzed,
Deventer,
Holland)
of
30%
B
at
0-10
min,
30-40%
B
at
10-20
min,
40%
B
at
20-30
min,
40-50%
B
at
30-40
min,
50%
B
at
40-50
min.
The
gradient
was
returned
to
initial
conditions
within
40
min
to
re-equilibrate
the
column.
The
flow
rate
was
0.8
ml/min,
aliquots
of
20µl
were
injected
and
the
column
was
maintained
at
25
°C
for
all
analysis.
On-line
UV
spectra
were
recorded
with
a
diode-array
detector
from
200
to
400
nm
and
the
absorbances
recorded
at
210
and
310
nm.
M.F.
Cometa
et
al
/
journal
of
Ethnopharmacology
123
(2009)
201-207
203
2.3.4.
Calibration
curves
The
calibration
curves
for
standard
iridoids
1-4,
were
prepared
for
the
present
study.
Standard
solutions
of
iridoids
1-4,
in
the
60-500
µg/ml
concentration
range,
were
prepared
and
injected
in
the
HPLC
system.
The
linearity
of
each
standard
curve
was
con-
firmed
by
plotting
the
peak
area
versus
the
concentrations
and
the
calibration
curves
were
constructed
by
means
of
the
least-square
method.
The
correlation
coefficient
calculated
for
each
calibra-
tion
curve
were:
R
2
=
0.9989
(1),
R
2
=
0.9998
(2),
R
2
=
0.9993
(3),
R
2
=
0.9996
(4).
2.3.5.
Sample
analysis
Aliquots
of
the
EtOAc
and
BuOH
fraction
samples,
after
suitable
dilution
in
methanol,
were
injected
into
HPLC
(at
the
concentrations
of
475,
950
and
190011g/rill).
Each
sample
solution
was
injected
in
triplicate
and
the
area
value
of
the
iridoid
peaks
were
interpolated
on
the
calibration
curves.
2.4.
Animals
Male
New
Zealand
albino
rabbits
(2.2-2.5
kg)
and
male
Dunkin-Hartley
guinea
pigs
(300-350
g),
were
purchased
from
Charles
River
(Calco,
Italy).
All
animals
were
acclimatised
to
a
12-h
light/dark
cycle
at
23
°C
with
food
and
water
available
ad
lib
for
at
least
3
days
before
starting
the
experiments.
The
directives
of
the
Council
of
the
European
Communities
(86/609/EEC)
on
animal
care
were
duly
observed.
2.5.
Drugs
and
solutions
for
pharmacological
assays
Acetylcholine
chloride,
carbachol
chloride,
dimethyl
sulfox-
ide
(DMSO),
(-
)-isoproterenol
(+)
bitartrate
salt
and
(S)-(-)-
propranolol
hydrochloride
were
purchased
from
Sigma
Chemical
Co.
(St.
Louis,
MO,
USA).
Dried
Me0H
extract,
EtOAc
fraction,
BuOH
fraction
and
com-
pounds
1-4
were
dissolved
in
DMSO
at
a
concentration
of
10
mg/ml
stock
solutions
and
stored
at
0-5
°C;
all
the
other
substances
were
dissolved
in
distilled
water
and
ethanol
(1:1)
as
1
mg/ml
stock
solu-
tions.
All
stock
solutions
were
diluted
in
distilled
water
on
the
day
of
the
experiment.
2.6.
Pharmacological
studies
2.6.1.
Rabbit
jejunum
The
animals
were
killed
by
a
blow
on
the
neck
and
bled.
Seg-
ments
of
jejunum
were
removed
and
suspended
in
a
10
ml
tissue
bath
containing
Tyrode
solution,
whose
composition
was
(mM):
NaCl,
137.0;
KC1,
2.7;
MgCl
2
,
1.1;
CaC12
x
2H
2
0,1.8;
NaHPO
4
x
H
2
O,
0.4;
NaHCO
3
,
11.9;
glucose,
5.0;
pH
7.4.
Tension
changes
were
recorded
by
an
isotonic
force
transducer
(U.
Basile
7050)
and
cali-
brated
before
each
experiment.
The
preparations
were
allowed
to
equilibrate
for
30
min
to
obtain
a
regular
spontaneous
activity.
At
the
start
of
each
experiment
the
sensitivity
of
the
tissues
was
tested
with
acetylcholine
(2.7
x
10
-7
M)
and
isoprenaline
(2.4
x
10
-8
M).
Then
the
following
experiments
were
performed:
2.6.1.1.
Relaxant
effect
of
Viburnum
prunifolium
on
jejunum
sponta-
neous
contractions.
Cumulative
concentrations
of
Me0H
extract,
EtOAc
fraction
and
BuOH
fraction
(1-100
µg/ml),
iridoids
1-4
(2
x
10
-5
to
4
x
10
-4
M)
and
isoprenaline
(10
-8
to
3
x
10
-6
M)
were
left
in
contact
with
the
tissue
for
2
min
(MeOH
extract,
fractions
and
compounds)
and
30
s
(isoprenaline)
for
each
concentration
in
the
presence
and
absence
of
propranolol
(10
-6
M)
and
then
washed
out.
The
antagonist
was
added
to
the
bath
15
min
before
repeating
the
concentration-response
determinations
of
either
isoprenaline
or
Viburnum
prunifolium
tested
components.
2.6.1.2.
Potentiating
effect
of
Viburnum
prunifolium
on
isoprenaline-
induced
relaxation.
The
jejunum
segments
were
equilibrated
for
5
min
with
non-relaxant
concentration
of
Me0H
extract,
EtOAc
fraction
and
BuOH
fraction
(0.5
µg/ml)
before
repeating
the
concentration-response
determination
of
isoprenaline
(10
-8
to
3
x
10
-6
M).
Responses
following
isoprenaline
or
Viburnum
prunifolium
tested
components
were
expressed
as
a
percentage
of
regular
spontaneous
jejunum
contractions
and
were
plotted
against
the
logarithm
of
agonist
concentration.
2.6.2.
Guinea-pig
trachea
Experiments
were
performed
as
previously
described
(Cometa
et
al.,
1998b;
Abdel-Haq
et
al.,
2000).
The
animals
were
killed
by
cervical
dislocation
and
the
trachea
was
rapidly
excised
and
placed
in
Krebs
solution
whose
composition
was
(mM):
NaCl
(118.0);
KC1
(4.7);
MgSO
4
(1.2);
CaC1
2
(2.5);
KH
2
PO
4
(1.2);
NaHCO
3
(25.0);
glucose
(10.5);
pH
7.4
at
room
temperature.
After
the
removal
of
adhering
fat
and
connective
tissue,
the
trachea
was
opened
longitudinally
by
cutting
through
the
cartilage
opposite
the
smooth
muscle
layer,
and
cut
again
in
transverse
segments
to
obtain
two
strips.
Each
strip
was
mounted
in
an
organ
bath
containing
5
ml
of
Krebs
solution,
kept
at
37
°C
and
gassed
continuously
with
95%
02,
5%
CO
2
under
a
tension
of
0.5
g,
measured
by
an
isotonic
transducer
(Ugo
Basile
7006,
Italy)
and
equilibrated
for
30-60
min
before
starting
the
following
experiments:
2.6.2.1.
Relaxant
effect
of
Viburnum
prunifolium
on
carbachol-
precontracted
trachea.
Tracheal
strips
were
maximally
contracted
by
carbachol
(5.5
x
10
-7
M)
and
relaxed
by
cumulative
con-
centrations
of
Me0H
extract,
EtOAc
fraction
and
BuOH
frac-
tion
(1-250
µg/ml),
iridoids
1-4
(2
x
10
-5
to
4
x
10
-4
M)
and
isoprenaline
(10
-9
to
3.0
x
10
-7
M).
In
another
set
of
experi-
ments,
tracheal
strips
were
maximally
contracted
by
carbachol
(5.5
x
10
-7
M)
and
when
they
reached
the
stable
tone,
propranolol
(10
-6
M)
was
left
in
incubation
for
10
min
before
repeating
the
concentration-response
relaxation
of
Viburnum
prunifolium
tested
components
and
isoprenaline.
2.6.2.2.
Potentiating
effect
of
Viburnum
prunifolium
on
isoprenaline-
induced
relaxation
of
carbachol-precontracted
trachea.
Carbachol-
precontracted
trachea
was
treated
with
non-relaxant
concentration
of
Viburnum
prunifolium
Me0H
extract
and
its
fractions
(0.5
µg/ml)
and
increasing
cumulative
concentrations
of
isoprenaline
(10
-9
to
3.0
x
10
-7
M)
were
added.
The
relaxant
effects
(E)
were
calculated
with
the
formula
E=
100
x
Tfi
na
l)/(Ti
n
i
t
i
a
l
T
O
),
where
T
was
the
tone
of
the
organ
before
(T
0
),
after
contraction
with
carbachol
(Tinitiai
),
and
after
the
addition
of
the
relaxant
agent
(T
fi
2.7.
Statistical
analysis
EC
50
values
(the
concentration
of
agonists
causing
half-maximal
relaxant
responses)
were
determined
from
individual
experiments
for
the
complete
agonist
concentration-response
curves
by
using
a
least
square
regression
analysis.
Mean
and
standard
error
(S.E.M.)
values
were
calculated
for
each
group
of
results
and
significance
of
differences
between
the
means
were
determined
by
one-way
analysis
of
variance
(ANOVA).
If
significant,
group
means
were
compared
by
post
hoc
analysis
using
Student-Newman-Keul's
or
Dunn's
methods
for
multiple
comparisons
of
means.
P<
0.05
or
less
was
considered
as
indicative
of
significance.
All
statistical
analy-
ses
were
performed
by
using
the
software
Sigma-Stat
(SPSS
Inc.,
Chicago,
IL,
USA).
(A)
450
ANALYSIS
COMPOUNDS
1
AND
2
400
mAU
EtOAc
fraction
WVL:210
300
200
2
14.72
100
0
50
921
c
o
0
40-
1
20
-
0-
-3,0
-2
,
0
-1,0
0,0
log
lig/m
I
a'
t
Bo-
b
100-
0,
z
O
2
ac
p
c
60
-
1,0
30
-
N-
Me0H
extract
-
BuOH
fraction
w•
-
E10Ac
fraction
4
isoprenaline
120
8
P
-•
100-
c
O
_
fi
t]
o
JD
a,
,
a
20
80-
60-
40-
o-
-9
8
-5
-3
log
(M)
Alt-
isoprenaline
-
N-
1
-
0-
2
-
a-
3
--
4
M.F.
Cometa
et
al
/
journal
of
Ethnopharmacology
123
(2009)
201-207
204
120
3.
Results
3.1.
Phytochemical
analysis
by
HPLC
Better
chromatographic
profiles
for
iridoidic
components
were
obtained
by
recording
the
absorbances
at
210
and
310
nm.
In
fact,
except
for
compound
2,
which
shows
an
absorbance
maximum
at
310
nm,
the
iridoids
isolated
from
the
extract
are
devoid
of
conju-
gated
double-bonds
and
do
not
present
any
absorbance
maximum
at
a
wavelength
greater
than
210
nm.
Due
to
this
feature,
eluents
with
an
adequate
cut-off
have
been
used
(see
Section
2.3.3).
The
single
iridoids
were
identified
through
their
retention
times
(tR),
i.e.
about
93,
14.7,
5.9,
and
4.7
min
for
1,
2,
3,
and
4,
respec-
tively.
HPLC
quantitative
analysis
of
iridoids
1-4
in
the
two
fractions
revealed
7.38%
(1),
14.90%
(2)
content
for
EtOAc
fraction,
and
18.47%
(3)
and
8.86%
(4)
for
BuOH
fraction.
The
chromatographic
profiles
of
the
most
active
fraction
(EtOAc
fraction)
recorded
at
210
and
310
nm,
are
reported
in
Fig.
2.
3.2.
Studies
on
isolated
rabbit
jejunum
Cumulative
concentrations
of
Viburnum
prunifolium
tested
components
(1-100µg/ml
for
Me0H
extract
and
fractions,
2
x
10
-5
to
4
x
10
-4
M
for
iridoids)
and
isoprenaline
(3.97
x
10
-3
M
to
1.19
µg/ml,
equivalent
to
10
-8
to
3
x
10
-6
M)
induced
a
concentration-dependent
inhibition
of
the
spontaneous
contrac-
tions
of
the
rabbit
jejunum
with
complete
inhibition
at
the
highest
concentration
(Figs.
3
and
4),
which
was
reversed
by
washing
(the
respective
EC
50
values
are
indicated
in
Table
1).
Propranolol
(10
-6
M)
abolished
the
isoprenaline
relaxant
effect
and
antagonised
the
effect
induced
by
all
Viburnum
prunifolium
tested
components
(Table
1).
Furthermore,
at
non-
relaxing
concentration
of
0.5
µg/ml
all
Viburnum
prunifolium
tested
extracts
produced
a
graded
shift
to
the
left
of
the
isoprenaline
concentration-response
curves
(Fig.
5).
0 0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30
0
(N)
EtOAc
fraction
350
300
NALYSIS
COMPOUND
2
mAU
2
WVL:310
nm
250
14.72
200
150
100
50
"
-50
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
Fig.
2.
HPLC
profiles
of
EtOAc,
recorded
at
210
nm
(panel
A)
and
at
310
nm
(panel
B).
Fig.
3.
The
inhibitory
effect
induced
by
cumulative
concentration
(1-100
µg/ml)
of
Viburnum
prunifolium
Me0H
extract,
BuOH
fraction
and
EtOAc
fraction
on
isolated
jejunum
spontaneous
contractions
in
comparison
with
isoprenaline
(3.97
x
10
-3
to
1.19
µg/ml).
Each
point
represents
mean
±
S.E.M.
of
four
or
more
experiments.
Sta-
tistical
comparison
was
performed
by
ANOVA
analysis
followed
by
post
hoc
test.
***P<
0.001
indicates
that
Me0H
extract,
BuOH
fraction
and
EtOAc
fraction
effects
were
statistically
different
from
each
other.
3.3.
Studies
on
guinea-pig
isolated
trachea
When
cumulative
concentrations
of
Me0H
extract,
EtOAc
fraction,
BuOH
fraction
(1-250µg/m1),
iridoids
(2
x
10
-5
to
4
x
10
-4
M)
and
isoprenaline
(10
-9
to
3.0
x
10
-7
M)
were
added
on
carbachol-precontracted
trachea
(5.5
x
10
-7
M)
a
concentration-
dependent
relaxation
was
observed
(Figs.
6
and
7).
The
order
of
potency
was:
EtOAc
fraction
>
BuOH
fraction
>
Me0H
extract
(Fig.
6)
and
1>
2
>3
>4
(Fig.
7).
Propranolol
(10
-6
M)
blocked
isoprenaline
responses
and
antagonised
the
relaxation
of
carbachol-precontracted
trachea
induced
by
all
Viburnum
pruni-
folium
tested
components
(Table
1).
Moreover,
Ext
and
its
fractions,
at
non-relaxant
concentration
(0.5
µg/ml),
potentiated
the
relax-
ant
effect
induced
by
isoprenaline
cumulative
concentration
(10
-9
to
3.0
x
10
-7
M)
on
carbachol-precontracted
trachea
(Fig.
8).
Wash-
ing
of
the
tissue
did
not
reverse
the
inhibitory
effect
induced
by
all
tested
extracts
for
about
30
min.
Fig.
4.
The
inhibitory
effect
induced
by
cumulative
concentration
(2
x
10
-5
to
4
x
10
-4
M)
of
Viburnum
prunifolium
isolated
iridoids
1-4
on
isolated
jejunum
spon-
taneous
contractions
in
comparison
with
isoprenaline
(10
-8
to
3
x
10
-6
M).
Each
point
represents
mean
±
S.E.M.
of
four
or
more
experiments.
Statistical
comparison
was
performed
by
ANOVA
analysis
followed
by
post
hoc
test.
*P<
0.05,
**P<0.01,
***P<
0.001
indicate
that
the
effects
of
the
single
iridoid
glucosides
(1-4)
were
sta-
tistically
different
from
each
other.
-
MeOH
extract
-
0-
BuOH
fraction
-
0-
ElOAc
fraction
--
isoprenaline
-3,0 -2,0
-1,0
0,0
log
Ng/m1
30
120
F
100-
0
c
.L.
)
8
60-
c
o
la
o
c
c
f
y
0-
20-
co)
80-
40-
isoprenaline
MeOH
extract
+
isoprenaline
-0-
BuOH
fraction
+
isoprenaline
EtOAc
fraction
isoprenaline
-8,0
-7,5
-7,0
-6,5
log
(M)
-6,0
-5,5
0
-8,5
-5,0
140
ID
120-
r6
.-
co
100-
15_
'al
Ea
o
a)
c
o
o
0)
60-
.
o
-
0
x
p2
40-
cl)
8
20-
-
M.F.
Cometa
et
al
/
journal
of
Ethnopharmacology
123
(2009)
201-207
205
Table
1
The
EC50
values
of
0-adrenergic
agonist
isoprenaline
and
Viburnum
prunifolium
methanolic
extract
(MeOH
extract),
its
fractions
soluble
in
ethylacetate
(EtOAc
fraction)
and
butanol
(BuOH
fraction)
and
iridoid
compounds
1-4,
on
rabbit
jejunum
and
guinea-pig
isolated
trachea
in
the
absence
and
presence
of
0-antagonist
propranolol
(10
-6
M).
Rabbit
jejunum
Guinea-pig
trachea
+Propranolol
+Propranolol
EC
50
(pg(ml)
Isoprenaline
0.07
±
131
nf
0.0316
±
1.43
nf
MeOH
extract
14.95
±
1.41
24.81
±
1.04'
>250
nf
BuOH
fraction
11.48
±
1.04
18.17
±
1.21'
103.7
±
3.44
159.65
±
2.48'
EtOAc
fraction
6.99
±
1.01
18.08
±
0.98"
15.8
±
132
28.44±
1.02"
ECso
(M)
Isoprenaline
1.74±0.20
x
10
-7
nf
1.73
±
0.21
x
10
-8
nf
9.12
±
6.89
x
10
-5
nf
1.09
±
0.03
x
10
-5
nf
2
1.14±0.07
x
10
-5
nf
154
±
0.08
x
10
-5
nf
3
2.29
±
0.13
x
10
-5
4.0
x
10
-5
"
2.69
±
0.17
x
10
-5
4.0
x
10
-5
"
4
nf
nf nf
nf
of=
not
found.
Values
are
expressed
as
means
±
S.E.M.
P<
0.05
by
ANOVA
test.
"
P<
0.01
by
ANOVA
test.
3.4.
Vehicle
effects
DMSO
(0-2.5%)
did
not
affect
the
amplitude
and
the
frequency
of
spontaneous
contractions
of
rabbit
jejunum
and
did
not
induce
relaxation
of
carbachol-precontracted
tone
of
guinea-pig
isolated
trachea.
4.
Discussion
Our
in
vitro
findings
extend
our
previous
studies
on
Viburnum
properties
(Cometa
et
al.,
1998a;
Tomassini
et
al.,
1998,
1999)
and
significantly
expand
the
pharmacological
evidence
on
Viburnum
prunifolium
in
suitable
biological
systems.
We
now
show
that
Viburnum
prunifolium
components
pro-
duce
relaxant
effects
of
rabbit
jejunum
spontaneous
contractions
and
guinea-pig
precontracted
trachea,
supporting
the
wide
appli-
cation
of
the
plant
in
traditional
medicine
(American
Herbal
Pharmacopoeia,
2000).
Our
new
information
specifies
the
effect
of
the
dried
stem
bark
methanolic
extract
of
this
plant
(MeOH
extract)
and
its
frac-
tions
(EtOAc
and
BuOH
fractions)
on
the
two
isolated
tissues
in
comparison
to
the
13-adrenergic
agonist
isoprenaline,
obtaining
maximal
tissues
relaxation
at
the
higher
concentrations.
Compari-
son
of
respective
potencies
showed
that
MeOH
extract
had
lower
relaxant
and
spasmolytic
effects
than
EtOAc
fraction
and
BuOH
fraction.
All
the
tested
iridoidic
compounds
1
>
2
>3
>
4,
in
order
of
decreasing
potency,
showed
the
same
biological
action
evoked
by
MeOH
extract
and
the
two
tested
fractions
in
both
isolated
tis-
sues.
Then
the
less
polar
iridoids
(1
and
2)
more
concentrated
in
the
more
active
fraction
(EtOAc
fraction),
showed
the
major
activity
in
comparison
with
the
more
polar
compounds
(3
and
4)
from
BuOH
fraction.
In
particular,
iridoid
4
is
by
far
the
least
active
compound,
suggesting
that
the
acylating
groups
(i.e.
acetyl
and
p-coumaroyl
groups)
could
be
involved
in
the
pharmacodynamic
properties
of
these
substances.
In
further
experiments
investigating
the
contribution
of
13-
adrenergic
stimulatory
effect
on
the
relaxant
properties
of
the
plant,
the
effects
of
MeOH
extract,
EtOAc
fraction,
BuOH
fraction
and
iridoids
were
re-examined
on
13-adrenergic
receptors
inhib-
ited
by
propranolol.
With
the
same
aim,
the
effect
of
non-relaxing
concentrations
of
the
plant
components
on
isoprenaline
activity
was
evaluated.
We
observed
a
significant
increase
of
EC
50
val-
ues
of
Viburnum
prunifolium
tested
components
in
the
presence
Fig.
5.
The
potentiating
effect
induced
by
Viburnum
prunifolium
MeOH
extract,
BuOH
fraction
and
EtOAc
fraction
(at
non-relaxing
concentration
of
0.5
µg/ml)
on
isoprenaline-induced
inhibition
(10
-8
to
3
x
10
-6
M)
of
jejunum
spontaneous
contractions.
Each
point
represents
mean
±
S.E.M.
of
four
or
more
experiments.
Statistical
comparison
was
performed
by
ANOVA
analysis
followed
by
post
hoc
test.
**P<
0.01,
***P<
0.001
indicate
that
inactive
concentrations
of
MeOH
extract,
BuOH
fraction
and
EtOAc
fraction
shift
significantly
to
leftward
isoprenaline
log
concentration-response
curve.
Fig.
6.
The
relaxant
effect
induced
by
cumulative
concentration
(1-250
µg/ml)
of
Viburnum
prunifolium
MeOH
extract,
BuOH
fraction
and
EtOAc
fraction
on
guinea-
pig
carbachol-precontracted
trachea
(55
x
10
-7
M)
in
comparison
with
isoprenaline
(1.8
x
10
-3
to
0.54
pgiml).
Each
point
represents
mean
±
S.E.M.
of
four
or
more
experiments.
Statistical
comparison
was
performed
by
ANOVA
analysis
followed
by
post
hoc
test.
*P<0.05,
**P<0.01,
***P
<
0.001
indicate
that
MeOH
extract,
BuOH
fraction
and
EtOAc
fraction
effects
were
statistically
different
from
each
other.
120
(7)
0
u
cp
loo-
m
.,..,
-2
or
ai
60
-
o
C
.
0
o
5
or
Ka
N
Q
)
8
9,
‹.)
eL
20
-
60
-
40
-
isoprenaline
N—
Me0H
extract
+
isoprenaline
—A—
BuOH
fraction
+
isoprenaline
--
EtOAc
fraction
+
isoprenaline
-9,00
-8,52
-7,96
-7
,
52
-8,96
-6,52
Isoprenalrne
log
(M)
0
206
M.F.
Cometa
et
al
/
journal
of
Ethnopharmacology
123
(2009)
201-207
120
tions
(Coruzzi
et
al.,
1989),
and
as
bronchodilators
clinically
used
in
the
treatment
of
asthma
(Thirstrup
et
al.,
1997;
Kume
et
al.,
2002;
Belvisi
et
al.,
2004).
Thus,
our
findings
strongly
support
the
wide
use
of
the
plant
with
these
indications
in
ethnomedicine.
However,
the
possibility
that
Viburnum
prunifolium
relaxant
properties
may
have
further
mechanisms
of
action
(e.g.
through
Ca
2
+
channel
inter-
actions)
remains
an
interesting
question
for
future
study
in
view
of
a
wider
use
of
this
medicinal
plant.
Regarding
the
activity
of
the
tested
compounds,
we
may
assume,
also
based
on
their
HPLC
quantification,
that
the
iridoids
isolated
significantly
contribute,
maybe
through
synergistic
action,
to
Vibur-
num
prunifolium
principal
properties.
In
our
opinion,
this
is
the
first
evidence
putting
light
on
the
possible
active
compounds
of
Viburnum
prunifolium,
indicating
a
new
interesting
approach
to
pharmacological
studies
on
the
plant
(Mariott
and
Munch,
1946;
Grote
and
Woods,
1947;
Horhammer
et
al.,
1966,
1967;
Jarboe
et
al.,
1966,
1967;
Ojewole,
1984).
-4-
isoprenaline
0—
1
—0—
2
A—
3
4
80
-
20
-
0
-
-20
-10
-7
-6
log
(M)
5
-4
Fig.
7.
The
relaxant
effect
induced
by
cumulative
concentration
(2
x
10
-5
to
4
x
10
-
a
M)
of
Viburnum
prunifolium
isolated
iridoids
1-4
on
guinea-pig
carbachol-
precontracted
trachea
(5.5
x
10
-7
M)
in
comparison
with
isoprenaline
(10
-9
to
3
x
10
-7
M)
relaxant
effect.
Each
point
represents
mean
±
S.E.M.
of
four
or
more
experiments.
Statistical
comparison
was
performed
by
ANOVA
analysis
followed
by
post
hoc
test.
*P<0.05,
**P<
0.01,
***P<
0.001
indicate
that
the
effects
of
the
single
iridoid
glucosides
(1-4)
were
statistically
different
from
each
other.
of
13-antagonist
receptors
and
a
leftward
shift
in
isoprenaline
log
concentration—response
curve
both
on
the
spontaneous
contrac-
tions
of
rabbit
jejunum
and
on
guinea-pig
carbachol-precontracted
trachea.
All
these
findings
allow
us
to
hypothesise
the
involve-
ment
of
the
13-adrenergic
system
on
the
relaxant
and
spasmolytic
effects
observed.
These
effects
could
be
confirmed
by
a
char-
acteristic
loss
of
tracheal
tone
(for
about
30
min)
to
continued
occupancy
of
13
2
-adrenoceptors
due
to
subsequent
agonist
stim-
ulation
(Katsunuma
et
al.,
2000).
The
13-adrenergic
desensitization
has
also
been
observed
in
vitro
rat
myometrium
(Johansson
and
Andersson,
1981;
Engstrom
et
al.,
2001).
In
conclusion,
based
on
our
results,
we
suggest
that
Viburnum
prunifolium
is
able
to
relax
isolated
rabbit
jejunum
and
guinea-pig
trachea
through
an
inter-
action
with
the
13-adrenergic
system.
13-Adrenomimetics
are
well
known
as
potent
inhibitors
of
jejunum
and
myometrial
contrac-
Fig.
8.
The
potentiating
effect
of
Viburnum
prunifolium
Me0H
extract,
BuOH
fraction
and
EtOAc
fraction
(at
non-relaxing
concentration
of
0511g/int)
on
isoprenaline-induced
inhibition
(10
-9
to
3
x
10
-7
M)
of
guinea-pig
carbachol-
precontracted
trachea
(55
x
10
-7
M).
Each
point
represents
mean
±
S.E.M.
of
four
or
more
experiments.
Statistical
comparison
was
performed
by
ANOVA
analysis
followed
by
post
hoc
test.
*P<
0.05,
**P<
0.01,
***P<
0.001
indicate
that
inactive
con-
centrations
of
Me0H
extract,
BuOH
fraction
and
EtOAc
fraction
shift
significantly
leftward
isoprenaline
log
concentration-response
curve.
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