J. Nutr.
Sci.
Vitaminol.,
36, 291-297,
1990
Note
Influence
of Oryzanol
on Platelet
Aggregation
in Rats
G. S. SEETHARAMAIAH,1 T. P. KRISHNAKANTHA,2 and N. CHANDRASEKHARA1,*
Departments of 1Food Chemistry and 2Nutrition and Food Safety, Central Food Technological Research Institute, Mysore-570 013, India (Received October 9, 1989)
Summary Platelet aggregation was measured using platelet rich plasma prepared from rats fed oryzanol in the control diet and those fed oryzanol in a 1 per cent cholesterol diet (HCD). Oryzanol with the control diet did not alter platelet aggregation induced by ADP and collagen. On the other hand, oryzanol fed along with HCD significantly inhibited platelet aggregation induced by ADP and totally inhibited aggregation induced by collagen. Key Words oryzanol, platelet, platelet aggregation
Platelets play an important role in the atherosclerotic condition. Patients with atherosclerotic disease are reported to exhibit enhanced platelet aggregating activity (1). Intraarterial platelet adhesion and aggregation is the key event in the initiation of arterial thrombosis (2). Further, there is considerable evidence to suggest that hypercholesterolemia, particularly the increase in LDL-cholesterol, is associated with increased platelet aggregation (3). Another observation of relevance is that epidemiological risk factors associated with coronary heart disease are known to activate platelets (4). Oryzanol which can be isolated from rice bran oil and which is a mixture of ferulic acid esters of triterpene alcohols and sterols has been investigated for its hypolipidemic activity by several workers in rats (5-10), in mice (11), and in human subjects (6, 12, 13). The efficacy of oryzanol in counteracting fructose induced hypertriglyceridemia was reported by Seetharamaiah and Chandrasekhara (14). Sharma and Rukmini (15, 16) investigated the hypocholesterolemic activity of rice bran oil and concluded that the unsaponifiable matter is responsible for the effect. The hypolipidemic effect of dietary rice bran oil has also been evaluated in humans (17). In view of the hypocholesterolemic and hypotriglyceridemic effects of oryza nol, the possibility of its being an inhibitor of platelet aggregation was studied by * To whom
correspondence
should
be addressed 291
.
292
G. S. SEETHARAMAIAH
us and the observations
are reported
in this paper.
MATERIALS Male
albino
used.
Four
(10%
protein, bile
control
and
A,
1 IU,
10;
D,
and
grade
purchased
locally.
After ether
(1:9v/v,
was
obtained
3,000
citrate
as
PRP
was
diluted
PPP
were
of
The
settings
was and
the
were
of
(hydrogenated
1; and received
elsewhere
fat-soluble daily
chemicals use.
by
cardiac
20,
10,
was
counts
thoroughly supernatant
centrifuged
at
plasma
(PPP).
3-4•~105/ƒÊl.
studies
glass citrate
the
was
to
under
mixed
and
of were
sodium
platelet-poor
aggregation
were garlic
a siliconized
infranatant as
from
sacrificed
3.8%
temperature The
and
into
was
was
solvents
were
Blood
room
platelet
for
rats
containing
supernatant the
and
puncture
tube
ADP
reagent
Onion
weeks,
(PRP).
(18).
collagen
before
seven
group
are
measured
to
bulbs
recording
of fed
within
1min
were
peeled,
measured
as
expressed means
as was
addition
of
prior
to
PRP 2-3h
the
to
and
after
the
and
in
was
the
of
or
ADP
and
adding
ADP
or
the
of as
system, solution
collagen.
mortar, of
at
calculated
aggregation ethanolic
Aliquots
cuvette
aggregation
a 20mM
a pestle
paper.
monitor
to
aggrometer
a stirred
aggregation
addition
before
described
of
dual
through
oryzanol
diet
crushed 1 filter
1min
of Fresh
homogenized, clear
extracts
collagen.
so
Platelet
above.
Mean•}SEM
evaluated
rate
(ĢT/min).
No. PRP
The
control
Chrono-log
light
adjusted
min
the
PRP
Whatman
Coulter
were
(19). per
rats
a
of
pen
Gerrard
to
in
transmission
influence
added
was
Results
the
from
added
aggregation
between
rat
other
for
at
used
the
by
through
obtained
each
anticoagulant.
adjust
until
transmission
garlic
and filtered
All
12min
the
to
was
described
prepared
oryzanol onion
PPP
of
studying
was
reported
plasma
measures
percent
For
as
Chronopar
as
for
separate
aggregation
37•Ž.
PRP
oil
and
centrifuge
blood)
at 37•Ž
which
in
bran
collected
rpm
to
with
440,
change
that
composition
fat
starch,
control
blood.
Platelet
as
so
diets
platelet-rich
10min
model
platelets
12;
were
(i)
cholester
The
casein,
thee diet
80-100g diets:
control+1%
vitaminized
distilled
a plastic to
1,200
incubated
collection
range
following
(iii)
65;
2;
USA.
were
wass to
at
for
PA,
respective
Blood
rpm
to
rice
solvents
the
sodium
centrifuged
the
oryzanol.
starch,
USA,
Haverton,
transferred
and
weight
on
HCD+0.5%
mixture
from Co.,
and
feeding
and
salt
body
oryzanol;
(iv)
added
isolated
anesthesia.
syringe
the
(10).
Corp.,
analytical
in
maintained
corn
10;
Chemical
Chrono-log
METHODS
control+0.5%
E were
was
Sigma
strain
and
sugar,
AND
were
(g/100g):
respectively
Oryzanol from
Wistar each
(HCD); was
oil),
vitamins
the
6 rats (ii)
salt
diet
vegetable
of
of
casein);
ol+0.15% the
rats
groups
et al.
and by
Student's
statistical t-test
significance
of
differences
(20).
J. Nutr.
Sci.
Vitaminol.
INFLUENCE
OF ORYZANOL
ON
RESULTS Preliminary optimal
experiments
for
adjusted
aggregation
to
ADP
and The
Table
be
as
effect
1
diet by
both
increase
in
the
collagen.
was
The Table
10ƒÊl
of
addition
of
garlic
used use
of
also
a
as
oryzanol
is
platelet in
in
ADP
as
in in
the
aggregation
(HCD) by
caused
both
an
ADP
and
inhibition
aggregation
in the
induced
highly
by
lower
in
in
with
of
collagen
as
aggregation
these
as
experiments (Table
confirmed
(Table
1).
earlier
collagen,
The
fact
observations
thrombin,
aggregation Since and
studies.
In
aggregation
totally
efficacy
increase
in
of
inhibited
of
oryzanol
relevant
collagen
oryzanol
as
the
epine
our
exper was
with
collagen
an
inhibitor
because
of
and
platelets
the
as is
increase
LDL-cholesterol
is one
would
could
not
which
was
whether
hypocholesterolemic
is not
agent.
aggregation
alcohol
all
ADP-induced
physiologically an
at
shown
right.
inducer
and
is
inhibition
in
under
compounds
con
pertinent
conditions.
earlier,
2
aggregation
platelet
with
system marked
HCD+oryzanol
all
the
(14)
Table
fed
collagen.
(3)
Consequently, its
both
indicated
percent
a significant
against was
was
and
associated
us
no
arachidonate,
inducers
disease
because
inhibition.
and
Oryzanol
diet
exerted
aggregation
HCD+oryzanol,
by
shown
and
aggregation
was
rats
system
is
the
solvent
inhibit
ADP,
hypercholesterolemia
of
results
to
the
hypotriglyceridemic
inhibitor
was
was
oryzanol. is
1.
induced
the
oryzanol
in
did
observation
reported
there of
than
with
fed
atherosclerotic As
rate
whereas
to
as
(•¢T/min)
ADP
inhibited
aggregation
ditions
and
like
been
This
aggregation
cholesterol
there
of
was
count
experiments
in Fig.
the
ADP,
oryzanol
effect
extracts
could
evident.
ADP
that
rats
platelet
high
by
used
aggregation
we
in
platelet
by inhibitory
have
inducer
of was
compounds
significantly
the
3-4•~105/ƒÊl
platelet
ability
aggregation
induced
which
established
iments,
all
shown to
The
HCD+oryzanol,
oryzanol)
and
and
phrine
due
of
rate
Several
under
fed
in
on are
of
Hence
anti-aggregating
oryzanol
percent
count
collagen.
possible
difference
the
293
inhibited.
20mM
onion
any
a platelet
or
the
collagen.
aggregation
The the
of
IN RATS
DISCUSSION
profiles
also
group
induced
by
(21)
as
Alcohol
inducer.
that
and
influence
2.
judged 2,
make
totally
aggregation the
not
percent
collagen
as
study
feeding
ADP rate
the
3.5•~105/ƒÊl,
aggregation
did
In
and
ADP
to
AGGREGATION
that
by
used
dietary
typical
induced
to
were
of
and
control
rate
close
AND
established induced
collagen
PLATELET
the activity
only
The add
fully
a hypocholesterolemic
present
to
its
potential
evaluate
used
as
or
not
itself
of
an The
efficacy
of
considerable
oryzanol
inferred
is
value.
caused
ability be
but
it
antiaggregating
solvent
cannot
that
therapeutic
the
anti-aggregating
(10)
observation
from
is wholly the
present
study. Another responsible observations Vol.
36, No.
possibility for that 3, 1990
its
is that
the
antioxidant
anti-aggregating (i)
platelet
aggregability
property
effect.
This was
(22,
23)
conclusion finds
enhanced
in
rats
of
oryzanol support
deficient
in
may
be
in
the
vitamin
294
G. S. SEETHARAMAIAH
Table
Values
1.
Effects
are
of dietary
Mean•}SEM
oryzanol
of
5
rats.
on platelet
*p
Sci.
Vitaminol.,
36, 291-297,
1990
Note
Influence
of Oryzanol
on Platelet
Aggregation
in Rats
G. S. SEETHARAMAIAH,1 T. P. KRISHNAKANTHA,2 and N. CHANDRASEKHARA1,*
Departments of 1Food Chemistry and 2Nutrition and Food Safety, Central Food Technological Research Institute, Mysore-570 013, India (Received October 9, 1989)
Summary Platelet aggregation was measured using platelet rich plasma prepared from rats fed oryzanol in the control diet and those fed oryzanol in a 1 per cent cholesterol diet (HCD). Oryzanol with the control diet did not alter platelet aggregation induced by ADP and collagen. On the other hand, oryzanol fed along with HCD significantly inhibited platelet aggregation induced by ADP and totally inhibited aggregation induced by collagen. Key Words oryzanol, platelet, platelet aggregation
Platelets play an important role in the atherosclerotic condition. Patients with atherosclerotic disease are reported to exhibit enhanced platelet aggregating activity (1). Intraarterial platelet adhesion and aggregation is the key event in the initiation of arterial thrombosis (2). Further, there is considerable evidence to suggest that hypercholesterolemia, particularly the increase in LDL-cholesterol, is associated with increased platelet aggregation (3). Another observation of relevance is that epidemiological risk factors associated with coronary heart disease are known to activate platelets (4). Oryzanol which can be isolated from rice bran oil and which is a mixture of ferulic acid esters of triterpene alcohols and sterols has been investigated for its hypolipidemic activity by several workers in rats (5-10), in mice (11), and in human subjects (6, 12, 13). The efficacy of oryzanol in counteracting fructose induced hypertriglyceridemia was reported by Seetharamaiah and Chandrasekhara (14). Sharma and Rukmini (15, 16) investigated the hypocholesterolemic activity of rice bran oil and concluded that the unsaponifiable matter is responsible for the effect. The hypolipidemic effect of dietary rice bran oil has also been evaluated in humans (17). In view of the hypocholesterolemic and hypotriglyceridemic effects of oryza nol, the possibility of its being an inhibitor of platelet aggregation was studied by * To whom
correspondence
should
be addressed 291
.
292
G. S. SEETHARAMAIAH
us and the observations
are reported
in this paper.
MATERIALS Male
albino
used.
Four
(10%
protein, bile
control
and
A,
1 IU,
10;
D,
and
grade
purchased
locally.
After ether
(1:9v/v,
was
obtained
3,000
citrate
as
PRP
was
diluted
PPP
were
of
The
settings
was and
the
were
of
(hydrogenated
1; and received
elsewhere
fat-soluble daily
chemicals use.
by
cardiac
20,
10,
was
counts
thoroughly supernatant
centrifuged
at
plasma
(PPP).
3-4•~105/ƒÊl.
studies
glass citrate
the
was
to
under
mixed
and
of were
sodium
platelet-poor
aggregation
were garlic
a siliconized
infranatant as
from
sacrificed
3.8%
temperature The
and
into
was
was
solvents
were
Blood
room
platelet
for
rats
containing
supernatant the
and
puncture
tube
ADP
reagent
Onion
weeks,
(PRP).
(18).
collagen
before
seven
group
are
measured
to
bulbs
recording
of fed
within
1min
were
peeled,
measured
as
expressed means
as was
addition
of
prior
to
PRP 2-3h
the
to
and
after
the
and
in
was
the
of
or
ADP
and
adding
ADP
or
the
of as
system, solution
collagen.
mortar, of
at
calculated
aggregation ethanolic
Aliquots
cuvette
aggregation
a 20mM
a pestle
paper.
monitor
to
aggrometer
a stirred
aggregation
addition
before
described
of
dual
through
oryzanol
diet
crushed 1 filter
1min
of Fresh
homogenized, clear
extracts
collagen.
so
Platelet
above.
Mean•}SEM
evaluated
rate
(ĢT/min).
No. PRP
The
control
Chrono-log
light
adjusted
min
the
PRP
Whatman
Coulter
were
(19). per
rats
a
of
pen
Gerrard
to
in
transmission
influence
added
was
Results
the
from
added
aggregation
between
rat
other
for
at
used
the
by
through
obtained
each
anticoagulant.
adjust
until
transmission
garlic
and filtered
All
12min
the
to
was
described
prepared
oryzanol onion
PPP
of
studying
was
reported
plasma
measures
percent
For
as
Chronopar
as
for
separate
aggregation
37•Ž.
PRP
oil
and
centrifuge
blood)
at 37•Ž
which
in
bran
collected
rpm
to
with
440,
change
that
composition
fat
starch,
control
blood.
Platelet
as
so
diets
platelet-rich
10min
model
platelets
12;
were
(i)
cholester
The
casein,
thee diet
80-100g diets:
control+1%
vitaminized
distilled
a plastic to
1,200
incubated
collection
range
following
(iii)
65;
2;
USA.
were
wass to
at
for
PA,
respective
Blood
rpm
to
rice
solvents
the
sodium
centrifuged
the
oryzanol.
starch,
USA,
Haverton,
transferred
and
weight
on
HCD+0.5%
mixture
from Co.,
and
feeding
and
salt
body
oryzanol;
(iv)
added
isolated
anesthesia.
syringe
the
(10).
Corp.,
analytical
in
maintained
corn
10;
Chemical
Chrono-log
METHODS
control+0.5%
E were
was
Sigma
strain
and
sugar,
AND
were
(g/100g):
respectively
Oryzanol from
Wistar each
(HCD); was
oil),
vitamins
the
6 rats (ii)
salt
diet
vegetable
of
of
casein);
ol+0.15% the
rats
groups
et al.
and by
Student's
statistical t-test
significance
of
differences
(20).
J. Nutr.
Sci.
Vitaminol.
INFLUENCE
OF ORYZANOL
ON
RESULTS Preliminary optimal
experiments
for
adjusted
aggregation
to
ADP
and The
Table
be
as
effect
1
diet by
both
increase
in
the
collagen.
was
The Table
10ƒÊl
of
addition
of
garlic
used use
of
also
a
as
oryzanol
is
platelet in
in
ADP
as
in in
the
aggregation
(HCD) by
caused
both
an
ADP
and
inhibition
aggregation
in the
induced
highly
by
lower
in
in
with
of
collagen
as
aggregation
these
as
experiments (Table
confirmed
(Table
1).
earlier
collagen,
The
fact
observations
thrombin,
aggregation Since and
studies.
In
aggregation
totally
efficacy
increase
in
of
inhibited
of
oryzanol
relevant
collagen
oryzanol
as
the
epine
our
exper was
with
collagen
an
inhibitor
because
of
and
platelets
the
as is
increase
LDL-cholesterol
is one
would
could
not
which
was
whether
hypocholesterolemic
is not
agent.
aggregation
alcohol
all
ADP-induced
physiologically an
at
shown
right.
inducer
and
is
inhibition
in
under
compounds
con
pertinent
conditions.
earlier,
2
aggregation
platelet
with
system marked
HCD+oryzanol
all
the
(14)
Table
fed
collagen.
(3)
Consequently, its
both
indicated
percent
a significant
against was
was
and
associated
us
no
arachidonate,
inducers
disease
because
inhibition.
and
Oryzanol
diet
exerted
aggregation
HCD+oryzanol,
by
shown
and
aggregation
was
rats
system
is
the
solvent
inhibit
ADP,
hypercholesterolemia
of
results
to
the
hypotriglyceridemic
inhibitor
was
was
oryzanol. is
1.
induced
the
oryzanol
in
did
observation
reported
there of
than
with
fed
atherosclerotic As
rate
whereas
to
as
(•¢T/min)
ADP
inhibited
aggregation
ditions
and
like
been
This
aggregation
cholesterol
there
of
was
count
experiments
in Fig.
the
ADP,
oryzanol
effect
extracts
could
evident.
ADP
that
rats
platelet
high
by
used
aggregation
we
in
platelet
by inhibitory
have
inducer
of was
compounds
significantly
the
3-4•~105/ƒÊl
platelet
ability
aggregation
induced
which
established
iments,
all
shown to
The
HCD+oryzanol,
oryzanol)
and
and
phrine
due
of
rate
Several
under
fed
in
on are
of
Hence
anti-aggregating
oryzanol
percent
count
collagen.
possible
difference
the
293
inhibited.
20mM
onion
any
a platelet
or
the
collagen.
aggregation
The the
of
IN RATS
DISCUSSION
profiles
also
group
induced
by
(21)
as
Alcohol
inducer.
that
and
influence
2.
judged 2,
make
totally
aggregation the
not
percent
collagen
as
study
feeding
ADP rate
the
3.5•~105/ƒÊl,
aggregation
did
In
and
ADP
to
AGGREGATION
that
by
used
dietary
typical
induced
to
were
of
and
control
rate
close
AND
established induced
collagen
PLATELET
the activity
only
The add
fully
a hypocholesterolemic
present
to
its
potential
evaluate
used
as
or
not
itself
of
an The
efficacy
of
considerable
oryzanol
inferred
is
value.
caused
ability be
but
it
antiaggregating
solvent
cannot
that
therapeutic
the
anti-aggregating
(10)
observation
from
is wholly the
present
study. Another responsible observations Vol.
36, No.
possibility for that 3, 1990
its
is that
the
antioxidant
anti-aggregating (i)
platelet
aggregability
property
effect.
This was
(22,
23)
conclusion finds
enhanced
in
rats
of
oryzanol support
deficient
in
may
be
in
the
vitamin
294
G. S. SEETHARAMAIAH
Table
Values
1.
Effects
are
of dietary
Mean•}SEM
oryzanol
of
5
rats.
on platelet
*p
