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5α-Reductase type 1 inhibition of Oryza sativa bran extract prepared by supercritical carbon dioxide fluid

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The three crude extracts including Oryza sativa (bran) from supercritical carbon dioxide fluid (scCO2) process which gave the highest unsaturated fatty acid contents and biological activities including the antioxidative, tyrosinase inhibition, stimulation index on human normal skin fibroblast were selected from ten edible plants to prepare the semi-purified fractions. Fraction No. 3 of the O. sativa bran crude extract gave the highest content of unsaturated fatty acids and 5α-reductase (type 1) inhibition activity (5AR). Its linoleic acid (LN) and total unsaturated fatty acid (TUC) contents were significantly positive and linear correlated to 5AR on DU-145 cell line (at r of 1.00, p0.9 (p
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J.
of
Supercritical
Fluids
59 (2011) 61–
71
Contents
lists
available
at
ScienceDirect
The
Journal
of
Supercritical
Fluids
jou
rn
al
h
om
epage:
www.elsevier.com/locate/supflu
5-Reductase
type
1
inhibition
of
Oryza
sativa
bran
extract
prepared
by
supercritical
carbon
dioxide
fluid
Warintorn
Ruksiriwanicha,
Jiradej
Manosroia,b,
Masahiko
Abec,
Worapaka
Manosroid,
Aranya
Manosroia,b,
aFaculty
of
Pharmacy,
Chiang
Mai
University,
Chiang
Mai
50200,
Thailand
bNatural
Products
Research
and
Development
Center
(NPRDC),
Science
and
Technology
Research
Institute
(STRI),
Chiang
Mai
University,
Chiang
Mai
50200,
Thailand
cDepartment
of
Pure
and
Applied
Chemistry,
Faculty
of
Science
and
Technology,
Tokyo
University
of
Science,
2641
Chiba,
Japan
dFaculty
of
Medicine,
Chiang
Mai
University,
Chiang
Mai
50200,
Thailand
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
2
March
2011
Received
in
revised
form
21
July
2011
Accepted
22
July
2011
Keywords:
Antioxidation
5-Reductase
inhibition
O.
sativa
crude
extract
Supercritical
carbon
dioxide
(scCO2)
Unsaturated
fatty
acids
a
b
s
t
r
a
c
t
The
three
crude
extracts
including
Oryza
sativa
(bran)
from
supercritical
carbon
dioxide
fluid
(scCO2)
process
which
gave
the
highest
unsaturated
fatty
acid
contents
and
biological
activities
including
the
antioxidative,
tyrosinase
inhibition,
stimulation
index
on
human
normal
skin
fibroblast
were
selected
from
ten
edible
plants
to
prepare
the
semi-purified
fractions.
Fraction
No.
3
of
the
O.
sativa
bran
crude
extract
gave
the
highest
content
of
unsaturated
fatty
acids
and
5-reductase
(type
1)
inhibition
activity
(5AR).
Its
linoleic
acid
(LN)
and
total
unsaturated
fatty
acid
(TUC)
contents
were
significantly
positive
and
linear
correlated
to
5AR
on
DU-145
cell
line
(at
r
of
1.00,
p
<
0.01).
Its
total
phenolic
contents
and
all
biological
activities
also
showed
positive
correlations
to
5AR
with
r
>
0.9
(p
<
0.05).
This
study
has
demonstrated
the
potential
of
fraction
No.
3
fractionated
from
the
O.
sativa
bran
crude
extract
prepared
by
scCO2to
be
developed
as
anti-androgenic
alopecia
products.
© 2011 Elsevier B.V. All rights reserved.
1.
Introduction
In
the
US,
an
estimated
of
40
million
men
and
20
million
women
suffer
from
baldness
and
have
spent
$1.5
billion
annually
on
hair
loss
therapies
[1].
Dihydrotestosterone
(DHT),
a
potent
male
hor-
mone,
is
the
cause
of
genetic
male
pattern
baldness
since
it
has
been
shown
that
the
DHT
levels,
numbers
of
the
DHT
receptors
on
the
hair
follicles
and
the
5-reductase
enzyme
activity
(which
converts
testosterone
to
DHT)
increase
in
the
balding
scalp
of
androgenic
alopecia
patients
[2–4].
DHT
has
three
times
greater
affinity
for
androgen
receptors
than
testosterone,
which
is
the
main
cause
of
the
androgenic
alopecia
leading
to
the
miniaturization
of
hair
follicle
and
hair
shedding
[5].
The
5-reductase
inhibitor
type
2,
finasteride
has
been
approved
by
the
US
FDA
to
use
in
male
pat-
tern
baldness,
whereas
dutasteride
(the
type
1
and
2
5-reductase
inhibitor)
has
been
approved
for
the
treatment
of
symptomatic
benign
prostatic
hyperplasia
(BPH),
but
still
hold
on
phase
III
for
the
treatment
of
male
pattern
hair
loss
[6].
The
type
1,
5-reductase
(SRD5A1)
related
to
genetic
male
pattern
baldness,
is
expressed
predominantly
in
the
skin,
scalp,
sebaceous
gland,
liver
and
brain,
whereas
the
type
2,
5-reductase
(SRD5A2)
is
found
predominantly
Corresponding
author
at:
Faculty
of
Pharmacy,
Chiang
Mai
University,
Chiang
Mai
50200,
Thailand.
Tel.:
+66
53
894806;
fax:
+66
53
894169.
E-mail
address:
pmpti005@chiangmai.ac.th
(A.
Manosroi).
in
androgen
target
organs
such
as
prostate,
genital
skin,
and
sem-
inal
vesicles
[7].
Finasteride
gives
several
side
effects,
such
as
the
decrease
of
libido,
erectile
dysfunction,
ejaculation
disorder
and
gynecomastia,
while
dutasteride
(5
weeks)
has
longer
half
life
than
finasteride
(5–6
h)
and
is
more
difficult
to
reverse
the
side
effects.
Although
finasteride
can
block
the
type
2,
5-reductase
in
andro-
gen
target
organs
more
than
that
in
the
hair
follicle,
it
has
been
approved
to
use
in
male
pattern
baldness
since
it
has
lower
side
effects
than
dutasteride.
Both
drugs
cannot
be
used
in
childbearing
age
females
with
the
pregnancy
category
X
which
show
the
risks
of
fetal
injury
or
birth
defects.
Nowadays,
natural
extracts
from
several
plants
have
been
used
for
hair
growth
promotion
such
as
Asiasari
radix
[8],
Eclipta
alba
[9],
essential
oil
of
Chamaecyparis
obtuse
[10],
Zizyphus
jujube
[11]
and
Sophora
flavescens
[12].
Most
extracts
have
targeted
on
the
induction
of
growth
factors
in
hair
follicle
cells,
such
as
insulin-
like
growth
factor-1
(IGF-1)
and
vascular
endothelial
growth
factor
(VEGF),
keratinocyte
growth
factor
(KGF)
and
epidermal
growth
factor
(EGF),
but
not
on
the
production
of
5-reductase
enzyme.
The
unsaturated
fatty
acids,
such
as
-linolenic
acid,
linoleic
acid
and
oleic
acid,
have
been
proved
to
have
anti-hair
loss
activity
by
inhibiting
5-reductase
enzyme
in
the
androgen
responsive
organs
[13].
In
fact,
several
edible
plants
contain
these
unsatu-
rated
fatty
acids
in
variable
amounts,
such
as
Carthamus
tinctorius
L.
(safflower),
Helianthus
annuus
L.
(sunflower),
Linum
usitatissimum
L.
(flaxseed),
Sorghum
bicolor
(L.)
Moench
(sorghum)
and
bran
of
0896-8446/$
see
front
matter ©
2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2011.07.017
62 W.
Ruksiriwanich
et
al.
/
J.
of
Supercritical
Fluids
59 (2011) 61–
71
Oryza
sativa
L.
(rice).
As
known,
rice
bran
is
discarded
or
used
as
livestock
feed
and
oil
production.
Rice
bran
oil
is
edible
and
has
been
claimed
for
improving
serum
cholesterol
levels
and
lipoprotein
profiles
similar
to
other
vegetable
oils,
such
as
corn
and
safflower
oil
[14,15].
Rice
bran
oil
has
been
extracted
from
rice
bran
using
scCO2by
both
pilot
[16]
and
lab
scale
[17].
The
health
promotion
properties
of
rice
bran
are
from
its
unsaturated
fatty
acid
contents
such
as
palmitic
acid,
oleic
acid,
linoleic
acid
and
-linolenic
acid,
which
are
known
as
antioxidants
and
anti-cancer
agents
by
stim-
ulating
the
production
of
the
substances
which
can
protect
cells
from
peroxides
[18]
and
anti-hair
loss
agents
[13].
The
raw
rice
bran
oil
contains
both
unsaturated
and
saturated
fatty
acids,
in
which
palmitic
acid
is
a
major
acid
(C18:0,
12–26%,
w/w,
typically
18%,
w/w).
The
unsaturated
fatty
acids
are
mainly
oleic
acid
(C18:1,
35–46%,
w/w,
typically
42%,
w/w)
and
linoleic
acid
(C18:2,
25–38%,
w/w,
typically
37%,
w/w)
with
the
traces
of
C18:3
acid
(0.4–3.8%,
w/w)
[19,20].
It
has
been
reported
that
the
quality
of
rice
bran
oil
extracted
by
SFE
was
far
superior
to
that
produced
by
hexane
extraction,
especially
at
80 C
[21].
The
phenolic
compounds
and
the
unsaturated
fatty
acids
containing
in
C.
tinctorius
have
been
shown
to
have
antioxidant
activity
[22]
which
can
recover
or
slow
down
the
miniaturization
of
hair
follicle.
Their
redox
properties
of
many
bioactive
compounds
can
lead
to
hair
shedding
and
act
as
reducing
agents,
hydrogen
donators
as
well
as
the
singlet
oxy-
gen
quenchers
in
the
hair
follicle
[23,24].
Also,
there
were
several
anti-hair
loss
medicinal
plant
recipes
which
have
been
traditionally
used
by
the
Chinese
and
Thai
people
for
over
100
years.
But,
most
of
recipes
and
plants
have
no
scientific
evidences
on
anti-hair
loss
activity.
The
relationship
between
this
activity
and
the
antioxidant
activity
as
well
as
the
bioactive
compounds
in
plants
still
never
been
explored.
Recently,
supercritical
fluids
have
been
introduced
as
an
alternative
one
step
at
low
temperature
for
the
preparation
of
plant
extracts.
At
the
critical
point,
supercritical
fluids
have
the
density
as
liquid,
but
low
viscosity
with
better
flow
property
as
gas.
Carbon
dioxide
is
a
widely
used
gas
to
produce
supercriti-
cal
fluid
because
of
its
low
critical
temperature
(Tc
=
31.1 C)
and
pressure
(Pc
=
73.8
bar).
It
has
high
solvating
power
at
near
critical
point.
Supercritical
carbon
dioxide
(scCO2)
has
been
used
for
the
substitution
of
organic
solvent
to
extract
many
plant
extracts
con-
taining
thermal
sensitive
constituents
with
the
advantages
of
not
only
being
environmental
friendly,
non-toxic
and
nonflammable,
but
also
inexpensive
[25,26].
It
has
been
used
as
an
alternative
to
organic
solvent
for
the
extraction
of
many
nuts
and
seeds,
such
as
bran
of
O.
sativa
(rice)
[27],
Arachis
hypogaea
L.
(peanuts)
[28]
and
Glycine
max
(L.)
Merr.
(soybean)
[29].
Although,
the
maceration
method
which
is
a
simply
and
low
cost
method,
it
requires
large
amount
and
high
purity
of
the
organic
solvents
which
are
usually
hazardous
and
flammable
solvent
wastes
and
are
generally
cum-
bersome
[26].
Moreover,
it
is
the
non-selective,
time
consuming
and
toxic
fume
emission
extraction
in
comparing
to
the
supercrit-
ical
carbon
dioxide
extraction
procedure.
This
present
study
has
compared
the
5-reductase
type
1
inhi-
bition
in
DU-145
cell
line
of
the
scCO2crude
extract
of
O.
sativa
bran,
C.
tinctorius
flowers
and
S.
bicolor
seeds
and
their
semi-purified
fractions.
The
relationship
between
the
5-reductase
type
1
inhibi-
tion
activity
and
other
biological
activities
as
well
as
the
bioactive
contents
in
the
crude
and
semi-purified
extracts
were
evaluated.
2.
Materials
and
methods
2.1.
Materials
Gallic
acid
(99.0%),
vitamin
C
(l-(+)-ascorbic
acid,
99.5%),
2,2-
diphenyl-1-picryhydrazyl
radical
(DPPH),
EDTA,
sulforhodamine
B
(SRB),
dimethyl
sulfoxide
(DMSO),
kojic
acid
(99.0%),
ferrozine,
finasteride
(99.5%),
Folin–Ciocalteu
reagent
and
ferric
chloride
(FeCl2)
were
purchased
from
Sigma
Chemical
Co.
(St.
Louis,
MO,
USA).
Mushroom
tyrosinase
(4187
U/mg)
and
l-tyrosine
were
pur-
chased
from
Fluka
(Buchs,
Switzerland).
Linoleic
acid
(99.0%),
oleic
acid
(98.5%)
and
1,6
diphenyl-1,3,5-hexatriene
were
from
Wako
Pure
Chemical
Industrial
Ltd.
(Osaka,
Japan).
-Linolenic
acid
(99.5%)
was
purchased
from
Tokyo
Chemical
Industrial
Ltd.
(Tokyo,
Japan).
The
standard
dutasteride
(99.5%)
was
purchased
from
Ka-
Shing
Business
Macau
Co.,
Ltd.
(Macau,
China).
Siliga
gel
60
was
purchased
from
Merck
(Damstadt,
Germany).
Dulbecco’s
modified
Eagle’s
culture
medium,
antibiotics
penicillin
and
streptomycin,
fetal
bovine
serum
and
trypsin
were
purchased
from
HyClone
(Logan,
UT,
USA).
All
other
reagents
and
solvents
were
of
analytical
grade.
2.2.
Plant
crude
extract
2.2.1.
Plant
sample
Parts
of
the
ten
edible
plants
which
have
been
searched
from
the
literature
reviews
to
contain
high
amount
of
unsaturated
fatty
acids
(-linolenic
acid,
linoleic
acid
and
oleic
acid)
were
collected
from
Chiang
Mai
Province
in
Thailand
during
October
to
November
in
2008
(Table
1).
The
plant
seeds
used
in
this
study
were
packed
in
vacuum
plastic
bags
and
purchased
from
Thai
Cereals
World
Co.,
Ltd.,
Bangkok,
Thailand.
The
%
moisture
contents
in
the
seeds
were
in
the
range
of
6–10%.
The
specimen
samples
were
authenticated
by
a
botanist
at
the
Natural
Products
Research
Development
Cen-
ter
(NPRDC),
Science
and
Technology
Research
Institute
(STRI)
at
Chiang
Mai
University,
Chiang
Mai
in
Thailand.
2.2.2.
Plant
preparation
All
plant
parts
were
seeds,
except
O.
sativa
and
C.
tinctorius
were
bran
and
flower,
respectively.
The
bran
of
O.
sativa
was
passed
through
sieve
No.
25
(0.707
mm).
Other
plant
parts
were
ground
into
small
pieces
by
a
blender
(Twist
HR
1701,
Philips,
Indonesia)
and
passed
through
sieve
No.
20
(0.841
mm).
The
plant
powder
was
kept
in
a
tight
container
at
4C
until
use.
2.2.3.
Maceration
method
Briefly,
200
g
of
the
plant
powder
were
macerated
with
1
l
of
95%
(v/v)
ethanol
at
room
temperature
(27
±
2C)
for
8
h
and
stirred
every
2
h.
The
extract
was
filtered
through
the
paper
filter
What-
man
No.
1,
connected
with
a
vacuum
pump.
The
residues
were
re-extracted
more
by
the
same
process
twice.
All
filtrates
were
col-
lected,
pooled
and
dried
by
a
rotary
evaporator
(Rotavapor
R210,
Buchi,
Switzerland)
at
40 C.
The
crude
extracts
were
kept
at
80 C
until
use.
2.2.4.
Supercritical
carbon
dioxide
fluid
extraction
Briefly,
200
g
of
the
plant
powder
were
put
in
the
supercritical
carbon
dioxide
fluid
apparatus
(scCO2)
(SFE-500MR-2-C50
System,
Thar
Instruments,
Inc.,
Pittsburgh,
USA)
together
with
25%
(w/v)
of
95%
(v/v)
ethanol
as
a
co-solvent,
in
the
chamber
at
40 C
and
200
bar
[30].
After
2
h,
the
pressure
was
released
and
the
extract
was
collected.
The
plant
extract
residues
were
re-extracted
more
by
the
same
procedure
3
times.
All
extracts
were
collected,
pooled,
mixed
and
dried
by
a
rotary
evaporator
at
40 C.
The
crude
extracts
were
kept
at
80 C
until
use.
2.2.5.
Determination
of
bioactive
compounds
and
biological
activities
of
the
crude
extract
The
resulting
extracts
were
determined
for
unsaturated
fatty
acid
and
total
phenolic
contents.
The
phytochemical
tests
were
also
investigated.
Briefly,
20
mg
of
the
crude
extracts
were
dis-
solved
in
80%
(v/v)
methanol
and
used
for
detecting
the
presence
of
alkaloids,
anthraquinones,
flavonoids,
glycosides,
carotenoids,
W.
Ruksiriwanich
et
al.
/
J.
of
Supercritical
Fluids
59 (2011) 61–
71 63
Table
1
Comparison
of
the
percentage
yields,
unsaturated
fatty
acid
contents
and
the
total
phenolic
contents
of
the
ten
edible
plant
crude
extracts
prepared
by
the
two
non-heated
processes
(scCO2and
ethanolic
maceration).
Plants Unsaturated
fatty
acid
contents
(%,
w/w) Total
phenolic
contents
(gallic
acid
equivalent;
GAE
mg/g
of
the
extract)
Scientific
name Common
name
Percentage
yields
(%w/w)
-linolenic
acid
Linoleic
acid
Oleic
acid
scCO2Maceration
scCO2Maceration
scCO2Maceration
scCO2Maceration
scCO2Maceration
Arachis
hypogea
L.
Peanut
16.94
12.88
0.17
1.08
0.24
0.72
0.33
0.39
Carthamus
tinctorius
L.
Safflower
20.23
33.88
9.36
4.45
4.02
2.13
0.65
0.03
1.36
1.44
Glycine
max
(L.)
Merr.
Soybean
5.42
26.28
0.14
0.05
1.08
0.56
0.12
0.08
0.53
0.46
Helianthus
annuus
L. Sunflower 14.01
29.93
0.02
0.01
1.97
0.64
0.42
0.82
2.41
1.76
Linum
usitatissimum
L. Flax 7.17
12.14
0.28
0.31
0.63
0.77
0.19
0.11
0.43
0.70
Nelumbo
nucifera
Gaertn.
Lotus
3.34
2.47
0.08
0.11
1.07
2.45
0.19
0.62
0.42
0.58
Oryza
sativa
L. Rice
11.21
17.90
5.67
4.41
23.34
20.03
27.28
19.48
0.65
0.51
Sesamum
indicum
L.
Sesame
12.01
15.88
0.05
0.09
3.96
3.19
2.75
1.89
0.71
0.86
Sorghum
bicolor
(L.)
Moench
Sorghum
2.54
5.12
0.02
0.15
5.56
2.66
1.78
0.51
1.05
1.13
Zea
may
L.
Corn
4.55
14.97
0.04
0.05
0.57
1.09
0.2
0.52
0.76
0.96
Note:
Values
represented
mean
(n
=
3).
“–”
represented
not
found
in
the
sample.
Total
phenolic
contents
were
presented
as
gallic
acid
equivalent
(GAE)
mg/g
of
the
crude
extracts.
tannins
and
xanthones
according
to
the
standard
methods
pre-
viously
described
[31–34].
The
biological
activities
[30]
including
DPPH
radical
scavenging,
lipid
peroxidation
inhibition,
metal
ion
chelating,
tyrosinase
inhibition
activities
and
cell
proliferation
on
aged
human
skin
fibroblasts
were
determined.
2.3.
Biological
and
anti-hair
loss
activities
of
the
semi-purified
fractions
from
the
selected
crude
extracts
2.3.1.
Preparation
of
the
semi-purified
fraction
containing
unsaturated
fatty
acids
from
O.
sativa,
C.
tinctorius
and
S.
bicolor
crude
extracts
The
semi-purified
fractions
of
the
three
selected
crude
extracts,
including
O.
sativa,
C.
tinctorius
and
S.
bicolor
from
the
scCO2pro-
cess
which
gave
the
highest
unsaturated
fatty
acid
contents
and
antioxidative
activities
were
prepared
as
previously
described
with
modification
[35].
Briefly,
25
g
of
the
crude
extracts
were
loaded
on
the
750
g
of
silica
gel
60
(Merck,
Germany)
column
(4
cm
×
100
cm)
and
eluted
with
petroleum
ether/ethyl
acetate
(8:1)
at
the
flow
rate
of
1
ml/min.
Each
fraction
of
100
ml
was
collected
and
the
solvent
was
evaporated
by
a
rotary
evaporator.
The
total
of
30
fractions
were
obtained.
Each
dried
fraction
was
analyzed
for
unsat-
urated
fatty
acid
contents
by
HPLC
with
the
previously
described
method
[30].
The
consecutive
fractions
which
contained
the
same
unsaturated
fatty
acids
were
pooled
and
evaporated.
There
were
four
dried
fractions
from
each
crude
plant
extracts.
The
percentages
yields,
the
unsaturated
fatty
acids
and
the
total
phenolic
contents
of
the
dried
fractions
were
determined.
2.3.2.
Biological
activities
of
the
semi-purified
fractions
The
semi-purified
fractions
were
tested
for
the
DPPH
radi-
cal
scavenging,
lipid
peroxidation
inhibition,
metal
ion
chelating,
tyrosinase
inhibition
and
cell
proliferation
activity
on
the
aged
nor-
mal
human
skin
fibroblasts
with
the
previously
described
methods
[30].
2.3.3.
Cytotoxicity
of
the
semi-purified
fractions
on
DU-145
cell
line
2.3.3.1.
Cell
culture.
The
human
prostate
carcinoma
cell
line
(DU-
145)
was
provided
by
Prof.
Dr.
Toshihiro
Akihisa
at
the
College
of
Science
and
Technology,
Nihon
University
in
Tokyo,
Japan.
Cells
were
cultured
under
the
standard
conditions
in
the
com-
plete
culture
medium
containing
RPMI
medium
supplemented
with
10%
(v/v)
fetal
bovine
serum
(FBS),
penicillin
(100
U/ml)
and
streptomycin
(100
mg/ml).
Cells
were
incubated
in
a
temperature-
controlled
and
humidified
incubator
(Shel
Lab,
model
2123TC,
USA)
with
5%
CO2at
37 C.
2.3.3.2.
Cytotoxicity
by
the
SRB
assay.
The
semi-purified
fractions
were
tested
for
cytotoxicity
on
DU-145
cells
by
the
SRB
assay
as
previous
described
[36].
The
standard
unsaturated
fatty
acids
(-
linolenic
acid,
linoleic
acid
and
oleic
acid),
the
standard
finasteride
and
dutasteride
at
0.0001–1
mg/ml
were
used
as
positive
controls.
The
cells
were
plated
at
the
density
of
1.0
×
104cells/well
in
96-well
plates
and
left
overnight
for
cell
attachment
on
the
plate
in
5%
CO2
at
37 C.
Cells
were
then
exposed
to
five
serial
concentrations
of
the
crude
extracts
and
their
semi-purified
fractions
(0.00001–1
mg/ml)
for
24
h.
After
incubation,
the
adherent
cells
were
fixed
in
situ,
washed
and
dyed
with
SRB.
The
bound
dye
was
solubilized
and
the
absorbance
was
measured
at
540
nm
by
a
microplate
reader.
The
experiments
were
done
in
triplicate.
The
percentages
of
cell
proliferation
were
calculated
according
to
the
following
equation:
Cell
viability
(%)
=
(Absorbancesample/Absorbancecontrol )
×
100.
The
concentrations
of
the
samples
which
gave
%
cell
viability
of
more
than
90%
were
used
in
the
5-reductase
inhibition
experiment.
2.3.4.
Inhibition
of
5˛-reductase
activity
2.3.4.1.
Cultivation
of
cells.
The
pellets
of
human
DU-145
cells
were
plated
onto
the
6-well
plates
separately
at
the
density
of
2.5
×
105
cells/well,
incubated
with
10%
(v/v)
FBS-DMEM
medium
containing
penicillin
(100
U/ml)
and
streptomycin
(100
mg/ml)
in
a
5%
CO2
incubator
(Shel
Lab,
model
2123TC,
USA)
at
37 C.
Cells
were
then
exposed
to
the
semi-purified
fractions,
the
standard
finasteride
and
dutasteride
at
the
final
concentration
of
0.1
mg/ml
and
the
standard
unsaturated
fatty
acids
at
0.1–0.001
mg/ml
for
24
h.
The
medium
were
removed,
and
the
cells
were
washed
with
PBS,
trypsinized
with
0.25%
trypsin
solution
for
2
min
and
suspended
in
PBS.
2.3.4.2.
Total
RNA
extraction.
The
total
RNA
from
the
cell
pellets
was
extracted
by
the
RNA
extraction
kit
(NucleoSpin®,
Macherey-
Nagel,
CA,
USA)
according
to
the
instructions
of
the
manufacturer.
The
concentration
of
the
total
RNA
was
quantified
by
Qubit
Flu-
orometer
and
Quant-iTTM RNA
BR
assay
kit
(Invitrogen,
CA,
USA).
The
total
RNA
solution
was
kept
at
20 C
until
used.
2.3.4.3.
Reverse
transcription-polymerase
chain
reaction
(RT-PCR).
The
5-reductase
type
1
and
2
genes
were
amplified
from
the
extracted
RNA
by
SuperScripTM One-Step
RT-PCR
with
Platinum®
64 W.
Ruksiriwanich
et
al.
/
J.
of
Supercritical
Fluids
59 (2011) 61–
71
Taq
kit
(Invitrogen,
CA,
USA)
according
to
the
manufacturer’s
pro-
tocol.
Briefly,
5
g
of
the
total
RNAs
were
reverse
transcribed
with
RT/Platinum
Taq®mix
and
subjected
to
PCR
cycles
with
the
primers
for
human
5-reductase
type
1
and
2
(SRD5A1
and
2)
as
follows:
94 C
for
15
s,
55 C
for
30
s,
72 C
for
45
s
for
35
cycles.
The
human
5-reductase
type
1
primers
were
designed
based
on
GenBank
accession
no.
NM
001047.2
and
NM
000348,
respectively,
with
a
forward
(5-CCA
TGT
TCC
TCG
TCC
ACT
AC-3)
and
reverse
(5-
TTC
AAC
CTC
CAT
TTC
AGC
GT-3),
produced
707
bp
amplicon
and
human
5-reductase
type
2
(SRD5A2)
forward
(5-GGG
TGG
TAC
ACA
GAC
ATA
CG-3)
and
reverse
(5-TCA
CGA
CTA
TGA
GGA
GAG
GG-3),
produced
938
bp
amplicon
[37].
The
RT-PCR
products
were
loaded
on
1%
agarose
gel
in
the
1×
tris-acetate-EDTA
(TAE)
buffer
chamber
at
100
V
for
30
min.
The
human
5-reductase
type
1
and
2
dsDNA
samples
were
quantified
by
the
Qubit
fluorometer
and
Quant-iTTM dsDNA
assay
kit
(Invitrogen,
CA,
USA).
2.4.
Statistical
analysis
The
results
were
presented
as
the
mean
of
three
independent
experiments
and
analyzed
by
SPSS
(version
16.0).
ANOVA
was
used
for
the
analysis
of
the
test
results
(LSD
test)
at
the
significance
level
of
p-value
<
0.05.
Correlation
coefficient
(r)
was
used
to
determine
the
relationship
between
the
variables
which
were
calculated
using
the
bivariate
correlation
statistical
function.
3.
Results
and
discussion
3.1.
Unsaturated
fatty
acid
and
total
phenolic
contents
in
the
crude
extracts
The
crude
extract
from
C.
tinctorius
by
both
ethanolic
maceration
and
scCO2showed
the
highest
percentage
yields
at
33.88
±
3.67
and
20.23
±
1.65%
(w/w),
respectively
(Table
1).
Although,
the
ethano-
lic
maceration
of
most
plants
gave
higher
percentage
yields
than
the
scCO2technique,
it
gave
lower
contents
of
the
main
target
compounds
(unsaturated
fatty
acids)
than
that
from
the
scCO2tech-
nique.
The
maceration
technique
gave
more
impurities
than
that
from
the
scCO2technique.
In
fact,
the
polar
organic
solvents
such
as
methanol
and
ethanol
can
also
extract
many
polar
compounds
such
as
phenolic
compounds,
flavonoids
and
anthraquinone
[38],
thereby
giving
the
extract
with
higher
impurities
and
percentage
yields.
The
high
lipophilic
property
and
solvating
power
of
the
scCO2fluid
can
dissolve
the
unsaturated
fatty
acids
especially
at
the
pressure
of
80–250
bar
and
the
temperature
range
of
40–80 C
[39].
Thus,
the
scCO2fluid
appears
to
be
the
best
solvent
choice
to
obtain
the
extract
with
high
content
of
unsaturated
fatty
acids.
For
other
methods
such
as
Soxhlet
extraction
and
maceration,
they
require
large
amount
and
high
purity
of
organic
solvents
(chloroform,
hex-
ane
or
methanol)
that
are
hazardous
and
can
be
the
flammable
solvent
wastes
and
are
generally
cumbersome
[26].
Moreover,
the
Soxhlet
extraction
was
the
non-selective,
time
consuming
and
toxic
fume
emission
technique
which
is
harmful
to
the
environments
[40].
The
crude
extract
of
O.
sativa
by
both
processes
exhibited
the
highest
contents
of
all
unsaturated
fatty
acids,
except
-linolenic
acid
in
comparing
to
the
crude
extracts
of
C.
tinctorius.
The
O.
sativa
crude
extract
from
the
scCO2technique
contained
the
-linolenic
acid,
linoleic
acid
and
oleic
acid
contents
at
5.67
±
0.52,
23.62
±
2.62
and
27.28
±
1.22%
(w/w),
which
were
more
than
those
from
the
maceration
method
(4.41
±
0.73,
20.03
±
1.89
and
19.48
±
1.78%,
w/w)
of
1.29,
1.18
and
1.40
times,
respectively.
Moreover,
by
scCO2
the
O.
sativa
crude
extract
exhibited
the
highest
total
unsatu-
rated
fatty
acids
(TUC)
at
56.57
±
3.73%
(w/w)
which
was
higher
than
those
in
the
C.
tinctorius
(14.03
±
2.17%,
w/w)
and
S.
bicolor
(7.36
±
1.18%,
w/w)
crude
extract
of
4.5
and
8.5
times,
respectively.
The
crude
extracts
which
gave
the
highest
total
phenolic
con-
tents
(TPC)
in
the
form
of
gallic
acid
were
observed
in
H.
annuus
from
both
by
the
scCO2and
the
maceration
methods
at
2.41
±
0.13
and
1.76
±
0.07
mg
gallic
acid
equivalent/gram
of
the
crude
extract
(GAE
mg/g),
respectively.
In
fact,
various
phenolic
compounds
were
found
in
H.
annuus,
such
as
5-O-caffeoylquinic
acid,
quercetin
and
kaempferol,
respectively
[41,42].
3.2.
Phytochemicals
in
the
crude
extracts
In
this
study,
95%
(w/v)
ethanol
was
used
as
a
co-solvent
in
the
scCO2extraction
at
25%
(w/v)
of
the
liquid
CO2and
as
a
sol-
vent
in
the
maceration
process.
Ethanol
is
a
safe
solvent
and
has
high
polarity
than
the
scCO2which
can
extract
polar
bioactive
compounds
from
the
rice
bran
such
as
-oryzanol,
glycosides
and
carotenoids.
The
phytochemical
compounds
were
shown
quali-
tatively
and
approximated
quantitatively
in
Table
2.
Carotenoids
which
are
antioxidants
were
found
in
all
crude
extracts
(data
not
shown).
The
C.
tinctorius,
S.
bicolor
and
G.
max
crude
extracts
by
both
processes
contained
most
phytochemicals
including
alka-
loids,
carotenoids,
glycosides,
xanthones
and
tannins.
Hence,
the
phytochemical
constituents
were
not
affected
by
the
extraction
processes,
but
depended
on
the
types
of
plants.
Xanthone
and
tannins
were
found
in
the
crude
extracts
of
C.
tinctorius,
G.
max,
Nelumbo
nucifera
Gaertn.,
O.
sativa,
S.
bicolor
and
Zea
may
L.
As
known,
natural
antioxidants
are
a
broad
range
of
phyto-
chemicals
including
phenolic,
nitrogen
and
carotenoids
[43].
The
antioxidative
activity
of
the
crude
extracts
which
contained
these
compounds
can
be
anticipated.
3.3.
Biological
activities
of
the
crude
extracts
The
biological
activities
including
DPPH
radical
scavenging,
lipid
peroxidation
inhibition,
metal
ion
chelation,
tyrosinase
inhibition
activities
and
stimulation
index
of
the
crude
extracts
of
the
ten
selected
edible
plants
including
bran
of
O.
sativa
prepared
by
the
two
non-heated
processes
were
compared
(Table
3).
Most
of
plant
crude
extracts
by
the
scCO2showed
slightly
lower
activities
than
those
by
the
ethanolic
maceration
technique.
This
might
be
from
the
higher
content
of
hydrophilic
substance,
phenolic
compound
as
shown
in
the
amount
of
total
phenolic
content
(TPC).
For
DPPH
radical
scavenging
activity,
the
C.
tinctorius
crude
extracts
(SC50 value
of
1.13
±
0.02
mg/ml)
gave
the
highest
DPPH
scavenging
activity,
which
higher
than
those
from
the
S.
bicolor
and
O.
sativa
crude
extracts
by
scCO2of
about
1.9
and
5.8
times,
respectively,
but
showed
lower
activity
than
the
standard
vita-
min
C
(SC50 value
of
0.044
±
0.004
mg/ml),
vitamin
E
(SC50 value
of
0.039
±
0.002
mg/ml)
of
about
25
and
29
times,
respectively.
The
highest
DPPH
scavenging
activity
of
the
C.
tinctorius
crude
extract
may
be
from
the
high
content
of
phenolic
compounds.
The
sig-
nificant
correlation
between
DPPH
scavenging
activity
(AF)
and
the
total
phenolic
contents
(TPC)
in
this
crude
extract
(r
=
0.99,
p
<
0.05)
was
observed.
The
chemical
constituents
in
this
plant
have
been
reported
to
be
flavonoids
[44],
lignans
[45],
triterpene
alcohols
[46],
and
polysaccharides
[47].
For
lipid
peroxidation
inhibition
activity,
the
three
crude
extracts
by
scCO2which
gave
the
highest
activity
were
H.
annuus,
A.
hypogaea
and
O.
sativa,
respectively.
The
O.
sativa
crude
extract
prepared
by
scCO2(IPC50 value
of
1.25
±
0.51
mg/ml)
showed
high
lipid
peroxidation
inhibition
activity
but
lower
than
vitamin
C
(IPC50 value
of
0.07
±
0.04
mg/ml)
and
vitamin
E
(IPC50 value
of
0.03
±
0.01
mg/ml)
of
17.9
and
41.7
times,
respectively.
By
scCO2,
this
crude
extract
gave
higher
lipid
peroxidation
inhibition
activity
than
C.
tinctorius
crude
extracts
(IPC50 value
of
2.27
±
0.76
mg/ml)
and
S.
bicolor
(IPC50 value
of
4.05
±
0.69
mg/ml)
of
about
1.8
and
3.2
times,
respectively.
W.
Ruksiriwanich
et
al.
/
J.
of
Supercritical
Fluids
59 (2011) 61–
71 65
Table
2
Comparison
of
phytochemical
compounds
of
the
ten
edible
plant
extracts
prepared
by
the
two
non-heated
methods
(scCO2and
ethanolic
maceration).
Scientific
name Common
name Part
used Alkaloid
Antraquinone
Carotenoid
Flavonoid
Glycoside
Xanthone
Tannin
Sucrose
Glucose
Fructose
SMSMSMSMS
M
S
M
S
M
S
M
S
M
Arachis
hypogaea
L. Peanut Seed −− −− ++ −− −− −− −− −−
Carthamus
tinctorius
L. Safflower Flower
+
+
+
+
+
+
+
+
+
+
+
+
Glycine
max
(L.)
Merr. Soybean Seed + + −− ++ −− ++
+
+
+
+
+
+
Helianthus
annuus
L.
Sunflower
Seed
+
+
+
Linum
usitatissimum
L. Flax Seed −− −− +
+
Nelumbo
nucifera
Gaertn. Lotus Seed +−− ++ −− ++ ++ −− −−
+
+
Oryza
sativa
L. Rice Bran
from
seed
+
+
+
+
Sesamum
indicum
L.
Sesame
Seed
+
+
Sorghum
bicolor
(L.)
Moench Sorghum
Seed
+
+
+
+
+
+
+
+
+
+
+
+
Zea
may
L. Corn Seed −− −− ++
+
+
+
Note:
“+”
represented
presence
in
the
extract.
represented
absence
in
the
extract.
“S”
represented
supercritical
carbon
dioxide
fluid
extraction.
“M”
represented
maceration
extraction.
Table
3
Comparison
of
antioxidative,
tyrosinase
inhibition
activities
and
the
stimulation
index
on
human
skin
fibroblasts
(30th
passage)
of
the
ten
edible
plant
extracts
prepared
by
the
two
non-heated
methods
(scCO2and
ethanolic
maceration).
Plant
scientific
name SC50 (mg/ml)
IPC50 (mg/ml)
CC50 (mg/ml)
IC50 (mg/ml)
Stimulation
index
(SI)
at
0.1
mg/ml
scCO2Maceration scCO2Maceration scCO2Maceration
scCO2Maceration
scCO2Maceration
Arachis
hypogaea
L.
151.23
52.56
1.23
1.44
ND
ND
ND
ND
1.03
0.76
Carthamus
tinctorius
L. 1.13 1.66
2.27
1.06
3.58
3.26
19.47
6.64
2.60
2.42
Glycine
max
(L.)
Merr.
7.26
5.77
6.66
10.11
93.05
55.35
ND
ND
2.12
1.77
Helianthus
annuus
L.
6.36
0.25
0.87
1.79
ND
37.33
ND
5.58
1.45
0.86
Linum
usitatissimum
L.
28.99
27.65
8.99
27.65
ND
ND
ND
ND
1.13
1.49
Nelumbo
nucifera
Gaertn.
40.69
6.57
3.19
1.79
10.3
5.05
ND
2.6
0.79
0.94
Oryza
sativa
L. 6.55 6.79 1.25
1.44
68.23
75.04
25.98
48.06
1.10
1.14
Sesamum
indicum
L.
24.3
8.32
9.3
0.87
ND
ND
ND
19.29
0.85
0.93
Sorghum
bicolor
(L.)
Moench
2.16
3.64
4.05
3.27
36.48
47.82
3.61
17.24
1.22
1.45
Zea
may
L.
28.48
22.6
8.48
22.6
7.88
10.41
1.53
3.53
0.73
0.69
Vitamin
C
0.044
0.07
NA
0.035
1.20
Vitamin
E 0.039 0.03 NA NA NA
EDTA
NA
NA
0.15
NA
NA
Kojic
acid
NA
NA
NA
0.024
NA
-Linolenic
acid
15.49
1.93
1.74
0.77
NA
Linoleic
acid 39.82
1.25
14.79
2.55
NA
Oleic
acid 55.58
3.51
26.34
7.11
NA
Note:
ND
represented
not
detected.
NA
represented
not
appreciable.
SC50 value
(mg/ml)
was
the
concentration
of
the
sample
that
scavenged
50%
of
the
DPPH
radicals.
IPC50 value
(mg/ml)
was
the
concentration
of
the
sample
that
inhibited
50%
of
the
lipid
peroxidation.
CC50 value
(mg/ml)
was
the
concentration
of
the
sample
that
chelated
50%
of
the
metal
ion.
IC50 value
(mg/ml)
was
the
concentration
of
the
sample
that
inhibited
50%
of
the
tyrosinase
enzyme.
Stimulation
index
(SI)
was
the
ratio
between
the
percentages
of
cell
proliferation
of
the
treated
sample
and
the
control
(no
treatment)
66 W.
Ruksiriwanich
et
al.
/
J.
of
Supercritical
Fluids
59 (2011) 61–
71
For
chelating
activity,
the
three
crude
extracts
by
scCO2which
gave
the
highest
activity
were
C.
tinctorius,
Z.
may
and
S.
bicolor,
respectively.
The
crude
extracts
from
C.
tinctorius
gave
the
highest
chelating
activity
(CC50 value
by
scCO2of
3.58
±
0.17
mg/ml),
which
was
higher
than
S.
bicolor
(CC50 value
of
36.48
±
3.69
mg/ml)
and
O.
sativa
crude
extracts
by
scCO2(CC50 value
of
68.23
±
8.06
mg/ml)
of
about
10.2
and
19
times,
respectively,
but
indicated
lower
activity
than
the
standard
chelating
agent,
EDTA
(CC50 value
of
0.15
±
0.04
mg/ml)
of
about
23
times.
For
tyrosinase
inhibition
activity,
the
three
crude
extracts
by
scCO2which
gave
the
highest
activity
were
Z.
may,
S.
bicolor
and
C.
tinctorius,
respectively.
The
S.
bicolor
crude
extract
by
scCO2showed
the
high
tyrosinase
inhibition
activity
(IC50 value
of
3.61
±
0.82
mg/ml)
which
was
higher
than
the
C.
tinctorius
(IC50
value
of
19.47
±
7.35
mg/ml)
and
O.
sativa
crude
extracts
by
scCO2
(IC50 value
of
25.98
±
13.42
mg/ml)
of
about
5.4
and
7.2
times,
respectively,
but
gave
lower
activity
than
the
standard
vitamin
C
(IC50 value
of
0.035
±
0.009
mg/ml),
kojic
acid
(IC50 value
of
0.024
±
0.003
mg/ml)
of
about
103
and
150
times,
respectively.
For
human
skin
fibroblast
stimulation
activity,
the
C.
tinctorius
crude
extract
by
scCO2(SI
value
of
2.60
±
0.20)
and
by
maceration
(SI
value
of
2.42
±
0.67)
at
0.1
mg/ml
gave
the
highest
stimulation
index,
which
was
higher
than
the
standard
vitamin
C
at
0.1
mg/ml
(SI
value
of
1.20
±
0.08)
and
S.
bicolor
(SI
value
of
1.22
±
0.19
mg/ml)
and
O.
sativa
crude
extracts
by
scCO2(SI
value
of
1.10
±
0.12
mg/ml)
of
2.2,
2.1
and
2.4
times,
respectively.
The
scCO2crude
extracts
from
the
three
plants
including
C.
tinctorius,
O.
sativa
and
S.
bicolor
were
selected
to
prepare
the
semi-
purified
fractions
because
of
the
highest
total
unsaturated
fatty
acid
contents
(TUC)
in
comparing
to
other
seven
edible
plants,
since
the
unsaturated
fatty
acids,
-linolenic
acid,
linoleic
acid
and
oleic
acid,
have
been
proved
to
have
anti-hair
loss
activity
by
inhibit-
ing
5-reductase
enzyme
in
the
androgen
responsive
organs
[13].
Although
extracts
from
the
scCO2indicated
only
slightly
lower
bio-
logical
activities
than
the
maceration
method,
the
scCO2process
gave
higher
unsaturated
fatty
acid
contents
than
by
the
macer-
ation
process.
Also,
the
scCO2method
is
not
only
a
non-toxic,
non-inflammable
and
inexpensive
process
but
also
has
high
sol-
vating
power
to
extract
unsaturated
fatty
acids.
In
addition,
the
crude
extract
of
C.
tinctorius
showed
the
highest
biological
activities,
followed
by
S.
bicolor
and
O.
sativa,
respec-
tively.
The
C.
tinctorius
crude
extracts
from
scCO2gave
the
highest
DPPH
scavenging,
chelating
and
stimulation
index,
high
tyrosinase
inhibition
activities
(in
the
3rd
rank),
moderate
lipid
peroxidation
inhibition
(in
the
4th
rank)
and
contained
the
high
total
unsaturated
fatty
acids
(in
the
2nd
rank).
The
crude
extract
of
O.
sativa
which
contained
the
highest
total
unsaturated
fatty
acids,
showed
the
moderate
biological
activities,
DPPH
scavenging
(in
the
4th
rank),
lipid
peroxidation
inhibition
(in
the
3rd
rank),
chelating
(in
the
4th
rank),
tyrosinase
inhibition
activities
(in
the
4th
rank)
and
stimu-
lation
index
(in
the
6th
rank).
The
S.
bicolor
crude
extracts
by
scCO2
showed
the
high
DPPH
scavenging
(in
the
2nd
rank),
chelating
(in
the
3rd
rank),
tyrosinase
inhibition
activities
(in
the
2nd
rank)
and
moderate
lipid
peroxidation
inhibition
(in
the
6th
rank)
and
stim-
ulation
index
(in
the
4th
rank)
and
contained
high
amount
of
total
unsaturated
fatty
acids
(in
the
3rd
rank).
3.4.
Biological
activities
and
5˛-reductase
inhibition
of
the
semi-purified
fractions
from
the
three
selected
plants
including
O.
sativa
crude
extracts
3.4.1.
Unsaturated
fatty
acid
and
total
phenolic
contents
Fraction
No.
3
of
O.
sativa
crude
extract
showed
the
highest
per-
centage
yields
(48.23
±
3.56%,
w/w),
followed
by
fraction
No.
1
of
S.
bicolor
(43.67
±
4.48%,
w/w)
and
fraction
No.
3
of
C.
tinctorius
(37.24
±
2.72%,
w/w)
crude
extracts.
However,
fraction
No.
3
of
the
O.
sativa
crude
extract
contained
the
highest
content
of
unsatu-
rated
fatty
acids
(-linolenic
acid
7.52
±
1.12%,
w/w;
linoleic
acid
49.25
±
3.67%,
w/w;
oleic
acid
42.17
±
4.12%,
w/w),
followed
by
the
crude
extract
of
O.
sativa
(-linolenic
acid
5.67
±
0.52%,
w/w;
linoleic
acid
23.62
±
2.62%,
w/w;
oleic
acid
27.28
±
1.22%,
w/w)
and
the
fraction
No.
4
of
the
O.
sativa
crude
extract
(-linolenic
acid
9.21
±
0.78%,
w/w;
linoleic
acid
32.07
±
1.31%,
w/w;
oleic
acid
13.71
±
1.34%,
w/w),
respectively.
The
higher
contents
of
unsatu-
rated
fatty
acids
were
found
in
fraction
No.
3
of
O.
sativa
crude
extract
than
in
the
crude
extracts
about
2
times
because
the
eluent
(petroleum
ether/ethyl
acetate
8:1)
was
more
non-polar
solvent
than
the
salvation
system
of
scCO2with
25%
(w/v)
of
ethanol.
As
known,
the
supercritical
carbon
dioxide
fluid
was
the
lipophilic
solvent
that
can
also
extract
the
colored
substances
from
the
plant
parts
such
as
safflower
and
sorghum.
So,
the
O.
sativa,
C.
tinctorius
and
S.
bicolor
crude
extract
were
in
green,
dark
orange
and
dark
yellow
appearances
which
will
be
undesirable
for
cos-
metic
formulation.
Therefore,
in
order
to
get
rid
of
the
colored
substances,
these
crude
extract
was
semi-purified
by
column
chro-
matography.
The
semi-purified
fraction
of
O.
sativa
extract
fraction
3
(OSF3),
C.
tinctorius
extract
fraction
3
and
S.
bicolor
extract
fraction
1
which
gave
the
highest
unsaturated
fatty
acid
contents
were
in
pale
yellow,
pale
orange
and
pale
yellow
appearances,
respectively.
In
addition,
the
crude
extract
of
C.
tinctorius,
fraction
No.
2
of
the
C.
tinctorius
crude
extract
and
the
crude
extract
of
S.
bicolor
con-
tained
high
total
phenolic
contents
at
1.36
±
0.11,
1.25
±
0.08
and
1.05
±
0.09
mg
gallic
acid
equivalent/gram
of
extract
(GAE
mg/g),
respectively.
These
crude
extracts
and
fractions
appeared
to
give
higher
phenolic
contents
because
of
the
slightly
polar
property
of
the
extraction
condition
of
25%
(w/v)
ethanol
in
scCO2.
3.4.2.
Biological
activities
of
the
semi-purified
fractions
3.4.2.1.
DPPH
radical
scavenging
assay.
The
three
extracts
and
fractions
which
gave
the
highest
scavenging
activity
were
the
crude
extract
of
C.
tinctorius,
fraction
No.
2
of
C.
tinctorius
crude
extract
and
the
crude
extract
of
S.
bicolor
with
the
SC50 val-
ues
of
1.13
±
0.12,
1.20
±
0.25
and
2.16
±
0.17
mg/ml,
respectively
(Table
4).
However,
the
C.
tinctorius
crude
extract
which
gave
the
highest
DPPH
scavenging
activity
showed
lower
activity
than
the
standard
vitamin
C
(SC50 value
of
0.04
±
0.004
mg/ml),
vita-
min
E
(SC50 value
of
0.04
±
0.002
mg/ml)
of
about
28
times,
but
exhibited
higher
activity
than
the
standard
unsaturated
fatty
acids,
-linolenic
acid
(SC50 value
of
15.49
±
0.02
mg/ml),
linoleic
acid
(SC50 value
of
39.82
±
0.05
mg/ml)
and
oleic
acid
(SC50 value
of
55.58
±
0.9
mg/ml)
of
about
14,
35
and
49
times,
respectively.
This
indicated
that
the
unsaturated
fatty
acids
in
the
fractions
and
the
extracts
may
not
be
the
only
bioactive
compounds
which
have
the
effect
on
this
activity,
but
also
other
bioactive
compounds
such
as
the
total
phenolic
compounds
existing
in
the
crude
extracts
and
fractions.
The
significant
positive
linear
correlation
of
free
radi-
cal
scavenging
activity
(AF)
and
the
total