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Curcuminoid Contents, Antioxidant and Anti-Inflammatory Activities of
Curcuma xanthorrhiza RoxB. and Curcuma domestica Val. Promising Lines
From Sukabumi of Indonesia

Waras Nurcholis
1, 2)
, Laksmi Ambarsari
1, 2)
, Ni Luh Putu Eka Kartika Sari
2
,
Latifah K Darusman
1, 3)


1)
Biopharmaca Research Center, Bogor Agricultural University, Indonesia
2)
Department of Biochemistry, Bogor Agricultural University, Indonesia
3)
Department of Chemistry, Bogor Agricultural University, Indonesia

E-mail: wnurcholis@gmail.com or bfarmaka@gmail.com

ABSTRACT
The main bioactive substances in the rhizomes of Curcuma xanthorrhiza and
Curcuma domestica that have eficacy as antioxidant and anti-inflamatory
activities are curcuminoids. In this study, ethanol extracts of C. xanthorrhiza and
C. domestica promising lines from Sukabumi of Indonesia were investigated for
the presence of curcuminoids, antioxidant and anti-inflamatory activities. HPLC
method was used to determined curcuminoids content. The antioxidant (radical
scavenging) potential of the samples was evaluated using 2,2-diphenyl-1-
picrylhydrazyl (DPPH) free radical method. While for the anti-inflamatory
activity, the in vitro cyclooxygenase 2 (COX2) inhibition method was used. The
curcuminoid content of C. xanthorrhiza and C. domestica were 31.27 and 66.32
mg/ g, respectively. IC
50
values for DPPH radical scavenging activity were 81.99
and 73.31 g/ mL, with C. domestica having lowest value and most potent than C.
xanthorrhiza. Percent inhibition values for COX2 inhibitor activity were 74.84
and 67.96 %, with C. domestica having the highest value. In this study, the
ethanol extracts of C. domestica promosing line from Sukabumi of Indonesia
exhibited most in curcuminoids content, antioxidants properties and anti-
inflamatory activity than C. xanthorrhiza promosing line.

Keywords: Curcuminoid, Antioxidant, Anti-Inflamatory, Curcuma xanthorrhiza,
Curcuma domestica, Promosing line


INTRODUCTION
Curcuma xanthorrhiza Roxb.,
also know as temulawak in
Indonesia, and Curcuma domestica
Val., known in Indonesia as
kunyit, are a medicinal plant from
the family Zingeberaceae distributed
in Indonesia. Traditionally, C.
xanthorrhiza rhizomes have been
used to treat stomach diseases, liver
disorders, constipation, bloody
diarrhea, dysentery, childrens
fevers, hemorrhoids, and skin
eruptions (Hwang et al., 2000).
Pharmacologically is has been
reported that C. xanthorrhiza has the
antimicrobial (Hwang et al., 2000),
anti-metastatic (Choi et al., 2004),
anti-cancer (Huang et al., 1998), anti-
candidal (Rukayadi et al., 2006),
anti-oxidant (Masuda et al., 1992)
and hypolipidemic activities (Yasni
et al., 1993). The rhizomes of C.
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domestica are commonly used as a
flavoring, coloring agent and
preservative (Thaikert &
Paisooksantivatana, 2009). The
traditional Indian medicine claims
the use of C. domestica powder
against biliary disorders, anorexia,
coryza, cough, diabetic wounds,
hepatic disorder, rheumatism and
sinusitis (Ammon et al., 1992).
Pharmacolological research has
demonstrated that C. domestica
possesses anti-inflammatory
(Mukophadhyay et al. 1982; Arora et
al., 1971; Chandra and Gupta, 1972),
antioxidant (Unnikrishnan and Rao,
1995; Pulla Reddy & Lokesh, 1992),
anti-protozoal (Rasmussen et al.,
2000), nematocidal (Kiuchi et al.,
1993), anti-bacterial (Chopra et al.,
1941; Bhavani Shankar & Murthy,
1979), antivenom (Ferreira et al.,
1992), anti-HIV (Mazumber et al.,
1995; Eigner and Scholz 1999), and
anti-tumor activities (Huang et al.,
1988).
The main yellow bioactive
substances in the rhizomes of C.
xanthorrhiza and C. domestica are
curcuminoid (Duke, 2002; Chainani-
Wu, 2003). The pharmacological
effect of curcuminoids has been
investigated, such as radical
scavenging (Sreejayan & Rao, 1996),
the inhibition of nitric oxide (NO)
(Pan et al., 2000; Onoda et al., 2000),
anti-inflammation (Banerjee et al.,
2003), anti-tumor (Khar et al., 1999),
anti-allergy (Ram et al., 2003) and
anti-dementia (Lim et al., 2001). This
study was undertaken to verify the
variation C. xanthorrhiza and C.
domestica promising lines from
Sukabumi of Indonesia for their total
curcuminoid, antioxidant and anti-
inflamatory activities.

MATERIALS AND METHODS
Materials
The rhizomes of C.
xanthorrhiza and C. domestica
promising lines were collected
during J uly 2011 from the area of
Sukabumi, West J ava, Indonesia.
The authentic chemical standards of
curcuminoid (curcumin,
demethoxycurcumin, and
bisdemethoxycurcumin) and 2,2-
diphenyl-1-picrylhydrazyl were
obtained from Sigma-Aldrich, USA.
The bioassay kit COX was purchased
from Cayman Chemicals. All the
chemicals and solvents used were
analytical grade.

Extraction of plans
Fresh rhizomes of plant
materials were washed with water,
cut into small pieces and dried for 5
days in the sun (the moisture: <
10%). They were then ground in a
grinder to be obtained in a powder
form (the size: 100 mesh). One kilo
grams of the powder of plants were
macerated using 1 x 10 L ethanol
70% in a tightly closed round bottom
flask at room temperature for a
period of 24 h and filtered with
Whatman filter paper (type 4). The
whole process was repeated one
times and the filtrate was
concentrated under reduced pressure
on rotavapor (BUCHI, R-250,
Switzerland) at 50 C temperature.
The concentrated extracts were then
used for the experiments.

Determination of curcuminoid
content
The curcuminoid content of
ethanol extract of C. xanthorrhiza
and C. domestica promising lines
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from Sukabumi was carried out
according to the principle and
protocol previously described by
J ayaprakasha et al., (2002) using
high performance liquid
chromatography (HPLC), with
modification. The systems condition
of HPLC are C18 column, UV-Vis
detector, 10 L volum injection, and
48 C temperature column. Its used
methanol as an additional mobile
phase, , which included solvents A:
methanol; B: 2% acetic acid; and C:
acetonitrile.

Antioxidant activity through
measurement of free radical
scavenging capacity by DPPH
assay
Free radical scavenging
capacity was investigated on the
basis of the scavenging activity of
DPPH by measuring the reduction of
absorbance at 517 nm. The method
was carried out as described by
Udenigwe et al., (2009) with a
modification. The different
concentrations of the plant extracts
(12.5200 g/ml; total volume of 40
L) in 96-well plates were mixed
with 160 L of 100 mM DPPH in
ethanol and then incubated in the
dark for 30 min at room temperature
prior to reading the absorbance at
517 nm in a micro plate reader. A
negative control, containing water
instead of the sample and blank
samples, using the same volume of
ethanol only in place of the DPPH
solution in ethanol, were all
evaluated at the same time per micro
titre plate.
The percentage of radical
scavenging was calculated as
follows;
% radical scavenging
=
(AcAcb) (As Asb)
(AcAcb)
x 100
where Ac is the absorbance of
water plus DPPH (in ethanol), Acb is
the absorbance of the blank (water
plus ethanol without DPPH), As is
the absorbance of the sample plus
DPPH (in ethanol) and Asb is the
absorbance of the sample plus
ethanol without DPPH. Different
sample concentrations were used in
order to obtain calibration curves and
to calculate the IC
50
values (IC
50
:
concentration required to obtain a
50% radical scavenging activity). All
test samples were conducted in
triplicate (n =3).

Determination of anti-
inflammatory activity
The ethanol extracts of C.
xanthorrhiza and C. domestica
promising lines from Sukabumi was
evaluated as anti-inflammatory with
cyclooxygenase 2 (COX2) inhibitory
activities. COX2 inhibition activity
was measured using the Cayman
Chemical Colorimetric COX (ovine)
Inhibitor Screening Assay Kit. All
procedures were performed as
indicated in the assay kit instructions.
The ethanol extracts were dissolved
in DMSO and added to the enzyme
reaction mixture.

RESULTS AND DISCUSSION
Curcuminoids content
Curcuminoids compound are
commonly in Curcuma species and
have been reported to have several
biological activities including
antioxidant and anti-inflammatory
properties (Itokawa et al., 2008). The
results of curcuminoids content on
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the ethanol extract of C. xanthorrhiza
and C. domestica promising lines
from Sukabumi are shown in Figure
1A. The curcuminoids content of C.
domestica (value, 66.32 mg/ g) most
highest than C. xanthorrhiza with
value 31.27 mg/ g. The rhizomes of
C. xanthorrhiza and C. domestica
show different profiles in the three
major curcuminoids. The
curcuminoids in C. domestica are
mainly curcumin, demethoxy-
curcumin, and bisdemethoxy-
curcumin (Figure 1B) (Lechtenberg
et al., 2004; Thaikert &
Paisooksantivatana, 2009). While for
the curcuminoids in C. xanthorrhiza
are curcumin and demethoxy-
curcumin (Figure 1C) (Lechtenberg
et al., 2004).

Antioxidant activity
The measures of antioxidant
activity were obtained using DPPH
(2,2-diphenyl-1-picrylhydrazyl)
method. Figure 2 shows the DPPH
radical scavenging activity of the
different successive extracts of C.
xanthorrhiza and C. domestica. This
activity was increased by increasing
the concentration of sample extracts.
DPPH antioxidant assay is based on
the ability of DPPH, a stable free
radical, to decolorize in the presence
of antioxidants. The DPPH radical
contains an odd electron, which is
responsible for the absorbance at 517
nm and also for a visible deep purple
color. When DPPH accepts an
electron donated by an antioxidant
compound, the DPPH is decolorized,
which can be quantitatively
measured from the changes in
absorbance. Thus, the DPPH radicals
were widely used to investigate the
scavenging activity of some natural
compounds. DPPH is widely used to
evaluate the antioxidant activity of
natural compounds (Udenigwe et al.,
2009). However, DPPHs scavenging
activity indicates the ability of the
antioxidant compound to donate
electrons or hydrogen, thereby
converting the radical to a more
stable species (Bougatef et al., 2009).


Figure 1. (A). Curcuminoids content of C. xanthorrhiza and C. domestica
promising lines from Sukabumi, West J ava, Indonesia. (B). the curcuminoids
component of C. domestica: curcumin, demethoxycurcumin, and
bisdemethoxycurcumin. (C). the curcuminoids component of C. xanthorrhiza:
curcumin and demethoxycurcumin.
0.
10.
20.
30.
40.
50.
60.
70.
Curcuma xanthorrhiza Curcuma domestica
31.27
66.32
C
u
r
c
u
m
i
n
o
i
d
s

c
o
n
t
e
n
t

(
m
g
/

g
)
PromisinglinesfromSukabumi
A
Curcumin
Demethoxycurcumin
Bisdemethoxycurcumin
B
C
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Figure 2. Scavenging effect of C. xanthorrhiza and C. domestica promising lines
from Sukabumi, West J ava, Indonesia on DPPH (2,2-diphenyl-1-picrylhydrazyl),
as measured by changes in absorbance at 517 nm.

Antioxidant activity using
DPPH radical scavenging assay
reported with IC
50
value is show in
Figure 3. The lower the IC
50
is the
higher the antioxidant activity of the
Curcuma promising lines.


Figure 3. DPPH free radical
scavenging activity of C.
xanthorrhiza and C. domestica
promising lines from Sukabumi,
West J ava, Indonesia.
The results showed that the ethanol
extracts of C. xanthorrhiza and C.
domestica have highest of
antioxidant activity (with IC
50
value
< 200 g/ mL, Blois, 1958). IC
50

values for DPPH radical scavenging
activity were 81.99 and 73.31 g/
mL, with C. domestica having lowest
value and most potent than C.
xanthorrhiza. Curcuminoids
compound are the major C.
xanthorrhiza and C. domestica
compounds with antioxidant activity
(J ayaprakasha et al., 2006; Masuda et
al., 1992). This study, we used
ethanol crude extract of C.
xanthorrhiza and C. domestica, so
its possible has pure compound
most stronger as DPPH free radical
scavenging activity than that
extracts.

Anti-inflammatory activity
Anti-inflammatory activity of C.
xanthorrhiza and C. domestica
promising lines from Sukabumi was
evaluated through the percentages of
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
12.5 25 50 100 200
A
b
s
o
r
b
a
n
c
e

m
e
a
s
u
r
e
d

a
t

5
1
7

n
m
Concentration (g/ mL)
C. xanthorrhiza
C. domestica
73.31
81.99
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
C. domestica C. xanthorrhiza
A
n
t
i
o
x
i
d
a
n
t

a
c
t
i
v
i
t
y
,

I
C
5
0

v
a
l
u
e

(

g
/
m
L
)
Promisinglines from Sukabumi
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cyclooxygenase 2 (COX2) inhibition.
Results are given in Figure 4. Percent
inhibition values for COX2 inhibitor
activity of C. domestica and C.
xanthorrhiza were 74.84 and 67.96
%, with C. domestica having the
highest value. Curcuminoids
compound are the major C.
xanthorrhiza and C. domestica
compounds with anti-inflammatory
properties (Chainani-Wu, 2003;
Tohda et al., 2006; Ozaki, 1990).
This study, we used ethanol crude
extract of C. xanthorrhiza and C.
domestica, so its possible has pure
compound most stronger as anti-
inflammatory activity than that
extracts.



Figure 4. Anti-inflammatory activity
of C. xanthorrhiza and C. domestica
promising lines from Sukabumi,
West J ava, Indonesia on
cyclooxigenase 2 (COX2) inhibition.

CONCLUSION
Curcuma domestica promising
line from Sukabumi, West J ava,
Indonesia is the highest
curcuminoids content compared to
Curcuma xanthorrhiza, whic are also
known to have antioxidant and anti-
inflammatory activities.

ACKNOWLEDGEMENTS
We express our gratitude to Dr.
Min Rahminiwati, dr. Lina Sulimin,
and Dr. Eka Intan Kumala Putri for
excellence technical support. This
research was supported by grants
from the Directorate General of
Higher Education, Ministry of
Education and Culture Republic of
Indonesia (Hibah Kompetitif
Penelitian Strategis Unggulan
Nasional Nomor: 476/SP2H/PL/
Dit.Litabmas/V/2011).

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