Biosci. Biotechnol. Biochem.
, 71 (6), 1428–1438, 2007
Immunostimulating Activity of Crude Polysaccharide Extract
Isolated from Curcuma xanthorrhiza Roxb.
Ah-Jin K IM,1 Yeon-O K IM,1 Jae-Seok S HIM,2 and Jae-Kwan H WANG1;2; y
1
Department of Biotechnology, Yonsei University, Seoul 120-749, Korea
2
Department of Biomaterials Science and Engineering,
Yonsei University, Seoul 120-749, Korea
Received April 28, 2006; Accepted March 20, 2007; Online Publication, June 7, 2007
[doi:10.1271/bbb.60241]
Curcuma xanthorrhiza Roxb., commonly known as
Javanese turmeric, has been reported to possess a va-
riety of biological activities, including anti-inflammatory
effects, anticarcinogenic effects, wound healing effects,
and serum cholesterol-lowering effects. CPE, crude
polysaccharide extract isolated from the rhizome of
C. xanthorrhiza using 0.1 N NaOH, consisted of arabi-
nose (18.69%), galactose (14.0%), glucose (50.67%),
mannose (12.97%), rhamnose (2.73%), and xylose
(0.94%), with an average molecular weight of 33,000
Da. In the present study, we investigated the effect of
CPE on nitric oxide (NO), hydrogen peroxide (H2 O2 ),
tumor necrosis factor- (TNF- ), and prostaglandin E2
(PGE2 ) production in RAW 264.7 cells. The uptake of
fluorescein-labeled Escherichia coli was measured to
determine whether CPE stimulates the phagocytic
activity of RAW 264.7 cells. CPE significantly increased
the phagocytosis of macrophages and the release of NO,
H2 O2 , TNF- , and PGE2 in a dose-dependent manner,
and showed a similar activity to lipopolysaccharide
(LPS). To study the mechanisms of CPE, we examined
induction of iNOS and COX-2. NO and PGE2 were
produced as a result of stimulation of inducible nitric
oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)
respectively. Both modulations of iNOS and COX-2
expression by CPE were evaluated by Western immu-
noblotting and RT-PCR. Since transcription of these
enzymes is under the control of nuclear factor-kappa B
(NF-B), we assessed the phosphorylation of inhibitor
B (IB ) through Western immunoblotting. CPE
clearly induced phosphorylation of IB , suggesting a
role as an NF-B activator. Taking all this together, we
conclude that CPE isolated from Curcuma xanthorrhiza
stimulates the immune functions of macrophages, which
is mediated in part by specific activation of NF-B.
Key words: Curcuma xanthorrhiza Roxb.; polysaccha-
ride; phagocytosis; immunostimulating
The plants used in traditional medicine constitute both
y
To whom correspondence should be addressed. Tel: +82-2-2123-5881; Fax: +82-2-362-7265; E-mail: jkhwang@yonsei.ac.kr
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low and high molecular weight compounds. Although
small bioactive compounds have been well studied, they
often do not account for all of the biological effects
achieved. In particular, the great majority of them,
which have been identified as cytotoxic to cancer cells,
are also toxic to normal cells.1) Hence the discovery of
new safe compounds, without side effects, has become
an important goal of research in the biomedical sciences.
Among the high molecular weight components of
medicinal plants, polysaccharides have attracted atten-
tion recently in the enhancement of host defense mech-
anisms, which has been recognized as a possible means
of immunomodulatory and antitumor effects without
doing harm.2) Several polysaccharides isolated from
Lentinus edodes, Schizophyllium commune, Angelica
gigas, Phellinus linteus, Platycodon grandiflorum, etc.
have been shown to possess immunostimulatory activ-
ities.3–5)
Macrophages play a major role in host defense against
infection and cancer.6) When pathogens cross an
epithelial barrier, they are engulfed by phagocytosis of
macrophages and are digested by lysosomal enzymes,
such as phosphatase, released from them.7) The other
effect of macrophages on pathogens is the secretion of
cytokines, including TNF-
, and inflammatory media-
tors, such as nitric oxide (NO), hydrogen peroxide
(H2 O2 ), and prostaglandins (PGs). The generation of NO
and H2 O2 by macrophage, plays an important role in
host defense against microbial infection. In particular,
nitric oxide has been identified as the major effector
molecule involved in the destruction of tumor cells by
activated macrophages.8,9) Nitric oxide and prostaglan-
dins are produced as a result of the stimulation of in-
ducible nitric oxide synthase (iNOS) and cyclooxyge-
nase-2 (COX-2) respectively. The expression of iNOS,
which catalyzes the production of nitric oxide from
L-arginine, is regulated by transcription factor NF-B
in murine macrophages. COX-2, a proinflammatory en-
zyme, is also regulated by NF-B. Cyclooxygenases
(COX) produce various types of prostaglandins (PGs),
 Immunostimulating Activity of Polysaccharide
which are implicated in various physiological events,
including progression of inflammation and immunomo-
dulation. NF-B exists in a latent form in cytoplasm,
presenting a dimer complex bound to an inhibitor pro-
tein, IB. Exposure of cells to such external stimuli as
mitogens, inflammatory cytokines, and bacterial lipo-
polysacharides (LPS) causes rapid phosphorylation of
IB, with subsequent degradation by proteosomes. Deg-
radation of IB allows the release and translocation of
NF-B to the nucleus to regulate the expression of
multiple target genes.
Curcuma xanthorrhiza Roxb., commonly known as
temu lawak or Javanese turmeric in Indonesia, has tra-
ditionally been used for medicinal purposes.10) This
plant possesses a variety of biological activities includ-
ing anti-inflammatory, anticarcinogenic, wound-healing,
and serum cholesterol-lowering effects.11–13) However,
no report to date has been published on whether poly-
saccharides in C. xanthorrhiza enhance various immune
responses. In this study, we found for the first time that
crude polysaccharide extract of C. xanthorrhiza induces
immunostimulating activity through the NF-B signal-
ing pathway.
Materials and Methods
Preparation of crude polysaccharide extract (CPE).
Dried rhizomes of Curcuma xanthorrhiza were collected
in Jakarta, Indonesia, and identified by Dr Nam-In Baek,
Institute of Life Science, Kyunghee University (Yongin,
Korea). A voucher specimen is deposited in the Depart-
ment of Biotechnology, Yonsei University (Seoul,
Korea).
Powdered rhizomes (15 g) of C. xanthorrhiza were
extracted with 750 ml of absolute ethanol and refluxed
for 2 h at 78
C. This procedure was repeated twice and
the suspension was filtered on Whatman
no. 2 paper.
The residues were suspended in 750 ml of 0.1 N NaOH
and refluxed for 2 h at 97
C. This extraction procedure
was repeated twice. After centrifugation at 700  g for
15 min at 4
C, the supernatant was treated with
-
amylase (Termamyl
120L, Novo Nordisk, Krogshoej-
vej, Denmark) and glucoamylase (AMG 300L, Novo
Nordisk) under optimum conditions to hydrolyze starch.
Exclusion of starch was confirmed with a glucose kit
(Sigma, St. Louis, MO, USA). The extract was treated
with 0.05% proteinase K (Sigma) at 37
C for 48 h in
50 mM Tris–HCl buffer (pH 7.5). The reaction mixture
was then heated at 100
C for 10 min to inactivate the
enzymes. The alkali extract was neutralized with 1 M
HCl and filtrated on Whatman
no. 2 paper to remove
insoluble materials. The filtrate was added to 4 volumes
of isopropyl alcohol to precipitate the polysaccharide
and maintained for 24 h at 4
C. The precipitate was
recovered by centrifugation at 700  g for 15 min,
washed with acetone, dried at room temperature, and
dissolved in distilled water. Low molecular weight com-
pounds were removed using a thin channel ultrafiltration
1429
system (Amicon TCF-10; Amicon, Billerica, MA, USA)
with an MWCO 1,000 membrane, and then dialyzed
against distilled water in spectrapor dialysis tubing
(Spectrum Laboratories, Rancho Dominguez, CA, USA)
with MWCO 500 Da. The solution was lyophilized, and
the resulting CPE was stored at 20
C prior to use.
Determination of the monosaccharide composition of
CPE. Polysaccharides (10 mg) were treated with 100 ml
of 24 N sulfuric acid at 25
C for 1 h, and 1.1 ml of
distilled water was added. The samples were hydrolyzed
at 100
C for 3 h under nitrogen and neutralized with 200
ml of 12 N ammonium hydroxide at room temperature.
Twenty ml of hydrolyzed polysaccharides, obtained by
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the procedures described above, were dissolved in 2.8 ml
of distilled water and injected into a high pH anion ex-
change chromatograph (HPAEC-PAD) consisting of a
Bio-LC DX-500 chromatograph (Dionex, Sunnyvale,
CA, USA) equipped with CarboPac PA1 (4  250
mm, Dionex) column and an electrochemical detector.
The column was eluted with 200 mM NaOH at a flow
rate of 0.5 ml/min. The constituting sugars, glucose, ara-
binose, galactose, mannose, xylose, and rhamnose, were
identified by their characteristic retention times as de-
termined by their respective standards using a DX-500
Bio-LC system (Dionex).
Determination of molecular weights. The molecular
weight of CPE was determined on Ultrahydrogel linear
column (7:8  300 mm, Waters, Boston, MA, USA) and
an Ultrahydrogel 500 column (7:8  300 mm, Waters)
coupled to a gel permeation chromatograph (GPC,
Younglin Instruments., Anyang, Korea) system by
elution with 0.1 N NaNO3 solution at a flow rate of
1.0 ml/min. Shodex pullulan standard (MW 788,000,
112,000, 22,800, 5,900 and 360; Showa denko, Tokyo,
Japan) was used as a calibration standard.
Cell culture. RAW 264.7 cells (KCLB no. 40071)
were purchased from the Korea Cell Line Bank (Seoul,
Korea). The cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM; Gibco BRL, Grand Island,
NY, USA), containing 10% fetal bovine serum (FBS;
Gibco BRL), 100 units/ml of penicillin, and 100 mg/ml
of streptomycin. The cells were incubated in the pres-
ence of 5% CO2 at 37
C.
Measurement of nitrite concentration. The nitrite
concentration in the culture medium was measured by
the Griess method.14) Briefly, RAW 264.7 cells (5  105
cells/ml) were treated with varying concentrations of
CPE (5, 10, 30, or 50 mg/ml) or LPS (10 mg/ml) at 37
C
for 24 h. Culture supernatant (100 ml) was mixed with an
equal volume of Griess reagent (1% sulfanilamide, 0.1%
naphthylethylenediamine dihydrochloride, and 2% phos-
phoric acid), and incubated at room temperature for
10 min. The nitrite concentration was determined at 540
nm using NaNO2 as a standard.
1430 A.-J. KIM et al.
Measurement of level of H2 O2 . The H2 O2 assay was
based on horseradish peroxidase (HRP)-dependent ox-
idation of phenol red by H2 O2 , leading to the formation
of a colored compound. H2 O2 production was measured
using Amplex Red reagent (10-acetyl-3,7-dihydroxy-
phenoxazine, Molecular Probes, Eugene, OR, USA).
Amplex Red reagent can be used to detect the release
of H2 O2 from RAW 264.7 cells. Briefly, a mixture of
50 mM Amplex Red reagent and 0.1 U/ml HRP in Krebs-
Ringer phosphate (KRPG: 145 mM NaCl, 5.7 mM sodium
phosphate, 4.86 mM KCl, 0.54 mM CaCl, 1.22 mM
MgSO4 , 5.5 mM glucose, pH 7.35) was introduced into
a microplate well. RAW 264.7 cells (2  104 cells/ml)
in KRPG were added to the reation mixture at 37
C for
20 h. H2 O2 production was determined using a fluo-
rescence microplate reader (PerSeptive Biosystems,
Framingham, MA, USA) equipped for excitation in a
range of 530–560 nm and emission detection at 590 nm.
Macrophage phagocytosis assay. RAW 264.7 cells
(1  106 cells/ml) were treated with varying concen-
trations of CPE (5, 10, 30, or 50 mg/ml) at 37
C for
24 h. Then heat-killed fluorescein isothiocyanate
(FITC)-labeled Escherichia coli BioParticles
(K-12
strain, Molecular Probes) were incubated with RAW
264.7 cells at 37
C for 2 h. The cells and bacteria were
rinsed with PBS, and extracellular fluorescence was
quenched with trypan blue (Molecular Probes). Quench-
ing prevents fluorescence of bacteria attached to the
outside of the cell, allowing determination of ingested
bacterial levels.15) Fluorescence was measured using a
CytoFluor Series 4000 multiwell fluorescence plate
reader (PerSeptive Biosystems, Framingham, MA,
USA).
Morphological observation. RAW 264.7 cells (1 
106 cells/ml) were grown on glass coverslips and treat-
ed with CPE. The cells were incubated with fluorescein-
labeled E. coli BioParticles
(K-12 strain, Molecular
Probes) for 2 h at 37
C. The cells were dyed with
phalloidin-tetramethylrhodamine B isothiocyanate (Sig-
ma), and were examined with a laser scanning confocal
microscope (Radiance 2100, Bio-Rad, Hercules, CA,
USA).
Measurement of PGE2 production. RAW 264.7 cells
were plated at 1  106 cells/ml and stimulated with
CPE (5, 10, 30, or 50 mg/ml) or LPS (10 mg/ml) for 24 h.
Culture supernatants were taken, and the concentration
of PGE2 was determined using ELISA kits (R&D Sys-
tem, Minneapolis, MN, USA) according to the manu-
facturer’s instructions. The assay is based on competi-
tion between unlabelled PGE2 and a fixed quantity
of peroxidase-labelled PGE2 for a limited number of
binding sites on a PGE2 specific antibody.
Western immunobloting. Cellular proteins were ex-
tracted from LPS and CPE treated RAW 264.7 cells
(2  106 cells/ml). The protein concentration was de-
termined by Bradford protein assay with bovine serum
albumin as a standard.16) Ten mg of cellular protein from
cell extract was loaded and electrophoresed on 10%
SDS–polyacrylamide gel. The separated proteins were
transferred to nitrocellulose membranes (Amersham
Biosciences). The nitrocellulose membranes were pre-
incubated for 1 h with blocking solution (5% skim milk)
at 4
C. They were incubated with a 1:1,000 dilution of
iNOS, TNF-
, COX-2, total IB, and phosphorylation
IB
antibody (Santa Cruz Biotechnology, Santa Cruz,
CA, USA) for 2 h at room temperature. The blots were
washed and then incubated for 1 h with anti-rabbit IgG
and anti-goat IgG antibody diluted to 1:2,000. The bands
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were visualized by ECL detection reagent (Amersham
Biosciences) using X-ray film. The density of the band
was calculated with an image analyzer (Vilber Lourmat,
Marne-la-Vallée Cedex, France).
Reverse transcription-polymerase chain reaction (RT-
PCR). Total RNA was isolated using TRIZOL Re-
agent (Sigma). The sequences used were as follows:
iNOS, sense: 50 -CTGCAGCACTTGGATCAGGAACC-
TG-30 , antisense: 50 -GGGAGTAGCCTGTGTGCACC-
TGGAA-30 ; TNF-
, sense: 50 -CCTGTAGCCCACGTC-
GTAGC-30 , antisense: 50 -TTGACCTCAGCGCTGAGT-
TG-30 ; COX-2, sense: 50 -GGAGAGACTATCAAGAT-
AGTGATC-30 , antisense: 50 -ATGGTCAGTAGACTTT-
TACAGCTC-30 ; -actin, sense: 50 -TGGAATCCTGTG-
GCATCCATGAAAC-30 , antisense: 50 -TAAAACGCA-
GCTCAG-TAACAGTCCG-30 . For PCR, the samples
were heated to 48
C for 45 min and cycled 30 times
(95
C for 30 s, 55
C for 1 min, and 72
C for 1 min),
and elongated at 72
C for 5 min. The PCR products
were electrophoresed on 1% agarose gel, followed by
ethidium bromide staining. The iNOS, TNF-
, COX-2,
and -actin primers produced amplified products at
311 bp, 374 bp, 861 bp, and 349 bp respectively.
Statistical analysis. Each experiment was perform-
ed at least in triplicate. All data are presented as the
mean  standard deviation (SD). Data analysis was
performed by one-way analysis of variance (ANOVA).
The differences between treated and control groups were
also analyzed by the Duncan test (SPSS 12.0).  p-
values < 0.05 and  p-values < 0.01 were considered
statistically significant.
Results
Isolation and characterization of CPE
In this study CPE was isolated from C. xanthorrhiza
using 0.1 N NaOH, and the yield was approximately
6.0%. The sugar analysis of CPE by HPAEC-PAD
showed that CPE was composed of arabinose (18.69%),
galactose (14.0%), glucose (50.67%), mannose
(12.97%), rhamnose (2.73%), and xylose (0.94%). The
molecular weight of CPE was determined to 33,000 Da
 Immunostimulating Activity of Polysaccharide 1431

180
160 **

NO production (% of control)
140
**
120
**
100 ** **
80
60
40
20
0

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LPS (10 µ g/ml) + - - - - -
CPE ( µ g/ml) - 0 5 10 30 50

Fig. 1. Effects of CPE on the NO Production of RAW 264.7 Cells.

RAW 264.7 cells (5  105 cells/ml) were treated with 5, 10, 30, or 50 mg/ml of CPE or LPS (10 mg/ml) for 24 h, and the nitrite levels of the
supernatant were determined. LPS was used as a positive control. Data are presented as mean  S.D. (n ¼ 3).  p-values < 0.01,  p-values <
0.05 compared to a control.

16
**
14

12 **
10
H2O2 (µM)

8
**
6

LPS (10 µg/ml) + - - - - -

CPE ( µg/ml) - 0 5 10 30 50

Fig. 2. Effects of CPE on the Production of H2 O2 .

RAW 264.7 cells were treated with 5, 10, 30, or 50 mg/ml of CPE or LPS (10 mg/ml) for 24 h at 37
C in a CO2 incubator. LPS is used as the
positive control. All experiments were done in triplicate.  p-values < 0.01,  p-values < 0.05 compared to control.

by comparing their elution patterns with those of pul- mediated in part through activation of NO genera-
lulans of known molecular weights. tion.17,18)

Effects of CPE on nitrite production (NO) in RAW
264.7 cells
Macrophages were incubated with different con-
centrations of CPE (5, 10, 30, or 50 mg/ml) or LPS (10
mg/ml) for 24 h, and NO concentrations in the culture
supernatants were assessed by the Griess reaction.14)
CPE increased NO production in RAW 264.7 cells in a
dose-dependent manner, and also showed comparable
activity with LPS (Fig. 1). Based on these results and
the relationship between NO and cytolytic function of
macrophages against a variety of pathogens, we suggest
that the immunostimulating effect of CPE might be
Effects of CPE on production of hydrogen peroxide
(H2 O2 )
H2 O2 is one of the oxygen metabolites produced by
phagocytes. The effect of CPE on H2 O2 production in
macrophages was studied. As shown in Fig. 2, a sig-
nificant increase in H2 O2 production was observed in the
groups treated with CPE and LPS. The level of H2 O2
production was higher than that of LPS as a positive
control. These results suggest that H2 O2 production of
CPE treated macrophages plays an important role as a
cytotoxic agent against invading microbes.
1432 A.-J. KIM et al.

400

350 **

Phagocytosis (% of effect)
300
**
250
**
200

150 **
100

50

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CPE ( µg/ml) 0 5 10 30 50

Fig. 3. Effects of CPE on Phagocytic Activity.

RAW 264.7 cells (5  105 cells/ml) were treated with 5, 10, 30, or 50 mg/ml of CPE for 24 h. After FITC-labeled E. coli was added, the cells
were incubated for 2 h. Supernatants were removed with unphagocytosed bacteria, and fluorescence was measured with a fluorecence microplate
reader. Data are presented as mean  S.D. of triplicate measurements.  p-values < 0.01,  p-values < 0.05 compared to control.

A B

5 µm 5 µm

Fig. 4. Phagocytosis of CPE.

A laser scanning confocal microscope image of RAW 264.7 cells was stained with TRITC after incubation with FITC-labeled E. coli for 2 h.
The cells are represented at 1;890 magnification. Scale bar indicates 5 mm. (A) control, (B) CPE 30 mg/ml.

Effects of CPE on phagocytic activity Table 1. Effects of CPE on the PGE2 Production of RAW 264.7
To determine the effects of CPE on the phagocytic Cells
activity of macrophages, the uptake of FITC-labeled Treatment PGE2 (ng/ml) % Control
E. coli between CPE treated and untreated macrophages
Control 114:51  3:81 100
was compared. Phagocytosis of macrophages was in- LPS alone 10 mg/ml 331:16  1:34 290.11
creased by CPE treatment in a dose-dependent manner CPE 5 mg/ml 324:64  0:92 284.64
(Fig. 3). Uptake of E. coli was also observed visually 10 mg/ml 346:64  1:94 303.67
with a confocal microscope (Fig. 4). The result was that 30 mg/ml 360:02  3:93 315.39
phagocytosis is one of the functions for cytotoxicity of 50 mg/ml 366:40  0:48 320.98
macrophages stimulated with CPE. The values are presented as means  S.D. (n ¼ 3). 
p-values < 0.01,

p-values < 0.05 as compared to control.

Effects of CPE on PGE2 production in RAW 264.7

cells
The amounts of PGE2 in the culture supernatants were
determined in order to study the effects of CPE on
COX-2 activity. Cells that were incubated with various
concentrations of CPE (5, 10, 30, or 50 mg/ml) for 24 h
caused 184, 203, 215, and 220% activation in the PGE2
level respectively, as compared to untreated cells
(Table 1). A concentration of 10 mg/ml LPS was used
as a positive control, and it resulted in 190% activation
of PGE2 production in the macrophages.
Activation of TNF-
in response to CPE in RAW
264.7 cells
TNF-
is a cytokine produced mainly by activated
macrophages that function to stimulate the recruitment
 Immunostimulating Activity of Polysaccharide
of neutrophils and monocytes to sites of infection and to
activate those cells to eradicate microbes.7,19) The levels
of TNF-
protein and TNF-
mRNA were analyzed by
Western immunoblotting and RT-PCR respectively. As
shown in Figs. 5 and 6, both the protein and mRNA
expression of TNF-
were enhanced by CPE in RAW
264.7 cells in a dose-dependent manner. Control -actin
was constitutively expressed and was not affected by
treatment with CPE. These results indicate that TNF-

expression of CPE exposed macrophages may be a
function to stimulate the recruitment of neutrophils and
monocytes to sites of infection.
Activation of iNOS in response to CPE in RAW 264.7
cells
To determine whether the increase in production of
NO was due to the increased protein and gene ex-
pression of iNOS expression, we examined the effect
of CPE using Western immunoblotting and RT-PCR
respectively. As shown in Figs. 7 and 8, the protein and
mRNA expression of iNOS were dose-dependently
increased. However, the protein expression of
-tubulin
and the mRNA expression of -actin were unaffected
by CPE. These results demonstrate that the increased
production of NO was due to the increased protein and
mRNA expression of this immunomodulating mediator.
Activation of COX-2 in response to CPE in RAW
264.7 cells
The levels of COX-2 protein and COX-2 mRNA were
analyzed by Western immunoblotting and RT-PCR re-
spectively to determine the increase in COX-2 produc-
tion by CPE in RAW 264.7 cells. As shown in Figs. 9
and 10, the protein and mRNA expression of COX-2
were dose-dependently increased. These results were in
good agreement with the above results that CPE in-
creased concentration-dependently PGE2 production in
RAW 264.7 macrophages.
Activation of pIB
in response to CPE in RAW 264.7
cells
To investigate whether CPE activates NF-B through
phosphorylation of IB
, we assessed the effect of CPE
on the amount of pIB
by Western immunobloting.
Treatment of RAW 264.7 cells with CPE or LPS in-
duced the phosphorylation of IB
(Fig. 11). Theses
results suggest that CPE can induce the degradation of
IB family members through phosphorylation of IB
,
and subsequently activate the nuclear translocation of
NF-B.
Discussion
Curcuma xanthorrhiza Roxb. is widely used in
Indonesia as a traditional medicine in the treatment of
various diseases. Although small compounds in C. xan-
thorrhiza, such as curcuminoids and xanthorrhizol, have
been extensively studied for their antibacterial, antiin-
1433
20–23)
flammatory, and anticarcinogenic activities, the
biological activities of polysaccharide remain unclear.
This study was undertaken to investigate the immunos-
timulating activity of high molecular weight polysac-
charide from C. xanthorrhiza.
CPE increased the phagocytic activity and the pro-
duction of NO and H2 O2 in RAW 264.7 cells (Figs. 1, 2,
and 3). Phagocytosis is the first step in the macrophage
response to invading microorganisms, and its activation
means elevation of the innate immune response. During
the phagocytic process, activated macrophages produced
reactive oxygen species (ROS) such as NO and H2 O2 .
These ROS play an important role as cytotoxic agents
against invading microbes. Since NO is related to the
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cytolytic function of macrophages against a variety of
pathogens, increased synthesis of NO can induce im-
munostimulating activity in macrophages. Moreover,
NO is recognized as an important messenger in diverse
pathophysiological functions, including vascular relax-
ation, vasodilatation, immune modulation, and cytotox-
icity against tumor cells.24) When activated, macro-
phages also inhibit the invasion of microorganisms by
releasing cytokines. Of the various cytokines, TNF-

plays a major role in defense against intracellular
pathogens. CPE also induced production of inflamma-
tory mediators such as PGE2 (Table 1) as strongly as
lipopolysaccharide (LPS). LPS was used as a positive
control, and it activated macrophages. Also, LPS is a
potent activator of NF-B in macrophages. PGE2 s are
involved in diverse functions, including nerve growth,
wound healing, and the immune response. Therefore,
CPE-mediated activation of macrophages and produc-
tion of ROS and cytokines can contribute to immunos-
timulating activity. These results might also bear on
antitumor activity. There have been many reports
demonstrating the antitumor activity of polysaccharides
isolated from various natural sources via stimulation of
the immune system.3)
CPE also induced production of iNOS (Figs. 7 and 8)
and COX-2 (Figs. 9 and 10) as strongly as LPS in RAW
264.7 cells. Administration of iNOS and COX-2 in-
hibitors promoted the growth of several transplantable
tumors.25) Expression of iNOS and COX-2 in macro-
phages is largely regulated by transcriptional activation.
Among these transcription factors, NF-B, which is a
primary transcription factor and regulates various genes,
is important in the immune response, inflammation, and
apoptosis. Activation of NF-B occurs mainly via IB
kinase (IKK)-mediated phosphorylation of inhibitory
molecules, including IB.26) Phosphorylation of IB was
clearly observed (Fig. 11), suggesting NF-B activation.
Therefore, it is suggested that CPE induces macrophage
activation through the NF-B signaling pathway. Both
CPE and LPS are large molecules that cannot penetrate
into cells. In the case of LPS, the molecule binds to
CD14 and activates PKC and PKA27,28) but the mem-
brane receptor in macrophages for CPE has not yet been
determined.
1434 A.-J. KIM et al.

LPS (10 µ g/ml) + - - - - -

CPE ( µ g/ml) - 0 5 10 30 50

TNF-α

α -tubulin

160
**
140
**
120
TNF-α (% of control)
**
100 ** **
80

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60

40

20

LPS (10 µg/ml) + - - - - -

CPE ( µ g/ml) - 0 5 10 30 50

Fig. 5. Effects of CPE on the Production of TNF-
.

RAW 264.7 cells were stimulated with CPE (5, 10, 30, or 50 mg/ml) or LPS (10 mg/ml) for 24 h. Cell extracts were then prepared and
subjected to Western immunoblotting using antibody for TNF-
. The results are representative of three independent experiments. Significance
was determined using the Duncan test versus the control group ( p-values < 0.01,  p < 0:05).

LPS (10 µg/ml) + - - - - -

CPE ( µg/ml) - 0 5 10 30 50

TNF-α

β-actin

120

100 ** **
**
TNF-α (% of control)

80

60

40

20

LPS (10 µ g/ml) + - - - - -

CPE ( µg/ml) - 0 5 10 30 50

Fig. 6. Effects of CPE on the mRNA Expression of TNF-
.

RAW 264.7 cells were stimulated with CPE (5, 10, 30, or 50 mg/ml) or LPS (10 mg/ml) for 24 h. Total RNA was isolated, and the gene
expression of TNF-
was analyzed by RT-PCR. All experiments were done in triplicate. Significance was determined by the Duncan test versus
the control group ( p-values < 0.01,  p < 0:05).

In summary, our study indicates that CPE stimulates 2). We also discovered phosphorylation and degradation
RAW 264.7 to secrete inflammatory mediators (such as of IB
, which is required for NF-B activation. These
NO and H2 O2 ) and cytokines (TNF-
, iNOS and COX- results indicate that CPE induces macrophage activation
 Immunostimulating Activity of Polysaccharide 1435

LPS (10 µg/ml) + - - - - -

CPE ( µ g/ml) - 0 5 10 30 50

iNOS

α-tubulin

250

iNOS (% of control) 200 **

150
**
**
**

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100
**

50

LPS (10 µ g/ml) + - - - - -

CPE (µ g/ml) - 0 5 10 30 50

Fig. 7. Effects of CPE on the Activation of iNOS.

RAW 264.7 cells were stimulated with CPE (5, 10, 30, or 50 mg/ml) or LPS (10 mg/ml) for 24 h. Cell extracts were then prepared and
subjected to Western immunoblotting using antibody for iNOS.
-Tubuline was used as an internal control. The density of the band was
calculated with an image analyzer. All experiments were done in triplicate. Significance was determined by the Duncan test versus the control
group ( p-values < 0.01,  p < 0:05).

LPS (10 µ g/ml) + - - - - -

CPE ( µg/ml) - 0 5 10 30 50

iNOS

β-actin

160

140 **
120 **
iNOS (% of control)

100 ** ** **

80

60

40

20

LPS (10 µ g/ml) + - - - - -

CPE ( µ g/ml) - 0 5 10 30 50

Fig. 8. Effects of CPE on the mRNA Expression of iNOS.

RAW 264.7 cells were stimulated with CPE (5, 10, 30, or 50 mg/ml) or LPS (10 mg/ml) for 24 h. Total RNA was isolated, and the gene
expression of iNOS was analyzed by RT-PCR. All experiments were done in triplicate. Significance was determined by the Duncan test versus
the control group ( p-values < 0.01,  p < 0:05).
1436 A.-J. KIM et al.

LPS (10 µg/ml) + - - - - -

CPE ( µg/ml) - 0 5 10 30 50

COX-2

α-tubulin

300

250 **
COX-2 (% of control)

200 **

150

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100 **
**
50

LPS (10 µ g/ml) + - - - - -

CPE ( µ g/ml) - 0 5 10 30 50

Fig. 9. Effects of CPE on the Activation of COX-2.

RAW 264.7 cells were stimulated with CPE (5, 10, 30, or 50 mg/ml) or LPS (10 mg/ml) for 24 h. Cell extracts were then prepared and
subjected to Western immunoblotting using antibody for COX-2.
-Tubuline was used as an internal control. The density of the band was
calculated with an image analyzer. All experiments were done in triplicate. Significance was determined using the Duncan test versus the control
group ( p-values < 0.01,  p < 0:05).

LPS (10 µg/ml) + - - - - -

CPE ( µg/ml) - 0 5 10 30 50

COX-2

α-actin

140

120 **
**
**
COX-2 (% of control)

100 ** **
80

60

40

20

LPS (10 µg/ml) + - - - - -

CPE ( µg/ml) - 0 5 10 30 50

Fig. 10. Effects of CPE on the mRNA Expression of COX-2.

RAW 264.7 cells were stimulated with CPE (5, 10, 30, or 50 mg/ml) or LPS (10 mg/ml) for 24 h. Total RNA was isolated, and the gene
expression of COX-2 was analyzed by RT-PCR. Significance was determined by the Duncan test versus the control group ( p-values < 0.01,

p < 0:05).
 Immunostimulating Activity of Polysaccharide 1437

LPS (10 µg/ml) + - - - - -

CPE ( µg/ml) - 0 5 10 30 50

pIκBα

Total IκBα

α-tubulin
200

180
** **
160
**
pIκBα (% of control)
140
**
120

100 **

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80

60

40

20

LPS (10 µg/ml) + - - - - -

CPE ( µg/ml) - 0 5 10 30 50

Fig. 11. Effects of CPE on the Phosphorylation of IB
.

RAW 264.7 cells were stimulated with CPE (5, 10, 30, or 50 mg/ml) or LPS (10 mg/ml) for 24 h. Cell lysates were then prepared and subjected
to Western immunoblotting using an antibody for phospho-IB.
-Tubulin was used as an internal control. The density of the band was
calculated with an image analyzer. All experiments were done in triplicate. Significance was determined using the Duncan test versus the control
group ( p-values < 0.01,  p < 0:05).

and immunostimulating activity through the NF-B
signaling pathway. Further work on the chemical puri-
fication and structural determination of CPE is pro-
gressing in order to find the structure-activity relation-
ship and to clarify the overall intracellular process as
related to macrophage activation.
Acknowledgments
This work was supported by the National Research
Lab Program of Korea through the Functional Bio-
polymer Lab at Yonsei University (2000-N-NL-01-C-
299), and partly by the Yonsei Biomolecule Research
Initiative of the Two-Step Brain Korea 21 Project.
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