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In vivo antitumor effects of bitter principles

from the antlered form of fruiting bodies of
Ganoderma lucidum

Article in Journal of Natural Medicines December 2005

Impact Factor: 1.59 DOI: 10.1007/s11418-005-0003-5

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J Nat Med (2006) 60: 4248
DOI 10.1007/s11418-005-0003-5

O R I GI N A L P A P E R

Jiang Jing Gao Akiko Hirakawa Byung Sun Min

Norio Nakamura Masao Hattori

In vivo antitumor effects of bitter principles from the antlered form

of fruiting bodies of Ganoderma lucidum

Received: 9 May 2005 / Accepted: 1 July 2005 / Published online: 23 September 2005

The Japanese Society of Pharmacognosy and Springer-Verlag 2005

Abstract Two triterpene fractions and a single gano-

derma alcohol obtained from an antlered form of the
Introduction
fruiting bodies of Ganoderma lucidum were examined for
Medicinal mushrooms have been used for a long time in
their antitumor eects on the growth of inoculated
traditional medicine. The potential medicinal value and
mouse Lewis lung carcinoma in mice by intraperitoneal
wide acceptability of these edible mushrooms have at-
administration. The ganoderma alcohol fraction signif-
tracted increasing interests in the search for biologically
icantly suppressed the tumor growth at doses of 50 and
active substances from them [1, 2]. Ganoderma lucidum
100 mg/kg in the treatment period, and even after the
Karst (Reishi in Japanese), a species of basidiomycetes
administration, showing antitumor activity with a T/C
that belongs to Ganodermataceae of Aphyllophorales
value of 70.6% at a dose of 100 mg/kg. On the other
[3], is one of the most popular chemopreventive mush-
hand, no obvious activity was shown at each dose in the
room in China, Japan, Korean, and other Asian coun-
ganoderma-acid-fraction-treated groups. Furthermore,
tries and has been under modern pharmacological
ganoderiol F, which exhibited the strongest cytotoxicity
research during the last 30 years. Nowadays, according
against four tumor cell lines among ve ganoderma
to the conrmation and elucidation of its pharmaco-
alcohols examined, remarkably inhibited the tumor
logical activities and clinical eects, G. lucidum and re-
growth, accounting for 63.7% and 78.7% of control
lated products are widely used as not only health foods
group at a dose of 5 mg/kg, 54.1% and 63.0% at a dose
but also clinical drugs for the prevention and treatment
of 10 mg/kg, and 47.7% and 53.9% at a dose of 20 mg/
of various kinds of diseases, such as hypertension, leu-
kg in and after the administration period, respectively, in
copenia, and cancer [4].
a dose-dependent manner. These results suggest that the
Many biological activities of components from fru-
antitumor eects of bitter principles in G. lucidum are
iting bodies, mycelia, and spores of G. lucidum have been
mainly due to ganoderma alcohols.
identied. The antitumor and immune-enhancing prop-
erties of polysaccharides have been deeply investigated
Keywords Antitumor eect Ganoderma lucidum
both in vitro and in vivo [58], and the water-soluble
Bitter principle Triterpene Ganoderiol F Lewis lung
extracts of this mushroom have been used in traditional
carcinoma Cytotoxicity
medicine as antitumor and immunomodulating agents
[9]. In contrast, for another major group of constituents,
triterpenes, which are bitter in taste and characteristic
principles in this mushroom, few studies have been
conducted about their antitumor activities in vivo al-
J. J. Gao A. Hirakawa B. S. Min N. Nakamura though over 140 highly oxygenated and pharmacologi-
M. Hattori (&) cally active lanostane-type triterpenes have been isolated
Institute of Natural Medicine,
Toyama Medical and Pharmaceutical University, 2630 Sugitani,
from this mushroom [10]. Extraordinarily, because of
Toyama 930-0194, Japan the diculty in obtaining sucient amounts of pure
E-mail: saibo421@ms.toyama-mpu.ac.jp compounds for animal experiments, no in vivo study on
Tel.: +81-76-4347630 the antitumor eect of G. lucidum using a single triter-
Fax: +81-76-4345060 pene component has been reported.
B. S. Min Recently, the triterpene-enriched fractions and ex-
Immunomodulator Laboratory, tracts from the umbrella form of the fruiting bodies and
Korea Research Institute of Bioscience and Biotechnology, mycelia of G. lucidum were reported to have antitumor
Taejon, Korea

activities in vitro or in vivo [11, 12]. Indeed, we have
found previously that the triterpenes isolated from G.
lucidum exhibited cytotoxicities against mouse sarcoma
Meth-A (lucidumols A and B, ganodermanondiol,
ganodermanontriol, and ganoderiol F) and S-180
(ganodermanonol and ganodermanondiol), mouse LLC
(lucidumol A, ganoderiol F, and ganodermanontriol),
and human breast carcinoma T-47D (ganodermanonol
and ganodermanondiol) cells [13, 14]. These results led us
to evaluate ganoderma triterpenes as antitumor drugs. In
this connection, we investigated the in vivo antitumor
eects of ganoderma alcohol and acid fractions obtained
from an antlered form of the fruiting bodies of G. luidum
using LLC-bearing mice. Furthermore, on the basis of
the cytotoxicity assay, ganderiol F was selected to
examine its antitumor eect in LLC-inoculated mice as a
representative ganoderma alcohol from this mushroom.
Materials and methods
Plant materials
An antlered form of the fruiting bodies of G. lucidum
was provided by TDK Co. Ltd (Akita, Japan), and their
specimens are deposited at Museum of Material Medica,
Toyama Med. Pharm. University, Toyama, Japan.
Chemicals and media
Column chromatography was carried out on Florisil
(100200 mesh, Nacalai Tesque, Kyoto, Japan) and sil-
ica gel (Kieselgel 60, 70230 mesh, Merck Co., Darms-
tadt, Germany). Thin-layer chromatography (TLC) was
done on pre-coated silica gel 60 F254 plates (0.25 mm,
Merck), and spots were detected under a UV light and
by spraying with 10% H2SO4 followed by heating.
Roswell Park Memorial Institute (RPMI) 1640 was
purchased from ICN Biomedicals Inc. (Ohio, USA) and
fetal bovine serum from JRH Biosciences Co. (Lenexa,
USA). Streptomycin and penicillin G potassium ob-
tained from Wako Pure Chemical Co. (Osaka, Japan),
sulforhodamine B (SRB) and adriamycin from Sigma
Chemical Co. (St. Louis, USA).
Preparation of ganoderma alcohol and acid fractions
Ganoderma alcohol and acid fractions were separated
according to our previous report [15]. One hundred and
ninety grams of pulverized fruiting bodies of G. lucidum
(antlered form) was extracted three times with 20 vol-
umes of CHCl3 by reuxing in a boiling water bath for
3 h, and the combined extracts were evaporated to
dryness in vacuo. Half of the CHCl3 extract (4.45 g) was
chromatographed on a Florisil column (25.0 g) eluted
with hexaneacetone (9:1 and 7:3), and CHCl3MeOH
(1:2) to give three fractions, monitoring by TLC. The
43
following two fractions, corresponding to ganoderma
alcohols (688.5 mg) and acids (2.15 g), respectively, were
evaporated in vacuo and used in this experiment as
alcohol and acid fractions.
Isolation of ganoderma alcohols
Eighteen grams of the CHCl3 extract obtained from
380 g of pulverized fruiting bodies of G. lucidum (ant-
lered form) mentioned as above was applied to a column
of Florisil (40 g). The elution was started with hexane
acetone (9:1, 8:2, and 7:3), then CHCl3MeOH (1:1) to
yield six fractions (fractions 16, 264 mg, 540 mg,
960 mg, 1.34 g, 8.14 g, and 444 mg, respectively). Flor-
isil chromatography of fraction 2 (hexaneacetone, 8:2)
gave ganodermadiol ( 6.0 mg) [16]. Repeated column
chromatography of fraction 3 on silica gel (hexane
acetone, 8:2) followed by preparative HPLC using a RP-
ODS-80TS column, and a linear gradient of CH3CN
(75% 95%) in 2% AcOH; 5 ml/min, aorded lu-
cidumol B ( 6.2 mg) and ganoderiol F ( 84.8 mg) [17].
Separation of fraction 4 by preparative HPLC (CH3CN
in 2% AcOH, 70% 90%) after chromatography on
Florisil and silica gel columns furnished ganoderm-
anontriol ( 48.2 mg) and ganodermatriol ( 28.0 mg) [18,
19]. The structures of these compounds were determined
by spectroscopic methods including 1D- and 2D-NMR,
UV, IR, and EIMS (Fig. 1).
Tumor cells
Meth A (mouse sarcoma) and Lewis lung carcinoma
(LLC, mouse) cells were purchased from RIKEN Cell
Line Bank (Tsukuba, Japan). Sarcoma 180 (mouse sar-
coma) and T-47D (human carcinoma) were obtained
from Dainippon Pharmaceutical Co. (Osaka, Japan).
The cells were maintained as monolayer cultures in
RPMI 1640 medium supplemented with 5% FBS, so-
dium bicarbonate (2 g), penicillin G (100,000 units), and
streptomycin (100 mg) in a humidied 5% CO2 atmo-
sphere at 37C.
Animals
Specic pathogen-free female BDF-1 mice, purchased
from Japan SLC Inc. (Shizuoka, Japan), were used at 4
weeks of age, weighing 1416 g. The animals were fed
with a commercial pellet chow (Clea Japan Inc., Tokyo,
Japan) in a temperature-controlled room at 25 2C
and water ad libitum.
Cytotoxicity assay
The in vitro tumor cell assay was carried out by SRB
method as described previously [14, 20]. The 50%
44

Fig. 1 Structures of ganoderma R2

alcohols isolated from
G. lucidum 25 R4
20
R
R5 3

R1

Compound R1 R2 R3 R4 R5

OH
Ganodermadiol (1) 24(25) CH2OH CH3
H
OH OH
Lucidumol B (2) OH CH3 CH3
H H

Ganoderiol F (3) O 24(25) CH2OH CH2OH

OH
Ganodermanontriol (4) O OH CH2OH CH3
H

OH
Ganodermatriol (5)
H
24(25) CH2OH CH2OH

inhibitory concentration (IC50) was calculated by the
Probit method [21].
Animal experiment
For the animal experiments, alcohol and acid fractions
and ganoderiol F were completely suspended in Tween
20 followed by dilution with 0.9% NaCl solution. The
nal concentration of Tween 20 in sample solution was
2%. Adriamycin was dissolved in 0.9% NaCl solution.
For the subcutaneous tumor assay [22], a suspension
of 5 105 LLC cells in 0.1 ml of 0.9% NaCl solution was
carefully inoculated intradermally into the left axilla of
mice. Then, the tumor-bearing mice were randomly as-
signed to treatment experimental groups (ve or six mice
in each group). Twenty-four hours after the tumor
inoculation, mice were intraperitoneally given ganoder-
ma alcohol and acid fractions (both of 50 and 100 mg/
kg), ganoderiol F (5, 10, and 20 mg/kg), and adriamycin
(2 mg/kg, as a positive control) once daily for 14 con-
secutive days. Control animals were given 0.1 ml of
0.9% NaCl solution.
The tumors were measured on each alternate day
using a vernier caliper from the initiation of treatment to
the time when grosses ulceration of the tumor was
developed in control mice. The tumor size was calcu-
lated from the following equation [22]:
tumor vol: mm3 0:5
a
b2
where a is the longest diameter and b is the shortest
diameter. The eects by treatments were represented as
follows:
T=C% mean value of a treated
group=mean value of a control group
100
Statistical analysis
The signicance of dierences between the experimental
groups and control group was calculated by Dunnetts t
test. P < 0.05 was considered signicant.
Results
Eects of triterpene fractions from the antlered form of
the fruiting bodies of G. lucidum on subcutaneously (s.c.)
inoculated LLC
The fractions of ganoderma alcohols and acids sepa-
rated from the antlered form of the fruiting bodies were
intraperitoneally administrated to LLC-bearing mice
with doses of 50 and 100 mg/kg per day for 14
 45

consecutive days starting from the day after the intra- Table 1 Cytotoxicity of ganoderma alcohols isolated from Gano-
derma lucidum on tumor cell growth. IC50 50% inhibitory con-
dermal inoculation of tumor cells. As shown in Fig. 2, centration, Meth-A mouse carcinoma, LLC Lewis lung carcinoma,
the tumor sizes of ganoderma-alcohol-fraction-treated T-47D human breast carcinoma
groups were 1,714245 and 1,451201 mm3 at doses
of 50 and 100 mg/kg per day, respectively, on day 13 in Compound IC50 (lg/ml)
the administration period while 2,542336 mm3 for the Meth-A S-180 LLC T-47D
saline control group. Administration of the ganoderma
alcohol fraction resulted in signicant suppression of Ganodermadiol >20 >20 >20 >20
tumor growth at both doses with T/C values of 67.5 and Lucidumol B >20 >20 >20 6.5
57.1%, compared with the control (data not shown). Ganoderiol F 10.1 7.1 2.0 3.0
Ganodermanontriol >20 >20 >20 >20
Moreover, after the administration period, the tumor Ganodermatriol >20 >20 15.0 >20
size of the 100 mg/kg group was 2,226374 to Adriamycina 0.13 0.11 0.06 0.02
5,0051,158 mm3 (on day 1620), accounting for 67.5 a
72.9% of the control level (3,297390 to Positive control
6,865390 mm3 on days 1620). As for the acid frac-
tion, no appreciable activity was observed at each dose.

activity in a cytotoxicity assay, was administrated to

Cytotoxicity of ganoderma alcohols isolated from
G. lucidum against four tumor cell lines
Five ganoderma alcohols, including ganodermadiol, lu-
cidumol B, ganoderiol F, ganodermanontriol, and
ganodermatriol, isolated from G. lucidum were tested for
their cytotoxic activities against LLC, T47D, S-180, and
Meth-A cells in vitro (Table 1). Among these com-
pounds, ganoderiol F showed the most appreciable
toxicity, with IC50 values of 2.0, 3.0, 7.1, and 10.1 lg/ml
against LLC, T47D, S-180, and Meth-A cell lines,
respectively.
Eect of ganoderiol F on subcutaneously (s.c.) im-
planted LLC
To evaluate the antitumor eect of a single ganoderma
alcohol, ganoderiol F, which exhibited the strongest
LLC-bearing mice at three doses of 5, 10, and 20 mg/kg/
day. When given intraperitoneally once daily for 2
weeks, the compound signicantly reduced the tumor
size to 1,502197, 1,275232, and 1,124180 mm3
compared with the control group (2,357188 mm3) at
three doses, respectively, and their T/C values were
63.7%, 54.1%, and 47.7% on day 14 in the adminis-
tration period (Fig. 3). Moreover, on day 20 after the
administration period, the tumor sizes of ganoderiol-F-
treated group were 4,086543, 3,272576, and
2,802265 mm3 at doses of 5, 10, and 20 mg/kg,
respectively, accounting for 78.7%, 63.0%, and 53.9%
of the control group (5,194573 mm3). Ganoderiol F
showed remarkable inhibitory eect on the tumor
growth in a dose-dependent manner both in and after
the administration period (Fig. 3). The T/C values of the
positive control treated with adriamycin were of 38.0%
and 44.5% in and after administration period, respec-
tively, at a dose of 2 mg/kg.

Fig. 2 In vivo antitumor eects

of alcohol and acid fractions
separated from the antlered
form of fruiting bodies of
Ganoderma lucidum on
intradermally inoculated Lewis
lung carcinoma (LLC) cells in
mice by intraperitoneally
administration. Data are
expressed as mean S.E.
(n=56). Statistical
signicance: *P<0.05,
**P<0.01, ***P<0.001 versus
control
46

Fig. 3 In vivo antitumor eects

of ganoderiol F isolated from
Ganoderma lucidum on
intradermally inoculated Lewis
lung carcinoma (LLC) cells in
mice by intraperitoneal
administration. Data are
expressed as mean S.E.
(n=56). Statistical
signicance: *P<0.05,
**P<0.01, ***P<0.001 versus
control

Fig. 4 Body-weight changes of

mice treated with alcohol and
acid fractions by intraperitoneal
administration (n=56)

Fig. 5 Body-weight changes of

mice treated with ganoderiol F
by intraperitoneal.
administration (n=56)

Toxic and side eects of intraperitoneally
administrated ganoderma alcohol and acid fractions and
ganoderiol F
No evident toxic or side eects were observed, such as
the weight reduction observed in the adriamycin-treated-
group at the end of the treatment. The mean body
weights of the mice treated with alcohol and acid frac-
tions decreased down to 5 days but then gradually in-
creased, becoming almost the same as control body
weights (Fig. 4). There was no signicant dierence in
body weights of mice administrated with ganoderiol F

(Fig. 5). The skin and hair texture, and behavioral pat-
tern did not reect any toxic reaction at experimental
doses.
Discussion
Basidiomycetes, such as Agaricus blazei, Lentinan ed-
odes, Polyporus umbellatus, and G. lucidum, have been
used by about 1,000,0002,000,000 people in Japan for
the prevention of cancer and/or as an adjuvant with
cancer chemotherapy drugs after the removal of malig-
nant tumors [12]. As regards G. lucidum, a large number
of research groups have proved the antitumor eects of
the polysaccharides and water extracts containing a- and
b-glucans, either in vitro or in vivo [6, 7, 23]. But the
ecacy on carcinogenesis of triterpenes is not yet well
proven.
In our recent studies on the comparison of biological
activity between two fruiting bodies of germinated and
mature antlered forms of G. lucidum, we found that the
aqueous extracts of the mature one, which have a much
higher content of total triterpenes, tended to be more
potent in antitumor activity than another in vivo al-
though the total polysaccharide contents of the two ex-
tracts were quite similar [24]. Hence, we speculate that
not only the polysaccharide content but also the triter-
pene content will inuence the antitumor activities of
G. lucidum. Consequently, we investigated the in vivo
antitumor eects of triterpene fractions from G. lucidum
in the present study. The intraperitoneal administration
for 14 consecutive days of ganoderma alcohol and acid
fractions separated from the antlered form of the fruit-
ing bodies of G. lucidum to LLC-bearing mice resulted in
signicant suppression of tumor growth both in and
after the administration period while no eect was ob-
served for the acid fraction.
This nding is very interesting when related to the
cytotoxic activities of triterpenes isolated from G. luci-
dum against several tumor cell lines, which have been
performed in our previous research [13, 14]. Of the 24
triterpenes tested, all seven alcohols (lucidumol A, lu-
cidumol B, ganodermanondiol, ganoderiol F, gano-
dermanontriol, ganodermanonol, and ganodermadiol)
exhibited cytotoxicities against several cell lines. Gano-
dermanonol and ganodermanondiol exhibited the most
potential cytotoxicity against Meth-A cells, with ED50
values of 2.8 and 3.4 lg/ml, respectively; and lucidumol
A, ganoderiol F, and ganodermanontriol had inhibitory
activity on LLC cells with ED50 values of 2.3, 6.0, and
9.6 lg/ml, respectively. As for the 17 ganoderma acids,
except for ganoderic acids h and G, none showed activ-
ities. Subsequently, we isolated ve ganoderma alcohols
including ganodermadiol, ganodermanontriol, ganoder-
matriol, lucidumol B, and ganoderiol F from the same
strain of G. lucidum in which ganoderiol F exhibited the
strongest cytotoxicity against LLC, T47D, S-180, and
Meth-A cells. We next examined the in vivo antitumor
eect of ganoderiol F. As shown in Fig. 3, when
47
administrated by the same method, ganoderiol F showed
remarkable inhibitory eect on the growth of inoculated
LLC both in and after the administration period in a
dose-dependent manner over the range tested.
The mechanism of the antitumor eects of G. lucidum
is not yet well understood. It has been found previously
that polysaccharides from G. lucidum exert their in vitro
and in vivo antitumor eects via immunomodulatory
properties, including the enhancement of lymphocyte
proliferation and antibody production [25], and pro-
duced both antigenotoxic and antitumor-promoting
activities [23, 26]. Triterpene components were reported
to exhibit the biological activities of hepatoprotection
[27], antihypertension [28], and inhibitory eects on the
cholesterol synthesis [29], mediated by inhibiting the
activities of enzymes such as b-galactosidase, angioten-
sion-converting enzyme, and cholesterol synthase. A
recent report pointed out that a triterpenoid fraction
from the fruiting bodies of G. lucidum had antitumor
and antimetastatic eects on liver through the inhibition
of tumor-induced angiogenesis, but the molecular
mechanism was not investigated [12]. More recently, Lin
et al. reported a triterpene-enriched extract from G. lu-
cidum inhibited the growth of hepatoma cells by sup-
pressing the protein kinase C, activating the c-Jun N-
terminal kinase/stress-activated protein kinase (JNK/
SAPK) and p38 mitogen-activated protein (MAP) ki-
nase, as well as prolonging the G2 cell-cycle phase in
Huh-7 cells [11].
It is noteworthy that this is the rst report about the
in vivo study on antitumor activity of G. lucidum using a
single triterpene component. It is said that the polysac-
charides of large molecular weights are not absorbed
within intestines, and the medicinal actions of mush-
rooms such as G. lucidum may be mediated by a com-
bination of the polysaccharides and other components
[30]. The present study emphasizes that in antitumor
activity, the other components may be ganoderma
alcohols, and suggests that they are potential antitumor
agents. Further studies, including the inhibitory mech-
anism of antitumor action, are necessary to understand
the antitumor eects of ganoderma alcohols in
G. lucidum.
Acknowledgements This paper is a part of the 21st COE program
sponsored by the Ministry of Education, Culture, Sports, Science,
and Technology, Japan. The authors are grateful to TDK Service
Corporation (Tokyo, Japan) and Reishi Sougou Kenkyu Jo (To-
kyo, Japan) for providing ganoderma samples and nancial
support.
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