PHYTOTHERAPY RESEARCH

Phytother. Res. 22, 1282–1291 (2008)

1282 Published online 20 June 2008 in Wiley InterScience
G. G. L. YUE ET AL.
(www.interscience.wiley.com) DOI: 10.1002/ptr.2478

Comparative Studies on the Immunomodulatory

and Antitumor Activities of the Different Parts
of Fruiting Body of Ganoderma lucidum and
Ganoderma Spores

Grace G. L. Yue1, Kwok-Pui Fung1,2, Ping-Chung Leung1 and Clara B. S. Lau3*

1
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China
2
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China
3
School of Pharmacy, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China

Ganoderma lucidum (GL, Lingzhi) has been suggested as a candidate for immunomodulation and cancer
treatment. The present study aimed at comparing the different parts of the fruiting body (whole fruiting body,
pileus and stipe) of GL as well as Ganoderma spores (sporoderm-broken and -unbroken), with regard to their
antitumor and immunomodulatory activities in S-180 sarcoma-bearing mice. The hot water extracts of differ-
ent parts of GL or the Ganoderma spores were orally administered to the sarcoma-bearing mice. The results
showed that GL whole fruiting body, stipe and sporoderm-broken spore possessed stronger inhibitory activities
on sarcoma growth when compared with the pileus extract. Higher immunomodulatory activities in terms of
enhancing the proliferative responses and the cytokines (IFN-γ, IL-4 and IL-6) production of spleen lymphocytes
were also found in GL stipe and sporoderm-broken spore treatment groups. The sporoderm-broken spores
had higher stimulatory effects on mitogen-activated spleen lymphocytes of healthy mice than those of sarcoma-
bearing mice. In addition, the immunostimulatory activities of GL hot water extracts and Ganoderma spores
were shown to be comparable; hence the latter did not show superiority in efficacy. This is the first com-
parative study on the immunomodulatory activities of Ganoderma spores and the fruiting body extracts.
Copyright © 2008 John Wiley & Sons, Ltd.

Keywords: Ganoderma lucidum; Ganoderma spores; antitumor; immunomodulatory; sarcoma.

polysaccharides isolated from spores could significantly

INTRODUCTION
In traditional Chinese medicine, Ganoderma was sug-
gested to perform the functions of strengthening body
resistance and lengthening life span (Zhao and Zhang,
2000; Chang and Buswell, 1999). Nowadays, it is the
main ingredient in a number of tonic formulations and
health supplements. In modern pharmacological studies,
Ganoderma lucidum (GL) was demonstrated to possess
antitumor and immunomodulatory properties. Previous
studies demonstrated that the polysaccharides isolated
from GL were able to inhibit the growth of sarcoma
transplanted in mice (Maruyama et al., 1989; Ohno
et al., 1998). The antitumor activities of GL were sug-
gested to act by modifying the host immune responses.
A mixture of GL fruiting body extract and spores as
well as the polysaccharides isolated from the antler-
shaped GL have been shown to have immunostimulat-
ing activity in vivo and in vitro (Kimura et al., 2002;
Bao et al., 2002a, 2002b; Kohguchi et al., 2004). The
ethanol extracts of G. lucidum spores were also shown
to have cytotoxic activities against human and mouse
tumor cells (Zhao et al., 2000; Zhu et al., 2000; Min
et al., 2000). In vivo studies suggested that the adminis-
tration of germination activated GL spores or the
* Correspondence to: Clara B. S. Lau, School of Pharmacy, Faculty
of Medicine, The Chinese University of Hong Kong, Shatin, New Territories,
Hong Kong SAR, China.
E-mail: claralau@cuhk.edu.hk
Copyright © 2008 John Wiley & Sons, Ltd.
Copyright © 2008 John Wiley & Sons, Ltd.
inhibit growth in mice transplanted with S180 sarcoma
(Jiang et al., 2005; Liu et al., 2002; Chen et al., 2002).
Ganoderma spores or their extracts were shown to
possess stimulating effects on lymphocyte proliferation
in vitro and antibody production in vivo (Bao et al.,
2001a, 2001b, 2002b; Fan et al., 2005; Lin et al., 2005).
As health supplements, different commercial products
of GL can be found, such as fruiting bodies in whole or
sliced pieces, extracts and spores in capsules. However,
detailed scientific research regarding the biological dif-
ferences among the different parts of the fruiting body,
i.e. the whole fruiting body, pileus and stipe, has not
been studied. A previous study showed the differential
antiproliferative effects and stimulating activities of the
different parts of GL fruiting body in human breast
cancer cells and mouse spleen lymphocytes, respectively
(Yue et al., 2006). In this study, the antitumor and
immunomodulatory activities of the hot water extracts
from the different parts of the GL fruiting body were
investigated in tumor-bearing mice. The effects of
Ganoderma spores were also examined and compared
with those of GL hot water extracts. The chemical com-
position of these extracts, including their soluble sugars
and amino acids were examined. The polysaccharides
were also characterized according to their molecular
size distribution. Through our comparative studies on
the biological activities of various types of Ganoderma,
more scientific evidence can be provided for the public
to assist them in choosing health supplements wisely.
Phytother. Res.Received 26 July(2008)
22, 1282–1291 2007
Revised 13DOI:
November 2007
10.1002/ptr
Accepted 28 November 2007
 ANTITUMOR AND IMMUNOMODULATORY EFFECTS OF GANODERMA STIPE
MATERIALS AND METHODS
Herbal materials. Ganoderma lucidum (GL) and Gano-
derma spores used in this study were purchased from
a herbal store in Hong Kong and in China, respec-
tively. Voucher specimens of the fungus and spores were
deposited in the museum of Institute of Chinese Medi-
cine, the Chinese University of Hong Kong. Fruiting
body samples and spores were authenticated using
morphological characters of the fruiting bodies by my-
cology experts. The breakage procedures of sporoderm
were completed by an ultra-fine grinding technique.
The fruiting bodies were dried in an oven (60 °C) for
24 h. The pilei, the stipes and the whole fruiting bodies
of GL (100 g) were extracted with distilled water
(1000 mL) by reflux for 2 h and repeated once. The
supernatant was collected, centrifuged and freeze-dried.
The percentage yield of the extracts from the pilei (GL-
P), stipes (GL-S) and whole fruiting bodies (GL-F) were
5.8, 4.3 and 5.0 (w/w), respectively. Two samples of
Ganoderma spores, namely sporoderm-broken spores
(BS) and sporoderm-unbroken spores (US) were used
in this study. The spores were observed microscopi-
cally with an Olympus inverted microscope (IX-71,
Olympus, Tokyo, Japan). The spores were suspended
in distilled water and the suspension was loaded into
the counting chamber of a hemocytometer. The number
of sporoderm-unbroken spores in a 16-square set of
the counting chamber was counted. There were 4–5
sporoderm-unbroken spores observed in the BS sample
in triplicate, whereas 240–264 sporoderm-unbroken
spores were observed in the US sample.
Animals. Inbred male BALB/c mice, 6–8 weeks of age,
24–26 g in weight, were fed and kept in the University
Laboratory Animal Services Centre, the Chinese Uni-
versity of Hong Kong. All experiments were performed
under licence from Department of Health of Hong Kong
SAR and endorsed by the University Animal Experi-
mentation Ethics Committee.
In vivo antitumor studies. Mice were inoculated sub-
cutaneously with S-180 mouse sarcoma cells (5 × 106 in
0.2 mL phosphate-buffered saline) into the right inguinal
region. The tumor cells were allowed to grow for 10 days,
then the mice were grouped randomly into different
treatment groups and each group contained 8–10 mice.
Each mouse was then given one oral dose of dissolved
GL extracts, spores suspension or distilled water (as
control) every day for 14 consecutive days.
The dosage of the hot water extracts and spores for
mice was converted from the daily dosage for humans
recommended in the Chinese Pharmacopoeia (2005)
and by the pharmaceutical company. Since the recom-
mended human daily dosage of the extracts and spores
are 5–10 and 60–100 mg/kg body weight, respectively,
the three testing dosages selected in this study were
100, 200, 400 mg/kg body weight for the extracts and 1,
2, 4 g/kg body weight for the spores.
The hot water extracts were dissolved in 0.3 mL of
distilled water and the spores were suspended in 0.3 mL
distilled water. The mice were killed on day 15. The
body weight was recorded on the first day of oral
administration of extracts and on the day of killing.
The S-180 tumors were removed and weighed. The
Copyright © 2008 John Wiley & Sons, Ltd.
1283
spleens were also removed and weighed. The spleen
lymphocytes were isolated from the spleen as described
previously (Ho et al., 2004).
Immunostimulatory studies. The isolated lymphocytes
were resuspended in culture medium. The cell number
was counted with a hemocytometer and the viability of
the cells was checked by trypan blue exclusion assay.
The concentration of lymphocytes was adjusted to
4 × 106 cells/mL for experiments. A volume of 100 μL
of cell suspension was applied onto each well of a 96-
well microplate with or without mitogens. The mitogens,
concanavalin A (Con A, a T-lymphocyte mitogen) or
lipopolysaccharide (LPS, a B-lymphocyte mitogen),
were added at a final concentration of 1 and 10 μg/mL
in 100 μL plain medium, respectively. Both Con A and
LPS were purchased from Sigma (St Louis, MO, USA).
Plain medium (100 μL) was added to the control
wells (as a non-stimulated cell control). All plates were
incubated in 5% CO2-humidified atmosphere at 37 °C
for 72 h. After the incubation period, the microplates
were centrifuged for 10 min at 300 × g and culture
supernatants were collected for cytokine ELISA. The
proliferation of the cells was estimated by [methyl-3H]-
thymidine incorporation. Tritiated thymidine (0.5 μCi/
well, GE Healthcare, UK) was added to each well and
further incubated for 6 h. Then the cells were harvested
on glass fiber filters. Radioactivity in the filters was
measured by a Packard TopCount NXT™ Microplate
Scintillation and Luminescence Counter (PerkinElmer
Inc., MA, USA). The proliferative response was ex-
pressed as the mean % ratio of counts per minute in
mitogen-treated and non-stimulated (control) wells. The
culture medium used was RPMI 1640 medium, which
was supplemented with 10% (v/v) fetal bovine serum,
100 IU/mL penicillin and 100 μg/mL streptomycin. Sterile
phosphate-buffered saline and all the supplements
mentioned above were purchased from Invitrogen
Gibco (Grand Island, NY, USA).
Determination of mouse IL-2, IL-4, IL-6, IFN-γ and
TNF-α production. The mouse spleen lymphocyte
supernatants were subjected to test for the presence of
cytokines such as IL-2, IL-4, IL-6, IFN-γ and TNF-α by
ELISA. The BD OptEIA™ ELISA assay procedures
were carried out as described in the kit manual recom-
mended. The ELISA sets and the tetramethylbenzidine
(TMB) substrate reagent sets for the detection of
mouse cytokines were purchased from BD Bioscience
PharMingen (CA, USA).
Determination of carbohydrate content (phenol-sulfuric
acid spectrophotometric method). Ganoderma extracts
and spores were weighed accurately and added to dis-
tilled water at 10 mg/mL and 50 mg/mL, respectively,
and extracted by sonication for 60 min. The mixtures
were centrifuged. The supernatant was added to 95%
ethanol. The samples were kept at 4 °C for 10 h. After
precipitation, the mixtures were centrifuged and the
pellets were dissolved in distilled water as test mater-
ials. Test materials, standard solution or water were
added with 5% phenol (w/v) and concentrated sulfuric
acid. The mixture was mixed immediately. After cool-
ing to room temperature for 5 min, 200 μL of each
reaction mixture was transferred to a 96-well plate in
triplicate. The absorbance at a wavelength of 492 nm
Phytother. Res. 22, 1282–1291 (2008)
DOI: 10.1002/ptr
1284 G. G. L. YUE ET AL.

was measured spectrophotometrically with a BMG
FLUOstarOptima microplate reader (BMG Labtech
GmbH, Germany). A calibration curve was constructed
using standard glucose solution at six concentrations
(0.075, 0.15, 0.25, 0.35, 0.425 and 0.5 mg/mL).
Determination of the molecular weight of polysaccha-
rides. The relative molecular weights and the mono-
saccharide compositions in the polysaccharides from
GL extracts and Ganoderma spores were analysed as
described by Huang et al. (2006). The GL extracts and
Ganoderma spores were dissolved or suspended in
0.05 M NaH2PO4-Na2HPO4 buffer (pH = 6.7, 0.05%
NaN3) and were filtered through a 0.45 μm filter. The
samples were run on a TSK-Gel G3000SWXL column
(5 μm, 7.8 × 300 mm, Tosoh Bioscience LLC, PA, USA)
eluted with 0.05 M NaH2PO4-Na2HPO4 buffer to deter-
mine the molecular weight by gel filtration. The flow
rate was 0.5 mL/min. The phenol–sulfuric acid method
was utilized to monitor the collected fractions. A cali-
bration curve was constructed using standard dextran
with molecular weights of 0.738 × 103, 5.8 × 103, 1.22 ×
104, 2.37 × 104, 4.8 × 104, 1.0 × 105, 1.86 × 105, 3.8 × 105,
8.35 × 105 Daltons (Da).
Determination of the monosaccharide composition of
polysaccharides. The monosaccharide composition in
the polysaccharide from GL extracts and the Ganoderma
spore suspension were determined by GC-MS. The
tested samples were hydrolysed (2 M H2SO4, 100 °C, 6 h)
and evaporated to dryness. The hydrolysed samples
were then acetylated as described by Huang et al. (2006)
and analysed by GC-MS on DB-1 capillary column
(15 m × 0.2 mm) with a flame ionization detector
(Agilent 6890GC/5973MS instrument) in a temperature
gradient 100–280 °C at 10 °C/min. Identification and
quantification of monosaccharides were made in com-
parison with standards (Huang et al., 2006).
Determination of amino acid content in GL extracts
and Ganoderma spores. The content of amino acids in
GL extracts and Ganoderma spores were determined
by HPLC (Zheng et al., 2005). The HP1050 HPLC
system consists of a quaternary pump, an auto sampler,
a HP 1046A fluorescence detector, with fluorescence
excitation at 340 nm or 260 nm and emission at 450 nm
or 305 nm, and a C18 column (Hypersil ODS, 5 μm,
125 mm × 4.0 mm, Cheshire, UK). Mobile phase A con-
sisted of 10 mM Na2HPO4-NaH2PO4 (PB, pH 7.2, 0.5%
tetrahydrofuran); mobile phase B was PB, methanol,
acetonitrile in a ratio of 50:35:15 (v/v). The gradient
was A:B (100:0 to 0:100) for 0–25 min. The flow rate
was 1 mL/min. Each amino acid was quantified by the
calibration curve of the authentic amino acid (Sigma,
MO, USA). Methanol and acetonitrile (HPLC grade)
was obtained from Merck (Darmstadt, Germany); and
sodium dihydrogen phosphate-2-hydrate and di-sodium
hydrogen orthophosphate (AR grade) from Sigma (MO,
USA).
Statistical analysis. Data were presented as the mean ±
SD. Statistical differences between the various experi-
mental groups and the control groups were assessed by
the two-tailed Mann-Whitney rank sum test for in vivo
experiments and by Student’s t-test for in vitro experi-
ments. The statistically significant differences among
different experimental groups were performed using
one-way analysis of variance (ANOVA) followed by
Bonferroni post-test. The statistical analyses were
carried out using GraphPad PRISM software version
3.0 (GraphPad Software, San Diego, CA, USA). In
all comparisons, p < 0.05 was considered statistically
significant.
RESULTS
Effects of GL hot water extracts and Ganoderma
spores on sarcoma growth and the proliferative
responses of spleen lymphocytes
The hot water extracts of the GL whole fruiting body
(GL-F) or the Ganoderma spores (BS) were admini-
stered orally to sarcoma-bearing mice. The final body
weights as well as spleen and tumor weight are shown
in Table 1. The GL-F at 100 and 200 mg/kg and BS at
1 and 2 g/kg were found to inhibit significantly the
growth of implanted S-180 sarcoma (p < 0.05, Table 1).
The spleen lymphocytes isolated from the mice
treated with GL-F or BS, were cultured in microplates.
As shown in Fig. 1A and 1B, the proliferative responses
of Con A-stimulated spleen lymphocytes from the mice
treated with 200 and 400 mg/kg of GL-F as well as 2
and 4 g/kg of BS were significantly different from those
of the untreated control mice.

Table 1. Effects of GL hot water extracts and Ganoderma spores on the weights of body, spleen and tumor in S-180 sarcoma-bearing
mice

Group No. of mice Dose (mg/kg) Body weight (g) Spleen weight (mg) Tumor weight (mg)

Control 18 0 26.6 ± 0.3 151.5 ± 47.6 561.7 ± 116.9

GL-F 18 100 26.6 ± 1.06 177.0 ± 51.2 300.2 ± 60.9a
GL-F 18 200 26.3 ± 0.68 163.1 ± 27.0 281.1 ± 46.8a
GL-F 18 400 26.6 ± 1.26 169.2 ± 61.2 352.4 ± 62.2
Control 19 0 25.7 ± 1.29 119.3 ± 13.8 426.1 ± 172.0
BS 19 1000 25.8 ± 0.89 121.4 ± 13.3 330.5 ± 191.4a
BS 19 2000 26.7 ± 1.31 115.0 ± 3.5 305.0 ± 184.0a
BS 19 4000 25.3 ± 1.46 106.4 ± 15.5 329.9 ± 195.8

Each value is presented as the mean ± SD of 18–19 mice in each group in duplicate experiments. Differences between each treatment
group and the control group were tested using two-tailed Mann-Whitney rank sum test, a p < 0.05.

Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 1282–1291 (2008)
DOI: 10.1002/ptr
 ANTITUMOR AND IMMUNOMODULATORY EFFECTS OF GANODERMA STIPE 1285

Figure 1. Effects of GL hot water extracts and Ganoderma spores on the proliferative responses of spleen lymphocytes to Con A.
Mice inoculated with S-180 cells were treated daily with the GL extract (A) at 100, 200, 400 mg/kg or Ganoderma spores (B) at 1, 2,
4 g/kg by oral administration for 14 days. After treatment, the spleen lymphocytes isolated from each treatment group were seeded
in 96-well microplates and stimulated in vitro for 72 h with 1 μg/mL Con A. The proliferative response was assessed by [methyl-3H]-
thymidine incorporation and expressed as the mean % ratio of counts per minute in mitogen-treated and non-stimulated (control)
wells + SD of two independent experiments with 8–9 mice each (with six wells each). ** p < 0.01, *** p < 0.001 compared with the
control group by Student’s t-test.

Differential antitumor and immunomodulatory
effects of different parts of GL fruiting body
in sarcoma-bearing mice
As the optimal dose of GL-F for both inhibiting tumor
growth and enhancing proliferative responses of spleen
lymphocytes has been determined, i.e. 200 mg/kg, the
efficacies of the extracts from different parts of the GL
fruiting body (F: whole fruiting body; P: pileus; S: stipe)
were then compared. In Table 2, the spleen weights of
GL-S-treated mice were significantly different from
those of the control (water-treated) mice. The GL-F
and GL-S treatments could significantly reduce tumor
weights by 35.8% and 37.1%, respectively. The tumor
weights of GL-P-treated mice were lower than those of
the control untreated mice (reduced 18.4%); however,
the difference was not statistically significant (Fig. 2A).
To evaluate the proliferative response and the cyto-
kine production, spleen lymphocytes of mice from
each group were cultured in microplates and stimu-
lated with either Con A or LPS. As shown in Fig. 2B,
the proliferative responses of Con A-stimulated spleen
lymphocytes from the mice treated with GL-F and GL-
S were significantly different from those observed in
lymphocytes of the control mice (p < 0.05) and GL-P-
treated mice (p < 0.01). Alternatively, the proliferation
responses of LPS-stimulated spleen lymphocytes in the
groups treated with all parts of GL fruiting body (F, P
and S) were significantly different from those from the
control group (p < 0.05) (Fig. 2C). These results sug-
gest that T lymphocytes were activated by oral admini-
stration of GL-S and GL-F, while macrophages and/or
B lymphocytes could be activated by all parts of the
fruiting body.
Different activities of sporoderm-broken and
sporoderm-unbroken Ganoderma spores in sarcoma-
bearing mice
The oral administration of 2 g/kg Ganoderma spores
was shown to be most effective; therefore, the efficacies
of sporoderm-broken spores (BS) and sporoderm-
unbroken spores (US) were also compared at this con-
centration. The final body weights and spleen weights
were not significantly different among the treatment
groups (Table 2). The sarcoma weights of BS- and US-
treated mice were reduced by 31.5% and 22.4% when
compared with the control untreated mice (Fig. 3A).
The oral administration of spores to the mice signifi-
cantly enhanced the Con A- and LPS-activated spleen
lymphocytes proliferation (Fig. 3B and 3C). The
stimulatory effects on Con A-activated lymphocytes
of BS-treated group were significantly higher than
the US-treated group. Furthermore, the proliferation
responses of LPS-stimulated spleen lymphocytes in
the US-treated group were comparable to those of the
BS-treated group (Fig. 3C). These results suggest that
macrophages and/or B lymphocytes could be activated
by US treatment.

Table 2. Effects of different parts of GL fruiting body and Ganoderma spores on the weights of body and spleen in S-180 sarcoma-
bearing mice

Group No. of mice Dose (mg/kg) Body weight (g) Spleen weight (mg)

Control 17 – 25.9 ± 0.5 105.8 ± 5.2

Whole fruiting body (GL-F) 17 200 25.8 ± 0.4 110.2 ± 10.7
Pileus (GL-P) 17 200 25.0 ± 0.4 106.2 ± 8.4
Stipe (GL-S) 17 200 25.9 ± 0.5 117.7 ± 15.5a
Control 16 – 28.2 ± 0.2 119.3 ± 13.8
Sporoderm-broken spores (BS) 16 2000 27.9 ± 1.0 121.3 ± 13.3
Sporoderm-unbroken spores (US) 16 2000 28.9 ± 0.3 116.5 ± 5.4

Each value is presented as the mean ± SD of 16–17 mice in each group. Differences between each treatment group and the control
group were tested using two-tailed Mann-Whitney rank sum test, a p < 0.05.

Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 1282–1291 (2008)
DOI: 10.1002/ptr
1286 G. G. L. YUE ET AL.

Figure 2. Effects of the extract of different parts of GL fruiting body on tumor growth and proliferative responses of spleen
lymphocytes to Con A or LPS of S-180 sarcoma in mice. (A) Mice inoculated with S-180 sarcoma were treated daily with the extracts
of whole fruiting body (F), pileus (P) or stipe (S) of GL at concentrations of 200 mg/kg by oral administration for 14 days. Differences
in tumor weights between each treatment group and the control group were tested using two-tailed Mann-Whitney test. * p < 0.05,
** p < 0.01. Spleen lymphocytes of sarcoma-bearing mice isolated from each treatment group were seeded in 96-well microplates
and stimulated in vitro for 72 h with 1 μg/mL Con A (B) or 10 μg/mL LPS (C). The proliferative response was assessed by [methyl-3H]-
thymidine incorporation and expressed as the mean % ratio of counts per minute in mitogen-treated and non-stimulated wells + SD
of two independent experiments with 8–9 mice each (with six wells each). Differences between each treatment group with control
group were tested using unpaired Student’s t-test, # p < 0.05, ## p < 0.01. Differences among each treatment group were determined
by one-way ANOVA followed by Bonferroni post-test, a p < 0.05, b p < 0.01, c p < 0.001, ns, not significant.

In another set of experiments, a batch of healthy
mice was orally administered with Ganoderma spores
for 14 days in order to evaluate the effects of spores on
the immune system of healthy mice. The spontaneous
proliferations (i.e. without mitogen stimulation) of the
resting lymphocytes were enhanced in both the tumor-
bearing and healthy mice, which were treated with
spores (both BS and US) (Fig. 4A). Furthermore, the
augmentation of proliferation in tumor-bearing mice
was greater than that in healthy mice. In contrast, the
increases of proliferative responses to Con A were
greater in the lymphocytes from healthy mice than those
from tumor-bearing mice (Fig. 4B).
Effects of GL extracts and Ganoderma spore
administration on the cytokine production of spleen
lymphocytes of the treated mice
In order to elucidate the stimulatory activities of
these supplements, the concentrations of five cytokines,
namely IL-2, IL-4, IL-6, TNF-α and IFN-γ, in the
culture medium of lymphocytes were determined by
ELISA. As shown in Fig. 5, the IFN-γ, TNF-α and IL-
Copyright © 2008 John Wiley & Sons, Ltd.
2 production of Con A-activated spleen lymphocytes of
GL-F-treated mice were increased significantly, suggest-
ing the treatment could enhance the function of T-helper
cells. The production of IL-6 in spleen lymphocytes
treated with GL-F could be activated by LPS but not by
Con A (Fig. 5E and F). As the differential proliferative
responses in lymphocytes from GL-P- and GL-S-treated
groups were observed, the cytokine production by the
lymphocytes from different treated groups in response
to mitogens were also compared. The releases of IFN-
γ, TNF-α, IL-4 and IL-6 from the Con A-activated
lymphocytes of GL-S-treated group were significantly
higher than those of the GL-P-treated group (Fig. 5A,
B, D and E). Also, the release of IL-6 in LPS-activated
lymphocytes of GL-S-treated group were significantly
higher than those of the GL-P-treated group (Fig. 5F).
The oral administration of spores (BS and US)
to tumor-bearing mice enhanced the proliferative
responses of spleen lymphocytes to mitogens (Con A
or LPS). As shown in Fig. 6, the IFN-γ, IL-2 and IL-4
production of Con A-activated spleen lymphocytes from
BS- and US-treated mice increased significantly. The
IL-6 production in Con A-activated spleen lymphocytes
was found to be significantly increased in the BS-treated
Phytother. Res. 22, 1282–1291 (2008)
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 ANTITUMOR AND IMMUNOMODULATORY EFFECTS OF GANODERMA STIPE 1287

Figure 3. Effects of the extract of Ganoderma spores on the tumor growth and the proliferative responses of spleen lymphocytes to
Con A or LPS of S-180 sarcoma in mice. (A) Mice inoculated with S-180 sarcoma were daily treated with sporoderm-broken spores
(BS) and sporoderm-unbroken spores (US) at concentrations of 2 g/kg by oral administration for 14 days. Differences in tumor
weights between each treatment group and the control group were tested using two-tailed Mann-Whitney test. ** p < 0.01. Spleen
lymphocytes of sarcoma-bearing mice isolated from each treatment group were seeded in 96-well microplates and stimulated in
vitro for 72 h with 1 μg/mL Con A (B) or 10 μg/mL LPS (C). Proliferative response was assessed by [methyl-3H]-thymidine incorpora-
tion and expressed as the mean % ratio of mean counts per minute in mitogen-treated and mitogen-untreated cells + SD of two
independent experiments with 8–9 mice each (with six wells each). Differences between each treatment group and the control group
were tested using unpaired Student’s t-test, # p < 0.05, ## p < 0.01, ### p < 0.001. Differences among each treatment group were
determined by one-way ANOVA followed by Bonferroni post-test, c p < 0.001, ns, not significant.

Figure 4. Effects of Ganoderma spores on the mitogenic activities of resting or Con A-stimulated spleen lymphocytes of healthy and
tumor-bearing mice. Healthy mice and S-180 tumor-bearing mice were treated daily with sporoderm-broken spores (BS) and sporoderm-
unbroken spores (US) at a concentration of 2 g/kg by oral administration for 14 days. Spleen lymphocytes isolated from each
treatment group were seeded in 96-well microplates and cultured without any mitogen (A) or stimulated in vitro for 72 h with 1 μg/
mL Con A (B) for 72 h. Mitogenic activities were assessed by [methyl-3H]-thymidine incorporation and expressed as the mean counts
per minute (cpm) + SD of two independent experiments with 8–9 mice each (with six wells each). Differences between each
treatment group and the control group were tested using unpaired Student’s t-test, * p < 0.05.

group, but not in the US-treated group. However,
both BS- and US-treatment could significantly stimu-
late IL-6 production in the LPS-activated spleen
lymphocytes. Besides, the Con A-activated cytokine
releases were higher in the BS-treated groups than those
in the US-treated groups.
Copyright © 2008 John Wiley & Sons, Ltd.
Chemical profiles of GL extracts and Ganoderma
spores
The carbohydrate content percentage in tested GL
extracts and Ganoderma spore samples were shown in
Table 3. The carbohydrate contents in the different parts
Phytother. Res. 22, 1282–1291 (2008)
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1288 G. G. L. YUE ET AL.

Figure 5. Production of (A) IFN-γ, (B) TNF-α, (C) IL-2, (D) IL-4 and (E) and (F) IL-6 by cultured spleen lymphocytes from GL extract-
treated mice in response to mitogens. After orally administering the GL extracts (F, whole fruiting body; P, pileus; S, stipe) for 14
days, the spleen lymphocytes isolated from each treatment group were seeded in 96-well microplates and stimulated in vitro for 72 h
with 1 μg/mL of Con A (A–E) or 10 μg/mL of LPS (F). Culture supernatants were collected and the cytokine concentrations were
specifically determined by ELISA. Results were expressed as mean concentration + SD of quadruplicates. Differences between the
extract-treated and untreated control group were determined by Student’s unpaired t-test. * p < 0.05, *** p < 0.001 compared with
the control group.

Table 3. Relative molecular weight distribution of polysaccharide in GL-extracts and Ganoderma spores

GL-F GL-P GL-S BS US

Carbohydrate content (%)a 3.59 ± 0.24 3.29 ± 0.16 3.45 ± 0.16 1.57 ± 0.06 0.41 ± 0.01
Peak 1 Mw 461 800 695 240 370 780 276 680 348 050
Mw/Mnb 1.322 1.342 1.07 1.888 2.354
Peak 2 Mw 1655 1593 2051 16 910 1638
Mw/Mn 1.08 1.053 1.316 1.031 1.032
Peak 3 Mw 1739
Mw/Mn 1.086

a
Each value is expressed as mean ± SD (n = 3).
b
Mw, weight average molecular weight; Mn, number average molecular weight.

Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 1282–1291 (2008)
DOI: 10.1002/ptr
 ANTITUMOR AND IMMUNOMODULATORY EFFECTS OF GANODERMA STIPE 1289

Figure 6. Production of (A) IFN-γ, (B) TNF-α, (C) IL-2, (D) IL-4 and (E) and (F) IL-6 by cultured spleen lymphocytes from Ganoderma
spores-treated mice in response to mitogens. After orally administering the spores (BS, sporoderm-broken spore; US, sporoderm-
unbroken spore) for 14 days, the spleen lymphocytes isolated from each treatment group were seeded in 96-well microplates and
stimulated in vitro for 72 h with 1 μg/mL of Con A (A–E) or 10 μg/mL of LPS (F). Culture supernatants were collected and the cytokine
concentrations were specifically determined by ELISA. Results were expressed as mean concentration + SD of quadruplicates.
Differences between the extract-treated and untreated control group were determined by Student’s unpaired t-test. * p < 0.05,
*** p < 0.001 compared with the control group.

Table 4. Sugar composition of polysaccharides of GL-extracts Table 5. Content of amino acids of GL-extracts and Ganoderma
and Ganoderma spores spores

Relative content (%) Content (g/100 g dry weight)

Sugar GL-F GL-P GL-S BS US Amino acid GL-F GL-P GL-S BS US

Ribose 3.752 1.150 0.577 0.441 1.680 Asp 2.32 2.44 1.22 0.74 0.74
Rhamnose 2.404 1.744 0.482 0.464 1.170 Glu 2.72 2.59 1.52 0.72 0.65
Arabinose 41.795 15.250 5.029 4.506 6.569 Ser 1.02 1.17 0.46 0.25 0.24
Xylose 2.413 2.786 3.415 1.262 3.329 His 0.25 0.28 0.10 0.11 0.10
Mannose 11.100 12.854 12.969 11.719 35.090 Gly 0.90 1.15 0.49 0.28 0.31
Glucose 36.034 60.994 73.098 74.624 49.646 Thr 1.41 1.60 0.63 0.36 0.37
Galactose 2.501 5.223 4.429 6.984 2.517 Arg 0.96 1.07 0.25 0.38 0.34
Ala 1.63 1.82 0.62 0.48 0.45
Tyr 0.29 0.19 0.19 0.18 0.16
Val 1.43 1.72 0.51 0.36 0.33
Met 0.17 0.21 0.036 0.11 0.086
of fruiting bodies were similar, while the content Phe 0.73 0.69 0.27 0.24 0.24
in sporoderm-broken spores (BS) was much higher Ile 0.99 1.17 0.34 0.25 0.24
than that in sporoderm-unbroken spores (US). The Leu 1.34 1.37 0.49 0.46 0.42
polysaccharide content of GL extracts and Ganoderma Lys 2.28 2.62 0.43 0.91 0.82
spores were characterized according to the molecular Pro 0.88 1.12 0.51 0.42 0.33
size distributions and sugar compositions. The mole- Total 19.3 21.2 8.1 6.3 5.8
cular weight distribution of each extracts is listed in
Table 3. There were three peaks exhibited by the BS
extract, whereas the other extracts exhibited only two
peaks. The molecular-weight among 370 780, 276 680 The GC-MS analysis results of sugar compositions
and 16 910 and low molecular weight of 2051 and are summarized in Table 4. The results showed that
1739 Da polysaccharides, detected in GL-S and BS, ribose, rhamnose, arabinose, xylose, mannose, glucose
might be critical for the immunomodulatory activity. In and galactose were natural sugars in the extracts. The
contrast, GL-P and US did not contain polysaccharides samples GL-S and BS consisted of glucose (>70 wt%)
of the corresponding molecular sizes. as a main component and trace ribose and rhamnose.
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 1282–1291 (2008)
DOI: 10.1002/ptr
1290 G. G. L. YUE ET AL.
The total amino acid contents in the tested extracts
ranged from 5.8 to 21.2 g/100 g dry weight, while the
lowest was found in US (Table 5). Glutamic acid was the
major amino acid found in GL-F and GL-S, while lysine
was the major amino acid found in GL-P, BS and US.
DISCUSSION
In this study, the antitumor and immunomodulatory
activities of GL hot water extracts and the Ganoderma
spores were evaluated using a tumor-bearing mice
model. The inhibitory effects of the extracts and the
spores on sarcoma growth, as well as the enhanced
spleen lymphocyte proliferation in response to mitogens
in the GL-F- or BS-treated mice were demonstrated.
The administration of GL-F and GL-S to mice signifi-
cantly increased the spleen lymphocyte proliferation
activated by Con A and LPS while the GL-P stimulated
the LPS-activated lymphocyte proliferation only. The
cytokine production of IFN-γ, TNF-α and IL-2 in Con
A-activated lymphocytes and IL-6 in LPS-stimulated
lymphocytes were enhanced by GL extracts treatment,
especially in GL-S-treated mice. These results suggested
that the GL-S might modulate the proliferation and
differentiation of T lymphocytes resembling other iso-
lated components of GL mentioned in previous studies
(Kohguchi et al., 2004; Wang et al., 2003; Ning et al.,
2004). However, the exclusive and high potency of
the stipe extract in immunomodulation has not been
demonstrated before. Previous phytochemical studies
showed that the distribution of ganoderic acid B con-
tents was different in different parts of the fruiting
bodies of G. lucidum (Ding et al., 1999). In this study,
the different parts of the fruiting body were found
to have differences in the amino acid contents and
the monosaccharide composition of polysaccharides.
Besides, the polydispersity (Mw/Mn) of the first peak
of GL-S polysaccharides were measured to be 1.07,
showing the approach uniform chain length of the
polymer chains. Based on both the chemical differ-
ences observed and the different potencies in the
pharmacological activities, attempts on the isolation
of active compounds from GL-S might be affected
accordingly.
The oral administration of spores markedly stimu-
lated the proliferation of spleen lymphocytes in response
to mitogens. These results corresponded well with the
previous studies (Fan et al., 2005; Lin et al., 2005; Wang
et al., 2001). The proliferation of spleen lymphocytes in
response to Con A of the BS-treated group was signifi-
cantly different from that of the US-treated group. The
findings were further supported by chemical composi-
tion analysis by thin layer chromatography (data not
shown). Besides, the total carbohydrate and amino acid
contents in the BS sample were found to be higher
than that in the US sample (Tables 3 and 5).
The efficacies of the stimulatory activities on spleen
lymphocyte proliferation were compared between the
GL-F and BS in this study. A slightly higher prolifera-
tive response was observed in BS-treated group than
that observed in GL-F-treated group (Fig. 1). On the
other hand, the inhibitory percentages of sarcoma
growth of BS-treated mice were found to be lower than
that observed in the GL-F-treated mice (Table 1). Taken
Copyright © 2008 John Wiley & Sons, Ltd.
together, the hot water extracts of G. lucidum may have
comparable immunomodulatory and antitumor activ-
ities to the sporoderm-broken spores in tumor-bearing
mice. Further research on the antitumor activities of
Ganoderma spores is being performed. Longer treat-
ment duration may provide more information regard-
ing the long-term use of these supplements.
The immunomodulatory effects of BS and US were
examined in both healthy and tumor-bearing mice in
this study. Most other studies placed emphasis on the
efficacy of their commercially available spore products
in a selected animal model. The present study demon-
strated for the first time that the immunostimulating
activities of BS and US were compared in different
health conditions of mice. It is interesting that the BS
treatment led to a 6.5-fold increase in proliferation in
tumor-bearing mice, while the same treatment led to a
2.8-fold increase in healthy mice lymphocyte prolifera-
tion. However, the levels of augmentation in Con A-
activated lymphocyte proliferation were much higher
in healthy mice than those in tumor-bearing mice. These
findings suggested that in healthy mice, the stimulatory
activities of BS were stronger in mitogen-activated
lymphocytes than in resting lymphocytes. Therefore,
BS may enhance immune function during infection. In
contrast, BS could markedly increase the proliferation
of lymphocytes in resting state in tumor-bearing mice,
implying that BS may enhance the immune function in
subjects with tumors. Previous studies have suggested
that the major mechanism for the antitumor activities
of G. lucidum water extracts or Ganoderma spores was
the stimulation of host defense responses (Gao et al.,
2005; Wang et al., 1997). This was supported by the
results of our present study.
In conclusion, G. lucidum was shown to possess
immunomodulatory activities, with the stipes of the fruit-
ing bodies of G. lucidum being the most potent. This
also confirmed that the stipes, which are usually dis-
carded during preparation processes, have significant
immunomodulatory effects just like or may be even
better than the pileus. The importance of using the
whole fruiting bodies of G. lucidum is now recognized.
In other words, the stipe, which is very often left out
during the preservation process, should be retained to
obtain the most comprehensive active components from
the fungi. Furthermore, both sporoderm-broken and
sporoderm-unbroken spores were found to be effective
in modulating the immune responses in healthy and
tumor-bearing mice, with sporoderm-broken spores
being the more potent. Last but not least, the efficacy
of immunostimulatory and antitumor activities of the
G. lucidum hot water extract was shown to be compar-
able to that of the Ganoderma spores, which certainly
did not demonstrate superiority. As health supplements,
the pharmacological activities of Ganoderma products
(dried fruiting bodies, extracts or spores) may not re-
flect in the retail price.
Acknowledgements
The authors would like to thank Professor Nian-lai Huang of San
Ming Institute of Mycology, Fu Jian, China for the authentication of
Ganoderma lucidum fruiting bodies. The authors are also grateful to
Dr Hui Cao of National Engineering Research Centre for Moderni-
zation of Traditional Chinese Medicine, Zhu Hai, China for the sup-
ply and authentication of Ganoderma spores.
Phytother. Res. 22, 1282–1291 (2008)
DOI: 10.1002/ptr
 ANTITUMOR AND IMMUNOMODULATORY EFFECTS OF GANODERMA STIPE
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