hf. J. Biochem.Cd Bid. Vol. 28, No. 5, DD.

60-607, 1996
Copyright 0 1996 Elkier ScienceLtd
Pergamon 1357-2725(95)00157-3 Printed in Great Britain. All rights reserved
1357-2725196$15.00+ 0.00

Myceiia of the Mushroom CO

their Cutture Me&urn Activate Masse L

H. X. WANG,’ T. B. NG,2 W. K. LIU,3 V. E. C. 001,“’ S. T. CHANG’

Departments of ’ Biology, 2Biochemistry and ‘Anatomy, The Chinese University of Hong Kong,
Shatin, Hong Kong

The aim of this study was to investigate the effects of the mushroom Corioks uersicdor on cells
of the hnmane system. Tbe caked mycelia of the - coriolw versicolor and tbeii culture
medium were separately extracted with
preparations were designated
separated by gel f&ration be
in vitro aad in v&o. After gel
weight of 13-19KDa was obtained. Gel filtration of LM and EM on x G-50 revealed
the presvwe of a larger peak of 28 KDa (from
of 3.5 KDa. IM, EM aad their large molecular
from BALB/c mice in vifro. Spknocytes from
IM and EM demoastrated an augmented mitoge& rqanse to Coo A. The
C57BL/6 mice that bad been
nitrite k. The resalts indic
by preparations of polyaaceharopeptide
versicolor. However, ao direct cytotoxic a
choriocarcinoma cells could be demonstrated. Copyright 0 1996 Ekvier S&ace Ltd
Keywords: Coriolus versicolor Polysaccharide Macrophage Mitogenic activity
Immtmomodtdation
Int. J. Biochem. Cell Biol. (1996) 28, 601-607

INTRODUCTION polysaccharide moiety is composed mainly of

glucose (74.6%) with the remainder being
A variety of compounds including polysaccha- galactose, mannose, xylose and fucose. Its
ride (Naruse and Takeda, 1974; Ueno et al., polypeptide moiety is relatively rich in glutamic
1980), Coriolan (Naruse and Takeda, 1974),
and aspartic acids. PSP is soluble and stable in
Krestin (PSK) (Hirase et al., 1970) and polysac-
hot water. Yang et al. (1992) have also demon-
charopeptide (PSP) (Yang et al., 1987) with
strated that PSP exhibited an antiproliferative
antitumor activity (Jong and Donovick, 1989)
or antitumor activity against Ehrlich ascites
have been reported to be present in mushrooms.
carcinoma cells, P388 leukemia cells and
Yang et al. (1987) have reported the physio-
sarcoma 180 inoculated into mice. PSP also
chemical characteristics of the polysaccharopep-
induced morphological changes in human lung
tide (PSP) isolated from deep-layer cultured
cancer cells including swelling, chromosomal
mycelia of the mushroom Coriolus versicolor. It
aggregation and abnormal nuclear division.
has a molecular weight of 100 KDa as judged by
SDS-polyacrylamide gel electrophoresis. Its Serum concentration of immunoglobulin IgG
was also elevated.
*To whom all correspondence should be addressed. Liu et al. (1993) observed that PSP adminis-
Received 24 May 1995; accepted 10 October 1995. tered in drinking water to C57 BL/6 mice
601
602 H. X. Wang et al.

activated the macrophages, as witnessed in an Kong. The culture medium used was made up
increased production of reactive oxygen and by dissolving 24 g potato dextrose broth, 5 g
nitrogen intermediates and tumor necrosis fac- peptone, 0.46g KH,PO,, l.Og K,HPO,, 0.5g
tor, and an enhanced transcription of the tumor MgSO,. 7H,O and 20 mg VBI in 1 1 of distilled
necrosis factor gene. However, PSP did not water, pH 6.0. The C. versicolor mycelium was
exert direct antiproliferative activity against cultured in an orbital shaking incubator at
a number of tumor cells including P388 Dl 150 r.p.m. and 27°C for 7 days.
(mouse monocyte-macrophage), mouse sarcoma
180, PU5- 1.8 (mouse monocyte-macrophage), Extraction
mouse melanoma B16 and human chorio- The culture broth was homogenized and
carcinoma JAR. boiled in distilled water for 6 hr. After filtration
Macrophages and lymphocytes are two to remove mycelial fragments, the filtrate was
major populations of cells in the host defense concentrated in a rotavapor and then
system, which act against invading pathogens. lyophilized. The resulting powder was desig-
Macrophages are responsible for the non- nated as crude powder (CP). The mycelium in
specific cellular response. They ingest infectious another sample of culture broth was filtered,
micro-organisms and digest them with lysoso- homogenized, extracted, concentrated and
ma1 enzymes. Macrophages can be activated by lyophilized. The resulting powder was desig-
lymphokines and other cell mediators to kill nated as intramycelial material (IM). After the
tumor cells by producing tumor necrosis factor culture broth was filtered, the used culture
and nitrite ions. When non-specific responses medium was again processed in the same way as
fail to check the invasion of pathogenic organ- the mycelium and designated as extramycelial
isms, specific immune responses are activated material (EM).
whereby the lymphocytes recognize a particular
pathogen and generate antibodies to promote Polysaccharide and protein contents
destruction of this pathogen. In addition, lym- The carbohydrate and protein contents of
phocytes and macrophages co-operate through intramycelial material, extramycelial material
the production of cell mediates (Nathan and
and their fractions were determined by the
Hibbs, 1991; Liu et al., 1993). Thus it was of anthrone and Lowry methods, respectively, as
interest to study the effect of the mushroom reported by Chang et al. (1988).
Coriolus versicolor on these two types of cell in
the immune system. We report herein that the Determination of molecular weight
cultured mycelia of the mushroom Coriolus
versicolor and their culture medium contained Gel filtration for determining the molecular
polysaccharide-peptide complexes with a mol- weights (MW) of intra- and extramycelial ma-
ecular weight much smaller than that found by terials was carried out on Sepharose 6B and
Yang et al. (1987). We have further shown that Sephadex G-50. Standard protein markers used
these complexes stimulate the mitogenic re- included bacitracin (MW 1,400), cytochrome
sponse of mouse splenocytes or T-cells, in vivo C (MW 12,400) and chymotrypsinogen A
and in vitro, and enhance macrophage pro- (MW 25,000). Blue dextran was used for
duction of nitrite ions. Because of the disparity determination of the void volume.
in findings between Yang et al. (1987) and Liu
et al. (1993) regarding the antiproliferative ac- Animals
tivity of PSP, the issue was re-examined in the Male inbred C57BL/6 and BALB/c mice were
present investigation. Consistent with our pre- used in this study. The animals were housed
vious observation (Liu et al., 1993), there was no under normal laboratory conditions (21 + 2°C
direct cytotoxic effect on fibroblast, hepatoma and a 12/12 hr light/dark cycle) and maintained
and choriocarcinoma cell lines. on standard rodent chow. For determination of
mitogenic activity in vivo, and production of
MATERIALS AND METHODS nitrite ions (NO;), the C57BL/6 mice were
pretreated by force-feeding using a stomach
Strain and culture condition tube with the intramycelial or extramycelial
The strain of Coriolus versicolor used in this material (50 mg/kg body weight/day). The con-
investigation was maintained in the Department trol group was similarly treated with phosphate-
of Biology of the Chinese University of Hong buffered saline (pH 7.2). For determination of
 Polysaccharide-peptide complexes from Coriolus uersicolor h03

EM-1

-s- O.D. 280

0 20 40 60 80 100 120 140

Elution volume (ml)

Fig. 1. Gel filtration of extramycelial material (EM) and intramycelial material (IM) on a Sephadex G-50
column (1 .O x 75 cm). The column was eluted with 50 mM ammonium bicarbonate buffer @H 9.0).

mitogenic activity in uitro, BALB/c mice were
used.
Cytotoxicity assay
The crude powder was re-dissolved in RPM1
1640, and its cytotoxicity and antitumor activity
were tested by incubation with L929 (mouse
fibroblast cell line), H3B (mouse hepatoma,
ATCC) and JAR (human placenta choriocar-
cinema, ATCC HTB 144) cell lines in a hu-
midified atmosphere of 5% CO, at 37°C for
24 hr. The viable cells were stained with crystal
violet and the optical density was measured by
a microplate ELISA reader (Liu and Ng, 1991).
Mitogenic activity
In vitro study. T-cells were isolated from
BALB/c mouse splenocytes in flasks coated with
anti-B cell antibody. Macrophages were re-
moved by adherence to a culture well for 2 hr.

100 ,

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

Ve/Vo
Fig. 2. Gel filtration of extramycelial material on a Sephadex G-50 column (1 .Ox 75 cm). The molecular
weight markers used were: (1) bacitracin (MW I.4 KDa), (2) cytochrome C (MW 12.4 KDa) aMi (3)
chyrnotrypsinogen A (MW 25 KDa). Ve = elution volume and Vo = void volume. 15 KDa and 3.5 KDa
refer to molecular weights of the peaks derived from extramycelial material.
604 H. X. Wang et al.

Table 1. The carbohydrate: protein ratios of intra- and extramycelial material and their fractions
IM EM IM- 1 EM-l IM-2 EM-2
Carbohydrate:protein ratio 1.77 1.85 2.69 2.84 1.17 1.06

The preparation of T-cells was more than 90%
pure when estimated by immunostaining. The
cells were diluted with RPM1 medium contain-
ing 10% fetal bovine serum and then seeded
(2 x IO6 cells/well/O.2 ml) in 96-well microplates.
Intra- and extramycelial material and their frac-
tions were then added at the concentration of
6-50 pg/ml. Cells cultured in the absence of
mycelial extracts served as control (CTL). The
cells were incubated at 37°C in a humidified
atmosphere of 5% carbon dioxide for 24 hr.
During the last 6 hr, the cells were pulsed with
OS pCi/lO PI/well of 3H-methyl-thymidine
(specific activity 5 pCi/mmole, Amersham, Eng-
land) and were then harvested on to a glass fiber
filter using a cell harvester. The radioactivity
was determined using a Beckman scintillation
counter. The proliferative response was ex-
pressed as mean counts per min (c.p.m.).
In vivo study. Splenocytes were isolated from
C57BL/6 mice, which had been pre-treated
with intra- or extramycelial material (mainten-
ance and procedure being the same as that
described for animals). Their mitogenic re-
sponse was determined as described above with
the addition of concanavalin A (Con A).
Production of nitrite ions
Male inbred C57BL/6 mice (8-12 weeks old)
were pretreated with IM and EM (50 mg/kg
body weight/day) for 1 week by force feeding.
After 3 days of eliciting by thioglycolate, the
peritoneal macrophages were collected from
the mice. The cells were rinsed, counted and
resuspended in DMEM medium without phenol
red, 10% fetal bovine serum, 100 III/ml peni-
cillin and 100 pg/ml streptomycin. The cells
(2 x 10’ cells/well/O.2 ml) were allowed to
adhere on to the surface of the wells of a 96-well
culture plate for 1 hr before being challenged
with lipopolysaccharide (LPS, 10 and
1000 rig/ml) for 24 hr. The amount of NO;
present in the culture medium was determined
by the calorimetric method of Keller et al.
(1990) using NaN02 as a standard. An aliquot
(100 ~1) of cell-free culture medium was re-
moved from each culture well and reacted with
50 ~1 of Griess reagent (1% sulfanilamide in 5%

120 I
I
100 { -L
, T
80 i
f
z mg/ml
z
P 6o 1
24 -__
> I
00.5
4o I!

20
T

0L
L929 H3B JAR
Fig. 3. Viability of L929 (mouse fibroblast cell line) and tumor cell lines H3B (mouse hepatoma) and JAR
(human placenta choriocarcinoma) after incubation with crude powder, which was dissolved in RPM1
1640 medium (0.5 and 1.0 mg/ml, respectively) for 24 hr. The data are presented as mean f standard
deviation (n = 6).
 Polysaccharide-peptide complexes from Coriolus z!ersicoLor 605

IM EM IM- 1 IM-2 EM-l EM-2

Fig. 4. In vitro mitogen ic activities of extramycelial material (EM), intramycelial material (IM). IM- I,
IM-2, EM-l and EM-2 at the concentrations of 650 pg/ml on T-cells from normal BALB/c mice. The
data are presented as mean &- standard deviation (n = 6). *P i 0.05 compared with respective control. i.e.
0 kc.dml.

H,PO,-O. 1% naphthalene-ethylenediamine di- peak and a small MW peak. The high MW

hydrochloride) for 10 min. The absorbance was
then measured at 540 nm using a microplate
ELISA reader (Bio Rad 3550).
RESULTS
When intramycelial and extramycelial ma-
terials were purified by gel filtration on Sepha-
rose 6B, in both cases there was only a single
peak (MW 13-19 KDa). After gel filtration on
Sephadex G-50 both intra- and extra-mycelial
materials separated into two peaks: a large MW
peaks possessed a MW of 15 KDa (from ex-
tramycelial material) and 28 KDa (from in-
tramycelial material), respectively. The low MW
peaks had a MW of 3.5 KDa. They were desig-
nated as EM- 1, IM-1, EM-2, and IM-2, respect-
ively (Figs 1 and 2). The carbohydrate:protein
ratios of intra- and extramycelial material and
their fractions are shown in Table 1.
Crude powder was subjected to the cytotox-
icity test in vitro and no cytotoxic effect was
found on a normal (fibroblast) cell line (L929)
or tumor (hepatoma H3B and choriocarcinoma

80

u *
8
0 60
I

CTL EM
Fig. 5. Mitogenic response of splenocytes from C57BL/6 mice, which had been pre-treated by force feeding
with extramycelial material (EM) and intramycelial material (IM) at 50 m&kg body weight/day for 7 days.
The data are presented as mean + standard deviation (n = 6). *P < 0.05 (against respective control).
CTL = control. Con A = concanavalin A.
606
CTL
Fig. 6. Production of NO; by macrophages of C57BL/6 mice, which had been pretreated by force feeding
with extramycelial material (EM) and intramycelial material (IM) at 50 mg/kg body weight/day for 7 days.
The data are presented as mean + standard deviation (n = 6). *P < 0.05 (against respective control).
CTL = control, LPS = lipopolysaccharide.
JAR) cell lines (Fig. 3). Intra- and extramycelial
material, EM-l and IM-1 enhanced the mito-
genie response of mouse T-cells in vitro, but
fractions IM-2 and EM-2 did not enhance the
proliferation of T-cells over the concentration
range of 6-50 fig/ml (Fig. 4). Intra- and
extramycelial materials also enhanced the mito-
genie response of mouse splenocytes (Fig. 5) and
stimulated the production of nitrite ions (Fig. 6)
in vivo.
DISCUSSION
It was demonstrated in the present investi-
gation that the mycelia of the mushroom Corio-
lus versicolor elaborated polysaccharide-peptide
complexes with a much lower molecular weight
than the value of 100KDa reported by Yang
et al. (1987). Apparently, these complexes could
be released by the mycelia into the culture
medium because similar complexes with similar
immunomodulatory activities were detected in
the culture medium. The discrepancy in the
molecular weight of Corioh versicolor polysac-
charide-peptide complex between the findings
in this investigation and the report of Yang et al.
(1987) may have been due to differences in the
method of molecular weight determination: gel
filtration in this study and electrophoresis in the
study of Yang ef al. (1987). We have attempted
H. X. Wang Ed al
LPS
hm')
b 00
la '0
1000
I
IM EM
to analyze the polysaccharide-peptide com-
plexes (IM and EM) with SDS-PAGE, but
found that their mobility was so slow that they
hardly moved. We believe that SDS-PAGE is
not suitable for molecular weight determination
of polysaccharide-peptide complexes because
their high carbohydrate content may contribute
to a very slow or negligible electrophoretic
mobility. There was also a difference in the
carbohydrate : protein ratio of the polysaccha-
ride-peptide complex in the two studies: it was
5.7: 1 according to Yang et al. (1987) and
1.8-2.8 : 1 in the present investigation.
Nitrite ions may play an important role in the
tumoricidal activity of macrophages (Kilbourn
et al., 1984; Ding et al., 1988; Nathan and
Hibbs, 1991) because they inhibit mitochondrial
respiration in tumor cells (Kilbourn et al., 1984;
Mauer et al., 1991; Takema et al., 1991). In
conformity with an earlier study using a
commercially available preparation of Coriolus
versicolor polysaccharopeptide showing the
ability of the complex to augment production of
nitrite ions by mouse macrophages (Liu et al.,
1993), the present study yielded similar results
using polysaccharidepeptide preparations from
the cultured mycelia of the same mushroom and
the culture medium. In addition, the mitogenic
response of the mouse splenocytes or T-cells was
enhanced following in vivo or in vitro treatment
 Polysaccharide-peptide
with the polysaccharide-peptide complex. The
splenocytes from the studied C57BL/6 mice
comprised a mixture of B and T lymphocytes,
while only T-cells from BALB/c mice were used.
The increased mitogenic responses of these cells
reflect their activation. Results of the cytotox-
icity assay in the present investigation, together
with data of an antiproliferative activity assay
from a previous study (Liu et af., 1993) suggest
that the polysaccharopeptide from C. versicolor
did not exert a direct cytotoxic effect against
fibroblast, hepatoma, choriocarcinoma, sar-
coma, melanoma and monocyte-macrophage
cell lines. Thus the polysaccharide-peptide com-
plex from C. versicolor is non-cytotoxic and
capable of stimulating both macrophages and
lymphocytes.
Acknowledgements-We thank MS Doris Yeung for her
expert secretarial assistance. This study was partially sup-
ported by an RGC (Research Grant Council) Research
Grant from UGC (University Grants Committee), Hong
Kong.
REFERENCES
Chang J. B., Park W. H., Choi E. C. and Kim B. K. (1988)
Studies on constituents of the higher fungi of Korea (LIV)
antitumor components of Favolus alveolar&s. Archiv.
Pharmacol. Res. 11, 203-212.
Ding A. H., Nathan C. F. and Stuehr D. J. (1988) Release
of reactive nitrogen intermediates and reactive oxygen
intermediates from mouse peritoneal macrophages.
J. Immunol. 141, 2401-2412.
Hirase S., Nakai S., Akatsu T., Kobayashi A., Oohara M.,
Matsunaga K., Fuji M. and Ohmura Y. (1970) Studies on
antitumor activity of polysaccharides. Proc. Jpn. Cant.
Assoc. 29th Annu. Meet., p. 288.
Jong S. C. and Donovick R. (1989) Antitumor and antiviral
substances from fungi. Adv. Appl. Microbial. 34,183-262.
Keller R., Keist R., Wechsler A., Leist T. P. and Van der
Meide P. H. (1990) Mechanisms of macrophage-mediated
tumor cell killing: a comparative analysis of the roles of
complexes from Coriolus versicolor 607
reactive nitrogen intermediates and tumor necrosis factor.
Int. J. Cancer 46, 682686.
Kilboum R. G., Klostergaard J. and Lopez-Berestein G.
(1984) Activated macrophages secrete a soluble factor
that inhibits mitochondrial respiration of tumor cells.
J. Immunol. 133, 2577-2581.
Liu W. K. and Ng T. B. (1991) Effects of methimazole-
induced hypothyroidism on alveolar macrophages.
Virchow Arch. B Cell Pathol. 60, 21-25.
Liu W. K., Ng T. B., Sze S. F. and Tsui K. W. (1993)
Activation of peritoneal macrophages by polysaccha-
ropeptide from the mushroom, Coriolus versirolor.
Immunopharmac. 76, 139-146.
Mauel J., Ransijn A. and Buchmuller-Rouiller Y. (1991)
Killing of Leischmania parasites in activated murine
macrophages is based on an L-arginine-dependent process
that produces nitrogen derivative. J. Leukocyte Riol. 49,
73-82.
Nathan C. F. and Hibbs J. B. Jr (1991) Role of nitric oxide
synthesis in macrophage antimicrobial activity. Curr.
Opin. Immunol. 3, 65-70.
Naruse S. and Takeda S. (1974) Studies on antitumor
activity of Basidiomycete polysaccharide. II. Antitumor
effects of polysaccharides prepared from cultures of
Basidiomycetes. Mie Med. J. 23, 207-231.
Sakagami H., Aoki T., Simpson A. and Tanuma S. I. (1991)
Induction of immunopotentiation activity by a protein-
bound polysaccharide, PSK (review). Anticanc. Res. 11,
993-1000.
Takema M., Inaba K., Uno K., Kakihara K., Taware K.
and Muramatsu S. (1991) Effect of L-arginine on the
retention of macrophage tumoricidal activity. J. Immunol.
146, 192881933.
Ueno S., Yoshikumi G., Hirose F., Imura Y., Wada T.,
Fuji T. and Takahashi E. (1980) Method of producing
nitrogen-containing polysaccharides. U.S. Patent 4, 202,
969.
Yang Q. Y., Yong S. C. and Yang X. T. (1987) The
physio-chemical characteristics of the polysaccharide-
peptide (PSP) of Coriolus versicolor (Yun-zhi). In Report
on the Polysaccharia’e-Peptide (PSP) of Coriolus versi-
color, pp. l-6. Landford Press, China.
Yang Q. Y., Tong S. C., Li X. Y., Zhou J. X., Chen R. T.
and Xu L. Z. (1992) Antitumor and immunomodulating
activities of the polysaccharopeptide-peptide (PSP) of
Coriolus versicolor. J. Immunol. Immunopharmac. 12,
29-34.