ΕΠΙΣΤΗΜΟΝΙΚΕΣ ΜΕΛΕΤΕΣ

Nerve Growth Factor-Inducing Activity of Hericium erinaceus

Nerve Growth Factor-Inducing Activity of Hericium erinaceus in 1321N1 Human Astrocytoma Cells

Koichiro Mori,“ c Yutaro Obara,“’1 Mitsuru Hirota/ Yoshihito Azumi,c
Satomi Kinugasa,c Satoshi Inatomi,c and Norimichi Nakahata*“’*


a
Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University; * 21st Century COE Program «CRESCENDO”, Graduate
School of Pharmaceutical Sciences, Tohoku University; Aoba 6-3, Aramaki, Aoba- ku, Sendai 980-8578, Japan: c Mushroom Laboratory, Hokuto
Corporation; 800-8 Shimokomazawa, Nagano 381-0008,


Japan: and dDepartment of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University; 8304 Minami- minowa, Kami-ina, Nagano
399-4598, Japan.

Received March 24, 2008; accepted June 20, 2008; published online June 23, 2008


Neurotrophic factors are essential to maintain and organize neurons functionally; thereby neurotrophic fac­tor-like substances or their inducers are
expected to be applied to the treatment of neurodegenerative diseases such as Alzheimer’s disease. In the present study, we firstly examined the
effects of ethanol extracts of four edible mushrooms, Hericium erinaceus (Yamabushitake), Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Agaricus blazei (Himematsutake), on nerve growth factor (NGF) gene expression in 1321N1 human
astrocytoma cells. Among the four mushroom extracts, only H. erinaceus extract promoted NGF mRNA expression in a con­centration-dependent
manner. In addition, secretion of NGF protein from 1321N1 cells was enhanced by H. eri­naceus extracts, and the conditioned medium of 1321N1
cells incubated with H. erinaceus extract enhanced the neurite outgrowth of PC12 cells. However, hericenones C, D and E, constituents of H. erinaceus, failed to pro­mote NGF gene expression in 1321N1 cells. The enhancement of NGF gene expression by H. erinaceus extracts
was inhibited by the c-jun N-terminal kinase (JNK) inhibitor SP600125. In addition, H. erinaceus extracts in­duced phosphorylation of JNK and
its downstream substrate c-Jun, and increased c-fos expression, suggesting that H. erinaceus promotes NGF gene expression via JNK
signaling. Furthermore we examined the efficacy of H. erinaceus in vivo. ddY mice given feed containing 5% H. erinaceus dry powder
for 7 d showed an increase in the level of NGF mRNA expression in the hippocampus. In conclusion, H. erinaceus contains active compounds that
stimulate NGF synthesis via activation of the JNK pathway; these compounds are not hericenones.

Key words nerve growth factor; Hericium erinaceus; astrocytoma; hericenone

Senile dementia is a serious social problem. In particular, Alzheimer’s disease, for which there is currently no effective therapy, is the most common
senile dementia. Alzheimer’s disease patients have notable abnormalities in cholinergic neurons in the basal forebrain.Neurotrophic factors have potent
biological activities, such as preventing neuronal death and promoting neurite outgrowth, and are essential to maintain and organize neurons functionally. 21 Glial cells sup­port neurons by releasing neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic
factor (BDNF), neurotrophin 3, and glial-derived neurotrophic fac­tor (GDNF). In particular, it is assumed that functional defi­ciency of NGF is related to
Alzheimer’s disease and plays a part in the etiology of the disease process.3-

It is known that NGF levels are decreased in the basal forebrains of Alzheimer’s disease patients, and in the frontal cortices of undemented patients with
senile plaques.4,5- Fur­thermore, intracerebroventricular administration of NGF eliminates degeneration and resultant cognitive deficits in rats
after brain injury,6- and it enhances the retention of pas­sive avoidance learning in developing mice.7) In aged rats, intracerebral
infusion of NGF partly reverses cholinergic cell body atrophy and improves the retention of spatial memory.8) In addition, intranasal
administration of NGF ameliorates neurodegeneration and reduces the numbers of amyloid plaques in transgenic anti-NGF mice (AD11 mice), in which have a
progressive neurodegenerative phenotype resembling Alzheimer’s disease.9) Therefore, NGF is expected to be ap­plied to the treatment of
Alzheimer’s disease.101

However, neurotrophic factors are proteins, and so are un­able to cross the blood-brain barrier; they are also easily me­tabolized by peptidases.
Therefore, their application as a medicine for the treatment of neurodegenerative disorders is assumed to be difficult. Alternatively, research has been
car­ried out on low-molecular weight compounds that promote NGF biosynthesis, such as catecholamines,11,121 benzo- quinones,13) fellutamides,14) idebenone,15) kansuinin, ingenol triacetate, jolkinolide B,16) dictyophorines, 17) scabronines,18) hericenones,18—21) erinacins,22—24) and cyrneines.25)

Hericium erinaceus
is a mushroom that grows on old or dead broadleaf trees. H. erinaceus is taken as a food in Japan and China without harmful effects. Hericenones
C—h2,19—21) and erinacines A—i22—24) were isolated from the fruit body and mycelium of H. erinaceus, respectively, all of
which pro­mote NGF synthesis in rodent cultured astrocytes. These re­sults suggest the usefulness of H. erinaceus for the treatment and prevention
of dementia. However, the detailed mecha­nism by which H. erinaceus induces NGF synthesis remains unknown.

In the present study, we examined the NGF-inducing activ­ity of ethanol extracts of H. erinaceus in 1321N1 human astrocytoma cells. The results
obtained indicate that H. eri­naceus has NGF-inducing activity, but that its active com­pounds are not hericenones. Furthermore, ICR mice given
feed containing 5% H. erinaceus dry powder for 7 d showed an increase in the level of NGF mRNA expression in the hip­pocampus.

© 2008 Pharmaceutical Society of Japan

MATERIALS AND METHODS

Materials
Dulbecco’s modified Eagle’s medium (DMEM) was from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). FCS was from Biological Industries (Kibbutz Beit Haemek,
Israel). HS was from ICN Biochemicals, Inc. (Costa Mesa, CA, U.S.A.). Tri Pure Isolation Reagent was from Roche Di­agnostics (Indianapolis, U.S.A.).
Oligo(dT)primer and NGF ELISA Kit Emax® Immunoassay System were from Promega Co., Ltd. (Madison, WI, U.S.A.). Rever Tra Ace was from Toyobo Co.,
Ltd. (Tokyo, Japan). Syber Premix Ex Taq was from Takara Bio Inc. (Shiga, Japan). U0126 was from Sigma Aldrich Japan (Tokyo, Japan). SP600125 was from
BIOMOL (Plymouth Meeting, PA, U.S.A.). A23187, SB203580 and Gf109203X were from Calbiochem (San Diego, CA, U.S.A.). H89 was from Seikagaku Corporation
(Tokyo, Japan). Anti- NGF was from Boehringer Mannheim (Mannheim, Ger­many). Anti-phospho-ERK (Thr202/Tyr204) antibody, Anti- ERK antibody, anti-phosho-JNK
(Thr183/Tyr185) antibody, anti-JNK antibody, anti-phospho-c-Jun (Ser63) antibody, and anti-c-Jun antibody were obtained from Cell Signaling Tech­nology
(Beverly, MA, U.S.A.). Real-time PCR was carried out using an Opticon real-time PCR system (Bio-Rad Labo­ratories Inc., Japan). Dextrin was from Wako Pure
Chemical Industries Ltd. (Tokyo, Japan). ddY mice were purchased from Japan SLC Inc. (Shizuoka, Japan). Hericium erinaceus, Pleurotus eryngii, Grifola frondosa, and Agaricus blazei were cultured by Hokuto Corporation in its facilities
(Nagano, Japan).

Cell Culture
1321N1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FCS, penicillin (50 units/ml), and streptomycin (50 mg/ml). PC-12
cells were grown in DMEM supplemented with 10% FCS, 5% HS, penicillin (50 units/ml), and streptomycin (50 mg/ml). The cells were cultivated in an incubator
contain­ing 5% CO2 at 37 °C.

Preparation of Mushroom Extracts
Fresh fruiting bod­ies of H. erinaceus, P eryngii, G. frondosa, and A. blazei were lyophilized and powdered. The dry powder (5 g) of
mushrooms was extracted with 150 ml of ethanol for 2h at room temperature, and H. erinaceus ethanol extract (499 mg), P eryngii ethanol
extract (386 mg), G. frondosa ethanol extract (328 mg) and A. blazei ethanol extract (426 mg) were obtained. Similarly,H. erinaceus was extracted with H2O and ethyl acetate, and H. erinaceus H2O extract (1734 mg), and H. erinaceus ethyl acetate extract (257 mg) were obtained. The extracts were stored at -30 °C before use.

RT-PCR
1321N1 cells were seeded into 12-well plates and allowed to grow to confluence. Twenty-four hours before the incubation with mushroom extracts, the medium
was re­placed with serum-free DMEM. The mushroom extract was dissolved in DMSO at 50 mg/ml and then further diluted with serum-free DMEM to the appropriate
concentration. 1321N1 cells were incubated with the mushroom extracts for 3 h. Total RNA of 1321N1 cells was extracted using Tri Pure Isolation Reagent
according to manufacturer’s protocol. First-strand cDNA primed by Oligo(dT) primer was prepared from total RNA (1 mg) using Rever Tra Ace, and was diluted
with water by 5 times to use as a template for the real-time PCR analysis. The primers for amplification and the sizes of respective PCR products were as
follows: NGF (sense: 5′-

CCAAGGGAGCAGTTTCTATCCTGG-3′, and antisense: 5′- GGCAGTTGTCAAGGGAATGCTGAAGTT-3′, for 189bp), b-actin (sense: 5′-AGGGAAATCGTGCGTGACAT-3′, anti­sense: 5
“-TCCTGCTTGCTGATCCACAT-3”, for 467bp), c­fos (sense: 5 “-GTTCTCGGGTTTCAACGCGGACTACGA- GGC-3”, antisense: 5 “-GGCACTAGAGACGGACAGATCT- GCGCAAAAGTCC-3”, for
922bp). Real-time PCR was car­ried out in a 20-ml solution volume containing SYBR Premix (10 ml), RT template (3 ml), water (6 ml) and primers (1 ml). The
amplification programs were as follows: NGF, 94 °C for 5 s, 61 °C for 20 s, and 72 °C for 15 s, for 35 cycles; b-actin, 94 °C for 10 s, 56 °C for 20 s, and
72 °C for 30 s, for 22 cy­cles; and c-fos, 94 °C for 10 s, 55 °C for 30 s, and 72 °C for 60 s, for 39 cycles. The levels of NGF and c-fos mRNA were
normalized to that of the corresponding b-actin mRNA.

Assay for Neurite Outgrowth in PC12 Cells
1321N1 cells were seeded into 24-well plates and allowed to grow to confluence- Twenty-four hours before the incubation with H. erinaceus extracts
or hericenone D, the medium was replaced with serum-free DMEM. After the incubation with H. eri- naceus extracts or hericenone D for 48 h, the
medium was collected, and centrifuged to remove cells.

PC-12 cells were seeded in a 24-well plate at a density of 7X104 cells/well and cultivated for 24h. After aspiration of the medium, DMEM
containing 10% FCS and 5% HS (100 ml), and 1321N1 cell culture medium prepared as described above (400 ml) were added and incubated for 48 h. On the other
hand, PC-12 cells were stimulated with H. erinaceus extract or NGF directly for 48 h. PC-12 cells were observed under a phase-contrast microscope
and neurite outgrowth was regarded as a sign of differentiation. About one hundred PC-12 cells in each well were evaluated. Cells bearing neu- rites longer
than one cell diameter were regarded as differen­tiated cells. Data are expressed as means±S.E.M. of the val­ues for three wells.

Enzyme Immunoassay of NGF
1321N1 cells were seeded into 24 well plates and allowed to grow to confluence. Twenty-four hours before the incubation with mushroom ex­tracts, the medium
was replaced with serum-free DMEM. The mushroom extract was dissolved in DMSO at a concentration of 50 mg/ml, and then diluted with serum-free DMEM to the
appropriate concentration. 1321N1 cells were incubated with mushroom extracts for 24 h, and then the culture medium was collected. The NGF content in the
medium was meas­ured by ELISA (Emax® Immunoassay System, Promega) ac­cording to the procedures of the manufacturer, except using anti-NGF
secondary antibody.

Immunoblot Analysis
After drug incubation, the medium was removed and cells were lysed in lysis buffer (75 mM Tris-HCl, 2% SDS, 10% glycerol, 3% 2-mercapto-ethanol, and 0.003%
bromophenol blue) for 15 min at room tempera­ture and scraped. Lysates were subjected to SDS-PAGE on a 10% polyacrylamide gel. Separated proteins were
transferred to a polyvinylidene difluoride membrane (Millipore, Biller­ica, MA, U.S.A.) using a semidry blot apparatus. The mem­brane was blocked 3% skim
milk in Tris-buffered saline, pH 7.4, containing 0.1% Tween 20 (TBST) for 40 min at room temperature. The membrane was washed with TBST and in­cubated with
primary antibody overnight at 4 °C. Then, the membrane was washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 2

h at room temperature. The immunoreactive proteins were detected using an ECL Western blotting detection system.

Fig. 1 . Effects of Mushroom Extracts on NGF mRNA Expression in 1321N1 Cells

Isolation and Analysis of Hericenones C, D, and E

1321N1 cells were stimulated with ethanol extract (100 mg/ml) of H. erinaceus (H), P. eryngii (P), G. frondosa (G), A. blazei (A), and A23187 (1 mM, as a positive control) for 3 h at 37 °C, and then NGF gene expression was examined by RT-PCR. Values rep­resent
the means±S.E.M. (n=3). *p<0.05 vs. control (Cont.).

Fig. 2. Effects of Mushroom Extracts on the Secretion of NGF from 1321N1 Cells

Fresh fruiting bodies of

H. erinaceus

(6.0 kg) were extracted with ethanol. The extract was concentrated and fractionated by solvent partition between chloroform and water. The chlo­roform
fraction (25.0 g) was subjected to silica gel column chromatography using toluene-acetone as the eluent. The toluene-acetone (9:1) eluate was subjected to
silica gel col­umn chromatography using hexane-ethyl acetate. The hexane-ethyl acetate (10:1) eluate was further purified by ODS column chromatography
using methanol to give heri- cenone C (210.9 mg), D (35.4 mg), and E (48.0 mg). Their spectral data (

1

H-NMR,

13

C-NMR, IR, and HR-FAB-MS) completely agreed with the data reported.

20

Hericenone C, D, and E in the extract of H. erinaceus were analyzed using HPLC (Shimadzu LC-10 series, SPD-6AV UV-VIS detector Shimadzu set at 260
nm, with a mbondapak C18 3.9X300mm, 10mm column (Waters)). The solvent used for separation was acetonitrile and the flow rate was 1.0 ml/min.

1321N1 cells were stimulated with ethanol extract (50, 100, 150 mg/ml) of H. eri­naceus (H), ethanol extract (100 mg/ml) of P eryngii
(P), G. frondosa (G), A. blazei (A), and A23187 (1 mM, as a positive control) for 24 h at 37 °C. The amount of NGF protein in the culture
media of 1321N1 cells was measured by ELISA. Values represent the means±S.E.M. (n=3). *m<0.05 vs. control (Cont.).

Fig. 3. Effects of H. erinaceus Extract on NGF mRNA Expression in 1321N1 Cells

NGF mRNA Expression in Mouse Brain Tissue

Male ddY mice aged 5 weeks and weighing 33—35 g at the begin­ning of the experiments, were used. Mice were divided into a control group and a

H. erinaceus

group, so that the average of their body weights was equalized. While the control group was given normal diet (MF, Oriental Yeast, Tokyo, Japan) containing
5% dextrin, the

H. erinaceus

group was given MF containing 5% freeze-dried powder of

H. erinaceus.

Animals were given feed and water freely and maintained under con­trolled conditions at a temperature of 24 ±1 °C, relative hu­midity of 45 ±5%, and 12-h
light cycle (09:00—21:00). Mice were fed an experimental diet for 1 or 7 d. At the end of the experiment, mice were killed by cervical dislocation, and the
cerebral cortex and hippocampus were dissected out. The tis­sues were rapidly homogenized in Tri Pure Isolation Reagent (Takara, Japan) and extracted
according to the manufacturer’s protocol. Then, NGF mRNA expression was examined by RT-PCR.

Statistical Methods
Data are expressed as means±S.E.M. Significant differences (^<0.05) were determined by one­way ANOVA, followed by a Tukey’s test.

RESULTS

Firstly, we investigated the NGF-inducing activity of four edible mushrooms, H. erinaceus, P eryngii, G. frondosa, and A. brazei, in
1321N1 human astrocytoma cells. The NGF mRNA level in 1321N1 cells was significantly increased by the calcium ionophore A23187 at a concentration of 1 mM (positive control), and by the ethanol extract of H. erinaceus at a concentration of 100 mg/ml. However, no significant in­crease in
NGF mRNA levels was induced by the ethanol ex­tracts of the other mushrooms (Fig. 1). In addition, the NGF protein level in the culture medium was also
increased by the ethanol extract of H. erinaceus, while the ethanol extracts of other three mushrooms failed to induce such an increase (Fig. 2).
Moreover, we investigated the effects of H2O and ethyl acetate extracts of H. erinaceus on NGF mRNA expres­sion compared with the
effects of the ethanol extract. The ethanol and ethyl acetate extracts promoted NGF mRNA ex-

1321N1 cells were stimulated with H2O extract (0.4, 2.0, 4.0 mg/ml), ethanol extract (50, 100, 150 mg/ml), and ethyl acetate extract (50, 100,
150 mg/ml) for 3h at 37 °C, and then NGF gene expression was examined by RT-PCR. Values represent the means±S.E.M. (n=3). *m<0.05 vs. control (Cont.).

pression in a concentration-dependent manner with similar potency. However, the H2O extract did not increase NGF mRNA expression (Fig. 3).

Next, we examined the NGF mRNA-inducing activity of hericenone C, D and E, components of H. erinaceus reported to be stimulators of NGF
biosynthesis in mouse astroglial cells at 33 mg/ml.20) However, hericenones C, D and E did not increase NGF mRNA expression at 10—100 mg/ml in
1321N1 cells (Fig. 4). Furthermore, we failed to demonstrate that hericenones C, D, and E stimulated NGF mRNA expression in primary cultured rat astroglial
cells (data not shown). Moreover, we analyzed the levels of hericenones C, D, and E

Fig. 4. Effects of Hericenones C, D, and E on NGF mRNA Expression in 1321N1 Cells



1321N1 cells were stimulated with hericenones C, D, and E (10, 30, 100 mg/ml) for 3h at 37 °C, then NGF gene expression was examined by RT-PCR. Values
represent the means±S.E.M. (n=3). *p<0.05 vs. control (Cont.).

in the ethanol extract of H. erinaceus by HPLC, and their concentrations in 100 mg/ml ethanol extract of H. erinaceus were 20 ng/ml for
hericenone C, 4 ng/ml for hericenone D, and 2 ng/ml for hericenone E.

To investigate the physiological effects of H. erinaceus on neurite outgrowth via NGF, PC-12 cells were cultivated for 2d in the
conditioned medium of 1321N1 cells that had been incubated with the ethanol extract of H. erinaceus for 24 h. Considering that NGF significantly
promoted neurite out­growth in PC-12 cells at concentrations of 10 and 100 ng/ml, but not at 0.1 and 1 ng/ml, NGF level in the conditioned medium of 1321N1
cells with H. erinaceus extract (Fig. 2) was too low to promote neurite outgrowth in PC-12 cells. Furthermore, the ethanol extract of H. erinaceus did not pro­mote neurite outgrowth in PC12 cells directly. However, neu- rite outgrowth was significantly promoted by the conditioned
medium of 1321N1 cells incubated with H. erinaceus ethanol extract at concentrations of 125 and 250 mg/ml for 24 h. On the other hand, hericenone
D did not show the effect such as ethanol extract of H. erinaceus (Fig. 5).

To clarify the mechanism underlying NGF induction by H. erinaceus, 1321N1 cells were pretreated with various kinase inhibitors, and then
stimulated with ethanol extract of H. eri- naceus. The promotion of NGF mRNA expression by the ethanol extract of H. erinaceus was
significantly inhibited by the c-Jun N-terminal kinase (JNK) inhibitor SP600125, but not by the MEK inhibitor U0126, the p38 MAPK inhibitor SB203580, the
PKA inhibitor H89, or the PKC inhibitor GF109203X (Fig. 6). In fact, the ethanol extract of H. eri- naceus induced phosphorylation of JNK and its
downstream substrate c-Jun in a time-dependent manner (Fig. 7). Further­more, the ethanol extract enhanced c-fos gene expression

(Fig. 8).

To investigate the effect of H. erinaceus in vivo, we meas­ured NGF mRNA expression in the cortex and hippocampus of mice administered H. erinaceus. The mice in the H. eri- naceus group were fed a diet containing 5% dried powder of H. erinaceus, and the mice of
control group were fed a diet containing 5% dextrin instead of H. erinaceus for 1 or 7 d. The level of NGF mRNA in the hippocampus of mice in the H. erinaceus group was significantly increased compared with that of mice in the control group at the 7th day. On the other hand, NGF mRNA
expression in cortex was not in­creased by H. erinaceus during the test period (Fig. 9).

Fig. 5. Morphological Differentiation of PC 12 Cells Induced by the Medium of 1321N1 Cells Conditioned with Ethanol Extract of H. erinaceus



(A) Morphological changes of PC12 cells. PC12 cells were directly stimulated with ethanol extract (100 mg/ml) of H. erinaceus (H) or NGF (100
ng/ml) for 2 d (upper fig­ures). PC12 cells were stimulated for 2d with 20% DMEM plus 80% 1321N1 culture medium conditioned with 125 or 250 mg/ml of
ethanol extract of H. erinaceus (lower figures). Scale bar: 100 mm. (B) Evaluation of neurite outgrowth. PC12 cells were di­rectly stimulated with
ethanol extract (100 mg/ml) of H. erinaceus (H) or NGF (0.1— 100 ng/ml) for 2 d (left). After 1321N1 cells were incubated for 2d in DMEM
contain­ing 125 or 250 mg/ml of ethanol extract of H. erinaceus (H) or 30 mg/ml of hericenone D (He.D), PC12 cells were cultivated for an
additional 2d in 20% DMEM plus 80% 1321N1 culture medium (right). Values represent the means±S.E.M. (n=3). * ,tp< 0.05.

Fig. 6. Effects of U0126, SP600125, SB203580, H89, and GF109203X on H. erinaceus-Induced NGF mRNA Expression



1321N1 cells were preincubated with U0126 (10 mM), SP600125 (30 mM), SB203580 (3 mM), H89 (10 mM), and GF109203X (5 mM for 20 m before the
addition of H. eri­naceus extract. After incubation with H. erinaceus ethanol extract (100 mg/ml) for 3 h, NGF mRNA expression was
examined by RT-PCR. Values represent the means ± S.E.M. (n=3). *,tp<0.05.

DISCUSSION

In the present study, we demonstrate that the ethanol ex­tract of H. erinaceus promotes the synthesis of NGF in 1321N1 human astrocytoma cells. H. erinaceus alone had NGF-inducing activity among the four mushrooms exam­ined. However, 100 mg/ml of the H. erinaceus ethanol extract

1321N1 cells were stimulated with ethanol extract of H. erinaceus (100 mg/ml) for 3h at 37 °C, and then c-fos gene expression was examined by
RT-PCR. Values repre­sent the means±S.E.M. (n=3). *p<0.05 vs. 0min.

Fig. 9. NGF Gene Expression in the Brains of Mice Fed H. erinaceus

Mice were fed a diet containing 5% H. erinaceus or dextrin (control), and then NGF mRNA expression in their hippocampus and cortex was analyzed by
RT-PCR. Values represent the means±S.E.M. (Day 1 and Day 7: n=8; Day 21: n=3). * p<0.05 vs. Control (- ).



significantly increased NGF mRNA expression but not NGF protein synthesis. Therefore, it is possible that effective con­centrations of H. erinaceus ethanol extract to induce NGF protein synthesis and secretion differs from that to induce NGF mRNA expression, because protein
synthesis/secretion is regulated by several factors.

Fig. 7. H. erinaceus–Induced Phosporylation of JNK and c-Jun in 1321N1 Cells

1321N1 cells were incubated with ethanol extract of H. erinaceus (100 mg/ml) for the indicated times, and then JNK and c-Jun phosphorylation were
analyzed by Western blotting. (A) Time-dependent increase in the level of phosphorylation of JNK induced by H. erinaceus. (B) Time-dependent
increase in the level of phosphorylation of c-Jun induced by H. erinaceus.

Fig. 8. Effects of H. erinaceus Ethanol Extracts on c-fos mRNA Expres­sion in 1321N1 Cells

Morphological differentiation of PC12 cells was promoted by the conditioned media of 1321N1 cells incubated with

H. erinaceus

ethanol extract, suggesting that

H. erinaceus

stim­ulates neuronal differentiation

via

an increment in the release of neurotrophic factors, including NGF, from glial cells. However, the NGF concentration elevated by

H. erinaceus

extract is assumed to be too low to promote neurite out­growth in PC-12 cells. Therefore, other factors might be in­volved in neurite outgrowth. In fact,
astrocytes secrete other neurotrophins, cytokines and growth factors, some of which influence morphological change or facilitate NGF-induced
differentiation in PC12 cells.

226—28)

It has been reported that phorbol esters enhance PKC-de- pendent NGF synthesis in primary mice astrocytes, and AP- 1, one of the targets for PKC, was
assumed to regulate NGF gene expression.20 In fact, there is an AP-1 consensus se­quence (TRE: TPA-response element) downstream of the TATA box
at the junction of the exon I/intron I region of the NGF gene.30) AP-1 consists of homo or hetero dimers of Jun/ Jun or Fos/Jun, which bind to
DNA at the AP-1 site TRE. In the present study, the enhancement of NGF gene expression by H. erinaceus was inhibited by the JNK inhibitor
SP600125, and H. erinaceus caused phosphorylation of JNK. These re­sults suggest that JNK is involved in the enhancement of NGF gene expression
induced by H. erinaceus. JNK is the predominant kinase to phosphorylate c-Jun.31) Furthermore, H. erinaceus enhanced c-Jun
phosphorylation and c-fos gene expression as well as JNK phosphorylation. The activation of AP-1 by H. erinaceus is assumed to participate in NGF
gene expression downstream of JNK, but PKC is not involved in this signaling pathway, because of a lack of inhibitory action with the PKC inhibitor
GF109203X.

It has been reported that the active components of H. eri- naceus are the hericenones C—H, which stimulate NGF pro­tein synthesis in mouse or rat
astrocytes.19—21) However, heri­cenones C, D, and E did not exhibit NGF-promoting activity at all under the present experimental condition using
1321N1 human astrocytoma cells. In addition, the concentrations of hericenones in the ethanol extract were very low (the concen­trations of hericenones C,
D, and E in the 100 mg/ml ethanol extract of H. erinaceus were 20, 4, and 2 ng/ml, respectively) compared to their effective concentration (33
mg/ml) as shown in a previous report.20) These results, therefore, raise the pos­sibility that H. erinaceus has unknown active
compounds that promote NGF expression, other than hericenones, which are lipid-soluble (soluble in ethanol and/or ethyl acetate).

Furthermore, the oral administration of H. erinaceus in­creased NGF mRNA expression in the mouse hippocampus. This result suggests the possibility
that the active compound could be absorbed into blood and delivered into the central nervous system through the blood-brain barrier. The hip­pocampus is
postulated to encode working memory.32) The increase in the level of NGF mRNA in the hippocampus sug­gests the potential of H. erinaceus to act on the central ner­vous system in vivo. However, we could not elucidate why

H. erinaceus
increased NGF mRNA expression in the hip­pocampus but not in the cortex. This difference might result from variations of expression level of signaling
molecules re­lated to JNK signaling pathway or kinetic difference of active components of H. erinaceus such as brain distribution and metabolism
in these tissues.

On the other hand, the mycelia of H. erinaceus are known to contain erinacines, which also stimulate NGF synthe- sis.22 24) It has been
reported that oral administration of eri- nacine A significantly increases the level of NGF in the rat locus coeruleus and hippocampus, but not in the
cerebral cortex.33) However, it has not yet been reported that the fruit body of H. erinaceus contains erinacines. Thus, it is
neces­sary to reevaluate whether fruit bodies contain erinacines, and to examine the existence of unknown derivatives with NGF-inducing activity in the
fruit bodies of H. erinaceus.

In conclusion, H. erinaceus contains active compounds that stimulate NGF synthesis via activation of the JNK path­way; these compounds
are not hericenones.

Acknowledgements
This work was supported in part by Grant-in-Aid for Scientific Research from the Japan Society for Promotion of Science (No. 18790039 to Y. O. and No.
19659011 to N. N.), from the Ministry of Education, Culture, Sports, Science and Technology (No. 18058002 to N. N.) of Japan, and from Hokuto Life Science
Foundation.

REFERENCES

  1. Collerton D., Neuroscience, 19, 1—28 (1986).

  2. Obara Y., Nakahata N., Drug News Perspect., 15, 290—298 (2002).

  3. Allen S. J., Dawbarn D., Clin. Sci. (London), 110, 175—191 (2006).

  4. Mufson E. J., Kroin J. S., Sendera T. J., Sobreviela T., Prog. Neurobiol., 57, 451—484 (1999).

  5. Hellweg R., Gericke C. A., Jendroska K., Hartung H. D., Cervos- Navarro J., Int. J. Dev. Neurosci., 16, 787—794 (1998).

  6. Kromer L. F., Science, 235, 214—216 (1987).

  7. Ricceri L., Alleva E., Chiarotti F., Calamandrei G., Brain Res. Bull., 39, 219—226 (1996).

  8. Fischer W., Wictorin K., Bjorklund A., Williams L. R., Varon S., Gage F. H., Nature (London), 329, 65—68 (1987).

  9. Capsoni S., Giannotta S., Cattaneo A., Proc. Natl. Acad. Sci. U.S.A., 99, 12432—12437 (2002).

10) Takei N., Tsukui H., Hatanaka H., J. Neurochem., 53, 1405—1410

(1989).

Furukawa Y., Furukawa S., Ikeda F., Satoyoshi E., Hayashi K., FEBS Lett., 208, 258—262 (1986).

Furukawa Y., Furukawa S., Satoyoshi E., Hayashi K., J. Biol. Chem., 261, 6039—6047 (1986).

Takeuchi R., Murase K., Furukawa Y, Furukawa S., Hayashi K., FEBS Lett., 261, 63—66 (1990).

Yamaguchi K., Tsuji T., Wakuri S., Yazawa K., Kondo K., Shigemori H., Kobayashi J., Biosci. Biotechnol. Biochem., 57, 195—199 (1993). Nitta A.,
Hasegawa T., Nabeshima T., Neurosci. Lett., 163, 219—222 (1993).

Yamaguchi K., Uemura D., Tsuji T., Kondo K., Biosci. Biotechnol. Biochem., 58, 1749—1751 (1994).

Kawagishi H., Ishiyama D., Mori H., Sakamoto H., Ishiguro Y., Fu­rukawa S., Li J. D., Phytochemistry, 45, 1203—1205 (1997).

Obara Y, Nakahata N., Kita T., Takaya Y., Kobayashi H., Hosoi S., Ki- uchi F., Ohta T., Oshima Y., Ohizumi Y. D., Eur. J. Pharmacol., 370, 79—84
(1999).

Kawagishi H., Ando M., Mizuno T., Tetrahedron Lett., 31, 373—376

(1990).

Kawagishi H., Ando M., Sakamoto H., Yoshida S., Ojima F., Ishiguro Y., Ukai N., Furukawa S., Tetrahedron Lett., 32, 4561—4564 (1991). Kawagishi
K., Ando M., Shinba K., Sakamoto H., Yoshida S., Ojima F., Ishiguro Y, Ukai N., Furukawa S., Phytochemistry, 32, 175—178 (1993).

Kawagishi H., Shimada A., Hosokawa S., Mori H., Sakamoto H., Ishiguro Y, Sakemi S., J B., Kojima N., Furukawa S., Tetrahedron Lett., 37, 7399—7402
(1996).

Kawagishi H., Shimada A., Shirai R., Okamoto K., Ojima F., Sakamoto H., Ishiguro Y., Furukawa S., Tetrahedron Lett., 35, 1569— 1572 (1994).

Lee E. W., Shizuki K., Hosokawa S., Suzuki M., Suganuma H., In- akuma T., Li J., Ohnishi-Kameyama M., Nagata T., Furukawa S., Kawagishi H., Biosci. Biotechnol. Biochem., 64, 2402—2405 (2000). Marcotullio M. C., Pagiotti R., Maltese F., Oball-Mond Mwankie G. N., Hoshino T., Obara Y.,
Nakahata N., Bioorg. Med. Chem., 15, 2878—2882 (2007).

Althaus H. H., Richter-Landsberg C., Int. Rev. Cytol., 197, 203—277 (2000).

Cho S. G., Yi S. Y., Yoo Y S., Neurosci. Lett., 378, 49—54 (2005).

Wu Y Y., Bradshaw R. A., J. Biol. Chem., 271, 13033—13039 (1996). Jehan F., Neveu I., Naveilhan P., Wion D., Brachet P., Brain Res., 672,
128—136 (1995).

Hengerer B., Lindholm D., Heumann R., Ruther U., Wagner E. F., Thoenen H., Proc. Natl. Acad. Sci. U.S.A., 87, 3899—3903 (1990). Minden A., Lin A.,
Claret F. X., Abo A., Karin M., Cell, 81, 1147— 1157 (1995).

White N. M., McDonald R. J., Neurobiol. Learn. Mem., 77, 125—184 (2002).

Shimbo M., Kawagishi H., Yokogoshi H., Nutr. Res., 25, 617—623 (2005).

1
To whom correspondence should be addressed. e-mail: nakahata@mail.pharm.tohoku.ac.jp

Efficacy of Orally Administered Superfine Dispresed Lentinan

Efficacy of Orally Administered Superfine Dispersed Lentinan (p-1,3-Glucan) for the Treatment of Advanced Colorectal Cancer

SHOICHI HAZAMA1, SEIJI WATANABE2, MANABU OHASHI3, MASASHI YAGI4,

MICHINARI SUZUKI5, KENJI MATSUDA6, TATSUHITO YAMAMOTO7,

YASUYO SUGA8, TETSUYA SUGA9, SABURO NAKAZAWA10 and MASAAKI OKA1

department of Surgery II, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi;

2Chemotherapy Division, Inoue Hospital, Nishi 2-Chome, Minami 7-Jo, Chuo-Ku, Sapporo, Hokkaido;


3
Digestive and General Surgery, Niigata University of Medicine, 1-754 Asahimachi-Dori, Chuo-Ku, Niigata; 4Department of Surgery, Public
Central Hospital of Matto-Ishikawa, 3-8 Kuramitsu, Hakusan, Ishikawa; 5Department of Surgery, Shunan City Shinnanyo Hospital, 2-3-15
Miyanomae, Shunan, Yamaguchi; 6Hashimoto Hospital, 4-31 Minaminocho, Horidome, Wakayama;


7
Surgery Division, Iseikai Tsushimi Hospital, 413-1 Emukai, Hagi, Yamaguchi; 8Pharmaceutical Laboratories, Ajinomoto Co., Inc., 1-1
Suzuki-Cho, Kawasaki-Ku, Kawasaki, Kanagawa;


9
Pharmaceutical Alliance Department, Ajinomoto Co., Inc., 2-1-1 Irifune, Chuo-Ku, Tokyo; 10Yamashita Hospital, 1-3-5 Nakamachi, Ichinomiya,
Aichi, Japan

2611

0250-7005/2009 $2.00+.40



Abstract.

Background: Lentinan (LNT) is an immune adjuvant medicine for advanced gastric cancer in Japan. Recently, an oral formulation of superfine dispersed
lentinan (SDL) has become clinically available. To investigate the safety and effectiveness of SDL, a multi center clinical study in patients with
advanced colorectal cancer was conducted. Patients and Methods: Adverse events were assessed and the patients’ quality of life (QOL) and the binding
ability of peripheral blood monocytes (PBM) to LNT were also evaluated. Results: Four grade 2 adverse events associated with SDL treatment were
observed among the 80 patients. Adverse events associated with chemotherapy were observed in 9 out of the 64 chemotherapy-treated patients. Among the
48 patients assessed for QOL, the patients with low QOL scores before SDL treatment (n=23) reported a significant improvement in their QOL scores after
12 weeks of SDL administration. The rates of LNT-binding PBM in the QOL- improved group were significantly higher than those in the QOL-not-improved
group (p<0.05). Conclusion: SDL was

Correspondence to:
Shoichi Hazama, Department of Surgery II, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami- Kogushi, Ube, Yamaguchi 755-8505, Japan. Tel: +81
0836222419, Fax: +81 0836222263, e-mail: hazama@po.cc.yamaguchi-u.ac.jp

Key Words:
Beta-1,3-glucan, colorectal cancer, lentinan, peripheral blood monocytes, quality of life, superfine dispersed lentinan.


safe and effective for suppressing the adverse effects of chemotherapy as well as improving QOL. The binding ability of PBM to LNT appears to be a
promising predictor of QOL improvement after SDL administration.

Lentinan (LNT) is a purified P-1,3-glucan with P-1,6- branches derived from the edible mushroom Lentinus edodes (Berk) Sing (1). LNT has immune
adjuvant effects and has been reported to increase host defense mechanisms against murine and human tumors (2-4). The clinical efficacy of LNT, such as its
effect on long-term survival and the improvement of the quality of life (QOL) status, were evaluated in patients with inoperable and recurrent gastric
cancer (5, 6). The mode of action of LNT consists of T-cell- dependent immunopotentiation mediated by macrophages and monocytes (7-9). Studies of some
P-glucan receptors such as CD11b, dectin-1 and toll-like receptor 2 (TLR2) have revealed that P-glucan binds to these receptors on macrophages and
monocytes (10-12). Furthermore, LNT- induced reductive macrophages are reportedly skewed toward Th1 as a result of the production of IL-12 (13-15), and the
binding ability of peripheral blood monocytes (PBM) to LNT might directly influence its in vivo effects (16). Intravenously administered LNT can
cancel a Th2-dominant condition in patients with digestive tract cancer and improve the balance between Th1 and Th2 (9). However, why orally administered
LNT is ineffective has been a long-standing puzzle. In aqueous solution, the particle size of LNT is

approximately 100 to 200 pm; this impedes the absorption of LNT particles through the intestinal mucosa. Recently, an oral formulation of superfine
dispersed lentinan (SDL) has been developed and is now clinically available. SDL reportedly enables the potentiation of intestinal mucosal immunity (17).

in the present study, we evaluated the safety and efficacy of SDL for suppressing the adverse effects of chemotherapy and the usability of the binding
ability of peripheral blood CD14+ monocytes to LNT as a promising predictor of QOL improvement in patients with advanced colorectal cancer who have been
treated with SDL.


Patients and Methods

Patients.
Between July 2004 and April 2005, patients with unresectable colorectal cancer were enrolled at 25 centers involved in a study group on Foods and
Lifestyle-related Disease, an affiliated organization of the Japanese Society of Geriatric Gastroenterology.

Eligibility.
Patients who fulfilled the following eligibility requirements were enrolled in the study: (i) a diagnosis of unresectable colorectal cancer; (ii) an age of
20 years or older; (iii) an Eastern cooperative Oncology Group (EcOG) performance status (PS) of Grade 2 or lower; (iv) an ability to drink at least 100 ml
of water per dose; (v) a life expectancy of at least 3 months; (vi) not receiving immunotherapy (the last immunotherapy session must have been completed at
least 4 weeks before enrollment); (vii) not taking immune adjuvant foods (the last immune adjuvant food treatment must have been completed at least 2 weeks
before enrollment); (viii) no allergies to Shiitake mushrooms (Lentinus edodes) or soybeans; (ix) adequate bone marrow function (hemoglobin
concentration >10 mg/dl, white blood cell (WBC) count >3,000/pl and <12,000/pl, neutrophil count >1,500/pl and platelet count >10×10 4/pl); (x) adequate liver function (serum bilirubin levels, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) < 2.5 times
the upper limit of normal); (xi) adequate renal function (serum creatinine levels within normal limits); (xii) no other severe medical conditions; and
(xiii) no other active malignancies. Written informed consent was obtained from all the patients. Pregnant women and patients receiving steroids or
immunosuppressive medicines for a prolonged period were excluded from the study. This study was approved by the Institutional Review Boards at all the
participating hospitals. The approvals were based on the 2000 revision of the Helsinki Declaration.

2612



Test sample containing SDL.

The SDL-containing test sample was supplied by Ajinomoto Co., Inc. and was prepared in 100 ml bags containing 15 mg of SDL per bag. According to the
manufacturer’s specification, the mean particle diameter of the LNT was 0.08 pm (17). No change in the distribution of particle sizes was noted after 18
months of storage at room temperature. The oral superfine dispersed formulation was confirmed to enable the LNT to adhere onto or to be taken up into
Peyer’s patches of the small intestine, and electron microscopy showed that the LNT particles were present in the vacuoles of epithelial cells (18). These
results indicate that LNT was not taken up into the Peyer’s patches unless it was dispersed as superfine particles.

Treatment schedule.
SDL was administered at a dose of 15 mg of LNT once daily for 12 weeks. During the period of SDL administration, the patients were also treated with
various chemotherapy regimens, such as 5-fluorouracil (5-FU); tegafur and uracil (UFT); tegafur, gimeracil and oteracil potassium (S-1); doxifluoridine
(5’-DFUR); levofolinate (LV); irinotecan (CPT-11); gemcitabine (GEM); or cisplatin (CDDP). Treatment was continued until disease progression, unacceptable
toxicity, an adverse event, or the withdrawal of patient consent. Compliance was assessed using data obtained from scheduled interviews with the patients
and regular monitoring.

Assessment of safety.
A complete blood cell count, liver function tests, renal function tests and urinalysis were performed at least every 4 weeks throughout the study period.
Adverse events were evaluated according to the CTCAE v3.0 (Common Terminology Criteria for Adverse Events version 3.0) (19).

Evaluation of QOL.
QOL was evaluated using the QOL Questionnaire for Cancer Patients Treated with Anticancer Drugs (QOL-ACD) in 48 patients who were able to answer the
questionnaire before and after 4, 8 and 12 weeks of SDL administration. The QOL-ACD was developed by Kurihara et al. in 1993 and is endorsed by
the Japanese Ministry of Health, Labor and Welfare. The questionnaire is the first patient­assessed QOL evaluation system for Japanese cancer patients for
which the reliability, validity and sensitivity to anticancer treatment have been verified (20). The QOL-ACD was primarily developed to assess outcome in
clinical trials. Briefly, the QOL-ACD consists of 22 items, 21 of which are investigated using Likert scales covering four domains, namely, activity (6
items), physical (5 items), psychological (5 items) and social (5 items) aspects. The remaining item covers global aspects of QOL as represented by a face
scale consisting of five different faces selected from the original ones used by Lorish and Maisiak (21). Patients were invited to answer all questions by
circling a number on the scale or the face that best described their state. The scores for all questions (1 to 5) were totaled to produce an overall QOL
score ranging from a minimum of 22 to a maximum of 110. A higher score represents a higher QOL status.

Binding ability of CD14+ monocytes to LNT.
Fluorescein-labeled LNT (F-LNT) was prepared using a reductive amination method (22). Briefly, the LNT solution plus 2 mM of sodium periodide was stirred
at 4°C overnight and then ethylene glycol was added. The mixture was dialyzed with distilled water. Fluorescein 5-thiosemicarbazide (40 pg/ml; Molecular
Probes, Eugene, OR, USA) was added under alkaline conditions and stirred at 4°C overnight. Schiff-bases were reduced using sodium borohydride. The
resulting F-LNT was stored at 4°C under light shielding before use.

Peripheral blood (PB) samples were collected into tubes containing sodium heparin anticoagulant prior to the initiation of SDL administration. The PB was
then incubated with F-LNT at 37°C for 75 minutes. During the last 30 minutes, phycoerythrin-labeled anti- CD14 antibody (CALTAG, Burlingame, CA, USA) was
added. The erythrocytes were lysed with FACS Lysing Solution (Becton Dickinson, San Jose, CA, USA) for 10 minutes at room temperature. After washing with
phosphate-buffered saline, the fluorescence intensity of the CD14+ monocytes was measured using a fluorescence- activated cell sorter (FACS Calibur; Becton
Dickinson).

Statistical snalysis.
The QOL scores of the patients before and after SDL administration were compared using a Wilcoxon signed rank test.

Table I. Patient characteristics.

No. of patients

Enrollment

80

Eligibility

Ineligible

9*

Eligible

71

Gender

Male

55

Female

16

Age (years)

Median, 65 (range, 32-82 years)

ECOG-performance status (PS)

0

41

1

23

2

4

Unknown

3

Chemotherapy

Yes

64

No

7

*Three patients were subsequently found to have had a low WBC count (<3,000/^l), a high WBC count (>12,000/^l), or a low hemoglobin concentration
(<10.0 g/dl) prior to the start of SDL administration. Two patients were given a biological response modifier (BRM) within the SDL treatment period.
Four patients became unable to drink at least 100 ml of water per dose within 2 weeks after enrollment.



The rates of LNT-binding PBM were compared using a Mann­Whitney U-test. P-values of less than 0.05 were considered statistically significant.


Results

Patient characteristics.
The demographic characteristics of the patients are shown in Table I. Between July 2004 and April 2005, 80 patients with unresectable colorectal cancer
were enrolled and treated with SDL. SDL-related adverse events were evaluated in all the patients. Three patients were later found to have had a low WBC
count (<3,000/|rl), a high WBC count (>12,000/|rl), and a low hemoglobin concentration (<10.0g/dl), respectively, prior to the initiation of SDL
administration. Two patients were given biological response modifiers (BRM) within the period of SDL treatment. Four patients became unable to drink at
least 100 ml of water per dose within 2 weeks after enrollment. These 9 patients were subsequently deemed to be ineligible for inclusion in this study and
were excluded from all analyses except for the safety analysis. Fifty-five men and 16 women with a median age of 65 years (range, 32-82 years) were
analyzed in all of the studies. Sixty-four of these 71 eligible patients also underwent chemotherapy, while the remaining 7 patients were only treated with
SDL during the study period.

2613



Safety of SDL and adverse events during chemotherapy.

Four adverse events (5%) that were or were suspected to have been related to SDL were observed within the period of SDL treatment: diarrhea occurred in 2
patients, and a rash and constipation occurred in 1 patient each. These events were grade 2 and disappeared or remitted within the study period. As shown
in Table II, chemotherapy-related adverse events were observed in 9 (14% ) out of the 64 patients who received chemotherapy among the 71 patients who were
eligible for inclusion in this study. Only one patient suffered from a grade 4 adverse event (neutropenia) after receiving 5-FU/LV chemotherapy. The other
adverse events in the 8 patients were grade 1 or 2. Among patients who received a CPT-11-based or a 5-FU-based regimen, adverse events were observed in 5
(19%) out of 27 patients and 4 (11%) out of 37 patients, respectively. Among the 24 patients who were treated with a chemotherapy regimen containing S-1,
only 1 (4%) patient exhibited an adverse event (grade 1).

Improvement of QOL status after SDL administration.
Forty- eight patients received SDL everyday for 12 weeks and were able to answer the questionnaire both before and after 4, 8 and 12 weeks of SDL
administration. The median overall QOL score before SDL administration was 85. Since no significant difference in the QOL scores was observed before and
after the administration of SDL, the overall QOL scores tended to remain constant throughout the 12 weeks of administration (Figure 1A). Among these 48
patients, 23 patients had a QOL score that was less than 85 before the start of SDL administration (QOL low-score group). The QOL scores of this group
improved significantly after 12 weeks of SDL administration. Figure 1B shows the changes in the overall QOL scores from before administration to after 12
weeks of SDL administration in the QOL low-score group (p=0.0199, Wilcoxon signed rank test). In 15 (65% ) out of the 23 patients in the QOL low-score
group, the overall QOL scores improved after 12 weeks of SDL administration.

Binding ability of CD14+ PBMs to LNT and its relation to QOL improvement.
A histogram showing the results of a FACS for CD14+ PBMs is shown in Figure 2. Two types of PBMs were present: bright-positive cells (strong F-LNT binding)
and dull-positive cells (weak F-LNT binding) cells. The binding ability of PBMs to LNT was measured before the initiation of SDL administration. The rates
of bright-positive cells among the CD14+ PBM cells varied individually, with a median of 3.6% , ranging from 0.2% to 44.4% among the 48 patients. The
patients were divided into three categories: namely, a high (>5.0% , n=20), moderate (1.0-4.9% , n=19) and low (<1.0% , n=9) group. Changes (A) in
the overall QOL scores from before to after 12 weeks of SDL administration were then evaluated and the patients were divided into three groups, namely: an
‘improved’ (A>0, n=22), a ‘high-score maintained’ (A<0 and QOL score at 12 weeks of SDL administration >85, n=12); and a ‘not-improved’ (A<0
and QOL score at 12 weeks <85, n=14) group. As shown in Figure 3, the binding abilities

of PBMs to LNT in the QOL ‘improved’ group were significantly greater than those in the QOL ‘not-improved’ group, according to a Mann-Whitney L-test
(p<0.05).


Discussion

2614


The present study demonstrates the safety and effectiveness of SDL for patients with advanced colorectal cancer. Four adverse events that were or were
suspected to have been related to SDL were observed in 4 (5%) out of the 80 patients. All of these symptoms were grade 2 and disappeared or remitted within
the study period. Thus, oral SDL treatment, like intravenous LNT treatment, appears to be safe for cancer patients. Regarding the onset of adverse events
associated with chemotherapy, 12 adverse events were observed in only 9 (14%) out of 64 patients who received chemotherapy. Among the CPT-11-based
regimens, neutropenia (grade 1) was observed in only one patient (4%) and no diarrhea (0%) was observed in the 27 patients. Neutropenia (incidence of all
grades, about 70%; grades 3/4, about 30%) and diarrhea (incidence of all grades, about 60% ; grades 3/4, about 20% ) are usually the main severe adverse
events associated with CPT-11-based regimens (23, 24). Among the 5-FU-based regimens, neutropenia and diarrhea were observed in 1 (3%) and 2 (5%) of the 37
patients, respectively. The respective incidences of neutropenia and diarrhea are reportedly 67% (grades 3/4, 31%) and 60%




Table II. Adverse events associated with chemotherapy.

Regimen

No. of patients

Non*

Symptoms (grade [G])

CPT-11-based

CPT

6

5

Neutropenia (G1)

regimens

CPT/5’-DFUR

8

6

Pruritus, nausea (G1)

CPT/S-1

6

5

Vomiting (G2)

CPT/5-FU

2

2

CPT/5-FU/LV

1

1

CPT/UFT

1

0

Nausea (G1)

CPT/5-FU /LV^S-1

2

2

UFT/LV^CPT/5’- DFUR

1

1

SubtotaI

27

22

5-FU-based

S-1

13

12

Thrombocytopenia (G1)/leukopenia (G1)

regimens

UFT/LV

9

8

Malaise (G2)/leukopenia (G1)

5-FU/LV

6

5

Diarrhea (G2)/neutropenia (G4)

UFT

1

1

5’-DFUR

2

2

5-FU

1

0

Diarrhea (G2)

S-1^5-FU/LV

1

1

UFT/GEM

1

1

5-FU/CDDP

1

1

S- 1/CDDP

2

2

SubtotaI

37

33

TotaI

64

55

9

CPT-11, Irinotecan; 5-FU, 5-fIuorouraciI; UFT, tegafur, uracil; S-1, tegafur, gimeracil, oteraci! potassium; 5’-DFUR, doxifluridine; LV, levofolinate; GEM,
gemcitabine; CDDP, cispIatin. *No. of patients with no adverse events.


Time of QOL evaluation

Figure 1.

Change in overall QOL scores from before SDL administration to after 12 weeks of SDL administration. A, Forty-eight patients were able to answer the
QOL questionnaires. Although no significant difference was observed, the QOL scores tended to remain constant during the 12-week treatment period. B,
Twenty-three patients’ QOL scores were initially less than the median (85). The QOL scores improved significantly (p=0.0199, Wilcoxon signed rank test)
with SDL. The QOL scores of 15 (65% ) of the 23 patients improved after 12 weeks of SDL administration.

A

f

\

ï

I

A
.j

1

B
,


100

x

I 80

60

40

20

01

be

a

e

b

o

Pm

0

10° 10

1

10

2

10

3
Fluorescence intensity Contour plot

A

Bl
B-2

IlSis

/

1


10° 10′ 102 103 10° 101 102 103 Fluorescence intensity

Figure 2.

LNT-binding ability of CD14+ monocytes by FACS Histogram. A, without F-LNT (LNT-negative) ; B, with F-LNT (B-1, dull-positive; B-2, bright-positive).

(grades 3/4, 11%) for IV 5-FU/LV and 11% (grades 3/4, 3%) and 54% (grades 3/4, 18% ) for oral UFT/LV (25). According to another report, the incidences of
neutropenia and diarrhea for oral UFT/LV were 34% (grades 3/4, 0%) and 39% (grades 3/4, 9% ), respectively (26). In addition, in the present study, the
incidence of adverse effects (thrombocytopenia/leukopenia) for regimens including S-1 was only 4% (1 out of 24 patients). The incidences of

2615


thrombocytopenia and leukopenia associated with S-1 in stage II and III gastric cancer patients (n=517) were reportedly 26% (grades 3/4, 0.2%) and 59%
(grades 3/4, 1%), respectively (27), while the incidence of leucopenia was 38% (grades 3/4, 2%) in advanced gastric cancer patients (n=150) (28). Several
reports have revealed that anticancer drugs such as CPT-11, paclitaxel, CDDP and 5-FU act by increasing intracellular reactive oxygen species (ROS), and
that N-acetylcysteine or mangafodipir (a superoxide dismutase (SOD) mimic) reduce the hematotoxicity of anticancer agents by reducing intracellular ROS
(29-31). LNT increases the intracellular reductive glutathione content as well as the N-acetylcysteine content and scavenges ROS by increasing reductive
glutathione (14, 15). Thus, the oral administration of SDL may be useful for the suppression of chemotherapy toxicity.

Figure 3.

LNT-binding abilities in QOL ‘improved” and ‘not-improved” groups. Forty-eight patients were divided into 3 categories according to their LNT-binding
abilities: >5%, high; 1.0-4.9%, moderate; <1.0%, low. The numbers in parentheses represent the number of patiens in each category. There was a
significant difference in the LNT-binding ability between the QOL ‘improved” and the ‘not-improved” groups (p<0.05, Mann-Whitney U-test).



In the present study, we also evaluated the patients’ QOL scores before and after SDL administration in 48 patients with unresectable colorectal cancer.
Usually, the QOL status of patients with advanced cancer tends to gradually decrease. In contrast, among the 48 patients in the present study, no
significant difference was seen between the QOL scores for before SDL administration and those for after 12 weeks of SDL administration. Thus, the QOL
status tended to remain constant for the 12 weeks of SDL administration. In particular, the QOL scores (Activity, Physical, Psychological, Social aspects
and Overall score) obtained after 12 weeks of SDL administration were significantly better than those obtained before SDL administration in the QOL
low-score group. The overall QOL scores were improved in 15 (65% ) of the 23 patients in this group. Some physical aspects (including appetite condition
and body weight loss) were significantly

improved after 8 weeks of SDL administration. The increased production of prostaglandin E2 (PGE2) in macrophages is reportedly involved in appetite loss
and reduced body weight in cachectic mice inoculated with colon cancer cells (32). A previous study demonstrated that LNT reduces PGE2 and increases IL-2
production in macrophages (33). In addition, an experiment confirmed that the administration of LNT significantly increased daily food intake in rats with
stress- induced anorexia (34). Moreover, LNT has been proven to inhibit the production of interleukin-6 (IL-6) in macrophages (35), which is known to cause
cachexia (36). These findings may support the present finding that SDL may effectively improve QOL in patients with advanced colorectal cancer.

To estimate more objective parameters, we evaluated the binding ability of CD 14+ monocytes to LNT. The binding abilities of PBMs in patients with seasonal
allergic symptoms reportedly exhibit individual variations, and the binding abilities of PBMs in patients whose symptoms were improved by SDL treatment
were significantly higher than those in patients whose symptoms were not improved (16). In the present study, the binding abilities of the PBMs to LNT also
showed individual variations, with a median of 3.6% (ranging from 0.2% to 44.4%) in the 48 patients whose QOL statuses were estimated. No significant
difference was seen between the QOL ‘improved’ group (n=22; median, 5.3%; range, 0.8­33.8%) and the ‘high-score maintained’ group (n=12; median, 5.0%;
range, 0.2-44.4%). The patients in the ‘high-score maintained’ group are patients who responded to SDL. On the other hand, the rate of LNT-bound PMBs in
the QOL ‘improved’ group was significantly higher than that in the QOL ‘not-improved’ group (n=14; median, 2.0%; range, 0.4­14.7%). There were several
patients whose PBMs exhibited variable (increased) LNT-binding abilities 1 or 2 months after the initiation of SDL treatment (data not shown). These
patients may have converted from LNT low-responders to high-responders as a result of the SDL treatment and/or chemotherapy. These results suggest that the
LNT-binding ability of CD 14+ monocytes should be estimated at 1 or 2 months after the initiation of SDL treatment to identify SDL responders. As mentioned
above, the mode of action of LNT is mediated by host immune competent cells and LNT is reported to bind to monocytes and macrophages, whose binding
abilities to LNT exhibit individual variations (16). Although the functional differences between F-LNT bright- positive monocytes and dull-positive
monocytes are still unknown and further investigations are required, the LNT- binding ability of cD14+ monocytes may be a promising predictor
for the selection of responders to SDL treatment.

2616


In conclusion, SDL was safe and effective for suppressing the adverse effects of chemotherapy as well as improving QOL. The binding ability of PBM to LNT
appears to be a promising predictor of QOL improvement after SDL administration.


Acknowledgements

This study was supported by grants from the Digestive Study Group on Foods and Lifestyle-related Disease, an affiliated organization of the Japanese
Society of Geriatric Gastroenterology.

We thank Ajinomoto Co., Inc., which provided the test samples containing SDL, and Dr. I. Makino, Dr. T. Bamba, Dr. Y. Arakawa, Dr. Y. Atomi and Dr. T.
Matsui, who are permanent organizers of the Digestive Study Group on Foods and Lifestyle-related Disease, for their general advice regarding this study. We
are also grateful to Dr. A. Kamiya of the Department of Pharmacy of Yamaguchi University Hospital for his kind advice regarding the preparation of the
clinical study protocol.


Conflict of Interest Statement

The authors did not have any assistance writing the present manuscript. T. Suga and Y. Suga are employees of Ajinomoto Co., Inc., which holds patents and
technologies for the production of SDL. Dr. Nakazawa is the representative organizer of the Digestive Study Group on Foods and Lifestyle-related Disease.
No other potential conflicts of interest relevant to this article are present.


References

  1. Chihara G, Maeda YY, Hamuro J, Sakaki T and Fukuoka F: Inhibition of mouse sarcoma 180 by polysaccharide from Lentinus edodes (Berk) Sing.
    Nature 222: 687-688, 1969.

  2. Zakany J, Chihara G and Fachet J: Effect of lentinan on the production of migration inhibitory factor induced by syngeneic tumor in mice. Int J
    Cancer 26: 783-788, 1980.

  3. Suga T, Shiio T, Maeda YY and Chihara G: Antitumor activity of lentinan in murine syngeneic and autochthonous hosts and its suppressive effect on
    3-methylcholanthrene-induced carcino­genesis. Cancer Res 44: 5132-5137, 1984.

  4. Ochiai T, Isono K, Suzuki T, Koide Y, Gunji Y, Nagata M and Ogawa N: Effect of immunotherapy with lentinan on patients’ survival and immunological
    parameters in patients with gastric cancer: Results of a multi-centre randomized controlled study. Int J Immunother VIII: 161-169, 1992.

  5. Taguchi T: Clinical efficacy of lentinan on patients with stomach cancer – End point results of a four-year follow-up survey. Cancer Detect Prev
    Suppl 1: 333-349, 1987.

  6. Nakano H, Namatame K, Nemoto H, Motohashi H, Nishiyama K and Kumada K: A multi-institutional prospective study of lentinan in advanced gastric
    cancer patients with unresectable and recurrent diseases: Effect on prolongation of survival and improvement of quality of life.
    Hepato-Gastroenterol 46: 2662-2668, 1999.

  7. Maeda YY and Chihara G: The effects of neonatal thymectomy on the antitumor activity of lentinan, carboxymethylpachymaran and zymosan, and their
    effects on various immune responses. Int J Cancer 11: 153-161, 1973.

  8. Hamuro J, Rollinghoff M and Wagner H: Induction of cytotoxic peritoneal exudates cells by T-cell immune adjuvants of the |3(1^3) glucan-type
    lentinan and its analogues. Immunology 39: 551-559, 1980.

  9. Yoshino S, Tabata T, Hazama S, Iizuka N, Yamamoto K, Hirayama M, Tangoku A and Oka M: Immunoregulatory effects of the antitumor polysaccharide
    lentinan on Th1/Th2 balance in patients with digestive cancer. Anticancer Res 20: 4707-4712, 2000.

  10. Thornton B P, Vetvicka V, Pitman M, Goldman R C and Ross G D: Analysis of the sugar specificity and molecular location of the ß-glucan-binding
    lectin site of complement receptor type 3 (CD11b/CD18). J Immunol 156: 1235-1246, 1996.

  11. Brown G D, Taylor P R, Reid D M, Willment J A, Williams D L, Pomares L M, Wong S Y C and Gordon S: Dectin-1 is a major ß- glucan receptor on
    macrophages. J Exp Med 196: 407-412, 2002.

  12. Gantner BN, Simmons RM, Canavera SJ, Akira S and Underhill DM: Collaborative induction of inflammatory responses by dectin- 1 and toll-like
    receptor 2. J Exp Med 197: 1107-1117, 2003.

  13. Hamuro J, Murata Y, Suzuki M, Takatsuki F and Suga T: The triggering and healing of tumor stromal inflammatory reactions regulated by oxidative and
    reductive macrophages. Gann Monograph Cancer Res 48: 153-164, 1999.

  14. Murata Y, Shimamura T, Tagami T, Takatsuki F and Hamuro J: The skewing to Th1 induced by lentinan is directed through the distinctive cytokine
    production by macrophages with elevated intracellular glutathione content. Int Immunopharmac 2: 673-689, 2002.

  15. Murata Y, Shimamura T and Hamuro J: The polarization of T(h)1/T(h)2 balance is dependent on the intracellular thiol redox status of macrophages due
    to the distinctive cytokine production. Int Immunol 14: 201-212, 2002.

  16. Yamada J, Hamuro J, Hatanaka H, Hamabata K and Kinoshita S: Alleviation of seasonal allergic symptoms with superfine ß-1,3- glucan – A randomized
    study. J Allergy Clin Immunol 119: 1119­1126,2007.

  17. Shen J, Ren H, Tomiyama MC, Suga Y, Suga T Kuwano Y, Iiai T, Hatakeyama K and Abo T: Potentiation of intestinal immunity by micellary mushroom
    extracts. Biomed Res 28: 71-77, 2007.

  18. Suga Y, Matsunaga Y, Sato Y, Murata M and Suga T: The importance of size for antitumor effects of beta-glucan. Biotherapy 19: 273-278, 2005 (in
    Japanese with English abstract).

  19. Trotti A, Colevas A D, Setser A, Rusch V, Jaques D, Budach V, Langer C, Murphy B, Cumberlin R, Coleman C N and Rubin P: CTCAE v3.0: development of
    a comprehensive grading system for the adverse events of cancer treatment. Semin Radiat Oncol 13: 176-181, 2003.

  20. Kurihara M, Shimizu H, Tsuboi K, Kobayashi K, Murakami M, Eguchi K and Shimozuma K: Development of quality of life questionnaire in Japan: quality
    of life assessment of cancer patients receiving chemotherapy. Psycho-oncology 8: 355-363, 1999.

  21. Lorish CD and Maisiak R: The face scale – a brief, nonverbal method for assessing patient mood. Arthritis Rheum 29: 906-909, 1986.

  22. Kishida E, Sone Y, Shibata S and Misaki A: Preparation and immunochemical characterization of antibody to branched ß-(1- 3)-D-glucan of Volvariella volvasea, and its use in studies of antitumor actions. J Agric Biol Chem 53: 1849-1859, 1989.

  23. Rosati G, Cordio S, Caputo G, Condorelli S, Germano D, Mattina M, Amadio P, Reggiardo G and Manzione L: Phase II trial of a biweekly regimen of
    fluorouracil and leucovorin plus irinotecan in patients with previously untreated advanced gastric cancer. J Chemother 19: 570-576, 2007.

  24. Bouzid K, Khalfallah S, Tujakowski J, Piko B, Purkalne G, Plate S, Padrik P, Serafy M, Pshevloutsky E M and Boussard B: A randomized phase II trial
    of irinotecan in combination with infusional or two different bolus 5-fluorouracil and folinic acid regimens as first-line therapy for advanced
    colorectal cancer. Ann Oncol 14: 1106-1114, 2003.

  25. Carmichael J, Popiela T, Radstone D, Falk S, Borner M, Oza A, Skovsgaard T, Munier S and Martin C: Randomized comparative study of tegafur/uracil
    and oral leucovorin versus parenteral fIuorouraciI and Ieucovorin in patients with previousIy untreated metastatic colorectal cancer. J
    Clin Oncol 20: 3617-3627, 2002.

  26. Shirao K, Hoff P M, Ohtsu A, Loehrer P J, Hyodo I, WadIer S, WadIeigh R G, O’Dwyer P J, Muro K, Yamada Y, Boku N, Nagashima F and Abbruzzese J L:
    Comparison of the efficacy, toxicity, and pharmacokinetics of a uraciI/tegafur (UFT) pIus oraI Ieucovorin (LV) regimen between Japanese and
    American patients with advanced coIorectaI cancer – Joint United States and Japan study of UFT/LV. J CIin OncoI 22: 3466-3474, 2004.

  27. Sakuramoto S, Sasako M, Yamaguchi T, Kinoshita T, Fujii M, Nashimoto A, Furukawa H, Nakajima T, Ohashi Y, Imamura H, Higashino M, Yamamura Y,
    Kurita A and Arai K, for the ACTS- GC Group: Adjuvant chemotherapy for gastric cancer with S-1, an oraI fluoropyrimidine. N EngI J Med 357:
    1810-1820, 2007.

  28. Koizumi W, Narahara H, Hara T, Takagane A, Akiya T, Takagi M, Miyashita K, Nishizaki T, Kobayashi O, Takiyama W, Toh Y, Nagaie T, Takagi S,
    Yamamura Y, Yanaoka K, Orita H and Takeuchi M: S-1 pIus cispIatin versus S-1 aIone for first-Iine treatment of advanced gastric cancer
    (SPIRITS triaI) – a phase III triaI. Lancet OncoI 9: 215-221, 2008.

  29. James H D: Redox moduIation of chemotherapy-induced tumor ceII kiIIing and normaI tissue toxicity. J NatI Cancer Inst 98: 223­225,2006.

  30. AIexandre J, Nicco C, Chereau C, Laurent A, WeiII B, GoIdwasser F and Batteux F: Improvement of the therapeutic index of anticancer drugs by the
    superoxide dismutase mimic mangafodipir. J NatI Cancer Inst 98: 236-244, 2006.

  31. Mantovani G, Maccio A, Madeddu C, Mura L, Gramignano G, Lusso M R, Murgia V, Camboni P, FerreIi L, Mocci M and Massa E: The impact of different
    antioxidant agents aIone or in combination on reactive oxgen species, antioxidant enzymes and cytokines in a series of advanced cancer patients at
    different sites – CorreIation with disease progression. Free RadicaI Res 37: 213­223,2003.

  32. Graves E, Hitt A, Pariza M W, Cook M E and McCarthy D O: Conjugated IinoIeic acid preserves gastrocnemius muscIe mass in mice bearing the coIon-26
    adenocarcinoma. Res Nurs HeaIth 28: 48-55, 2005.

  33. Hamuro J, Kikuchi T, Takatsuki F and Suzuki M: Cancer ceII progression and chemoimmunotherapy – DuaI effects in the induction of resistance to
    therapy. Br J Cancer 73: 465-471, 1996.

  34. Aou S, Ma J and Hori T: Effects of Ientinan on food intake and pIasma caIcium IeveI. Biotherapy 5: 1728-1731, 1991 (in Japanese with EngIish
    abstract).

  35. Suzuki M, Takatsuki F, Maeda Y Y, Hamuro J and Chihara G: Antitumor and immunoIogicaI activity of Ientinan in comparison with LPS. Int J
    ImmunopharmacoI 16: 462-468, 1996.

  36. Strassmann G, Masui Y, Chizzonite R and Fong M: Mechanisms of experimentaI cancer cachexia: LocaI invoIvement of IL-1 in coIon-26 tumor. J ImmunoI 150: 2341-2345, 1993.

Received December 29, 2008 Revised March 9, 2009 Accepted May 5, 2009

Anticancer Effects of Ganoderma lucidum

Anticancer Effects of Ganoderma lucidum: A Review of Scientific Evidence

Authors: John W. M. Yuen ;Mayur Danny I. Gohel DOI: 10.1207/s15327914nc5301_2 Publication Frequency: 8 issues per year

Published in: v Nutrition and Cancer, Volume 53, Issue 1 September 2005 , pages 11 -17

Formats available: PDF (English)

View Article: 9 View Article (PDF)

Abstract

«Lingzhi» (Ganoderma lucidum), a popular medicinal mushroom, has been used in China for longevity and health promotion since ancient times. Investigations
into the anticancer activity of lingzhi have been performed in both in vitro and in vivo studies, supporting its application for cancer treatment and
prevention. The proposed anticancer activity of lingzhi has prompted its usage by cancer patients. It remains debatable as to whether lingzhi is a food
supplement for health maintenance or actually a therapeutic «drug» for medical proposes. Thus far there has been no report of human trials using lingzhi as
a direct anticancer agent, despite some evidence showing the usage of lingzhi as a potential supplement to cancer patients. Cellular immune responses and
mitogenic reactivity of cancer patients have been enhanced by lingzhi, as reported in two randomized and one nonrandomized trials, and the quality of life
of 65% of lung cancer patients improved in one study. The direct cytotoxic and anti-angiogenesis mechanisms of lingzhi have been established by in vitro
studies; however, clinical studies should not be neglected to define the applicable dosage in vivo. At present, lingzhi is a health food supplement to
support cancer patients, yet the evidence supporting the potential of direct in vivo anticancer effects should not be underestimated. Lingzhi or its
products can be classified as an anticancer agent when current and more direct scientific evidence becomes available.

Immunomodulating Activity of Agaricus brasiliensis

Original Article

Immunomodufating Activity of Agaricus brasiliensis KA21 in Mice and in Human Volunteers


Ying Liu1, Yasushi Fukuwatari1, Ko Okumura2, Kazuyoshi Takeda2, Ken-ichi Ishibashi3, Mai Furukawa3, Naohito Ohno3, Kazu Mori 4, Ming Gao
4 and Masuro Motoi5

1 Mibyou Medical Research Center, Institute of Preventive Medicine, Tokyo, Japan, department of Immunology, School of Medicine, Juntendo University School of
Medicine, Tokyo, Japan, laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo,
Japan, department of Acupuncture and Moxibustion, Suzuka University of Medical Science and Mie, Japan, and 5Toei Pharmaceutioa! Co., Ltd.,
Tokyo, Japan

We performed studies on murine models and human volunteers to examine the immunoen- hancing effects of the naturally outdoor-cultivated fruit body of Agaricus brasiliensis KA21 (i.e. Agaricus blazef). Antilumor, leukocyte-enhancing, hepatopathy-alleviating and endotoxin
shock-alleviating effects were found in mice. In the human study, percentage body fat, percentage visceral fat, blood cholesterol level and blood glucose
level were decreased, and natural killer cell activity was increased. Taken together, the results strongly suggest that the A. brasiliensis fruit
body is useful as a health-promoting food.

Keywords: A. brasiliensis-clinical research -cold water extract-NK activity – outdoor-cultivated – safety

® 2007 The Author(s),

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (htLp:/7crcativecommons.org/ 1
iccnscs/by-nc/2.0/tik/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly
cited.



Alternative medicine is the general term for ‘medicine and treatment that have not been verified scientifically or applied clinically in modern Western
medicine’ (I 12). The range of alternative medicine varies widely to include traditional medicine and folk remedies as well as new therapies that are not
covered by health insurance. Considering the current world population, the percentage of people utilizing modern Western medicine is surpris­ingly low,
with the World Health Organization (WHO) indicating that 65-80% of health management is by traditional medicine. ‘Mibyou’ is a recently established term
that means a half-sick person having clinical laboratory data that borders healthy individuals and patients. Education of the mibyou population about
eating habits is also significantly important for maintain­ing public health by the government.

In Japan, an increasing number of people are turning to alternative medicine mainly in the form of health foods such as amino acids, lipids, carbohydrates,
plants, seaweeds, insects, bacteria, yeasts and mushrooms. Such mushrooms as Lenilnula edodes, Ganoderma htcidum and Grifola frondosa are commercially available. Agaricus brasiliensis (A. blazei ss. Heinemann) is a health food that has received recent
attention. A. brasiliensis has been reported to improve symptoms of lifestyle-related diseases including obesity, hypertension and diabetes, and
to have anti-inflammatory, anti tumor, cancer inhibitory and immuno-enhancing effects (13-18). However, many reports were either animal studies or clinical
studies with few eases.

Many mushrooms, also called as macrofungi, are clas­sified as higher-order microorganisms, Basidiomycota. To discuss the functions of Basidiomycota, it is
important to compare them under the same conditions, including not only the species but also the strain, as well as methods of cultivation and processing.
Basidiomycota products involve mycelia, spores and fruit bodies in

each species. The fruit body and the mycelium are distributed widely in foods. To maintain the manufactur­ing process, the mycelium is superior to the
fruit body; however, its components arc known to be quite different. There are many ways to obtain the fruit body, e.g. collecting naturally grown
mushrooms from hills and fields, and outdoor or indoor cultivation.

Agaricus brasiliensis
KA21 used in this study is a fruit body cultivated outdoors in Brazil. Fruit bodies were air dried by a ventilator with a blowing temperature lower than
60°C to maintain their enzyme activities. We have recently examined the structure and antitumor activity of polysaccharide fractions of the fruit body and
concluded significant contribution of the highly branched 1,3-P-glucan moiety on the activity. We also prepared the cold and the hot water extracts (AgCWE
and AgHWE) and examined on a murine diabetic model C57B1 Ksj-db/ db, and found that AgCWE showed much stronger pharmacological activity to this model.
These facts strongly suggested that pharmacological action of cold water extract differ from that of hot water extract. We have also shown that the cold
water extract contains enzymes such as polyphenol oxidase and peroxidase (19-25), Table 1 shows the general constituents of A. brasiliensis KA21.
K.A21 has high protein and fiber content. It also has high levels of vitamins Bl, B2, B6, niacin, pantothenic acid, folic acid and biotin. It contains many
minerals including large amounts of iron, potas­sium, phosphorus, magnesium, zinc and copper, and certain amounts of manganese and selenium. In addition,
it contains detectable concentrations of vitamin D as it is cultivated under the sunlight.

To successfully achieve and maintain food safety for citizens, laws related to foods have become strictly controlled. Recently, medical doctors in National
Cancer Center Hospital East in Japan reported three cases of severe hepatic damage, taking A. blazei extract (26), They mentioned it is necessary
to evaluate many modes of complementary and alternative medicines, including the A. blazei extract, in rigorous, scientifically designed and
peer-reviewed clinical trials. Very recently we have experienced evacuation of one health food originated from A. brazei, because of inducing
genotoxicity in experi­mental animals. Ministry of Health, Labor and Welfare reported it is only the case of one product and the molecular mechanisms are
under investigation. It is also simultaneously reported that other related products did not show such toxicity. Agaritine is a well known toxic metabolite
of agaricaceae, such as Agaricus bisporus, and the relationship between agaritine content and the toxicity has attracted attention. In any case,
function as well as safety of products originated from macrofungi, especially agaricaceae should be precisely examined as much as possible.

Thus, to safely and effectively use alternative medicine including A. brasiliensis, analysis at the molecular level

Tabic 1. Composition of A. brasiliensis KA21

Energy

288.00 kcal

Protein

38.50 g

Fat

2.60 g

Carbohydrate

27.70g

fi-glucan

12.4 g

Fiber

20.60 g

Sodium

8.40 mg

Calcium

22.50 mg

Iron

lO.lOmg

Potassium

2920.00 mg

Phosphorus

952.00 mg

Magnesium

96.50 mg

Zinc

7.87 mg

Copper

7.67 mg

Manganese

0.825 mg

Iodine

0

Selenium

88.00 pg

Arsenicum

0 .48 ppm

Cadmium

2.01 ppm

Plumbum

0.13 ppm

Hydrargyrum

0,18 ppm

Total chromium

Opg

Vitamin in A (total caronene)

0

Vitamin B (total caronene)

Vitamin Bl (Thiamin)

0.63 mg

Vitamin B2 (Riboflavin)

3.04 mg

Vitamin B6

0.54 mg

Vitamin B12

ûpg

Niacin

33.50 mg

Pantothenic acid

22.90 mg

Folic acid

230.00 pg

Biotin

123.00 pg

Total vitamin C (Total c acid)

0 mg

Vitamin D

56.7 pg

Vitamin E (Total tocopherol)

0

Vitamin K1 loquinone)

0

Agaritine

15.3 ppm

Nate:
In 100g dry weight, measured by Japan Food Research laboratories.

Agaritine was measured by MASIS laboratories by HPLC method.



by basic research and proving their effects by clinical research are important. In a human safety study, we found that long-term intake of the fruit bodies
of A. brasiliensis KA21 cultivated outdoors had no adverse effects (22). In the present study, we demonstrated the immunomodulating effect of A. brasiliensis KA21 both by animal and human studies. As described earlier, the fruit body contained many enzymes even after the drying process,
and cold and hot water extracts were prepared and administered orally to examine immunomodulation in mouse models. Drinking such cold water extracts of A. brasiliensis is a traditional custom in Brazil. In the clinical study, we determined the weight, body mass index (BMI), percentage body fat,
percentage visceral fat and blood biochemical levels [total protein, blood glucose, cholesterol, neutral fat, glutamate oxalo- acetate transaminase (GOT),
glutamate pyrvic transami­nase (GPT) and glutamyl transferase (y-GTP)], and natural killer (NK) cell activity before and after admin­istration of A. brasiliensis KA21. Analysis of the data from the viewpoint of mibyou is also included.


Methods

Agaricus brasiliensis
Fruit Bodies

Strain KA21 was cultivated outdoors in Brazil, and its fruit bodies were washed and dried using hot air at 60°C or lower.

Measurement of Ingredients

All ingredients except for agaritine were measured by Japan Food Research Laboratories, Shibuya, Tokyo using the standard protocols recommended by the
Resources Council, the Science and Technology Agency of Japan. The concentration of agaritine was measured by HPLC/MS/MS by MASIS Inc, Minamitusgaru,
Aomori.

Preparation of Hot Water Extract (AgHWE) and Cold Water Extract (AgCWE) of A. brasiliensis

The fruit bodies of KA21 (lOOg each) were ground using a domestic coffee mill, suspended in O.lg/ml physiological saline (Otsuka Pharmaceutical Co., Ltd),
and extracted in an autoclave (120CC, 20 mm) or with cold water (4°C, 1 day). The supernatant after centrifuga­tion was designated as AgHWE or
AgCWE. The extracts were kept frozen at — 20°C until use.

Oral Administration to Mice

AgHWE and AgCWE prepared by the earlier-described method were administered to mice orally for 2 weeks, and cell count and cell population were determined.

Murine Tumor Mode!

Solid form tumor: Sarcoma ISO cells (1 x 106/mouse) were subculaneously administered to the groin of ICR mice on day 0. AgHWE or AgCWE was
orally administered (p.o.) daily for 35 days. Standard p-glucan, sonifilan (SPG) was administered intraperito- neally on days 7, 9 and 11. After
35 days, the mice were sacrificed and the weight of the solid tumor was measured.

Inflammatory Cytokine Production in Primed Mice

Balb/c mice were primed with a standard p-glucan, SCG (200 jig/mouse} from Spar ass is crispa on day 0, and AgHWE or AgCWE was orally administered
daily for 1 week. One week later, bacterial lipopolysaccharide (LPS, 10 pg/mouse) was administered intravenously, serum was collected 90min after the LPS
administration, and serum TNF-a and IL-6 expression levels were measured with ELISA. Antibodies and standards were purchased from Pharmingen Ltd.

Concanavalin A-Induccd Hepatic Injury in Mice

AgHWE or AgCWE were orally administered for several days in mice. One day after the final administration, Concanavalin A (Con A) was intravenously
administered to induce liver injury. Interleukin 6 levels in sera were measured 3 h after Con A administration. GOT and GPT were measured 24 h after Con A
administration.

Clinical Research in Humans

Research was performed on 31 healthy subjects who were not taking any medication prior to or at the time of the study. We explained the study to them in
writing, and obtained informed consent to use the test results. The subjects were divided into three groups, group 2 and group 3 (total 20 subjects) were
administered the normal dose, and group 1(11 subjects) were administered a 3-fold higher dose (safety clinical study group) of A. brasiliensis.

Group 1. For 6 months from May 31 to November 26, 2004, the 11 subjects (mean age 43.6± 12.6 years, male 6, female 5) were asked to take 30 tablets/day
(divided into three administrations; each tablet contained 300 mg of A. brasiliensis), which is three limes the normal dose. Then, we measured and
analyzed the subjective changes in their condition, liver function (GOT, GPT. y-GTP), renal function [blood urea nitrogen (BUN), creatinine] and
nutritional status (total protein).

Group 2, For 3 months from April 12 to July 8, 2005, 12 subjects (mean age 45.3±8.1 years, male 9, female 3) were asked to take the normal dose of 10
lablets/day (divided into two administrations; each tablet contained 300mg of A. brasiliensis). Then, we measured body weight, BMI, percentage
body fat, percentage visceral fat and blood biochemical levels (total protein, blood glucose, cholesterol, neutral fat, GOT, GPT and y-GTP).

Group 3. For 3 months from May to August. 2005,

8 subjects (mean age 22.3 ±0.5 years, male 6, female 2) were asked to take the normal dose, and immune function (NK cell count, NK cell activity) was
measured. In the measurement of immune function, we divided the eight subjects into two groups in a double-blind manner, A. brasiliensis group and
placebo group, administered

10 tablets/day (divided into two administrations; each tablet contained 300 mg of A. brasiliensis) for 7 days, and determined NK cell count and NK
cell activity in peripheral blood. After two-month drug withdrawal, the same study was conducted with the tablets exchanged (crossover). We analyzed the
cell fraction in peripheral blood and regarded mononuclear cells with CD3″CD16 + CD56+ as NK cells. Following the usual method, we
measured NK cell activity by 4h 51Cr-release assay using K562 tumor cells as targets, at an effector/ target ratio (E/’T) = 20 or 10 (the mixing
ratio of mononuclear cells and K562 cells is 20 or 10),

Statistical Analysis

Paired /-test was used to evaluate statistical significance. PcO.OS was considered significant in all analyses.


Results

Chemical Analysis of A. brasiliensis KA21 for Safety Assessment

Before starting animal and human experiments, the chemical composition and additives were screened. The chemical composition and nutrients are shown in
Table 1. Recently, a major toxic compound of

agaricaceae ‘agaritine’ has attracted attention by showing tumor-promoting activity in rats. The agaritine content of A. brasiliensis
KA21 was measured and it was as low as

  1. ppm. Heavy metals, such as lead and mercury were

lower than the detection limit. Three hundred types of pesticides were measured and none was detected (data not shown).

[3-gIucan content of A. brasiliensis KA21 was n.dglOOg“1 measured by Japan food research labora­tories. We have already precisely
examined the structure of polysaccharide fractions of KA21, and the major structure of P-glucan showing immunomodulating activity was determined to be
(3-1,6-linked glucan with highly branched p- 1,3-segment (20).

Vitamin D is a well known vitamin of macrofungi and KA21 contained 56.7 pg 100 g~! (=ca. 2250IU 100 g-!). Same strain, cultured
inside the house did not contain detectable concentration of vitamin D (data not shown). It is well known that concentration of vitamin D is strongly
dependent on sunlight exposure. Vitamin D content of KA21 well reflected the culture condition of outdoor and under the sunlight.

From these data, A. brasiliensis KA21 was found to be chemically and analytically safe for animal and human studies.

Parameters and Effects on Experimental Animals

Effect on Normal Inbred Strains of Mice

For the animal experiments, AgCWE, AgHWE were prepared and examined. When AgCWE or AgHWE was administered orally at the dose of 20 mg/mouse to healthy mice
(C3H/HeN) for 2 weeks, cell count in the thymus was not changed (data not shown), but that in the spleen was increased in the AgCWE group (Fig. 1).

Cells were doubly stained with CD4/CD8ct, aP/y<L or CD3/B220, and the ratios of cell populations were calculated after measurement with a flow
cytometer. No notable changes were seen in the thymus (data not shown), whereas the ratio of CD4+ in the spleen was increased significantly in
the AgHWE group (Fig. 1).

And tumor Activity of Orally Administered AgCWE and AgHWE in Sarcoma ISO Transplanted Mice

We evaluated the antitumor effect of A. brasiliensis on Sarcoma 180 solid tumor, which is the standard system to measure antitumor effects in
mice, Sonifilan (SPG) was used as standard material. Oral administration of

Figure 1. Cell number and population of splénocytes from AgHWE or CWE p.o. mice. AgHWE, CWE or saline (200pi/mouse, J day, 1 shot), was p.o.
administered to C3H/HcN mice for 14 days. The splénocytes were collected from each group of mice on day 14. Total cell number was counted with a
hcmocyiomctcr (left). CD4/CD8a were measured by flow cylomclory (right). The results represent the means±S.D. *P<0.05, **P<0.01 compared with control by Student’s /-test.



CWE

Saline

HWE

AgCWE or AgHWE for 35 days led to the suppression of tumor growth (Table 2).

Protection against Concanavalin A-Induced Liver Injury by Orally Administered AgCWE and AgHWE in Mice

The intravenous administration of Con A, a plant lectin, triggers acute hepalopalhy in mice. We administered oral AgCWE or AgHWE as pretreatment, and
then assessed the effects of Con A on hepatopathy. When 200 pi of

Table 2. Anliminor cffccl of A. brasiliensis extracts on solid form of Sarcoma 180 in ICR mice

Name

Dose

0»g)

Times

Route

CR/n

Tumor weight mcan/SD (g)

%

Inhibition

/-test

Control

0/12

8.6 ±4.3

0.0

SPG

0.1

3

i.p.

7/11

0.4 ± 1.1

95

<0.001

Control

0/10

15.0 ±6.5

0

AgCWE

2

35

p.o.

0/10

9.6 ±6.5

36

<0.05

AgHWE

2

35

p.o.

0/10

7.9 ±2,5

47

<0.01

Note\
Dose, per mouse; times, day 7,9, II; CR/n, Number of tumor free mice/total mouse. SPG, Standard [Lglucan as positive control.

AgCWE or AgHWE was administered for 7 days as pretreatment, GOT was found to decrease significantly in the AgCWE group. A similar trend was seen in the
AgHWE group. When the dose was increased to 600 pi and administration was continued for 7 days, the effect became more notable (Fig. 2). GPT was
decreased in a similar manner (data not shown). Similar studies were performed using different forms of administration and several mouse lines, and all
cases showed a decreasing trend. Together, the results show that A. brasiliensis KA21 protects mice from hepatic injury.

Protection of Multiple Organ Failure Induced by Lipopolysaccharide by Oral Administration of
A. brasiliensis KA2Ï

Next, we investigated cytokine production induced by the administration of bacterial endotoxiti, EPS, an agent that induces multiple organ failure in
severe infections, to determine the hepatocellular protective effect of AgCWE and AgHWE, The levels of TNF-a and IL-6 generated by LPS administration
were decreased in both groups (Fig. 3), indicating that A. brasiliensis controls the level of cytokine production to protect organs.

140

120

100

80

60

40

20

0

Normal

Figure 2. Effect of AgCWE or HWE p.o. on Con A-lnduccd liver injury. (Left) AgHWE or CWE (200ftl/mousc) was p.o. administered to Balb/c mice for 7
days. Con A {20 mg kg“1) was iv administered on day 7 and the sera were prepared 24 h later from each group of mice. Results arc expressed
as the mean ±SD */-’<0.05 compared with control by Student’s Most. 7). (right) AgHWE or CWE (600pi/monsc) was p.o,

administered to Balb/c mice for 7 days. Con A (20 mg kg“1} was iv administered on day 7 and the sera were prepared 24 h later from each
group of mice. Results arc expressed as the mean±SD ***P<0.001 compared with control by Student’s /-lest.

Figure 3. Effect of oral A, brasiliensis on LPS-induccd cytokine production. fi-Glucan (SCG, 200 pg/mousc) was i.p. administered to Ba!b/e
mice on day 0. AgHWE or CWE was p.o. administered to these mice for 7 days. LPS (10 pg/mouse) was iv administered as a triggering reagent on day 7 and
the sera were prepared 1.5 h later from each group of mice. IL-6 and TNF-a was measured by ELISA. Results arc expressed as the mcan±SD */5
<0.05 compared with control by Student’s /-test, (left) TNF-a, (right) IL-6.



After

Clinical Research

Safety of
A, brasiliensis

Before determining the safety of A. brasiliensis KA2Î, a normal dose was administered for 3 months to 13 subjects as a preliminary
experiment and measured changes of general clinical parameters. Mean body weight (71.2-» 70.9kg), size of waist (85.4—> 83.5cm), percentage
body fat (34.4-33.0%) and BMI (27.8-27.6) did not show any clinical sign of illness by taking it. Thus to precisely determine the safety of A. brasiliensis KA21, a dose of three times higher than the normal dose was administered for 6 months to 11 subjects (group i, see “Methods’), and
subjective changes in conditions, liver function, renal function and nutritional conditions were measured and analyzed. After measuring the biochemical
parameters, we confirmed no statistically significant difference before and after administration, and no side effects caused by long-term administration
(Table 3).

Effect of
A. brasiliensis on Biochemical Parameters related to Adiposis and Diabetes

In order to evaluate the effect of A. brasiliensis KA21 on lifestyle-related diseases, the normal dose was administered to 12 subjects (group 2,
see ‘Methods’) for 3 months and comparison of clinical biochemical data was made. The results are as follows: (i) Significant decreases were seen in body
weight and BMI (P<0.01 each) after administra­tion (Figs 4 and 5). (ii) Significant decreases were observed in percentage body fat (Pc 0.01)
and percentage visceral fat (TcO.Ol) after administration (Figs 6 and 7). (iii) Significant increase was found in total protein level (T<0.03) after
administration (Fig. 8). (iv) Significant reduction was seen in blood glucose level (T<0.02) after administration (Fig. 9).

P<
0.01

{N=12)

Before

Figure 5. Effect of A. brasiliensis on BMI. Experimental protocol was shown in “Methods’.

Biochemical

parameters

Before

(mean ±SD)

After

(mean ±SD)

Statistics

(/”-value)

Total protein (gdl

!
) 7.50 ±0.16

7.41 ±0,25

0.31

BUN (mgdl“)

15.81 ±5.93

] 3.45 ±2.25

0.12

Creatinine (mgdl-1

0.92 ±0.21

0.90 ±0.20

0.19

GOT (ill“1)

18.8 ±4.75

19.8 ±4.40

0.10

GPT (pi»“)

15.7 ±6.90

16.3 ±4.90

0,52

y-GTP {pi'»1)

35.4 ± 29.6GTP

35.9 ±30.1

0.89

(*=11).

j— P < 0,01

n

Table 3. Safety of A. brasitien sis KA2I in Iiu man volunteers


120


100


Before

P<0.01

(N=12)

After

Before

Figure 6. Effect of A- brasiliensis on percentage body fat. Experimental protocol was shown in ‘Methods’.

P<
0.01

n

O)

ie

r 80

.c

OJ

’(D

* 60 >,

T3

o

CO

{N=
12)

40

P<0.03

“1

Figure 8. Effect of A. brasiliensis on total protein level. Experimental protocol was shown in “Methods’,

(W=12)

Alter

In order to analyze the data more precisely, the subjects were divided according to total cholesterol level into a normal value group (T-CHO<200mg/dl)
and a mibyou (slightly sick) value group (T-CHO > 200 mg/dl)

P<
0.02

150 r

120

90

60

30

Before

Figure 9. Effect of A. brasiliensis on blood glucose level. Experimental protocol was shown in “Methods’.

T-CHO<200 mgdl1 (W=4)

for comparison. No change was observed in the T-CHO < 200 mg/dl group before and after adminis­tration, whereas a decrease was seen in the T-CHO >
200 mg/dl group after administration (Fig, 10).

The subjects were divided according to blood neutral fat level into a normal value group (TG < 120 mg/dl) and a mibyou value group (TG > 120 mg/dl)
for comparison. No change was observed in the former, whereas a decrease was observed in the latter after administration (Fig, 11).

Improvement of Liver Function by
A. brasiliensis

To determine liver function, we compared GOT, GPT and y~GTP values of the earlier mentioned subjects shown in the previous section. When comparison was
made among all 12 subjects, no differences were seen before and after administration (Fig. 12). By contrast, after the subjects were divided into normal
and mibyou according to GOT level, the average value of GOT in the normal value group (GOT<25IUU1) was found to increase slightly after
administration, whereas that in the mibyou value group (GOT > 25 IU I”1) was found to decrease after administration, although the difference
was not statistically significant (Fig. 13). The average value of GPT was increased in the normal value group (GPT<25IU)“1) after
administration, whereas that in the mibyou value group (GPT > 25 IU1“1) was decreased slightly after administration, the difference being not
statistically significant (Fig. 14). The average value of y-GTP was decreased slightly in the normal value group (y-GTP <30 IU l“1) after
administration, whereas that in the mibyou value group (y-GTP > 30 IU I“1) was almost unchanged (Fig. 15).

Taken together, we determined that both lipid and blood glucose levels showed a decreasing trend for

T-CHO>200 mgdl“1 (A/=8)

210

300

After

250

200

150

100

O

50

Before

After

Before

B 140

P 105

175

70

35

TG>l20mgdr5 (W=8)

TG<120 mgdl»1 (W=4)

300

250

120

100

80

60

40

20

0

After

200

After

150

100

50

Before

Before

Figure 11. Effect of A. brasiliensis on neutral fat love! from the viewpoint of Mi by cm. Experimental protocol was shown in “Methods

y-GTP

GPT

GOT

(IUE1)

(IUE1)

(!U! )

42.62*35.40 38.46*29,48

38,23*31,94 38,54*28,48

„ 29.07*16.37 26.31 ±9,30

80 r

100 r

75

50

25

Before After

Before After

Before After

Fimu-e 12. Effect of A. brasiliensis on liver function. Experimental protocol was shown in “Methods’.

GOT>25 (UE1 (N=7)

GOT<25 lUf 1 (A/=5)

(IUI 1) 60

50

40

30

20

10

0

(IUE1) 30

25

20

15

10

After

After

Before

Before

GPT<25 IUr1 (N=3)

GPTS25 lUf1 (N=9)

(tur1)

Before After Before After

Figure 14. Effect of A. brasiliensis on liver function (GPT Value) from the viewpoint of mibyou. Experimental protocol was shown in “Methods’

(fur1)

30

20

15

10

0

V-GTP<30 IUr (A/=5)

y-GTP£30 IUr {N=7)

(lUf1)

120

100

80

(IUI

30

60

40

20 ■

20

15

0

0

Before After Before After

Figure 15, Effect of A. brasiliensis 011 liver function {y-GTP Value) from the viewpoint of mibyou. Experimental protocol was shown in
‘Methods’

Figure 16. Comparison of NK cell count between groups before and after administration of A. brasiliensis. Experimental protocol was shown in
“Methods’.

lifestyle-related diseases. In addition, an improvement in liver function was noted.

Modulation of Natural Killer CelI by
A. brasiliensis

In order to evaluate the effect of A. brasiliensis KA2Î on immune function, NK cell number and function were examined by eight subjects in a
double-blinded experimental protocol shown in “Methods’ (group 3, see ‘Methods’). The normal dose or placebo was administered to eight subjects for 7 days
and NK cell number and activity in peripheral blood was compared as follows.

Effect of
A. brasiliensis on NK Cell Count

Comparison of NK cell count before and after admin­istration, and comparison between the A. brasiliensis group and placebo group were made, and no
statistically significant differences were observed (Fig. 16).

Augmentation of NK Cell Activity by A.
brasiliensis KA21

Before administration, no significant differences were observed between A. brasiliensis group and placebo group

E/T=10


A. brasiliensis
placebo □


Figure 17. Effecl of A. brcwliensis 011 NK cell activity, (Comparison between A. blazei group and placebo group). Experimental
protocol was shown in ‘Methods”,


E/T=2û

E/T=10 P< 0,001

E/T=20 P< 0.001

30

?

25

«E

0

20

Z

15

O

10

>

0

<

5

40

g 35

CA

%
30 o

z 25 Ô

S” 20 >

o , c

< 15 10

After

Before

Before

After

Figure 18. Comparison of NK cell activity before and after administration of A. brasilienxis. Experimental protocol was shown in ‘Methods”.

E/T=10 N S

E/T=20

Figure 19. Comparison of NK cell activity before and after administration of placebo. Experimental protocol was shown in ‘Methods’.

35 s? 30

jA

o 25 X

z

Is 20

< 15

10

21 19 E 17

Ai

® 15

u

z 13 Ô ~ 11

s

“o 9 <

7

5

After

Before

Before

Atter

(Fig. 17 left). After administration, there were significant differences between the two groups, with P<0.01 for the H/T = 20% group and P<
0.001 for the E/T = 10% group (Fig. 17 right). Figs 18 and 19 show individual changes in NK cell activity after administration of A. brasiliensis
(Fig. 18) and placebo (Pig, 19) groups. NK cell activity was increased significantly in A. brasiliensis groups, with PcO.001 for the E/T = 20%
group and PcO.OOl for the E/T =10% group. Meanwhile, NK cell activity was not increased significantly in the placebo group after administration.


Discussion

Japan is rapidly becoming a super-aging society, and such issues as decreased workforce, consumption and tax revenues, and increased international
competition

among neighboring Asian nations are emerging. As a dramatic increase in the number of elderly patients is inevitable, the social security system is
expected to become financially strained, and patient and consumer awareness of their rights will be enhanced because of the increased financial burden
levied on them. Whereas genetic disposition is said to be involved in the development of lifestyle-related conditions and diseases, such as diabetes,
hyperlipidemia and cancer, several other factors also determine their development; therefore, lifestyle is closely related to the development of such
conditions and diseases. On the other hand, there is a need to reduce the significantly elevated medical expenses in the future. There are discussions as
to whether we should pay medical expenses to aid people who do not practice a healthy lifestyle. The number of people who are not sick yet not healthy,
that is, ‘in poor health’ or ‘mibyou’, is increasing at an accelerated pace (27,28). It is difficult to maintain regular eating habits in stress-laden
daily life. Improvement of diet by consuming functional foods seems to contribute to the health improvement of people with poor health, as well as to the
prevention of the development of lifestyle-related diseases.

There are many functional foods in Japan and they are expensive for customers, thus accurate information is needed to select the best food for each
customer. All the parameters of safety, cost performance, evidence of function, as well as taste are important to disclose.

Mushrooms have been a part of oriental medicine for hundreds of years as being beneficial for health. Most traditional knowledge about the medicinal
properties of mushrooms comes from the Far East, Japan, China, Korea and Russia. The most striking evidence is that lentinan from L, edocks,
sonifilan from Sc/nzophyllum commune and krestin from Con’orus versicolor have been approved for anticancer drugs mediated by immune
stimulation. A great many mushroom products are on the market as health promoting foods, and basic and clinical researches of these products have been
performed continuously (29-41).

Currently, there are 80 000 known fungal species in the world. It is surmised that 1 500000 species exist, including undiscovered species. These fungi are
classified by king­dom, phylum/division, class, genus and species. Many fungi are classified into Basidiomycota or Ascomycota, whereas others are also
classified into the kingdom Protozoa or kingdom Chromista. Fungi include mush­rooms, molds and yeasts, which have significantly different appearance and
sizes. As mushrooms are too large to be considered microorganisms, they are referred to as macrofungi. Lichens of which two or more microorganisms live in
a symbiotic relationship are also included. Fungi exhibit both the sexual form (for example, morphology of mushroom) and the asexual form for regeneration
(for ex a tuple, morphology of mycelium) and either form is used depending on surrounding environmental changes; however, the exis­tence of both forms
(holomorph) is not known for all fungi. Their nomenclature is also characteristic. The background of the discovery of a fungus is reflected in its name and
different names may be given depending on whether the fungus exhibits the sexual form (teleomorph) or the asexual form (anamorph) of regeneration. Fungi,
particularly mushrooms, are ‘cultivated’ and distributed products, and detailed analysis of their components lias been performed. In the Standard Tables of
Food Composition in Japan (Fifth Edition), 36 foods are classified as ‘mushrooms’. The representative nutritional composition of mushrooms includes fiber,
glucose and sugar alcohols, organic acids, fatty acids, inorganic substances, vitamins, free amino acids, bitter and pungent components, flavor components,
enzymes, biophylactic substances, pharmacologically active substances and toxic components. Moreover, molds and yeasts are related to some fermented foods,
A variety of foods including sake (rice wine), miso (bean paste), soy sauce, cheese and katsuobushi (dried bonito) are manufactured with the help of
eukaryotic microorganisms. Fungus produces many secondary metabolites that are used as drugs or raw material for drugs, an example of which is penicillin.

As regards edible mushrooms, some are consumed raw, and cultivated hypha and culture broth are distributed as supplements after processing. Although they
are from the same fungus, there is no proof that they contain the same components as the cultivated fruit bodies. In the early 1980s, we performed animal
studies to compare the macromolecular components of G. frondosa fruit bodies, mycelia and fermented products. That the quantities and quality of
components contained in each extract differed considerably was also reflected in the activity (29-32). Grifola frondosa has been well studied in
Japan and in other countries. Interestingly, the major active compo­nent differs depending on the study group (33-37). Comparing mushrooms and mycelia at
the product level, it was found that live fungus differs from dried products. From the viewpoint of stable supply, the dried product is desirable, but its
components change accord­ing to the drying method. It is likely that the components differ if the ‘fungal strain’ differs. Thus, one type of mushroom may
vary greatly when processed as food or other products. When we want to discuss or evaluate components and pharmacologic action, we need to conduct
comparisons under detailed conditions, especially if we perform animal experiments.

Agaritine (N-[y-L-(+)-glutamyl]-4-hydroxymethylphe- nylhydrazine) was identified in fruit bodies of cultivated mushrooms belonging to the genus Agaricus, including commerce A. bisporus and closely related species (42-46), 4-(hydroxymethyl) benzenediazonium ion that had
mutagenicity is believed to be formed when agaritine is metabolized. Agaritine is most prevalent, usually occurring in quantities between 200 and 400pgg _1 as

fresh weight, 1000-2500j-igg”1 as dry weight in culti­vated mushroom. Recently, agaritine in A. brasiliensis (A. blazed) sample and
products was measured. These samples contained 112-1791 pgg-1 of agaritine as dry weight (47). In the present study, we have detected only low
concentrations of agaritine (15.3 ppm; 15.3pgg~”1) in the preparation made of A. brasiliensis KA21. This value was <1/100 of the
quantity of average values of A. bisporus, Agaritine content is known to be significantly varied depending on processing. Household processing
(e.g. boiling, frying, microwave heating or drying) will reduce the agaritine content in A. bisporus by up to 50% or even more (48). Also,
agaritine has recently been shown to be degraded oxygen dependent in water (42,43). There have been long discussing the toxicity and carcinogenicity of
agaritine (44,45). However, the conclusion is still controversial. Toth and co-workers (46,49-51) undertook the work to assess the possible carcinogenic
activity of the phenylhydrazines and related compounds in A. bisporus. Their studies indicated that most of phenylhydrazine and related compounds
in the mushroom are carcinogenic in Swiss albino mice. The only compound that was tested negative was agaritine, a finding that significantly muddied the
interpretation of the carcinogenicity data. Also, these studies were the conservative risk model. In the absence of epidemiologi­cal data, no evaluation of
carcinogenicity of agaritine to humans could be made.

We have analyzed A. brasiliensis KA21 from various aspects and reported the p-glucan, the enzymes of polyphenol oxidase, peroxidase and
p-l,3-Glucanase. P-glucan content of A. brasiliensis I<A21 was

  1. g 100 g“1 measured by Japan food research labora­tories. We have already precisely examined the structure of polysaccharide fractions of
    KA21, and the major structure of p-glucan showing immunomodulating activity was determined to be p-l,6-linked glucan with highly branched
    p-1,3-segment (20). During that study we have prepared hot water extract, cold alkaline extract, and hot alkaline extracts and analyzed
    polysaccharide structure of all these fractions. Of much interest, all the fraction showed quite similar structural features that major linkage is
    p-1,6-linked glucan. From these data, major polysaccharide component in A. brasiliensis is p-1, 6-linked glucan, and it is consistent with
    the previous study. However, we have mentioned that antitumor activity needs p-1,3-linkages in addition to p-1,6-linkage based on the results of
    the limited chemical degradation study. However, this conclusion is still temporal and structural activity relations needed human studies.

This study showed that the fungus is rich in vitamins; as it is cultured outdoors, it contains detectable concentrations of vitamin D. Vitamin D is a well-
known vitamin of macrofungi and KA21 contained 56.7 pg 100 g“1 dry weight. In the parallel experiments, vitamin D was contained lower than the
detection limit (0.7 pg 100g-1) in the mycelium of this fungi cultured in the liquid medium and the fruit body of A. hlazei imported
from China. Much differences of vitamin D in these products well reflected the culture condition of outdoors and under the sunlight. Relationship between
vitamin D content and sunlight exposure has been demonstrated in various macrofungi (52). Based on the definition in the manual of Health Food Regulation
in Japan, the food containing more than l,5pgl00g»! (= 60IU 100g“!) of vitamin D is defined as the food containing high vitamin D
content. Considering the rule, KA21 is the food containing high concentration of vitamin D. Micronutrients such as vitamins and minerals promote the
metabolism of waste products, carbohy­drates and lipids via cellular activation, and improved insulin resistance by decreasing blood glucose. Fiber and
unsaturated fatly acids decrease blood pressure and promote decholeslerolization. KA21 also contained other micronutrients, thus it is good for health for
variety of reasons.

Meanwhile, in an analysis of the active components in bupleurum root, a crude drug, we found that polyphenols polymerized by enzymes have a strong
immunoenhancing effect (53-55). A. brasiliensis also has a number of enzymes related to the polymerization of polyphenols (23,24). Polyphenols
polymerized by these enzymes may be active components in this fungus. In our clinical research, decreases in body weight, BMI, percentage body fat,
percentage visceral fat and blood glucose level were noted and a tendency to decrease blood cholesterol level, blood neutral fat level. GOT, GPT and y-GTP
was observed in the mibyou value group. On the basis of the earlier results, among the components of this fungus, all the polysaccharides, enzymes,
vitamins and minerals may be involved in the normalization of biochemical test results.

This study measured immune function in mice. When we compared the number and population of immunocompetent cells after administration of AgCWE or AgHWE to
healthy mice orally for 2 weeks, it was found that the percentage of spleen CD4+T cells was increased in the AgHWE group and the number of
spleen cells was increased in the AgCWE group. Furthermore, both AgCWE and AgHWE showed antitumor effects and AgCWE prevented Con A-induced hepatopathy and
suppressed cytokine production induced by LPS. CD4+T cells are divided into type 1 helper T cells (Thl) and type 2 helper T cells (Th2) based on
T-cell antigen stimulation, and Thl is considered to be a more important contributor to the antitumor effect, Thl is thought to infiltrate local sites
well, demonstrate strong cytotoxicity and cytokine production ability, and induce complete tumor regression by locally inducing CTL, which has the ability
to produce IFN-y (56-58).

It is likely that the antitumor effect of A. brasiliensis is closely related to the increase in CD4+T cell count.

As changes in immunocytes were demonstrated by the oral administration of A. brasUiensis in healthy mice, it is expected that the daily intake of A. brasUiensis may have preventive effects on immunoregulation failure.

Agaricus brasUiensis
suppressed organ dysfunction accompanied by blood with excessively high cytokine levels, which is related to multiple organ failure. It is desirable that
cytokines be produced at certain levels as needed. In these models, such as LPS-elicited cytokine production, A. brasUiensis controlled excessive
cytokine production (Fig. 3). A. brasUiensis can not only promote but also control immunity, which is considered a desirable effect.

Among the effects of A. brasUiensis on immune function, we examined changes in the ratio of NK cells to peripheral mononuclear ceils and NK cell
activity in humans. Both the A. brasUiensis group and the placebo group showed no significant changes in the ratio and number of NK cells to
peripheral mononuclear cells after 1-week administration. On the other hand, comparing the A. brasUiensis and placebo groups, NK cell activity was
significantly enhanced by the administration of A. brasUiensis. When individual cases were examined, almost all cases showed increasing NK cell
activity with the administration of A. brasUiensis, although there were differences in the degree of increase (Fig. 18).

The measurement of NK cell activity has been most widely used in both animal and human experiments,

because NK cells play a critical role in natural

immunology, and measurement of cytotoxicity is reliable for evaluation with good reproducibility (5). The immune function is affected by NK cells as well
as various

lymphocyte and humoral factors including antibodies, complement and cytokines. There have been several publications demonstrating products of macrofungi
enhanced NK activity (59-63).

The effect of A. brasUiensis on the degree of NK cell activity enhancement varied significantly among individuals. It was recently clarified that
effectiveness as well as the appearance of side effects with each

medication were significantly different in each individual. This is explained partly by polymorphism and the link­age of CYP-related genes, a
drug-metabolizing enzyme group (64,65). On the other hand, many causative genes have been discovered in immunity-related diseases, some of which are
polymorphic. It is possible that polymorph­ism may be related to individual differences observed in the effects of A. brasUiensis. Research into
receptors for mushroom components is not extensive. Dectin-1 was recently determined to be the receptor for cell wall p-giucan, a major component of
mushrooms (66 68). The relationship between polymorphism of the receptor for pathogens and disease has been elucidated (69,70). The effects of A. brasUiensis and receptor gene poly­morphism may be related. Further analysis is necessary in the future.

Through basic and clinical research, we confirmed that A. brasUiensis can help to improve symptoms of lifestyle-related diseases because of its
anti-infiammalory, anti tumor and immunoenhancing effects, and that A. brasUiensis is a useful health food to treat mibyou (primary prevention).

Very recently we have experienced recall of one health food originated from A. brazei, because of inducing genotoxicily in experimental animals.
Ministry of Health, Labor and Welfare reported it is only the case of one product and the molecular mechanisms are under

investigation. Based on the clinical examination shown in this study, KA21 is very safe for human health. Any

adverse effect could not be detected in our study.

We have also stated that content as well as pharmaco­logical action is significantly influenced by culture

conditions even in the same fungi, such as vitamin D content. In addition, proteins may be decomposed during processing. Much restricted regulation for
each of the health foods might be needed for increasing human health. In any case, agaricaceae contained many species for functional foods, thus, much
study should be needed continuously. This study helped to understand the mushrooms of agaricaceae are very safe and useful for human health.


Conclusion

(i) In basic research using a mouse model, we determined that A. brasUiensis has anti tumor, anti-inflammatory and hepatocellular protective
effects. It was suggested that the increase in the number of helper T cells and the enhancement of NK cell activity are related to these effects.

(ii) In clinical research on human volunteers, we found that A. brasUiensis decreased body weight, BMI, percentage body fat, percentage visceral
fat and blood glucose level significantly, and reduced obesity. It also decreased blood cholesterol level and neutral fat level, normalized liver function
and activated the immune function in mibyou patients (people with poor health).


References

  1. Kidd PM. The use of mushroom glucans and proteoglycans in cancer treatment. Ahem Med Rev 2000;5:4-27.

  2. Mayell M. Maitakc extracts and their therapeutic potential. Alieni Med Rev 2001;6:48-60.

  3. Ventura C. CAM and cell fate targeting: molecular and energetic insights into cell growth and differentiation. Evid Boxed Complement Alternat Med 2005;2:277-83.

  4. Cooper EL. Bioprospecting: a CAM Frontier. Evid Boxed Complement Aitemai Med 2005;2:1-3.

  5. Takeda K, Okumura K. CAM and NK cells. Evid Boxed Complement Alternat Med 2004;1:17-27.

  6. Shimazawa M, Chikamalsti S, Moriinoto N, Mishima S, Nagai H, Hara H. Neuroproleclion by Brazilian green propolis against in vitro

and in vivo ischemic neuronal damage. Evid Based Complement A hermit Med 2005;2:201-7.

  1. Cooper EL. CAM. eCAM, bioprospecting: the 2isi century pyramid, Evid Based Complement Alternat Med 2005;2:125-7.

  2. Lindcquist U, Timo H, Nicdermcycr J, Jülich WD. The pharmacological potential of mushrooms. Evid Based Complement Alternat Med 2005;2:285-99,

  3. Tcntsawa K. Evidcncc-bascd reconstruction of kampo medicine: Part I Is kampo CAM? Evid Based Complement Alternat Med
    2004:1:11-16.

10. Kaminogawa S, Nanno M. Modulation of immune functions by foods. Evid Based Complement Alternat Med 2004;1:241-50.

Jl.Alsumi K. is alternative medicine really effective? Alternative Medicine 2000 (in Japanese).

  1. Atsumi K. Recommendations of Alternative Medicine. Japan Medical Planning 2000 (in Japanese),

  2. Huan .SJ, Mau JL. Antioxidant properties of mcthanolic extracts from Agaricus bhtzei with various doses of y-irradiation. Food Set Techno! 2006;39:707-16.

  3. Bellini MF, Angcli JPF, Matuo R, Terczan AP, Ribciro LR, Manlovani MS, Antigcnotoxicity of Agaricus blazci mushroom organic and aqueous extracts in
    chromosomal aberration and cytokinesis block micronuclcus assays in CHO-k) and HTC cells, Toxicol in Vitro 2006;20:355-60.

  4. Zhong M, “I’ai A, Yamamoto 1. In vitro augmentation of natural killer activity and intcrfcron-y production in murine spleen cells with
    agaricus blazci fruiting body fractions. Biosei Biotechnoi Biochem 2005;69:2466-9.

  5. Ellcrtscn LK, He Hand G, Johnson E, Grinde B. Effect of a medicinal extract from Agaricus blazci Mmill on gene expression in a human monocyte cell
    line as examined by microarrays and immuno assays, hit Jmmunapharmacoi 2005;6:133-43,

  6. Kobayashi H, Yoshida R, Kanada Y. Fukuda Y, Yagyu T, Inagaki K, el al. Suppressing effects of daily oral supplementation of beta-glucan extracted
    from Agaricus blazci Murill on sponta­neous and peritoneal disseminated metastasis in mouse model. J Cancer Res Clin Oncol
    2005;131:527-38.

IS, Ker YB, Chen KC, Chyau CC, Chen CC, Guo JH, Hsich CL, cl al. Antioxidant capability of polysaccharides fractionated from submcrgc-ciilturcd Agaricus
blazci mycclia. J Agrie Food Client 2005;53:7052-8,

  1. Ohno N, Furukawa M, Miura NN, Adachi Y, Motoi M,

Yadomae T. Antitumor bcta-giucan from the cultured fruit body of Agaricus hlazei. Biol Phann Bulletin 2001;24:820-8.

  1. Ohno N, Akanuma AM, Miura NN, Adachi Y, Motoi M.

(i-3)-beta-glucan in the fruit bodoes of Agaricus blazci. Phann Pharmacol Lett 2001; 11:87-90.

  1. Moloi M, Ishibashi K, Mizukami O, Miura NN, Adachi Y,

Ohno N. Anti bcta-glucan antibody in cancer patients (preliminary

report). Int J Med Mushrooms 2004;6:41-48.

  1. Liu Y, Fukuwatari Y, Okumura K, Takeda K, Ohno N, Mori K, ct al. Basic and clinical research on immunorcgtilatory activity of Agaricus blazci. Toho Igakit 2004;20:29-36.

  2. Akanuma AM, Yamagishi A, Motoi M, Ohno N. Cloning and characterization of polyphcnoloxidase DNA from Agaricus brasiliensis S. Wasser ct ah
    (Agaricomycciidcac). hit J Med Mushrooms 2006;8:67-76,

  3. Hashimoto S, Akanuma AM, Moloi M, Imai N, Rodrigues CA, ct al. Effect of culture conditions on chemical composition and biological activities of Agaricus hraziiiensis. Int J Med Mushrooms, in press.

  4. Furukawa M, Miura NN. Adachi Y, Motoi M, Ohno N. Effect of Agaricus brasiliensis on Murine Diabetic Model C57Bl/Ksj-db/db. Int J Med Mushrooms 2006;8:115-28.

  5. Mukai H, Wa tana be T, Ando M, Katsumata N. An alternative medicine, Agaricus blazci, may have induced severe hepatic dysfunction in
    cancer patients. J Clin oncologv 2006;36:808-10.

  6. Christine KC, Mark AR, Jay Olshansky S. The price of success: health care in an aging society. Health off 2002;21:87-99.

  7. Kaneko H, Nakanishi K. Proof of the mysterious efficacy of ginseng: basic and clinical trials: clinical effects of

medical Ginseng, Korean red Ginseng: specifically, its anti-stress action for prevention of disease. J Pharmacol Sei 2004;95:158-62.

  1. lino
    K, Ohno N, Suzuki I, Sato K, Oikawa S, Yadomae T. Structurc-function relationship of antitumor beta~l,3-glucan obtained from matted mycelium of
    cultured Grifola frondosa, Chem Phann Bull 1985;33:4950-6.

  2. Ohno N, Adachi Y, Suzuki I, Oikawa S, Sato K, Ohsawa M, ct al. Antitumor activity of a beta-1,3-glucan obtained from liquid cultured mycelium of Grifola frondosa, J Pharmacobiodyn 1986;9:861-4.

  3. Takcyama T, Suzuki I, Ohno N, Oikawa S, Sato K, Ohsawa M, ct al. Host-mediated amiiumor effect of grifolan NMF-5N, a polysaccharide obtained from
    Grifola frondosa. J Pharmacobiodyn 1987;10:644-51.

  4. Suzuki I, Takcyama T, Ohno N, Oikawa S, Sato K, Suzuki Y, ct al. Antitumor effect of polysaccharide grifolan NMF-5N on syngeneic tumor in mice. J Pharmacobiodyn 1987;10:72-7.

  5. Kodama N, Asakawa A, Iitui A, Masuda Y, Nanba H. Enhancement of cytotoxicity of NK cells by D-Fraction, a polysaccharide from Grifola frondosa. Oncol Rep 2005; 13:497-502.

  6. Kodama N, Komula K, Nanba H. Effect of maitakc (Grifola frondosa) D-Fraction on the activation of NK ceils in cancer patients. J Med Food 2003;6:371-7.

  7. Harada N, Kodama N, Nanba H. Relationship between dendritic cells and the D-fraciion-induccd Th-1 dominant response in BALB/c tumor-bearing mice. Cancer Lett 2003;192:181-7.

  8. Kodama N, Komula K, Nanba H. Can maitakc MD-fraclion aid cancer patients? Aitern Med Rev 2002;7:236-9.

  9. Inoue A, Kodama N, Nanba H. Effect of maitakc (Grifola frondosa) D-fraction on the control of the T lymph node Th-l/Th-2 proportion. Biol Phann Bui! 2002;25:536-40.

  10. Masaki K, Hirotakc K. Delayed ceil cycle progression and apoptosis induced by hem iceilu I asc-treated Agaricus blazci. Evid Based Complement Alternat Med 2006; in press, available on-line.

  11. Kasai HL, He M, Kawamura M, Yang PT, Deng XW, Mimkania M, et a). IL-12 production induced by Agaricus blazci fraction H (ABH) involves toll-like
    receptor (TLR). Evid Based Complement Ahern Med 2004;1:259-67.

  12. Inagaki N, Shibala T, Itoh T, Suzuki T, Tanaka H, Nakamura T, ct al. Inhibition of IgE-depcndcnl mouse triphasic cutaneous reaction by a boiling
    water fraction separated from mycelium of Phcllinus 1 in Letts. Evid Based Complement Ahern Med 2005;2:369-74.

  13. Al-Fatimi MAA, Jülich W-D, Jansen R, Lindcquist U. Bioaciivc components of the traditionally used mushroom pod ax is pistillaris. Evid Based Complement Ahern Med 2006;3:87-92.

  14. Amlcrsson HC, Hajslova J, Sclmlzova V, Panovska Z, Hajkova L, Gry J. Agaritine content in processed foods containing the cultivated mushroom
    (Agaricus bisporus) on the Nordic and the Czech market. J Food Addit Contain 1999;16:439-46.

  15. Sclmlzova V, Hajslova J, Pcroulka R, Gry J, Andcrsson HC. Influence of storage and household processing on the agaritine content of the cultivated
    Agaricus mushroom. Food Addil Contain 2002;19:853-62.

  16. Fricdcrich U, Fischer B, Lulhy J, Hann D, Schlatter C, Wurglcr FE. The mutagenic activity of agaritinc-a constituent of the cultivated
    mushroom Agaricus bisporus and its derivatives detected wit It the Salmonella/mammalian

microsome assay (Ames Test). Z Lehensm Unters Forsch 1986; 183:85-9.

  1. Papaparaskeva C, foannidcs C, Walker R. Agaritine docs not mediate the mutagenicity of the edible mushroom Agaricus bisporus. Mutagenesis
    1991 ;6:213-7.

  2. Toth B, Gannett P, Rogan E, Williamson J. Bacterial mutagenicity of extracts of the baked and raw Agaricus bisporus mushroom. In Vivo
    1992;6:487-90.

  3. Nagaokaa MH, Nagaoka H, Kondo K, Akiyama H, Mailani T. Measurement of a genoloxic hydrazine, agaritine, and its derivatives by HPLC with
    fluorescence dcrivaiizalion in the agaricus mushroom and its products. Citem Phann Bull 2006;54:922-4.

  4. Hajslova J, Hajkova L, Schulzova V, Frandscn H, Gry J, Andcrsson HC. Stability of agaritine – a natural toxicant of Agaricus mushrooms. Food Addit Contain 2002;19:1028-33.

  5. Toth B. Carcinogenic fungal hydrazines. In Vivo 1991;5:95 -100.

  6. Toth B, Sornson H. Lack of carcinogenicity of agaritine by subcutaneous administration in mice. M vcopathologia 1984;85:75-9.

  7. Toth B, Taylor J, Mattson B, Gannett P. Tumor induction by 4-(mclhyl)benzcnediazonkim sulfate in mice. In vivo 1989;3:17-22.

  8. Stamcts P. Notes on nutritional properties of culinary-medicinal mushrooms, hit J Med Mushrooms 2005;7:103-10.

  9. Oh no N, Yadomac T. Milogcnie subs lances of Bupicuri radix, in traditional herbal medicines for modern times, Bupieurum species, scientific
    evaluation and clinical applications. In; Shcng-Li (cd). CRC Taylor & Francis, 2006, 159-76.

  10. Izumi S, Oh no N, Kawakila T, Nomoto K, Yadomac T. Wide range of molecular weight distribution of mitogcnic substancc(s) in the hot water extract
    of a Chinese herbal medicine, Bupieurum chincnsc. Bio! Pharm Bid! 1997;20:759-64.

  11. Ohlsu S, Izumi S, Iwanaga S, Ohno N, Yadomac T. Analysis of mitogcnic substances in Bupieurum chincnsc by ESR spectroscopy. Biol Pharm Bull 1997;20:97-100.

  12. Kidd P. Thl/Th2 balance: the hypothesis, its limitations, and implications for health and disease. A Item Med Rev 2003;8:223-46.

  13. Okamolo M, Hascgawa Y, Hara T, Hashimoto N, Imaizumi K, Shimokata K, ct al. T-hclpcr type I/T-helper type 2 balance in malignant pleural effusions
    compared to tuberculous pleural effusions. Chest 2005;128:4030-5,

  14. Knutson KL, Disis ML. Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol hnnnmother 2005;54:721-8.

  15. Sarangi I, Ghosh D, Bhulia SK, Mailick SK, Maiti TK. Anti-tumor and immunomodulating effects of Plcurotus oslrcaius mycelia-derived proteoglycans, hit hnimmopharmacol 2006;6:1287-97.

  16. Kim GY, Lee JY, Lee JO, Ryu CH, Choi BT, Jeong YK, ct al. Partial characterization and immunostimulatory effect of a novel polysaccharidc-protcin
    complex extracted from Phcllimis lintcus. Biosci Biotechnol Bine hem 2006;70:1218-26,

  17. Aim WS, Kim DJ, Chac GT, Lee JM, Bac SM, Sin JI, ct al. Natural killer cel! activity and quality of life were improved by consumption of a mushroom
    extract, A got-tens blazei M mill Kyowa, in gynecological cancer patients undergoing chemotherapy. Int J Gynecol Cancer
    2004;14:589-94.

  18. Kancno R, Fontanari LM, Santos SA, Di Stasi LC, Rodrigues FE, Lira AF. Effects of extracts from Brazilian sun-nmshroom (Agaricus blazei) on the NK
    activity and Iymphoproliferative responsiveness of Ebrlich tumor-bearing mice. Food Che in Toxicol 2004;42:909-16,

  19. Fujimiya Y, Suzuki Y, Os hi man K, Kobori H, Moriguchi K, Nakashima H, ct al. Selective tumoricidal effect of soluble prolcoglucan extracted from
    the basidiomycctc, Agaricus blazei Murill, mediated via natural killer cell activation and apoptosis. Cancer hnnntno! hnnnmother
    1998;46:147-59,

  20. Bosch TM, Meijcrman 1, Bcijnen JH, Schcllcns JH. Genetic polymorphisms of drug-metabolising enzymes and drug transporters in the chemotherapeutic
    treatment of cancer. Clin Pharmacokinet 2006;45:253-85.

  21. Musana AK, Wilke RA. Gene-based drug prescribing: clinical implications of the cytochrome P450 genes. WMJ 2005;104:61-6.

  22. Nctea MG, Gow NA, Mu mo CA, Bates S, Collins C, Fcnverda G, el al. Immune sensing of Candida albicans requires cooperative recognition of
    man nans and glucans by lectin and To II-1 ike receptors. J Clin Invest 2006;! 16:1642-50.

  23. Brown GD, Dcctin-1: a signalling non-TLR paltcrn-rccognilion receptor. Nat Rev Immunol 2006;6:33-43.

  24. Saijo S, Fujikado N, Furula T, Chung S, Kotaki H, Scki K, ct al. Declin-1 is required for host defense against Pneumocystis carinii but not Candida
    albicans. Nat I minimal 2007;8:39-46.

  25. Sutherland AM, Walley KR, Russell JA. Polymorphisms in CD 14, maimosc-binding lectin, and ToII-like receptor-2 arc associated with increased
    prevalence of infection in critically ill adults. Crit Care Med 2005;33:638-44.

  26. Mullighan CG, Hcaticv S, Doherty K, Szabo F, Grigg A, Hughes TP, ct al. Mannosc-binding lectin gene polymorphisms arc associated with major
    infection following allogeneic hemopoietic stem cell transplantation. Blood 2002;99:3524 -9,

Received July 1, 2006; accepted January 16, 2007

20

After

1
For reprints and all correspondence; Naohito Ohno, Professor,

2
Tokyo University of Pharmacy and Life Science, School of

3
Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

4
Tel; +81-426-76-5570; Fax; +81-426-76-5570;

Effects of D-Fraction, a Polysaccharide from Grifola frondosa on Tumor

Effects of D-Fraction, a Polysaccharide from Grifola frondosa on Tumor Growth Involve Activation of NK Cells

Noriko Kodama,*a Kiyoshi Komuta,* Norio Sakai,c and Hiroaki NANBAa

a
Department of Microbial Chemistry, Kobe Pharmaceutical University; 4-19-1 Motoyama-kitamachi, Higashinada-ku,


Kobe 658-8558, Japan: b International Department, Osaka Police Hospital; 10-31 Kitayamacho, Tennoji, Osaka 543-0035, Japan: and c
Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University; 1-2-3 Kasumi, Minami-ku,
Hiroshima 734-8551, Japan.

Received July 12, 2002; accepted August 16, 2002


Natural killer (NK) cells are directly cytotoxic for tumor cells and play a primary role in regulating immune responses. We monitored levels of NK cell
cytotoxic activity in cancer patients receiving D-Fraction extracted from maitake mushrooms (Grifola frondosa). Elevated levels of cytotoxic
activity were maintained for one year. To elucidate the mechanisms underlying long-term activation of NK cells during treatment with D-Fraction, we
examined tumor volume and levels of IFN-

y and TNF-a

in MM46-bearing C3H/HeN mice to which D-Fraction was administered for 19 d. D-Fraction markedly suppressed tumor growth, corresponding with increases
in TNF-

a and IFN-y released from spleen cells and a significant increase in TNF-a

expressed in NK cells. This sug­gests that the D-Fraction activates NK cells even on the 20th day after treatment. Furthermore, D-Fraction in­creased
macrophage-derived interleukin (IL)-12, which serves to activate NK cells. These results suggest that NK cells are not only responsible for the early
effects of D-Fraction on tumor growth, but also for the long-term tumor-suppressive effects of D-Fraction through increased IL-12 released from
macrophages.

Key words IL-12 production; macrophage; NK cell

tients for long periods of time, exceeding one year (Table 1). To investigate maintenance of NK cell activation by D-Frac­tion, we examined NK cell
activation in MM-46 carcinoma- bearing mice treated with D-Fraction for 19 d. Our data in this paper may support a critical role for NK cell activation in
immunotherapy with D-Fraction for cancer patients.

MATERIALS AND methods

Materials
For the detection of human NK activity, chromium-51 (Daiichi Kagaku Yakuhin Co., Tokyo) and lymphopacel (d= 1.077) (IBL Co., U.S.A.) were prepared.

Animals
Male C3H/HeN mice (4-weeks-old) were ob­tained (Japan Crea Co., Osaka) and were raised for one week before being used for experiments. Food and water were
given freely to these mice until used for experiments.

Cells
MM-46 carcinoma cells were kindly donated by Dr. Kanki Komiyama. 51Cr labeled K-562 cells were used as target cells of human NK.

Preparation of D-Fraction
D-Fraction was prepared from the dried powder of the fruit body of maitake mush­rooms (Grifola frondosa) (Yukiguni Maitake Co., Niigata),
according to the method described in our previous paper.6) The level of LPS contained in D-Fraction was determined by using Endospecy ES-20S Set
(Seikagaku Industry Co., Tokyo), and the ratio (%) of LPS in D-Fraction was less than

We previously reported that the (1®3)-branched (1®6)-^- glucan termed D-Fraction, extracted from the fruit body of the maitake mushroom

(Grifola frondosa)

(Fig. 1), is a bio­logical response modifier (BRM) like lentinan, which is found in

Letinus

edodes.

1—3)

These polysaccharides enhance the activity of immunocompetent cells such as macrophages, helper T cells, and cytotoxic T cells, which attack tumor cells.
These effects of BRMs led us to hypothesize that oral administration of D-Fraction would be an effective treatment for cancer patients. Orally administered
D-Fraction was shown to reduce tumor size in mice, without causing un­wanted side effects.

2

1

Therefore, D-Fraction has been com­mercialized as a health material, the safety of which has been confirmed by the Consumer Product Testing Co. (New
Jer­sey, U.S.A.) We have already tried a non-randomized clinical trial of D-Fraction therapy for patients with lung and breast cancer in stages II—IV

4)

In this trial, we found that IL-2 pro­duction was enhanced in D-Fraction-treated patients, sug­gesting that macrophages and T cells were activated by D-
Fraction. In addition, D-Fraction is revealed to be effective for human immunodeficiency virus (HIV) infection

5)

and lis­teriosis,

6

1

by reinforcing the immune system. These results indicate that one network of the immuno-response pathway by which D-Fraction enhances anti-tumor effects is
depen­dent on an adaptive immunity associated with cytotoxic T cell activation.

Fig. 1. Chemical structure of D-Fraction

© 2002 Pharmaceutical Society of Japan

* To whom correspondence should be addressed. e-mail: n-kodama@kobepharma-u.ac.jp


Natural killer (NK) cells rapidly recognize and lyse a large variety of tumor or virus-infected cells, without the need for either prior sensitization or
MHC-dependent recognition, which is different from cytotoxic T cell.

7)

Pham-Nguyen

et al.

have reported that the early NK cell response alone is not sufficient for tumor clearance, and that both T cells and NK cells are required in the
development of the long-term sur­vival in the hepatic tumor model by IL-12-mediated gene therapy.

8)

In our clinical study, we also found that D-Fraction activated NK cells in lung, breast and liver carcinoma pa­

0.000007%. In the experiment using macrophage cell line RAW 264.7 cells, D-Fraction was pretreated with polymixin B (30 mg/ml) for 2h.

Administration of D-Fraction to Cancer Patients
Dr. Kiyoshi Komuta of the Osaka Police Hospital gave informed consent to 8 cancer patients. Eight stage II—IV cancer pa­tients between 43- and 74-years-old
were orally administered 100 mg of D-Fraction on consecutive days for 34 months and their NK activity was examined.

Administration of D-Fraction to MM46 Carcinoma- Bearing C3H/HeN Mice
MM-46 carcinoma cells (2X106) were implanted in the right axillary region of 5-week-old male C3H/HeN mice. After 24 h, D-Fraction (5 mg/kg/d)
was administered to MM-46 carcinoma-bearing mice intraperi- toneally (i.p.) for 19 consecutive days. Tumor inhibition ratio (T.I.R.) was calculated as
follows: [1-(weight of tumor mass from mice treated with D-Fraction)/(weight of tumor mass from mice treated with PBS)]X 100.

Detection of Human NK Cell Activity
NK cells (1 X 106) obtained from blood using the Conray-Ficoll method9) were mixed with 2X107 of 51Cr-labelled
K-562 (target cell of NK cell) cells in a tube. After incubation for 1h, release of 51Cr into the culture supernatant was detected using a g-
counter. NK activity was calculated as: NK activity (%) = [(experimental release-spontaneous release) cpm/(max-re- lease-spontaneous release) cpm (by 1n
HCl treatment)] X 100.

RESULTS AND DISCUSSION


Effects of D-Fraction on MM46-Carcinoma Cell Growth

On the 20th day of D-Fraction administration to MM-46 tumor-bearing mice, D-Fraction significantly decreased tumor growth as compared with mice
administered phosphate bufferent saline (PBS), when the T.I.R. was 82% (Fig. 2). The effect of D-Fraction was also investigated in C3H/HeJ mice that are
non-response to LPS; the T.I.R. was 78%. These results indicated that D-Fraction inhibited the growth of tumor.

Effects of D-Fraction on NK Cell Activation
D-Frac- tion has already been reported to enhance cellular immunity by stimulating cells such as macrophages, helper T cells and cytotoxic T cells. 2) To detect NK cell activation, we exam­ined IFN-g and TNF-a release from whole spleen cells on the 20th day of D-Fraction administration by
mouse IFN-g and TNF-a ELISA kits (Genzyme Co., Minneapolis, U.S.A.). As shown in Figs. 3A and 3B, levels of IFN-g and TNF-a were significantly increased by
1.5- and 2.5-fold, re­spectively, compared to control mice. Intracellular expression of TNF-a in splenic NK cells under D-Fraction administra­tion was also
investigated using flow cytometric analysis. To detect NK cells, 2X106 cells were stained with R-PE-conju- gated PanNK and Cy-Chrom™-conjugated
CD3e antibodies (PharMingen Co., San Diego, CA, U.S.A.) and CD3e nega­tive and PanNK positive was determined to represent the NK cell. Fluorescein
isothiocyanate (FITC)-conjugated TNF-a antibody (PharMingen Co.) was used for detecting TNF-a expression in NK cells. After staining, NK cells expressing
TNF-a were analyzed using a FACSCalibur analyzer (Beck- ton Dickinson Co., Grenoble, France) and histograms were calculated with CellQuest software (Becton
Dickinson,

Fig. 2. Effect of D-Fraction on MM-46 Carcinoma Cell Growth


D-Fraction (5 mg/kg/d) was administered to MM-46 carcinoma-bearing mice i.p. for 19 consecutive days, and then tumor volume (mm3=longest
diameterXshortest diame- ter2/2) was measured on day 20.16) Data are expressed as mean±S.E.M. of 8 experi­ments. **,
/><0.01; ***, _p<0.001 compared to control mice (Student’s Mest).


Figs. 3A and 3B. Effects of D-Fraction on Release of IFN-g or TNF-a from Whole Spleen Cells in MM-46 Carcinoma-Bearing Mice

On day 20 in mice administered D-Fraction (5 mg/kg/d) or PBS (—) i.p., splenocytes were collected according to previously described methods. 6) Splenocytes were plated at 1X106 cells/well in a 96-well plate and cultured with Con A (10 mg/ml) for 24h in 5% CO

2

at 37 °C. After activation, levels of INF-g and TNF-a in culture supernatant were assessed using ELISA. Data are expressed as mean±S.E.M. of 4—8
experiments. **, /><0.01; ***, _p<0.001 compared to control mice (Student’s Mest).



Fig. 3C. Effect of D-Fraction on Intracellular TNF-a Expression in NK Cells of MM-46 Carcinoma-Bearing Mice


On day 20 in mice administered D-Fraction (5mg/kg/d) or PBS (—), intracellular TNF-a expression in splenic NK cells was investigated using flow
cytometric analysis. Shown are representative histograms of triple-color flow cytometric analysis of spleno­cytes stained with R-PE-conjugated PanNK,
Cy-ChromTM-conjugated CD3£, and FITC-conjugated TNF

-a
antibodies.

Mountain View, CA, U.S.A.). As shown in Fig. 3C, D-Frac- tion administration resulted in a 1.3-fold increase in intracel­lular TNF-aexpression in
splenic NK cells compared to con­trol mice. TNF-a is a cytokine released from activated NK cells in the same way as IFN-g,10,11) and
has various func­tions relating to inflammatory and cytotoxic reactions, in ad­dition to cytotoxicity and the ability to directly cause hemor-

Fig. 4. IL-12 Release from RAW 264.7 Cells Stimulated with D-Fraction RAW 264.7 cells (1X

106 cells/well in a 24-well plate) were stimulated for 18h with D-Fraction. To remove any possible LPS contamination, D-Fraction was treated
with polymyxin B (30 mg/ml) for 2h before use. At the indicated time of incubation, IL-12 in the culture supernatants was measured using ELISA. Data
are expressed as mean

±S.E.M. of 3 experiments. *, p<0.05 as compared with the basal (0 mg/ml of D- Fraction) (Scheffe’s F-test).




Table 1. Activities of NK Cells in Cancer Patients

Patient

Treatments (100 mg of D-Fraction)

Beforea) (date)

Aftera) (date)

SCLC (small cell lung carcinoma) (74-year-old female, stage III) SCLC (small cell lung carcinoma) (54-year-old male, stage III)
Alveolar cell carcinoma (54-year-old female, stage III-B) Pulmonary adenomatosis (54-year-old male, stage-IV) Gallbladder carcinoma
(48-year-old female, stage II) Bronchogenic cancer (58-year-old male, stage III-B)

Masto carcinoma (breast cancer) (43-year-old female, stage III) Parathyroid carcinoma (69-year-old male, stage III-B)

23% (’99.6) 21% (’98.5) 24% (’98.4) 21% (’99.7) 28% (’98.7) 26% (’99.5) 30% (’98.10) 23% (’99.6)

44% (’99.9), 51% (’00.2)

42% (’99.7), 47% (’00.9)

48% (’98.9), 44% (’99.4), 42% (’00.5), 44% (’01.2) 42% (’00.9), 47% (’01.2)

40% (’98.8), 39% (’00.1), 48% (’01.5)

33% (’00.10), 39% (’01.1), 31% (’01.6)

47% (’99.9), 53% (’00.12)

38% (’99.9), 44% (’00.4), 62% (’01.6)

a)
NK activity (%) examined. Standard level of NK activity on human, 18—40%.


rhagic tumor necrosis.

12)

Consequently, significant increases in release of IFN-g and TNF-a suggest activation of NK cells by D-Fraction.

Effects of D-Fraction on IL-12 Release from Macro­phages
IL-12 is a cytokine released by monocytes and macrophages, and is critical to the functions of NK and T cells. It induces IFN-g release from both NK and T
cells.12—14) NK cells can lyse a variety of different tumor cells by exocytosis of perforin-containing granules, and sub­sequent formation of
lytic pores by perforin on the target cell membrane. Activation of NK cells with IL-2 and IL-12 has recently been reported to increase the binding of
perforin to the target cell membrane and subsequent lysis of tumor cells.15) IL-12 is released from monocytes and macrophages. To investigate
the effect of D-Fraction on IL-12 release, macrophage cell line RAW 264.7 cells were incubated with various concentrations of D-Fraction and secretion of
IL-12 was assessed. When cells were treated with D-Fraction (500—1000 mg/ml) for 24 h, the amount of IL-12 released significantly exceeded basal levels (0 mg/ml D-Fraction) (Fig. 4). Also, the effect of D-Fraction on IL-12 production was investigated in vitro using peritoneal macrophages
from nor­mal C3H/HeJ mice. When the normal peritoneal macro­phages were stimulated with D-Fraction (500 mg/ml) for 24 h, IL-12 production showed
an increase compared with that of the control (data not shown). These results suggested that the long-term anti-tumor effects of D-Fraction are
attrib­utable to activation of NK cells via macrophage-derived IL- 12, and to T cell activation. NK cells may be further acti­vated by INF-g released from
not only themselves but also activated T cells.

Effect of D-Fraction on the Activation of NK Cells in Cancer Patients
We concluded that D-Fraction was safe with no toxicity according to data confirmed by the Con­sumer Product Testing Co. Then, a non-randomized clinical
trial of D-Fraction was conducted to check the effect in NK cells of 8 cancer patients, who agreed to the trial. As shown in Table 1, the NK cell
activities of these patients were en­hanced 1.2—2.7 times by treatment with D-Fraction. Even though these tests were limited and in a non-controlled trial,
these results indicated that D-Fraction was effective for acti­vation of NK in cancer patients.

In conclusion, D-Fraction represents an important BRM for NK cells by enhancing IL-12 release from macrophages. In immunotherapy using D-Faction for cancer
patients, NK cells are responsible for early anti-tumor responses, while both NK and T cells are responsible for long-term anti-tumor responses. Although
the dependence of the immune system on NK cells requires further study, our results also indicate the possibility of immunotherapy by various agents, which
enhance the activity of NK cells for cancer patients.

Acknowledgements
We wish to thank Dr. Kanki Komi- yama of Kitasato Institute for the tumor cells utilized in our study, and also Mr. Yasuo Ohdaira, Manager of the
Develop­ment Division of Yukiguni Maitake Co. who contributed maitake to our studies.

REFERENCES

  1. Adachi K., Nanba H., Kuroda H., Chem. Pharm. Bull., 35, 262—270 (1987).

  2. Hishida I., Nanba H., Kuroda H., Chem. Pharm. Bull., 36, 1819— 1827 (1988).

  3. Nanba H., Hamaguchi A., Kuroda H., Chem. Pharm. Bull., 35, 1162— 1168 (1987).

  4. Kodama N., Komuta K., Nanba H., Altern. Med. Rev., 7, 236—239 (2002).

  5. Nanba H., Kodama N., Schar D., Turner D., Mycoscience, 41, 293— 295 (2000).

  6. Kodama N., Yamada M., Nanba H., Jpn. J. Pharmacol., 87, 327—332 (2001).

  7. Schattner A., Duggan D. B., Am. J. Hematol., 18, 435—443 (1985).

  8. Pham-Nguyen K. B., Yang W., Saxena R., Thung S. N., Woo S. L., Chen S. H., Int. J. Cancer, 81, 813—819 (1999).

  9. Kou K., Sawada S., Med. Tech., 21, 574—580 (1993).

  10. Tchorzewski H., Acta Haematol. Pol., 25, 56—61 (1994).

  11. Nagashima S., Mailliard R., Kashii Y., Reichert T. E., Herberman R.

B.’ Robbins P., Whiteside T. L., Blood, 91, 3850—3861 (1998).

  1. Haranaka K., Satomi N., Sakurai A., Int. J. Cancer, 34, 263—267 (1984).

  2. Trinchieri G., Gerosa F., J Leukoc Biol., 59, 505—511 (1996).

  3. Lee S. M., Suen Y., Qian J., Knoppel E., Cairo M. S., Leuk Lymphoma, 29, 427—438 (1998).

  4. Lehman C., Zeis M., Uharek L., Br J. Haematol., 114, 660—665 (2001).

  5. Ingber D., Fuzita T., Kishimoto S., Nature (London), 348, 555 (1990).