Immunomodulating 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 Gao4 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

Show more details – table 1 >>>

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 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.

Show more details - table 2 >>>

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).

Show more details - table 3 >>>

P<
0.01

{N=12)

Before

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).


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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;

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