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RES E A R C H A R T I C L E Open Access
Differences in antioxidant activities of
outdoor- and indoor-cultivated Agaricus brasiliensis,
and protective effects against carbon
tetrachloride-indu ce d acute hepatic injury in mic e
Daisuke Yamanaka
1
, Masuro Motoi
1,2
, Akitomo Motoi
2
and Naohito Ohno
1*
Abstract
Background: Agaricus brasiliensis (A. brasiliensis) is a medicinal mushroom that exerts various pharmacological
actions. We previously demonstrated that different cultivation conditions altered the activity of the polyphenol-related
enzymes from t his mushroom. However, the influence of cultivation conditions on the antioxidant acti vity of the
fruiting bodies remains unclear. Therefore, in this study we compared the antioxidative effects of fruiting bodies of
A. brasiliensis cultivated outd oors and indoors. In additi on, we assessed wh ether different cultivation methods
aff ected the he patoprotective effects against CCl
4
-induced liver inj ury.
Methods: We assessed the antioxi dative effects of mushroo ms cultivated in open-air or indoors using the DPPH
radical-scavenging assay. Furthermore, we prepared experimental feeds containing outdoor- o r indoor-cultivated
A. brasiliensis. Acute li ver injury was in duced by CCl
4
injection in mi ce that consumed feed containin g outdoor- or
indoor-cultivated A. brasiliensis. The hepatoprotective effects of these mushrooms were then evaluated by monitoring
the reduction in the circulating levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase.
The significance of the differences between the means was assessed using Student’s t-test. Finally, histopathological
analysis of liver was performed.
Results: In the DPPH assay, the antioxidant activity of outdoor-cultivated A. brasiliensis was higher than t hat of
indoor-cultivated mushroom. Moreover, in the mouse model of CCl
4
-induced hepatitis, the oral administration of
outdoor-cultivated A. brasiliensis reduced liver damage significantly, but indoor-cultivated mushrooms failed to inhibit
hep atitis. The hepatop rotective effects of outdoor-cultivated A. brasiliensis were observed even when ingestion
commenced only 1 day before CCl
4
injection, and these effects were not affected by excessive heat treatment.
Conclusions: Ou tdoor cultivation significantly enhanced the anti oxidative activity of A. brasiliensis fruiting bodies.
In addition, outdoor-cultivated A. brasiliensis was mo re effective at protecting against CCl
4
-induced liver injury in
mice than mushroom s grown in a greenhouse.
Keywords: Agaricus blazei, Cul tivation condition, Sunlight, Hepatitis, Antioxidative activity
* Correspondence: ohnonao@toyaku.ac.jp
1
Laboratory for Immunopharmacology of Microbial Products, School of
Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1
Horinouchi, Hachioji, Tokyo 192-0392, Japan
Full list of author information is available at the end of the article
© 2014 Yamanaka et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Yamanaka et al. BMC Complementary and Alternative Medicine 2014, 14:454
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Background
Agaricus brasiliensis (A. brasiliensis) S. Wasser et al. (also
known as A. blazei Murrill sensu Heinemann, A. rufbtegu-
lis and A. subrufescens) [1-3] is a medicinal mushroom
used worldwide as a biological response modifier [4,5]. To
date, many reports demonstrated beneficial effects of A.
brasiliensis on human health. For example, natural killer
cell activity was upregulated after the intake of the fruiting
body of A. brasiliensis without any adverse effects [6]. The
immunoenhancing effects of this mushroom are princi-
pally exerted by abundant β-glucans [7,8], and several in-
nate immune receptors such as Toll-like receptor (TLR) 2
[9], TLR4 [10], and dectin-1 [11] also contribute to the
augmentation of host immune systems stimulated by A.
brasiliensis. Evidence of pharmaceutical activity has im-
proved the reliability of A. brasiliensis as a functional food.
In addition to immunoenhancing activities, we identified
additional beneficial effects of A. brasiliensis,suchasthe
protection against concanavalin A- or LPS-induced mur-
ine liver injury, after oral administration in water extracts
[6]. The oral administration of extracts of A. brasiliensis
fruiting bodies or mycelium also exhibited hepatoprotec-
tive effects in a model of CCl
4
-induced hepatitis [12,13].
Although the underlying mechanism(s) behind its hepato-
protective effects remain unclear, the antioxidative activity
of A. brasiliensis could contribute to its activity because
inhibiting the generation of free radicals is important for
protecting against CCl
4
-induced hepatic injury [13,14].
Therefore, the antioxidative properties of this mushroom
may be important for the treatment of free radical-
induced hepatitis.
Hepatitis remains an incurable disease. In particular,
viral hepatitis is highly infectious, and so is a global health
issue. Many patients who are unable receive effe ctive
pharmaceutical agents choose to use traditional medi-
cines such as A. brasiliensis due to it s hepatoprot e ct ive
activities. Today, many commercial products ba sed on
this mushroom are available, and patients can select one
of these products. However, we previously demonstrated
that different A. brasiliensis cultiv ation conditio ns (such
as open air or indoors) affected its protein and chemical
components significantly. In particular, outdoor cultiva-
tion strongly upregulated the activity of peroxida se and
laccase [15]. These enzymes contribute to the degradation
or composition of phenolic compounds, and, thus, cultiva-
tion conditions may influence their antioxidative properties
and related biological activities. Therefore, to assess the
optimum use of A. brasiliensis, we investigated the effect
of cultivation conditions on its antioxidative activity.
In the present study, we a ssessed the influence of cul-
tivation conditions on the antioxidant ac tivities of three
kinds of A. brasiliensis mushroomandcomparedthe
hepatoprotective effects of outdoor- and indoor-cultivated
A. brasiliensis on CCl
4
-induced murine hepatic injury.
Methods
Animals and materials
Male ICR mice (4 weeks of age) were purchased from
Japan SLC (S hizuoka, Japan). The mice w ere housed in
a specific pathogen-free environment, and w ere used at
5–6 weeks of age. All experiment s were performed in
accordance with the guidelines for laboratory animal ex-
periments provided by the Tokyo University of Pharmacy
and Life Sciences, and the Committee for Laboratory Ani-
mal Experiments at Tokyo University of Pharmacy and
Life Sciences (P13-47) approved each experimental proto-
col. 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical was
purchased from Tokyo Chemical Industry Co., Ltd, Tokyo,
Japan. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox) was purchased from Calbiochem Inc., CA,
USA. Carbon tetrachloride was purchased from Wako Pure
Chemical Co. (Osaka, Japan), and olive oil was obtained
from Nikko Pharmaceutical Co., Ltd. (Gifu, Japan).
A. brasiliensis fruiting bodies
A. brasiliensis strain KA21, deposited at National Institute
of Technology and Evaluation, Japan with deposition
number: FERM P-17695 was cultivated outdoors with
sunlight o r indoors avoiding sunshine, using the same
compost. The fruiting bodies were har vested in Brazil,
washed, and dried using hot air at 60°C or lower ( Toei
Shinyaku Co., Ltd., Tokyo, Japan). The Japanese commer-
cial product of dried fruiting bodies from another strain
of A. brasiliensis , cultivated indoors in Japan, was also
purchased and tested as another indoor-cultivated sample.
Outdoor- and indoor-cultivated A. brasiliensis KA21, and
the indoor-cultivated Japanese sample were named KAOD,
KAID, and JAID, respectively. The ITS-5.8S rDNA se-
quence of KAOD and JAID was analyzed using ITS4 and
ITS5 primers at Techno-Suruga Laboratory Co., Ltd.
(Shizuoka, Japan). KAOD and JAID showed 99.6% and
100% homology with A. subrufescens (GenBank accession
number AY818654.1 and KJ541796.1, respectively).
Measurement of ingredients
Japan Food Research Laboratories (Shibuya, Tokyo)
measured the ingredients of A. brasiliensis fruiting bod-
ies using the standard protocols recommended by the
Resources Council, the Science and Technology Agency
of Japan.
Antioxidant assay
The ability of A. brasiliensis to scavenge DPPH free radicals
was assessed using the previously described decolorization
method, with minor modifications [16,17]. Briefly, pow-
dered mushrooms were extracted with 50% methanol
(50 mg/mL) at 60°C for 60 min. Trolox dissolved in 50%
methanol was used as a standard antioxidant. Diluted ex-
tracts (20 μL) were combined with 200 μL of 150 μmol/mL
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DPPH in 50% methanol. All samples were prepared in
triplicate. Following incubation in the dark at room
temperature for 30 min, the absorbance at 517 nm wa s
read using a Safire microplate reader (Te ca n, Salzburg ,
Austria). The effect of natural color changes o f the
samples was controlled for by using a blank , and the
data wa s expressed as equivalent s of Trolox.
Feed preparation
Various doses of powdered fruiting bodies from A. brasi-
liensis were mixed into the basic synthetic diet AIN-93G
(Oriental Yeast Co., Ltd, Tokyo, Japan), and cornstarch
was substituted for A. brasiliensis. The doses of K AOD
tested were 1, 3, and 10%, whereas KAID and JAID were
tested at 3 and 10%, respectively. AIN-93G supplemented
with cornstarch was used as the control diet. All feeds
were admixed by Oriental Yeast Co., Ltd, and the molding
and drying processes were carried out at 60°C or lower.
Each test diet was irradiated with 30 kGy γ-rays for
sterilization. The ingredients in the solid feed are shown
in Table 1. To evaluate the heat stability of K AOD,
AIN-93G and K AOD-10% were autoclaved and air-
dried at 50°C , and named heat-treated AIN-93G and
heat-treated K AOD, respectively.
Experimental protocol
For the chemical hepatic injury experiment s, mice were
divided randomly into 13 groups. All groups of mice
were fed an experimental diet for 11 days. On the 10th
day, hepatotoxicity was induced by intraperitoneal (i.p.)
injection of CCl
4
(4 ml/kg body weight of 1% CCl
4
solu-
tion in olive oil). In the control group, mice re ceived an
i.p. injection of olive oil alone. The feeding design is
Table 1 Ingredients in solid feed
Ingredient Content (g/kg diet)
Corn starch 397.486 - R
Casein 200.000
α-Corn starch 132.000
Sucrose 100.000
Soybean oil 70.000
Cellulose powder 50.000
AIN-93G Mineral Mix 35.000
AIN-93G Vitamin Mix 10.000
L-Cystine 3.000
Choline bitartrate 2.500
tert-butylhydroquinone 0.014
KAOD, KAID, or JAID R
Control diet (AIN-93G): R =0.
KAOD-1%: R =10.
KAOD-3%: R =30.
KAOD-10%: R =100.
KAID-3%: R =30.
JAID-10%: R =100.
Figure 1 Experimental protocol.
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shown in Figure 1. Mice in groups I and II received AIN-
93G and KAOD-10%, respectively, during the experiment,
and were treated with olive oil. Mice in groups III–VI re-
ceived AIN-93G, KAOD-10%, KAOD-3%, and KAOD-1%,
respectively. Mice in Groups VII and VIII received KAID-
3% and JAID-10%, respectively. Groups IX were fed
AIN-93G, but then received K AOD-10% on day 9 until
the end of the experiment. Mice in Groups X or XI were
fed AIN-93G, and then received K AOD-10% for 24 h
before or after the injection of CCl
4
, respe ctively. Mice
in Groups XII or XIII received AIN-93G, followed by
heat-treated AIN-93G or heat-treated KA OD, respect-
ively, from day 7 until the end of the experiment. All
mice in Groups III– XIII were treated with CCl
4
on day
10. Twenty-four hours after t he i.p. administration of
CCl
4
or olive oil, mice were s acrificed by CO
2
inhal-
ation. Blood samples were collected by heart puncture,
and the liver was removed and use d to analyze biochem-
ical markers of early acute hepatic damage and for
histopathological e valuation.
Measurement of serum ALT, AST, and LDH activities
Collected whole blood wa s allowed to stand at room
temperature for 90 min, and then centrifuged (12,000 rpm
for 10 min at 4°C) to obtain serum. The enzyme activities
of alanine aminotransferase (ALT ) and a spartate amino-
transferase (AST ) in serum were then mea sured using a
Wako Transaminase CII-Test kit (Wako Pure Ch emical
Co.) following the manufacturer’s instructions. The color
development was stopped using citric acid solution, and
the optical density at 550 nm was measured using a mi-
croplate reader (MTP450; Corona Electric, Ibaraki, Japan).
Lactate dehydrogenase (LDH) activity was assayed accord-
ing to the instructions supplied with the commercial assay
kit (NescoVL LD, Alfresa Pharma Co., Osaka, Japan). The
absorbance at 340 nm was read using a Safire microplate
reader (Tecan).
Histopathological analysis
Livers were fixed in 10% formalin neutral buffered solution
(pH 7.4) (Wako Pure Chemical Co.), embedded in paraffin
blocks, and thinly sectioned. Tissue sections were stained
Table 2 Basic nutrient composition comparison of
cultivation methods
KAOD KAID JAID Unit
Moisture 7.5 5.5 5.9 g
Energy 179 184 183 kcal
Protein 39.8 43.3 41.6 g
Fat 2.9 3.7 3.1 g
Carbohydrate 24.2 21.5 20.2 g
Fiber 18.9 19 22.7 g
Sodium 3.4 7.5 9.7 mg
Vitamin A (total caronene) - - -
Vitamin B1 (Thiamin) 0.79 1.41 0.52 mg
Vitamin B2 (Riboflavin) 3.5 4.01 5.49 mg
Vitamin B6 0.49 0.87 0.76 mg
Vitamin B12 0.27 - - μg
Niacin 48.8 47.7 53.4 mg
Pantothenic acid 20.2 23.9 15.9 mg
Folic acid 0.18 0.36 0.21 mg
Biotin 150 174 128 μg
Total vitamin C (Total c acid) - - -
Vitamin D 30.7 - 0.9 μg
Vitamin E (Total tocopherol) - - - mg
Vitamin K1 (Phylloquinone) - - - μg
Calcium 42.5 36.6 1.6 mg
Iron 11 11.8 7.76 mg
Phosphorus 994 987 1110 mg
Magnesium 98.9 105 114 mg
Potassium 2.84 2.96 2.67 g
Copper 16.5 12.3 1.65 mg
Iodine - - -
Manganese 0.67 0.7 0.82 mg
Selenium 51 120 24 μg
Zinc 10.9 19.7 16.4 mg
Total chromium 0.05 - - mg
Molybdenum - - -
-; not detected.
All data are shown for 100 g dry weight, and were measured by Japan Food
Research laboratories.
Figure 2 Free radical scavenging activities of various A.
brasiliensis. The antioxidant capacities of KAOD, KAID, and JAID
extracts (powder 50 mg/mL, supernatant) were measured using
the DPPH radical-decolorization assay. Each sample was diluted
and reacted with the DPPH r eagent. Results are presented as
mean (n =3) ± SD. Significant difference from KAOD extract:
***p <0.001.
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with hematoxylin and eosin (HE). The Biopathology
Institute Co., Ltd. (Oita, Japan) prepared paraffin blocks
and carried out HE staining. The sections were then ob-
served under a normal light microscope (Biozero BZ-8100
microscope, Keyence, Osaka, Japan).
Statistical analysis
The significance of the differences between the means
was assessed by Student ’s t-test.
Results
Influence of cultivation conditions of the antioxidative
activities of A. brasiliensis
We demonstrated previously that the enzymatic activities
of A. brasiliensis fruiting bodies can be modulated by cul-
tivation conditions, such as open-air or indoor growth
[15]. Therefore, we first compared the ingredients in the
fruiting bodies of A. brasiliensis and studied the antioxi-
dant activities of KAOD, KAID, and JAID. As shown in
Table 2, the basic composition of KAOD and JAID was
similar (except for vitamin D and calcium), and there were
no major differences between KAOD and KAID. Never-
theless, in the DPPH radical-scavenging assay, the antioxi-
dant activity of KAOD extract was higher than that of
KAID or JAID (Figure 2). These data suggest that outdoor
cultivation augments the antioxidant capacity of A. brasi-
liensis fruiting bodies.
Effect of outdoor cultivated-A. brasiliensis on CCl
4
-induced
hepatic injury
Because KAOD yielded increased antioxidative activity,
we next focused on its pharmacological activity. To evalu-
ate whether the oral administration of A. brasiliensis
whole fruiting bodies exerted hepatoprotective properties,
we carried out an in vivo experiment using a mouse model
of CCl
4
-induced hepatitis. As shown in Figure 3A, al-
though K AOD-1% did not suppress the blood relea se of
liver enzymes , 10 days of administration of KAOD-10%
Figure 3 Hepatoprotective effects of KAOD. Serum and livers were isolated from ICR mice fed KAOD-1%, KAOD-3%, or KAOD-10% ad libitum
for 11 days. (A) ALT, AST, and LDH activities in the serum from mice fed (a) KAOD-1% and injected with CCl
4
, (b) KAOD-3% and injected with
CCl
4
or (c) KAOD-10% and injected with CCl
4
or olive oil were measured using commercially available kits. Data are presented as mean ± standard
deviation, (a) n =6, (b) n =12, (c) n =7 (CCl
4
), n =3 (olive oil). Significant difference from AIN-93G: **p <0.01; ***p <0.001; n.s.: not significant.
(B) HE-stained liver sections from mice administered with (a-1) AIN-93G with CCl
4
(control for KAOD-1%), (a-2) KAOD-1% with CCl
4
, (b-1) AIN-93G with
CCl
4
(control for KAOD-3%), (b-2) KAOD-3% with CCl
4
, (c-1) AIN-93G with CCl
4
(control for KAOD-10%), (c-2) KAOD-10% with CCl
4
, (c-3) AIN-93G with
olive oil, or (c-4) KAOD-10% with olive oil were observed under a normal light microscope. Scale bar =100 μm.
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or KAOD-3% significantly inhibited CCl
4
-induced changes
in the circulating levels of enzymes including ALT, AST,
and LDH. In particular, mice fed KAOD-10% showed
comparable levels of biochemical markers a s olive oil-
injected mice. Data obtained from histopathological
studies supported the results of the biochemical analyses.
(Figure 3B). Liver sections from control and KAOD-alone
treated mice showed normal liver architecture. Injection
with CCl
4
-induced obvious liver injury, which presented
as large areas of extensive necrosis and a loss of hepatic
architecture. K AOD prevented the development of CCl
4
-
induced histological changes in a dose-dependent manner.
The effect of cultivation conditions of the
hepatoprotective activities of A. brasiliensis
The above experiment s defined the optimal dose of
KAOD for hepatoprotection, therefore, we next prepared
experimental diets containing KAID and compared its ef-
fect s on outdoor- and indoor-cultivated A. brasiliensis
KA21. Although K AOD-3% exerted hepatoprotective
effects , K AID-3% did not inhibit the release of hepatic
enzymes or prevent histopathological changes in the
liver ( Figure 4).
To confirm these effects, we prepared a commercially
available product from another strain of A. brasiliensis
cultivated indoors in Japan, and mixed it into the experi-
mental diet as JAID-10%. Oral administration of JAID-
10% did not suppress the acute liver injury induced by
injection of CCl
4
(Figure 4). These results strongly sug-
gest that indoor cultivation significantly reduces the he-
patoprotective activity of A. brasiliensis fruiting bodies.
Optimization of the hepatoprotective properties of A.
brasiliensis
To determine the optimal use of A. brasiliensis ,we
assessed the length of time taken for orally administered
KAOD-10% to protect against CCl
4
-induced liver injury.
Administration of KAOD-10% for 2 days wa s sufficient
for hepatoprotective effects, assessed by the suppression
of enzyme alterations (Figure 5A). Moreover, to a ssess
whether the hepatoprotective effects of A. brasiliensis
Figure 4 Effect of cultivation conditions of the hepatoprotective activities of A. brasiliensis. Serum and livers were isolated from ICR mice
fed AIN-93G, KAOD-3%, KAOD-10%, KAID-3%, or JAID-10% ad libitum for 11 days. (A) ALT, AST, and LDH activities in the sera from (a) KAID-3% or
KAOD-3% administered mice, or (b) KAOD-10% or JAID-10% administered mice were measured using commercially available kits. Data are presented
as mean ± standard deviation, (a) n =6, (b) n =7. Significant difference from AIN-93G or (a) between KAID-3% and KAOD-3% or (b) JAID-10% or
KAOD-10% : *p <0.05; **p <0.01; ***p <0.001; n.s.: not significant. (B) HE stained liver sections from mice administered (a-1) AIN-93G with CCl
4
(control for KAOD-3% and KAID-3%), (a-2) KAOD-3% with CCl
4
, (a-3) KAID-3% with CCl
4
,(b-1)AIN-93GwithCCl
4
(control for KAOD-10% a nd
JAID-10%), (b-2) KAOD-10% with CCl
4
, (b-3) JAID-10% with CCl
4
,. Scale bar =100 μm.
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were pre ventive or curative, mice were treated with
KAOD-10% for 24 h before or after inje ction with CCl
4
.
Hepatoprotection was observed only in the pre-ingestion
group. In ad dition, histopathological analyses strongly
supported a hepatoprotective effect of K AOD-10%
after 24 h of administration in the pre-ingestion group
(Figure 5). These data suggest that the oral administra-
tion of K AOD exerts preventative effe cts against acute
hepatic injury, and applies raid effe cts.
During the preparation of experimental diets containing
KAOD, we avoided high temperatures to preserve a variety
of heat-sensitive properties. Therefore, we next assessed
the effects of heat processing on the hepatoprotective
properties of A. brasiliensis. As shown in Figure 6, the he-
patoprotective effects of orally administered KAOD-10%
on the release of hepatic enzymes and the histopatho-
logical changes in the liver were not affected by autoclav-
ing, suggesting that the hepatoprotective properties of A.
brasiliensis are heat stable.
Discussion
In 2008, a clinical study reported that A. brasiliensis
extracts normalized liver function in hepatitis B patients
[18]. In addition, several animal experiments have inves-
tigated the hepatoprotective effects of these mushroom
extracts [19,20]. However, the process of extraction not
only concentrates the active ingredients, but also in-
creases the potential for side effects. In 2006, possible
adverse effects of A. bra siliensis were reported, where it
was suggested that intake of these mushroom extracts
may induce hepatic dysfunction [21]. Therefore, in this
study we did not make extracts, and demonstrated the
hepatoprotective activities of non-extracted fruiting bod-
ies of A. brasiliensis using outdoor cultivation (Figures 3).
In addition, because indoor-cultivated mushrooms failed
to protect against liver damage (Figure 4), these mush-
rooms may require extraction or higher doses to exert
hepatoprotective activities, which may increase the pos-
sibility of adverse effects. The safety of the normal use of
Figure 5 Effective dosing period of KAOD on hepatoprotection. Serum and livers were harvested from ICR mice fed KAOD-10% ad libitum
from day 9 until the end of the experiment, from day 9 until CCl
4
injection or from CCl
4
injection until the end of the experiment. (A) The
activities of ALT, AST, and LDH in the serum of KAOD-10%-administered mice (a) for 2 days, (b) for 24 h from day 9 until CCl
4
injection or (c) for
24 h from CCl
4
injection until the end of the experiment were measured using commercially available kits. Data are presented as mean ± standard
deviation, n =6. Significant difference from AIN-93G: *p <0.05; **p <0.01; n.s.: not significant. (B) HE stained liver sections from mice administered
(a-1) AIN-93G with CCl
4
(control for 2 days), (a-2) KAOD-10% (2 days) with CCl
4
, (b-1) AIN-93G with CCl
4
(control for pre-ingestion group), (b-2)
KAOD-10% (pre-ingestion group) with CCl
4
, (c-1) AIN-93G with CCl
4
(control for post-ingestion group), (c-2) KAOD-10% (post-ingestion group)
with CCl
4
. Scale bar =100 μm.
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non-extracted KAOD was demonstrated in our previous
clinical study [6]. Therefore, outdoor cultivation could
be better a method for reducing the risk of side effects.
In addition, because hepatoprotective activities of KAOD
were observed even when it was administered 24 h
before CCl
4
injection (Figure 5) and was not affected by
autoclaving (Figure 6), the key molecules for hepatopro-
tection could be rapid-acting and heat-tolerant. Thus,
these characteristics could be exploited to help use this
mushroom for inhibiting hepatic damage.
Although indoor systematic cultivation has the advan-
tage of being able to provide a stable supply, we believe
that outdoor growth, and particularly sunshi ne, is one
of the most important factors for the b iological activity
of this mushroom. Sunlight increased the levels of in-
gredientssuchasvitaminDandβ-glucan, and enhanced
the activity of several enzymes [15]. This study showed
that although outdoor-cultivated A. brasiliensis contained
more abundant β-glucan than that grown in the shade,
the immunomodulatory effects of these mushrooms may
be similar because the primary structure of β-glucan was
unchanged. Ne vertheless, many report s have suggested
beneficial effects of vitamin D [22,23]. In addition, other
biological properties including enzymes and mole cules
produced by the enzymes could be affected by the cultiva-
tion conditions. Consistent with this, our study demon-
strated that KAOD wields increased antioxidative activity
compared with other mushrooms (Figure 2). Therefore,
it is possible that outdoor cultivation could augment the
antioxidative properties of A. brasiliensis,becausethe
mushrooms protect themselves from sunlight-induced
oxidative damage.
Many reports have described hepatoprotective proper-
ties of A. brasiliensis [24,25], but the underlying mechan-
ism(s) of these effects are yet to be elucidated. Because
both JAID-10% and KAOD-1% failed to exert hepatopro-
tection, we considered if KAOD contained factors that
would explain the 10-fold great er hepatopro tective
properties than JAID. However, the basic ingredients
of KAOD, K AID, and JAID were similar, except for
vitamin D (Table 2), and we failed to demonstrate any
contribution of vitamin D2 to hepatoprotection (data
not shown). To clearly define the pharmacologic
actions of A. brasiliensis , the molecular mechanism(s)
behind the hepatoprote ctive effects of this mushroom
must also be investigated.
Our current research focuses on the antioxidative com-
ponents of A. brasiliensis, because the oral administration
Figure 6 Effect of heat treatment of KAOD on hepatoprotection. Serum and livers were isolated from ICR mice fed heat-treated AIN-93G or
heat-treated KAOD ad libitum from day 7. (A) The activities of ALT, AST, and LDH in serum were assessed using commercially available kits. Data
are presented as mean ± standard deviation, n =6. Significant difference between heat-treated AIN-93G and heat-treated KAOD: *p <0.05; **p <0.01.
(B) HE stained liver sections from mice administered (a) heat-treated AIN-93G with CCl
4
, and (b) heat-treated KAOD with CCl
4
were observed under a
normal light microscope. Scale bar =100 μm.
Yamanaka et al. BMC Complementary and Alternative Medicine 2014, 14:454 Page 8 of 10
http://www.biomedcentral.com/1472-6882/14/454
of several antioxidants protects against liver injury in ani-
mal models [26,27]. Therefore, we will attempt to identify
the key molecules that exert these hepatoprote ctive
activities using both indoor- a nd outdoor-cultivated A.
brasiliensis in future studies. The antioxidant activity
of this mushroom is modulated not only by cultivation
method, but also by different maturation pha ses [17].
Therefore, we propose t hat the pharmaceutical activity
of A. brasiliensis could be optimized by the de velop-
ment of a basic manufacturing method.
Conclusions
In the present study, we demonstrated that open-air cul-
tivation strongly upregulated the total antioxidative ac-
tivity of A. brasiliensis. In addition, outdoor-cultivated A.
brasiliensis wa s more effective than mushrooms grown
in the shade at protecting against CCl
4
-induced acute
hepatic injury in mice. Therefore, we concluded that
open-air cultivation could augment the antioxidative and
hepatoprotective properties of the fruiting bodies of A.
brasiliensis.
Abbreviations
ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; DPPH: 1,1-
diphenyl-2-picrylhydrazyl; HE: Hematoxylin and eosin; LDH: Lactate
dehydrogenase; TLR: Toll-like receptor.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors conceived of the study, and supervised the data collection. DY
participated in the design of the study. DY led the statistical analysis. All
authors were involved in and contributed to the data analysis. All authors
participated in drafting and revising the manuscript, and all authors
approved the final manuscript.
Acknowledgments
We wish to thank Ms. Yuina Ishimoto for her excellent technical assistance
and Dr. Akiko Matsumoto-Akanuma for her helpful suggestions during
manuscript preparation. This work was supported in part by the Programme
for Promotion of Basic and Applied Researches for Innovations in Bio-oriented
Industry (BRAIN) [H20-08-02 to NO].
Author details
1
Laboratory for Immunopharmacology of Microbial Products, School of
Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1
Horinouchi, Hachioji, Tokyo 192-0392, Japan.
2
Toei Shinyaku Co., Ltd, 1-11-23
Shimorenjyaku, Mitaka, Tokyo 181-0013, Japan.
Received: 24 October 2013 Accepted: 12 November 2014
Published: 24 November 2014
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doi:10.1186/1472-6882-14-454
Cite this article as: Yamanaka et al.: Differences in antioxidant
activities of outdoor- and indoor-cultivated Agaricus bras iliensis,and
protective effects against c arbon tetra chloride-indu ced acute hepa tic
injury in mice. BMC Complementary and Altern ative Medici ne 2014 14:454.
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