Continuous intake of the
Chaga mushroom (Inonotus
obliquus) aqueous extract
suppresses cancer progression
and maintains body
temperature in mice
*, Jun Watanabe
, Masako Maeda
, Masato Yamamoto
, Mamiko Mochizuki
, Nobuyuki Kagami
, Kazuho Honda
Center for Biotechnology, Showa University, Tokyo, Japan
Center for Laboratory Animal Science, Showa University, Tokyo, Japan
College of Art and Science at Fujiyoshida, Showa University, Tokyo, Japan
Department of Anatomy, School of Medicine, Showa University, Tokyo, Japan
* Corresponding author at: Center for Biotechnology, Showa University, 1-5-8 Hatanodai, Shinagawa-ku,
Tokyo 142-8555, Japan.
E-mail address: firstname.lastname@example.org (S. Arata).
Aims: Cancer is a leading cause of morbidity and mortality worldwide; therefore,
effective measures for cancer prevention and treatment are in constant demand.
The extracts of Inonotus obliquus (Chaga mushroom) demonstrate potent anti-
tumor activities and have been used to treat cancer in several countries; however,
the actual effect and underlying mechanisms are still unclear. In the present
study, we aimed to investigate the effects of continuous intake of aqueous extract
from I. obliquus on tumor suppression.
Main methods: Anticancer activity of the I. obliquus extract was examined in
mouse models of Lewis lung carcinoma growth and spontaneous metastasis after
15 March 2016
20 April 2016
6 May 2016
Heliyon 2 (2016) e00111
2405-8440/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
3 weeks of continuous extract intake at the dose of 6 mg/kg/day, which
corresponded to that ingested daily with Chaga infusion in Japan.
Key findings: The extract of I. obliquus caused significant tumor suppressive
effects in both models. Thus, in tumor-bearing mice, 60% tumor reduction was
observed, while in metastatic mice, the number of nodules decreased by 25%
compared to the control group. Moreover, I. obliquus extract-treated mice
demonstrated the increase in tumor agglomeration and inhibition of vasculariza-
tion. Interestingly, I. obliquus intake decreased body weight in middle-aged mice
and increased body temperature in response to light-dark switching in mature adult
mice. Furthermore, I. obliquus prevented temperature drop in mice after tumor
Significance: Our findings suggest that the I. obliquus extract could be used as a
natural remedy for cancer suppression by promoting energy metabolism.
Keywords: Biochemistry, Biological sciences
Cancer is one of the leading causes of morbidity and mortality worldwide, with
approximately 14 million new cases and 8.2 million cancer-related deaths
documented in 2012 . While discovering novel therapeutics and providing
adequate care to cancer patients is very important for decreasing cancer burden,
prevention is probably a more critical aspect which was somewhat neglected .
Cancer prevention through socioeconomic interventions, environmental changes,
and lifestyle modifications could be a solution for reducing constantly increasing
cancer incidence. Recent epidemiological data demonstrate that obesity increases
the risk of cancer development . Cao et al. [4,5] have reported that mice
housing with increased space, physical activity, and social interactions (enriched
environment, EE) which stimulated physical and mental activities, demonstrated
the suppression of both tumor growth and obesity via inhibition of leptin secretion
from white adipose tissue. EE is also known to decrease adiposity, stimulate
energy expenditure, and induce brown-like (beige) cells in white fat .
Furthermore, it has been reported that cancer patients are characterized with
hypothermia and hyperglycemia  and that physiologic responses to high body
temperature improve the tumor microenvironment . These findings indicate that
the induction of lipid metabolism and maintenance of proper body temperature
could be important aspects in cancer prevention.
Medicinal mushrooms have an established history of use in traditional oriental
therapy and nutritionally functional foods. Inonotus obliquus (Chaga mushroom)
belonging to the family Hymenochaetaceae of Basidiomycetes, preferably grows
on the trunks of mature live birch trees . The extracts of I. obliquus have been
used in China, Korea, Japan, Russia, and the Baltics for their favorable effects on
2405-8440/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
lipid metabolism and cardiac function, as well as for anti-bacterial, anti-
inflammatory, anti-oxidant, and anti-tumor activities .I. obliquus extracts were
found to inhibit hepatitis C virus  and human immunodeficiency virus [11,
12] and demonstrated strong anti-oxidant and immunostimulatory activities in
vitro [13,14]. At the same time, animal studies revealed that aqueous extracts of
I. obliquus exhibited anti-inflammatory effects in experimental colitis [15,16]
and promoted lipid metabolism . Several studies investigated the anti-tumor
activity of the I. obliquus aqueous extract and found that it suppressed the
proliferation  and induced apoptosis  of various carcinoma cell lines.
Furthermore, the compounds isolated from I. obliquus extracts were shown to
inhibit skin carcinogenesis  and tumor growth in Sarcoma-180 cell-bearing
mice . However, despite increasing evidence of anticancer activity exhibited
by the I. obliquus extract and its individual components [9,22], the underlying
mechanisms are still unclear and the effects of I. obliquus on cancer prevention
are not understood.
In this study, we examined anti-cancer effects of the continuous intake of the
I. obliquus extract using mouse models of tumorigenesis and spontaneous
metastasis. The dose of I. obliquus extract (6 mg/kg/day) was calculated based
on the daily intake of the extract as tea infusion in Japan. We also tested the real
time body temperature using an implanted nano-thermometer. This is the first
study showing that continuous intake of the I. obliquus aqueous extract suppresses
cancer progression and maintains body temperature.
2. Material and methods
C57BL/6 mice obtained from Japan SLC Inc. (Shizuoka, Japan) at the age of 8
weeks were kept in the room with controlled temperature (23 ± 2 °C) and a 12-h
light/dark cycle (lights on at 8 am). All experimental procedures involving animals
were approved by the Institutional Animal Care and Use Committee of Showa
University (Permit Number: 55019), which operates in accordance with the
Japanese Government for the care and use of laboratory animals.
2.2. Preparation of aqueous extract from I. obliquus
I. obliquus sclerotia were collected from birch trees in Fujiyoshida city,
Yamanashi prefecture, Japan, and identified by Drs. Mashasi Osawa and Hisasi
Shibata of Yamanashi Forest Research Institute, Japan. The material was
powdered and 32.0 g was suspended in 1 L of water, boiled for 90 min to be
concentrated to 200 ∼300 ml. The extract was filtered through filter paper (No.
101, Advantec, Tokyo, Japan) and freeze-dried, yielding 1.3 g (4.1% of raw
2405-8440/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
material). It was re-dissolved in sterilized distilled water to the concentration of
2.4 mg/ml and stored at −80 °C as stock solution.
2.3. Cancer cell culture
Lewis lung carcinoma cell line (3LL) was obtained from National Institutes of
Biomedical Innovation (Osaka, Japan) and maintained in RPMI 1640 medium
supplemented with glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/
mL), and 10% (v/v) heat-inactivated FBS (Thermo Fisher, Waltham, MA, USA).
2.4. I. obliquus anticancer activity in mouse models of carcinoma
and spontaneous metastasis
Mice were given water with or without 24 μg/mL extracts from I. obliquus (1% of
stock solution) for 3 weeks before tumor inoculation and throughout the
experimental period; approximately 6 mg/kg of I. obliquus extract was ingested
in 5 ml of drinking water per day. Cancer models were established as described
previously . Briefly, 5 × 10
live 3LL cells were suspended in 0.2 ml of serum-
free MEM and injected subcutaneously in the right flanks of mice to develop solid
intra-abdominal tumors (tumor-bearing model) or into the tail vein to produce
colonies of metastatic cells in the lung (spontaneous metastasis model). In the
tumor-bearing model, tumor size was measured with calipers every day and tumor
volume was calculated as (width)
× length × 0.52. Mice were sacrificed at day 16
after tumor cell inoculation, and solid tumors were collected and weighted. For
histological examination, some mice were anesthetized at day 7 of cancer cell
implantation by intraperitoneal injection of sodium pentobarbital (50 mg/kg), and
perfused transcardially with saline, followed by 4% paraformaldehyde in 50 mM
phosphate buffer (pH 7.2). Pulmonary nodules in the metastasis model were
counted using 3D lung images acquired by micro-CT and visually confirmed in the
lungs fixed with neutral formalin and observed under a stereomicroscope. For
micro-CT scanning, animals were anesthetized with isoflurane at day 9 after
cancer cell inoculation and analyzed using an R_mCT2 micro-CT scanner
(Rigaku Corporation, Tokyo, Japan) under the following conditions: FOV24, ϕ24
mm × H19 mm; tube voltage, 90 kV; tube current, 160 μA. Some mice were
sacrificed 2 weeks after cancer cell injection, and the extracted lung tissues were
fixed with neutral formalin and used for histology.
Tissues were immersed in 20% sucrose in 0.1 M phosphate buffer (pH 7.2) at 4 °C
and embedded in O.C.T. compound (Sakura Finetek, Tokyo, Japan). Frozen
sections were cut with a microtome at the thickness of 8 μm, washed in PBS
(pH 7.2), and stained with Gill's hematoxylin and eosin (HE). For immunostaining,
tissue sections were treated with 0.3% H
in PBS to quench endogenous
peroxidase activity, blocked in 5% normal horse serum in PBS for 60 min, and
incubated overnight with rat anti-CD31 polyclonal antibodies (1:500; 550274, BD
Biosciences, East Rutherford, NJ, USA) at 4 °C. Sections were washed with PBS
and incubated with biotinylated goat anti-rat IgG (1:200; BA-4001, Vector,
Burlingame, CA, USA) for 2 h at room temperature, followed by the reaction with
avidin-biotin complex solution (Vector) and chromogen diaminobenzidine
(Vector). Images were captured using an AX70 microscope (Olympus, Tokyo,
2.6. Body temperature measurement
Mouse body temperature was monitored using the DST nano-T temperature
logger (17 mm × 6 mm, 1 g; STAR ODDI, Gardabaer, Iceland). The loggers were
implanted in the abdomen and body temperature was continuously measured at
2.7. Statistical analysis
Unpaired T-test was performed to assess the significance of independent
experiments after statistical outliers were removed using the Smirnoff-Grubbs
rejection test. Three groups (Fig. 5B and C) were compared using one-way
ANOVA with a Tukey multiple comparison test. The Ekuseru-Toukei software
(Social Survey Research Information Co. Ltd., Tokyo, Japan) was used for
statistical analysis. P values <0.05 were considered statistically significant.
3.1. I. obliquus extract promoted a decrease of body weight in
First, we examined the effect of continuous intake of the I. obliquus aqueous
extract on body weight of mature adult mice (12–15 weeks) and middle-aged mice
(30 weeks). The results indicated that middle-aged mice lost about 8% of weight
after 17 days of drinking I. obliquus extracts compared to the control group
(Fig. 1A); there was no difference in water intake between the two groups
(Fig. 1B). Interestingly, the I. obliquus extract caused no changes in body weight or
the amount of consumed water in mature adult mice (Fig. 1C and D). These data
suggest that the intake of the I. obliquus extract promoted lipolysis in developed
fatty tissue; therefore, in further experiments we used mature adult mice (12–15
weeks) to avoid the influence of body weight difference between control and
treated mice observed prior to tumor implantation on subsequent tumor growth.
3.2. Continuous intake of the I. obliquus extract slowed tumor
progression in mice with implanted Lewis lung carcinoma cells
To explore whether I. obliquus had the ability to suppress tumor growth, mice
received water supplemented with I. obliquus extract for 3 weeks prior to and 16
days after the implantation of 3LL cells for the development of solid intra-
abdominal tumors. The images of carcinomas from the treated and control mice
showed that the I. obliquus extract suppressed tumor growth (Fig. 2A).
Furthermore, quantitative analysis revealed significant retardation of tumor
development in the I. obliquus group starting from day 14 after cancer cell
implantation (Fig. 2B); at day 16, the average tumor size in the treatment group
was 60.3% less than in the control group (Fig. 2C). These results indicate that
continuous uptake of the I. obliquus extract produced a strong anti-tumor effect.
Fig. 1. The intake of the I. obliquus extract promoted body weight loss in middle-aged mice. Middle-
aged and mature adult mice received water without (control) or with the I. obliquus extract for the
indicated times and were analyzed for body weight. (A) Body weight of middle-aged mice (weight at
day 0: 37.3 ± 3.5 g and 37.9 ± 5.1 g for the water and I. obliquus group, respectively; n = 4–5). (B)
Amount of water drunk by middle-aged mice. (C) Body weight of mature adult mice (weight at day 0:
22.7 ± 1.8 g and 23.3 ± 2.1 g for the water and I. obliquus group, respectively; n = 8–9). (D) Amount of
water drunk by mature adult mice. The data were normalized to the body weight at day 0 and expressed
as the mean ± SD; *P <0.05.
3.3. I. obliquus extract decreased tumor vascularization
Next, we analyzed the effect of the I. obliquus extract by histochemistry. HE
staining revealed agglomerated tumor morphology and decreased tumor size in
the I. obliquus group (Fig. 3A). Furthermore, immunostaining for CD31, a
marker of vascular endothelial cells, revealed that the presence of CD31-positive
cells in I. obliquus-treated mice tended to decrease compared to the control
group, indicating that tumor vascularization could be suppressed by the intake of
the I. obliquus extract (Fig. 3B). These results suggest that the continuous intake
of the I. obliquus extract could decrease tumor vascularization and, consequently,
suppress cancer progression.
Fig. 2. The intake of the I. obliquus extract suppressed tumor development in mice implanted Lewis
lung carcinoma cells. Mice drinking water with or without the I. obliquus aqueous extract were injected
3LL cells subcutaneously in the right flanks and analyzed for tumor size. (A) Representative images of
carcinomas. (B) Quantification of tumor size at the indicated times after 3LL cell implantation. (C) The
weight of solid tumors at day 16 after 3LL cell implantation. Each dot represents a single mouse and
lines show mean values (n = 8 per group; *P <0.05).
3.4. Continuous intake of the I. obliquus extract suppressed
To further explore anti-tumor effects of the I. obliquus extract, it was tested in a
model of spontaneous metastasis to lung tissue induced by intravenous injection
Fig. 3. I. obliquus extract caused tumor agglomeration and suppressed vascularization. 3LL cells were
injected subcutaneously at the left abdomen of mice drinking water with or without the I. obliquus
aqueous and tumors were analyzed by histology 7 days after cells implantation. (A) HE staining. (B)
Immunostaining for CD31.
of 3LL cells. First, we analyzed lung metastasis under a stereomicroscope 14
days after tumor implantation. As shown in Fig. 4A and B, lung nodules in
I. obliquus-treated mice tended to decrease compared to the control group. Then,
the lungs from animals anesthetized at day 9 after cancer cell inoculation were
analyzed for the number of pulmonary nodules using micro-CT. The results
indicate that the intake of the I. obliquus extract significantly decreased the
number of tumor nodules in the lungs (Fig. 4C and D). Histology analysis (HE
staining) of tumor nodules demonstrated that I. obliquus intake decreased the size
Fig. 4. I. obliquus extract suppressed metastasis in mice injected with Lewis lung carcinoma cells. Mice
drinking water with or without the I. obliquus aqueous extract received intravenous injection of 3LL
cells. (A) Representative stereomicroscopic images of fixed mouse lungs containing carcinoma nodules.
Arrowheads mark 3LL nodules on the lung. (B) The number of metastatic nodules counted under a
stereomicroscope. Each dot represents a single mouse and lines show mean values (n = 4–6 per group).
(C) Representative CT images of mouse lungs containing carcinoma nodules. Arrowheads mark 3LL
nodules visible as dents on the lung. (D) The number of nodules measured in CT images of the lungs
extracted at day 9 after cancer cell injection. Each dot represents a single mouse and lines show mean
values (n = 4–5 per group; *P <0.05).
and induced agglomerated morphology of tumor nodules (Fig. 5). These data
suggest that continuous intake of the I. obliquus extract could suppress tumor
3.5. Continuous intake of the I. obliquus extract prevented body
temperature decrease after tumor implantation
We hypothesized that the maintenance of the body temperature could be a key
factor in suppressing tumor development by the I. obliquus extract. To test this
hypothesis, we subcutaneously implanted nano-temperature loggers in the
abdomen and measured the body temperature in a real-time format. Interestingly,
tumor-free mice continuously drinking the I. obliquus extract showed higher body
temperatures at the switch from darkness to light (Fig. 6A). As there was no
difference in water intake between the I. obliquus and control groups (Fig. 1D),
these data suggest that I. obliquus could upregulate energy metabolism. While
body temperature gradually decreased after tumor implantation in the control
group at the light-to-dark switching (Fig. 6B), it was not the case in the group
taking the I. obliquus extract. These data suggest that I. obliquus may suppress
tumor growth by regulating energy metabolism.
Fig. 5. The intake of the I. obliquus extract agglomerated tumor cells in the mouse model of
spontaneous metastasis. Mice drinking water with or without the I. obliquus aqueous extract received
intravenous injection of 3LL cells. Representative HE-stained images of lung sections 2 weeks after
cancer cell injection are shown.
The results of this study indicate that daily intake of the I. obliquus extract has anti-
cancer effects. We also showed, for the first time, that I. obliquus maintains the
body temperature in a mouse model of tumorigenesis. Although previous studies
Fig. 6. Continuous intake of the I. obliquus extract prevented body temperature decrease in mice
implanted Lewis lung carcinoma cells. (A) Average body temperature of mice measured from week 2 to
3 after the intake of drinking water without or with the aqueous extract of I. obliquus. (B, C) Average
body temperature of mice receiving water (B) or I. obliquus extract (C) measured 1 week before (0
week), and 1 week and 2 weeks after cancer cell injection. The data are expressed as the mean (n = 3
per group); *P <0.05 versus water (A) or 0 week (B, C).
have reported anti-tumor effects of I. obliquus [9,22], most of them analyzed
cancer treatment and not cancer prevention, as I. obliquus extracts were
administered immediately before or after tumor transplantation [19,24,25]. We
hypothesized that long-term continuous intake of the I. obliquus extract could
suppress tumorigenesis by supporting normal metabolic reactions in the
organism, including thermogenesis. Continuous intake of the I. obliquus extract
at the dose similar to that received by Japanese people through daily Chaga tea
drinking (6 mg/kg/day) significantly reduced body weight in middle-aged mice
(Fig. 1A), but not in mature adult mice (Fig. 1C), suggesting that I. obliquus
intake promotes lipolysis in age-accumulated adipose tissue. A previous study
showed that water-soluble components extracted from I. obliquus improved
insulin sensitivity and reduced adiposity in obese mice fed a high-fat diet ,
suggesting beneficial anti-hyperglycemic effects and lipid metabolism enhance-
ment. Consistent with our result, that study detected no difference in body weight
between control and I. obliquus extract-treated mice fed a normal diet. In view of
the link revealed between obesity and multiple types of cancer , continuous
intake of the I. obliquus aqueous extract could have a potential for tumor
suppression by promoting lipid metabolism.
As the next step, we examined the anticancer activity in mice taking the I. obliquus
extracts daily for 3 weeks prior to tumor implantation. As shown in Fig. 2 and
Fig. 3, a significant suppression of cancer development was observed both in the
tumor growth model and spontaneous metastasis model after the ingestion of the
I. obliquus extract at the dose corresponding to human daily uptake. Though
the mechanisms underlying anti-tumor effects of I. obliquus are still unclear,
previous studies suggest that apoptosis-inducing activity, changes in tumor
microenvironemt such as suppression of angiogenesis, and the improvement of
chronic inflammation in adipose tissue could be involved in I. obliquus-mediated
cancer suppression. Thus, oral administration of the I. obliquus aqueous extract
ameliorated acute inflammation in dextran sodium sulfate-induced colitis in mice
. Here, we observed a tendency for tumor cell agglomeration (Fig. 3A and
Fig. 5) and reduced vascularization (Fig. 3B) in mice drinking the I. obliquus
extract, which can be explained by anti-inflammatory effects exerted by I. obliquus
. Metabolic regulation may also be an important aspect of the anti-cancer effects of
I. obliquus. Obesity and hypernutrition may affect different tissues simultaneously
resulting in a systemic increase in non-esterified fatty acids, insulin, glucose,
leptin, and inflammatory cytokines, and decrease in adiponectin , which can
directly promote cancer cell survival, proliferation, and malignant progression. We
measured serum levels of glucose, leptin, and adiponectin in mice drinking the
I. obliquus extract; however, no difference was found (data not shown). Further
studies are needed to understand the molecular mechanism underlying I. obliquus
As recent findings revealed a correlation between tumor development and body
temperature [8,27], we measured the body temperature in mice receiving the
I. obliquus extract continuously before and after tumor implantation. The results
revealed a significantly lower body temperature in the control group at the light-to-
dark switching (Fig. 6B), while the body temperature in the I. obliquus group
remained constant. Although there is no direct evidence of causal relationship
between the maintenance of body temperature and tumor suppression, mice
drinking the I. obliquus extract showed higher body temperature before tumor
implantation (Fig. 6A), indicating that the effects occurred prior to tumor
suppression and may be involved in the anti-tumor activity. It has been suggested
that brown adipose tissue (BAT) plays a key role in energy homeostasis and
thermogenesis not only in infants but also in adults . BAT activity is known to
positively correlate with energy expenditure during cold exposure and negatively
with age, body mass index, and fasting glycemia, suggesting the association
between BAT, cold-induced thermogenesis, and energy metabolism . The
increase in the body temperature by I. obliquus may be mediated by BAT activity
and stimulation of lipid metabolism. As hypothermia is known to activate
adipocytes to stimulate tumor growth , the effect of I. obliquus on maintaining
the body temperature could play a critical role in tumor suppression. Future studies
addressing metabolic and inflammatory mechanisms underlying the responses of
the tumor microenvironment to I. obliquus are required to substantiate our
We showed that the continuous intake of the I. obliquus extract can potentially
suppress cancer development through the maintenance of the body temperature. In
addition, middle-aged mice drinking the I. obliquus extract exhibited body weight
loss. Our findings suggest that the aqueous extract of I. obliquus could be used as a
natural product for cancer suppression and general health care.
Author contribution statement
Satoru Arata: Conceived and designed the experiments; Performed the experi-
ments; Analyzed and interpreted the data; Wrote the paper.
Jun Watanabe: Performed the experiments; Wrote the paper.
Masako Maeda, Masato Yamamoto: Contributed reagents, materials, analysis tools
Hideto Matsuhashi, Mamiko Mochizuki, Nobuyuki Kagami: Performed the
Kazuho Honda: Conceived and designed the experiments.
Masahiro Inagaki: Conceived and designed the experiments; Contributed reagents,
materials, analysis tools or data.
This research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors.
Competing interest statement
The authors declare no conflict of interest.
No additional information is available for this paper.
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