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Integrative Cancer Therapies
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Research Article
Introduction
Inonotus obliquus is a parasitic Polyporus from the
Hymenochetaceae family. This fungus infects hardwood
trees, mostly those from the genus Betula (birches), and to a
lesser extent, those from the genera Quercus (oaks), Populus
(poplars), Alnus (alders), Fagus (ashes), and Acer (maples).1
It was first identified and described by Persoon2 (1801),
who named it Boletus obliquus. Then, it was renamed
757912ICTXXX10.1177/1534735418757912Integrative Cancer TherapiesGéry et al
research-article20182018
1Normandie University, UNICAEN, Centre F. Baclesse, Caen, France
2Labéo Frank Duncombe, Saint-Contest, France
3Normandie University, UNIROUEN, France
4National University of Architecture and Construction, Kyiv, Ukraine
Corresponding Author:
David Garon, UR ABTE EA4651, Cancer Treatment Center F. Baclesse,
Avenue Général Harris, BP 5026, 14076 Caen Cedex 05, France.
Email: david.garon@unicaen.fr
Chaga (Inonotus obliquus), a Future Potential
Medicinal Fungus in Oncology? A Chemical
Study and a Comparison of the Cytotoxicity
Against Human Lung Adenocarcinoma Cells
(A549) and Human Bronchial Epithelial Cells
(BEAS-2B)
Antoine Géry, PharmD1, Christelle Dubreule, MSc2,
Véronique André, PharmD, PhD1, Jean-Philippe Rioult, PharmD, PhD1,
Valérie Bouchart, PhD2, Natacha Heutte, PhD3,
Philippe Eldin de Pécoulas, PharmD, PhD1,
Tetyana Krivomaz, PhD4, and David Garon, PharmD, PhD1
Abstract
Background: Inonotus obliquus, also known as Chaga, is a parasitic fungus growing on birches and used in traditional
medicine (especially by Khanty people) to treat various health problems. In this study, we aimed to quantify the 3
metabolites frequently cited in literature, that is, betulin, betulinic acid, and inotodiol in the Chaga recently discovered
in forests located in Normandy (France), and to compare their concentrations with Ukrainian and Canadian Chaga. This
study also explores the cytotoxicity of the French Chaga against cancer-derived cells and transformed cells. Methods: A
quantification method by HPLC-MS-MS (high-performance liquid chromatography–tandem mass spectrometry) of betulin,
betulinic acid, and inotodiol was developed to study the French Chaga and compare the concentration of these metabolites
with extracts provided from Chaga growing in Canada and Ukraine. This method was also used to identify and quantify
those 3 compounds in other traditional preparations of Chaga (aqueous extract, infusion, and decoction). Among these
preparations, the aqueous extract that contains betulin, betulinic acid, and inotodiol was chosen to evaluate and compare
its cytotoxic activity toward human lung adenocarcinoma cells (A549 line) and human bronchial epithelial cells (BEAS-
2B line). Results: French Chaga contains betulin and betulinic acid at higher levels than in other Chaga, whereas the
concentration of inotodiol is greater in the Canadian Chaga. Moreover, the results highlighted a cytotoxic activity of the
Chaga’s aqueous extract after 48 and 72 hours of exposure with a higher effect on cancer-derived cells A549 than on
normal transformed cells BEAS-2B (P = 0.025 after 48 hours of exposure and P = 0.004 after 72 hours of exposure).
Keywords
Inonotus obliquus, cytotoxicity, cancer, chromatography, betulin, betulinic acid, inotodiol, quantification, traditional medicine
Submitted August 23, 2017; revised November 8, 2017; accepted December 18, 2017
2 Integrative Cancer Therapies
Polyporus obliquus by Fries3 (1830), followed by Quélet4
(1888), who called it Poria obliqua (under the bark of dry
Fagus). In 1927, Bourdot and Galzin5 called it Xanthochrous
obliquus, and its current name, Inonotus obliquus, was
given by Pilàt6,7 (1936, 1942), who studied it thoroughly.
Chaga has oblique pores—the origin of its species name
obliquus.
Currently, this fungal species has only been described in
the northern hemisphere. We can particularly find it in
Canada, in the north of the United States of America, in
Kazakhstan, in Siberia, in Ukraine, in Japan, in South
Korea, in China, as well as in Europe (mostly in the north-
ern and eastern parts of the continent). Its description in
France is more recent, the first one dating from 1953 in
Seine-et-Marne under the name of Xanthochrous obliquus8;
it has also been found and described even more recently in
Normandy. Chaga has been used since the 12th century in
Eastern Europe. From historical chronicles, it is known that
Kiev Prince (Knyaz) Vladimir Monomakh had a lip tumor
and got rid of the disease thanks to treatment with Chaga
mushroom.9 The Khanty people, an ethnic group from
Siberia formerly called the Ostyaks,10 also used it in tradi-
tional medicine for different therapeutic indications: as an
anthelminthic, as an antitubercular, to cure digestive disor-
ders (gastritis, ulcers, etc), or even to prevent cardiac or
hepatic illnesses. They used the crushed asexual form of the
Chaga in several ways: by infusion, inhalation, or macera-
tion in water of the charcoal obtained after burning to make
body soap, which was used as an antiseptic.11
In the middle of the 20th century, it was still used in
Siberia for its properties by Russian farmers and workers too
poor to buy tea: they crushed it and drank it as an infusion.
This use of the Chaga in Siberian gulags is mentioned in
Alexandre Soljénitsyne’s book Le Pavillon des cancéreux
(Cancer Ward).12 Soviet health authorities noticed a
decrease of the incidence of cancer cases in this population
and assumed that the consumption of this infusion was a
protective factor against cancer. In 1955, the USSR Ministry
of Health recognized the therapeutic interest of I obliquus
used as a decoction and wrote it down in the Soviet
Pharmacopeia under the name of Befunginum.
The extracts of I obliquus have been used in China,
Korea, Japan, Russia, and the Baltics for their favorable
effects on lipid metabolism and cardiac function, as well as
for antibacterial, anti-inflammatory, antioxidant, and antitu-
mor activities.13
Inonotus obliquus extracts were found to inhibit hepati-
tis C virus14 and human immunodeficiency virus15 and dem-
onstrated strong antioxidant and immunostimulatory
activities in vitro.16,17 At the same time, animal studies
revealed that aqueous extracts of I. obliquus exhibited anti-
inflammatory effects in experimental colitis18-21 and pro-
moted lipid metabolism.22 The mushroom has the ability to
increase peroxisome proliferator-activated receptors γ
transcriptional activities, which are expected to be thera-
peutic targets for dyslipidemia and type 2 diabetes.23
Its biological activities explain why it is used as an adju-
vant in oncology, especially in anticancer chemotherapies
in Asian pharmacopoeias.
The chemical analysis of Chaga in scientific literature
revealed several compounds such as polysaccharides, triter-
penes, and polyphenols, which might be responsible for
most of the therapeutic effects previously mentioned.24 A
tetracyclic triterpene called inotodiol produced following
the lanosterols biosynthetic pathway elicited the interest of
the international scientific community.25-29 Inotodiol has
antiproliferative properties, demonstrated in vitro with
human lung adenocarcinoma cells (A549) cancer-derived
cells or HeLa.30,31
In addition, 2 other components derived from birch are
frequently described in Chaga: betulin (or betulinol) and
betulinic acid.32 Several species of birch are, indeed, used in
traditional medicine with a very wide geographical distribu-
tion. The spectrum of pharmacological properties associ-
ated with their uses is important: antimicrobial, antidiabetic,
hepatoprotective, antiarthritic, and anticancer activities.32
These last 2 activities were the most studied, especially
from betulin and betulinic acid. In traditional medicine, the
use of birch against rheumatism is reported, for example, in
Bosnia-Herzegovina and Lebanon.33,34 From an experimen-
tal point of view, the study by Gründemann et al35 confirms
the anti-inflammatory effect of the extract of Betula pen-
dula by its action on the lymphocytes. Different species
belonging to the genus Betula have also been tested to eval-
uate their anticancer potential. The compounds betulin and
betulinic acid were tested in vitro on different models of
cancer cells (cutaneous, ovarian, and pulmonary) demon-
strating their antiproliferative potential.36-38
Our work is the first experimental approach on Chaga
collected in France. This contribution aims, on the one
hand, to develop a quantification method of betulin, betu-
linic acid, and inotodiol in Chaga extracts, and on the other
hand, to evaluate and compare the biological activity of
these extracts toward A549 line and human bronchial epi-
thelial cells (BEAS-2B line).
Materials and Methods
Collection and Identification of Chaga (Inonotus
obliquus)
Chaga can be described as an irregular cracked, black-
brownish, hard, brittle fungus, looking like charcoal on the
outside, with a diameter ranging from 10 to 20 cm; inside it
is brown in color. The microscopic examination shows a
monomitic structure with brown hyphae and a thick parti-
tion with a diameter from 2.5 to 6-7 µm. These hyphae are
however separated but without loops.39,40
Géry et al 3
The fungal infestation results from a contamination of the
duramen of the host trees by basidiospores via unhealed inju-
ries, which have left this matrix uncovered. The asexual form
grows as long as the tree lives and causes a fibrous white rot
of the central cylinder of the tree, degrading the cellulose,
hemicellulose, and lignin. The sexual form (fruiting body)
appears between the bark and the sapwood as a yellowish
crust turning brown 2 to 12 years after the death of the host
tree.41,42 It shows oblique and elongated pores upholstered by
a hymenium with bi- to tetrasporic basidium without basal
loop. It is this sexual form that releases elliptic to globular,
smooth, yellowish basidiospores measuring from 8 to 10 µm
× 5 to 7.5 µm ensuring the dissemination of the fungus.43
Asexual forms of I. obliquus were respectively purchased
in Canada (Gaspésie Sauvage Produits Forestiers Inc), har-
vested in Ukraine (the Karpatsky National Park, Ivano-
Frankivsk region), and in France (the Forest of Grimbosq,
Normandy, France). These forms resembling blackish
growths (Figure 1), with a circumference from 15 to 28 cm
and measuring about 10 m, were harvested to a height from
0.8 to 2.5 m on trunks of birch (Betula pendula).
Preparation of the Extracts
Chaga was dried in a desiccator (Drying Device Dönex
SIGG AG 1978) for 5 days at 35°C. The dry fungal material
was crushed and then sprayed with Blender BB90E
(Waring) before sieving to 2 mm to prepare aqueous, cyclo-
hexane, and ethyl acetate extracts. The extractions were
performed in the darkness at room temperature (20°C).
Cyclohexane Extract. The powder (180 g) was first stirred in
contact with cyclohexane (1 L) for 90 minutes on an orbital
agitator at 180 rpm. After filtration on Whatman No. 3
paper the recovered solid residue was extracted again with
cyclohexane (500 mL) for 2 hours with the same stirring
system and then filtered on Whatman No. 3 filter paper. The
2 cyclohexanic extracts were pooled and evaporated with
Rotavapor at 40°C until the obtainment of a yellow residue
of 2.6294, 2.4744, and 2.5166 g, respectively, for the Cana-
dian, French, and Ukrainian Chaga.
Ethyl Acetate Extract. The dried solid residue was then con-
tacted with ethyl acetate (1 L) for 4 days on an orbital agita-
tor at 180 rpm. At the end of these 4 days, filtration was
carried out in 3 steps: on carded cotton, on Büchner, and on
filter paper Whatman No. 3.
Extracts were evaporated with Rotavapor at 40°C until
the obtainment of a yellow powdery residue of 0.9837,
0.9248, and 0.9315 g, respectively, for the Canadian,
French, and Ukrainian Chaga.
Aqueous Extract. The powder (100 g) was stirred on contact
with ultrapure water (500 mL) for 22 hours at room tem-
perature (20°C) on an orbital agitator at 180 rpm. Filtration
was then carried out on Whatman No. 3 paper in order to
obtain 250 mL of aqueous extract. A 100 mL aliquot of this
extract was concentrated with Rotavapor at 40°C until a
volume of 40 mL was obtained.
Decoction by Khanty Method.11 The asexual form was cut in
small pieces from 5 to 10 g then put in boiling water for 15
minutes. We used carded cotton and filter paper Whatman
No. 3 for filtration.
Infusion According to the Canadian Method (Gaspésie Sauvage
Produits Forestiers Inc). We put 3 chunks (from 5 to 10 g) in 1
L of cold water and let it rest for 30 minutes, then we heated
it without boiling it for 30 additional minutes. We used carded
cotton and filter paper Whatman No. 3 for filtration.
Before their use, all extracts obtained were stored in the
refrigerator at +4°C and protected from light.
Quantification by High-Performance Liquid
Chromatography Coupled to a Mass
Spectrometer (HPLC-ESI-QTOF MS/MS)
Reagents and Materials. Acetonitrile (ULC-MS grade) and
acetone (pesticides grade) were purchased from Biosolve
Chimie (Dieuze, France), while formic acid (98% purity)
Figure 1. Asexual form of Inonotus obliquus (Chaga) on birch
trunk.
4 Integrative Cancer Therapies
and ethyl acetate (HPLC HiPerSolv Chromanorm grade)
were purchased from VWR (Radnor, PA). Reverse osmosis
water (HPLC grade) prepared using a Millipore water puri-
fication system was used for all the preparations. The inter-
nal standard (Atrazine D5; 99% purity) was obtained from
Dr Ehrenstörfer. Betulin (97.5% purity) and betulinic acid
(97.5% purity) standards were purchased from Sigma
Aldrich (Saint-Louis, MO), while inotodiol standard (95%
purity) was purchased from Chemfaces (Hubei, China). All
the solutions were filtered through a 0.45 µm PVDF Milli-
pore Millex HV (Merck-Millipore, Billerica, MA).
Standard Solutions and Samples Preparation. Standard stock
solutions of atrazine D5, betulin, betulinic acid, and inoto-
diol at a concentration of 1000 mg/L were prepared in ethyl
acetate (2 mg of powder in 2 mL of ethyl acetate). A work-
ing solution containing inotodiol, betulin, betulinic acid
standards at a concentration of 10 mg/mL was prepared in
acetone (50 µL of each stock solution qs 5 mL of acetone).
We also prepared a working solution of our internal stan-
dard (atrazine D5) at a concentration of 500 µg/L in aceto-
nitrile (5 µL of stock solution in 10 mL of acetonitrile). The
range calibration made with these working solutions was
from 0.001 to 5 mg/L.
Ethyl acetate and cyclohexane dry extracts were taken
up in 10 mL (for the French Chaga) or 30 mL of acetonitrile
(for the Canadian and Ukrainian Chagas), passed through
ultrasound for 15 minutes, and then filtered through PVDF
0.45 µm filters. The cyclohexane extract was then diluted to
10% and the ethyl acetate extract to 20% (for the French
Chaga) or to 1% (for the Canadian and Ukrainian Chagas)
in a solution containing acetonitrile, distilled water, and the
internal standard (atrazine D5). The water extract, infusion,
and decoction were filtered through PVDF 0.45 µm filters
and then diluted to 20% (water extract and infusion) or 50%
(decoction) in a solution containing acetonitrile, distilled
water, and the internal standard (atrazine D5).
HPLC-ESI-QTOF MS/MS Analysis. A HPLC analysis was
applied on an Agilent 1290 Infinity LC instrument (Agilent,
Santa Clara, CA) consisting of a binary pump, a thermostat-
ted autosampler, and a column compartment. The samples
were separated on Waters Acquity UPLC BEH C18 15
µmm × 2.1 mm × 1.7 µm (Waters, Milford, MA). The
mobile phase was a stepwise gradient of water (containing
0.01% formic acid, v/v) and acetonitrile (containing 0.01%
formic acid, v/v; 0 minute, 97:3; 4 minutes, 70:30; 12min-
utes, 30:70; 15 minutes, 5:95; 17 minutes, 5:95; and 20 min-
utes, 97:3). The column temperature was set at 40°C and the
flow rate was 0.45 mL/min. The HPLC system was con-
nected to an Agilent 6470 MS-MS triple quadrupole (Santa
Clara, CA) equipped with an ESI interface using the follow-
ing operation parameters: capillary voltage, 3.5 kV ((+) ESI
mode); nebulizer, 40 psig; drying gas (nitrogen) flow rate,
10.0 L/min; sheath gas flow rate, 10.0 L/min; gas tempera-
ture, 350°C; sheath gas temperature, 350°C; and V charg-
ing, 500 V. The multiple reaction monitoring transitions
used for the 3 target analytes and internal standards are
shown in Table 1. The quantification data were processed
with Agilent Mass Hunter Quantitative Workstation Soft-
ware Version B.07.01 (Agilent Technologies).
Method Validation. The limits of detection, limits of quantifi-
cation, regression equation, and correlation coefficient of
calibration curves (r²) for each standard are reported in
Table 2.
Cytotoxicity Assay
The A549 cells (human alveolar epithelial cells derived
from an adenocarcinoma) having a doubling time of 24
hours were cultured in 96-well microplates (BD Falcon) in
a DMEM medium (Gibco) supplemented with 1% bicar-
bonate solution at 7.5% (Gibco) and 10% decomplemented
fetal calf serum.
The BEAS-2B cells (immortalized human bronchial epi-
thelial cells) having a doubling time of 26 hours were cul-
tured in 96-well microplates (BD Falcon) in 500 mL of
BEBM medium (Gibco) supplemented with 2 mL bovine
pituitary extract, 0.5 mL of hydrocortisone, 0.5 mL of
Table 1. Multiple Reaction Monitoring Transitions Used for the 3 Target Analytes and Internal Standards.
Compound Name
Precollision
Ion Mass MS1
Produced
Ions MS2 Res Dwell Frag (V)
Collision
Energy (V)
Cell Acc
(V) Polarity
Retention
Time
Betulinic acid 439.3 Unit 95 Unit 20 100 48 7 Positive 14.545
minutes
81.1 100 48 Positive
Atrazine D5: ISTD 221.1 Unit 179.1 Unit 20 115 16 Positive 7.419
minutes
69.1 115 40 Positive
Betulin 425.3 Unit 95 Unit 20 100 48 Positive 14.781
minutes
81 100 48 Positive
Inotodiol 425.3 Unit 246.9 Unit 20 100 12 Positive 15.66
minutes
Abbreviation: MS, mass spectrometer.
Géry et al 5
human epidermal growth factor, 0.5 mL of epinephrine, 0.5
mL of transferrin, 0.5 mL of insulin, 0.5 mL of retinoic acid,
and 0.5 mL of triiodothyronine to obtain BEGM medium.
Each well was seeded 24 hours before exposure with 10 000
cells for A549 cells line and 15 000 cells for BEAS-2B cells
line suspended in 200 µL of medium, and then the microplates
are incubated in a stove at 37°C in a 5% CO2 atmosphere.
Before dilution, the aqueous extract was passed over a
hydrophilic filter with a PES membrane with a porosity of
0.22 µm. The cells were then exposed to 6 different dilu-
tions of aqueous extract (expressed in %, vol/vol): 25%,
10%, 5%, 2%, 1%, and 0.5%. Eight replicates were made
by dilution and exposure duration: 24, 48, and 72 hours.
After the end of the exposure time, the cells were stained
with sulforhodamine B and the absorbance reading was per-
formed by spectrophotometry at 570 and 655 nm. From the
obtained absorbance means, the percentage of cell growth
inhibition was calculated for each concentration.
Statistical Analysis
Student’s t test was used to compare cytotoxicity on A549 and
BEAS-2B cells. P < 0.05 was considered as statistically sig-
nificant. Analyses were conducted using the SAS version 9.4.
Results
Quantification of Metabolites by HPLC-ESI-
QTOF MS/MS
We first used organic extracts made from French Chaga to
develop the method of detection and quantification of the 3
metabolites searched in this study (betulin, betulinic acid,
and inotodiol).
An analysis of the chromatograms by extracted ion chro-
matograms allowed to demonstrate the presence of betulin,
betulinic acid, and inotodiol by searching for their masses
in cyclohexane and ethyl acetate extracts. Figure 2A to C
shows the mass spectra and the chromatograms obtained for
these 3 metabolites in the different organic extracts.
Then, we applied this method of detection and quantifi-
cation to other preparations of French Chaga, that is, an
aqueous extract, an infusion, and a decoction. Betulin, betu-
linic acid, and inotodiol were found (to a lesser extent than
in organic extracts) in the aqueous extract, but not in the
infusion or in the decoction (Table 3).
We also searched for these 3 metabolites in organic
extracts prepared from the Canadian and Ukrainian Chaga
to compare their concentration depending on the geographi-
cal origin of the samples. The results presented in Table 3
show that there are greater concentrations of birch metabo-
lites in French Chaga and more inotodiol in Canadian
Chaga. It is important to note, however, that the difference
in concentration of these metabolites may be due not only to
environmental factors (climate, host tree, etc) but also to the
conservation technique and the lapse of time between har-
vesting and the production of the extracts.
Biological Activity of the Aqueous Extract From
French Chaga
Figure 3A to C shows that cytotoxic activity exists for aque-
ous extract and was greater on cancerous cells than on nor-
mal transformed cells.
Table 2. LOD, LOQ, Regression Equation, and Correlation Coefficient of Calibration Curves.
Cyclohexane
Extract
Ethyl Acetate
Extract
Aqueous
Extract Infusion Decoction
Betulinic acid LOD (µg/L) 0.3 0.3 0.3 1.7 0.7
LOQ (µg/L) 1 1 1 5 2
Regression equation/
correlation
coefficient
French Chaga (February 8, 2016), dilution range = 1-1000 µg/L: y = (−6.09 × 10−8) ×
x2 + (2.52 × 10−4) × x + (6.58 × 10−4)/r2 = 0.9998
Canadian/Ukrainian Chaga (December 23, 2016), dilution range = 1-500 µg/L: y =
(−2.19 × 10−7) × x2 + (4.55 × 10−4) × x + (3.61 × 10−5)/r2 = 0.9994
Betulin LOD (µg/L) 0.3 0.3 0.3 1.7 0.7
LOQ (µg/L) 1 1 1 5 2
Regression equation/
correlation
coefficient
French Chaga (February 2, 2016), dilution range = 1-5000 µg/L: y = (−3.44 × 10−9) ×
x2 + (6.56 × 10−5) × x + (3.62 × 10−4)/r2 = 0.9998
Canadian/Ukrainian Chaga (December 23, 2016), dilution range = 1-1000 µg/L: y =
(−3.55 × 10−8) × x2 + (1.77 × 10−4) × x + (5.11 × 10−5)/r2 = 0.9989
Inotodiol LOD (µg/L) 1.7 1.7 1.7 8.3 3
LOQ (µg/L) 5 5 5 25 10
Regression equation/
correlation
coefficient
French Chaga (February 2, 2016), dilution range = 10-5000 µg/L: y = (−1.52 × 10−10) ×
x2 + (1.30 × 10−5) × x + (2.53 × 10−4)/r2 = 0.9998
Canadian/Ukrainian Chaga (December 23, 2016), dilution range = 10-5000 µg/L: y =
(−6.63 × 10−10) × x2 + (2.51 × 10−5) × x + (5.60 × 10−4)/r2 = 0.9992
Abbreviations: LOD, limits of detection; LOQ, limits of quantification.
6
Betulin
Betulinic acid
Inotodiol
A
(continued)
7
(continued)
Figure 2. (continued)
B
8
C
Figure 2. (A) Mass spectra of betulin, betulinic acid, and inotodiol. (B) Chromatograms of betulin, betulinic acid, and inotodiol in extracts of French Chaga. (C) Chromatograms
of betulin, betulinic acid, and inotodiol obtained in extracts of Ukrainian (blue) and Canadian Chaga (orange).
Figure 2. (continued)
Géry et al 9
The cytotoxicity on A549 and BEAS-2B cells was char-
acterized by a dose-dependent and time-dependent effect.
Our results showed that after 48 and 72 hours of exposure,
the cytotoxic activity was significantly reduced or lesser on
the BEAS-2B cells than on the A549 cells (P = 0.025 after
48 hours of exposure and P = 0.004 after 72 hours of expo-
sure). These observations underline the greater sensibility
of highly proliferative cells compared with normal ones,
which could be kept in mind for therapeutic uses.
Discussion and Conclusions
The identification of betulin and betulinic acid in all fungal
extracts can be considered as a signature of the link between
the parasitic fungus and its plant host. Indeed, these 2
metabolites, known to be present in the birch bark, are also
concentrated in the Chaga. This observation has previously
been made with other plant-fungus associations such as
pine and Polyporus pinicola.44,45
Our study confirms the presence of inotodiol in the
French Chaga. This secondary metabolite has previously
been identified in extracts obtained from Chaga samples
from China, Finland, Thailand, and Russia.24,46,47 More gen-
erally, I. obliquus is characterized by the presence of several
lanostane-type triterpenes, in particular inonotsuoxide A,
inotodiol, trametenolic acid, and lanostérol.48
The absence of inotodiol in infusion and decoction
could be because of the concentrations below the limits
of quantification of our method. This could also suggest
that the properties attributed to this fungus in traditional
medicine are not because of the metabolites we have
sought to assay, but rather to the more polar molecules
and/or lower molecular masses found in Chaga. Indeed,
several studies have already demonstrated other types of
molecules such as polysaccharides,49 melanin com-
plexes,50 lignin derivatives,51 or polyphenols such as gal-
lic acid52 in aqueous extracts.
For the cytotoxicity tests, we have chosen to use the
aqueous extract, because of the following:
The presence of compounds known for their cyto-
toxic activity: inotodiol, betulin, and betulinic
acid.31,53,54
The presence of water-soluble compounds at the ori-
gin of properties recognized in traditional medicine.
The safety of water used as a solvent in cell culture.
The cytotoxic activity of Chaga extracts appears to be
related to its diversity of active secondary metabolites.
Compounds such as betulin and betulinic acid are known
for their anticancer activity.38 Lanostanes such as inotodiol
are also studied for their cytotoxic effects.48 Thus, com-
pounds present in the aqueous extract could explain or at
least partly account for these effects.
The aqueous extract of Chaga showed antiproliferative
activity on different cellular models. For example,
Mazurkiewicz et al55 demonstrated the action of an extract
of Polish Chaga on A549 lung cells as in our study.
Antiproliferative activity has also been demonstrated in
melanoma cells,56 hepatic cancer cells,57 as well as in sar-
coma cells.58
Chung et al59 showed that different fractions from
extracts of Russian Chaga showed cytotoxicity on various
cellular models including A549 cells. These fractions cor-
responded to lanostanes including inotodiol. The study by
Zhao et al46 also shows the efficacy of certain lanostanes
such as inonotsutriol against A549 cancer cells.
In conclusion, the analysis of the organic extracts of
Chaga revealed birch compounds such as betulin, betulinic
acid, and a characteristic fungal molecule inotodiol. The
Chaga recently discovered in the forests of Normandy con-
tains inotodiol as do those collected in Canada and Ukraine,
but our quantification method showed that the geographical
origin of the fungus has an impact on the concentration of
these metabolites. The biological results confirm a cyto-
toxic activity of the French Chaga on normal transformed
BEAS-2B cells but to a far lesser extent than on lung cancer
cells (A549). These observations allow us to consider the
therapeutic interest of the Chaga, its chemical complexity,
Table 3. Quantification of Betulin, Betulinic Acid, and Inotodiol in Different Preparations of Chaga.
Preparations Betulin (mg/L) Betulinic Acid (mg/L) Inotodiol (mg/L)
Canadian Chaga Cyclohexane extract 0.15 0.12 8.27
Ethyl acetate extract 13.45 5.12 464.86
Ukrainian Chaga Cyclohexane extract 0.78 0.14 41.37
Ethyl acetate extract 0.88 1.12 147.56
French Chaga Cyclohexane extract 6.48 0.37 52.70
Ethyl acetate extract 1100 52.92 373.02
Aqueous extract 0.23 0.16 11.70
Infusion <0.005a<0.005a<0.005a
Decoction <0.002a<0.002a<0.002a
aThe values are less than limits of quantification.
10 Integrative Cancer Therapies
Figure 3. (A) Cytotoxicity of the aqueous extract from French Chaga on A549 and BEAS-2B cells lines after 24 hours of exposure.
(B) Cytotoxicity of the aqueous extract from French Chaga on A549 and BEAS-2B cells lines after 48 hours of exposure. (C)
Cytotoxicity of the aqueous extract from French Chaga on A549 and BEAS-2B cells lines after 72 hours of exposure.
Géry et al 11
and to emphasize the interest of continuing to investigate
mycotherapy potential by associating both chemical and
biological approaches.
Acknowledgments
We would like to thank Pr Boris Czerny (ERLIS EA4254,
UNICAEN) for his help in the collection of Chaga. We would also
like to acknowledge Margaret Dearing for improving the English
of the article.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
study was supported by the Ligue Nationale Contre le Cancer
(Comité de la Manche).
ORCID iD
David Garon https://orcid.org/0000-0003-3545-6641
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