Chad hunting for Chaga on horseback in the Northern Canadian Forest 


We at Canadian Chaga Company have only one product to sell and that is Canadian Chaga. You can be sure we use the freshest and best Chaga we can find and we never put any additives in our Chaga. What you get is 100% pure Chaga processed in sanitary conditions and you purchase it from right where it is grown in Northern Canada.


chaga mushroom tea

Recipe 

 
Fill a Slow cooker (Crock Pot) with water and add  1/4 ounce of Chaga chunks and two teaspoons of Chaga powder. Cook for 4 to 8 hours at a medium setting. When finished drink as tea or add it to your morning coffee using a ladle, half Chaga, half Coffee. 
The Chaga will take the acid out of the coffee and add an interesting flavor.
When you are down to 2/3s of the crock pot add water to the top and two teaspoons of Chaga powder and reheat for 2 hours.
You can repeat this many times as the Chaga goodness will keep making tea or until the color of the tea is gone.
If you want to add more flavor to a cup of Chaga tea then add a bit of Cinnamon, vanilla or honey.


Chaga Capsules

chaga mushroom powder benefits


Chaga Capsules is one of the best ways to take Chaga as you are more likely to take Chaga every day. 


The King of Herbs


Chaga's Scientific name is  Inonotus Obliquus and it is a non-toxic mushroom that has many medicinal qualities. Chaga is known as one of the highest food antioxidants in the world and it has the highest level of superoxide dismutase or (SOD) that has been detected in any food or herb. 
The three most beneficial ways to consume the properties of Chaga are grinding it into a powder and brewing it as a tea, also known as a hot water extraction, taking it in a capsule or tincture.



Chaga 124 Capsules

This is the best for starting out in order to get Chaga's benefits into your system fast.

​​Now only

$98. USD + $8 shipping ​​

 Our Chaga capsules are free from artificial flavors, artificial colors, corn, gluten, wheat, lactose. Dairy, preservatives, added sugar, yeast, and any GMO materials.


We use Capsule med cards to helps you keep track of what you are taking and record the results.  A jar of capsules in a cupboard is to easy to forget or misplace.

 Proprietor

Chad  Smith

  Cleanliness, full disclosure, and safety first is our motto.


We do quality production

 not

mass production.


This is how we build a relationship of trust with each other.  We at  CanadianChaga.com  company provide only 100% pure local dehydrated Chaga processed under the most strict sanitary conditions. None of our Chaga is ever touched by human hands. Our Chaga is the cleanest as we even use a steam spray for the outside.

We wear disposable plastic gloves during the entire process of weighing, mixing, and making our Chaga.  We wear a dust mask over our mouth and nose (this protects you as much as it protects the quality of the Chaga. We ALWAYS wash our hands.  Once opened, we store Chaga in airtight containers to avoid contamination.  Everyone wears a hair net while in the processing room.  All Chaga is steamed cleaned on the exterior when brought in from the forest 

We at Canadian Chaga Company have only one product to sell and that is Chaga. You can be sure we use the freshest and best Chaga we can find and we never put any additives in our Chaga. What you get is 100% pure Chaga processed in sanitary conditions and you purchase it from right where it is grown in Northern Canada.
 

CanadianChaga.Com 

Home of the finest Canadian Chaga Mushroom

 Chaga helps to cure CANCER

Here is the proof

Chad

7research-arti5cle201820187912ICTXXX10.1177/1534735418757912Integrative Cancer TherapiesGéry et al
Research Article

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 medicineSubmitted August 23, 2017; revised November 8, 2017; accepted December 18, 2017

[page1image2279582528] [page1image2279582816] [page1image2279586720] [page1image2279584928] [page1image2279576464] [page1image2279576800] [page1image2279577072] [page1image2279561152] [page1image2279561808] [page1image2279562080] [page1image2279562352]

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

1Normandie University, UNICAEN, Centre F. Baclesse, Caen, France2Labéo 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

[page1image2279448032] [page1image2279448304] [page1image2279448576]

Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-

NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

Integrative Cancer Therapies
1–12
© The Author(s) 2018
Reprints and permissions: sagepub.com/journalsPermissions.navhDttOpsI://1d0o.i.1o1rg7/71/01.15137477/13553417385745178971572912journals.sagepub.com/home/ict

2

Integrative Cancer Therapies

[page2image1942454416]

Polyporus obliquus by Fries3 (1830), followed by Quélet4(1888), who called it Poria obliqua (under the bark of dryFagus). 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 nameobliquus.

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 obliquusused 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.32These 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

[page3image1940934128] [page3image1940934400]

Figure 1. Asexual form of Inonotus obliquus (Chaga) on birch trunk.

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)

4 Integrative Cancer TherapiesTable 1. Multiple Reaction Monitoring Transitions Used for the 3 Target Analytes and Internal Standards.

[page4image2279058288] [page4image2279058560]

Compound Name

Betulinic acid Atrazine D5: ISTD Betulin
Inotodiol

Precollision Produced Ion Mass MS1 Ions

439.3 Unit 95 81.1

221.1 Unit 179.1 69.1

425.3 Unit 95 81

425.3 Unit 246.9

MS2 Res

Unit Unit Unit Unit

Dwell

20 20 20 20

Frag (V)

100 100 115 115 100 100 100

Collision Cell Acc Energy (V) (V)

48 7 48
16
40

48 48 12

Polarity

Positive Positive Positive Positive Positive Positive Positive

Retention Time

14.545 minutes 7.419 minutes 14.781 minutes 15.66 minutes

[page4image2278989408] [page4image2278972224]

Abbreviation: MS, mass spectrometer.

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,
ted autosampler, and a column compartment.
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

a thermostat- The samples

Géry et al

5

[page5image2168911760]

Table 2. LOD, LOQ, Regression Equation, and Correlation Coefficient of Calibration Curves.

[page5image1943505824]

Betulinic acid

Betulin

Inotodiol

LOD (μg/L) 0.3 0.3
LOQ (μg/L) 1 1 1 5 2

Regression equation/ correlation coefficient

Regression equation/ correlation coefficient

LOD (μg/L)
LOQ (μg/L) 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

1.7 1.7 1.7 8.3 3 5 5 5 25 10

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

Cyclohexane Ethyl Acetate Extract Extract

Aqueous Extract

Infusion

Decoction

[page5image1943161248]

0.3

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

0.3 0.3 0.3 1.7 0.7

LOD (μg/L)
LOQ (μg/L) 1 1 1 5 2

1.7

0.7

[page5image1943637696] [page5image1943637968]

Abbreviations: LOD, limits of detection; LOQ, limits of quantification.

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.

[page6image1902028688]

A

Betulin

Betulinic acid

Inotodiol

(continued)

6

[page7image2229717856]

Figure 2. (continued)

B

(continued)

7

[page8image2213082800]

Figure 2. (continued)

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).

8

Géry et al

9

[page9image2213246592]

Table 3. Quantification of Betulin, Betulinic Acid, and Inotodiol in Different Preparations of Chaga.

[page9image2212833888] [page9image2212830512]

Canadian Chaga Ukrainian Chaga French Chaga

Preparations

Cyclohexane extract Ethyl acetate extract Cyclohexane extract Ethyl acetate extract Cyclohexane extract Ethyl acetate extract Aqueous extract Infusion

Decoction

Betulin (mg/L)

0.15 13.45 0.78 0.88 6.48

1100 0.23

<0.005a<0.002a

Betulinic Acid (mg/L)

0.12 5.12 0.14 1.12 0.37

52.92 0.16

<0.005a<0.002a

Inotodiol (mg/L)

8.27 464.86 41.37 147.56 52.70 373.02 11.70

<0.005a<0.002a

[page9image1901994176] [page9image1901796144]

aThe values are less than limits of quantification.

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 betulinicacid.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,

[page10image2354259472]

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

[page11image2276879568]

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  [page11image2276863040] https://orcid.org/0000-0003-3545-6641

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