Chemical Content and Cytotoxic Activity on Various Cancer Cell Lines of Chaga (Inonotus obliquus) Growing on Betula pendula and Betula pubescens

Chaga mushroom (Inonotus obliquus) is a pathogenic fungus that grows mostly on birch species (Betula pendula Roth and B. pubescens Ehrh.) and has traditionally been used as an anticancer medicine. This study aimed to compare the chemical composition and cytotoxic activity of chagas growing on both Betula spp. on various cancer cell lines. The freeze-dried extracts contained triterpenes inotodiol, lanosterol betulin, and betulinic acid typical to conks growing on Betula species. The cytotoxic activity of chaga growing on Betula pendula and B. pubescens 80% ethanolic extracts against 31 human cancer cell lines was evaluated by a sulforhodamine B assay. Chaga extract showed moderate activity against all cancer cell lines examined; it did not result in high cytotoxicity (IC50 ≤ 20 µg/mL). The strongest inhibitions were observed with chaga (growing on B. pendula) extract on the HepG2 and CAL-62 cell line and with chaga (from B. pubescens) extract on the HepG2 cell line, with IC50 values of 37.71, 43.30, and 49.99 μg/mL, respectively. The chaga extracts from B. pendula exert somewhat stronger effects on most cancer cell lines studied than B. pubescens extracts, which can be attributed to a higher content of inotodiol in B. pendula extracts. This study highlights the potential of chaga as a source of bioactive compounds with selective anticancer properties. To the best of our knowledge, this study is the first investigation of the chemical composition of I. obliquus parasitizing on B. pubescens.


Introduction
A basidiomycete fungus chaga (Inonotus obliquus) typically occurs on the trunks of trees growing in the Northern Hemisphere [1].This pathogenic fungus grows mostly on birch species (Betula spp.) and is less known on other trees [2,3].As a real parasite, chaga kills the host organism to fulfil its lifecycle [4].Due to being found mainly on birch trunks, the chaga drug is known in pharmacy as Fungus betulinus, not to be confused with another species, Piptoporus betulinus.
As a natural source of biologically active substances, the chaga has been traditionally used as an antitumour, anti-inflammatory, antibacterial, hepatoprotective, and antioxidant natural remedy [10].Chaga has been recognised for its medicinal uses since the sixteenth century [11].Owing to containing a range of pharmaceutical and nutraceutical value, including polyphenols, triterpenoids, polysaccharides, and lignin, chaga has demonstrated diverse therapeutic effects.These encompass antioxidant, anti-inflammatory, antibacterial, antiviral, hepatoprotective, antidiabetic, anti-obesity, renoprotective, hepatoprotective, immunomodulatory, antitumour, and anti-fatigue activities.Significantly, chaga has shown efficacy in anticancer activities [11][12][13].The conks of I. obliquus have been the best-known and most popular anticancer remedy in Estonian ethnomedicine among other 43 natural drugs [14].Nowadays, chaga is rather well studied and primarily known as a promising natural material against cancer and immunotherapy [6,9,[15][16][17].The anticancer activity of different chaga extracts has been studied by several researchers [15,[18][19][20][21][22][23][24].The water extract of chaga exhibited a potential anticancer activity against B16-F10 melanoma cells in vitro and in vivo through the inhibition of proliferation and induction of differentiation and apoptosis of cancer cells [25].Szychowski et al. (2018) compared the 80% ethanol and methanol extracts and showed their different biological activities.The ethanol extract exhibited the highest properties inhibiting the activity of xanthine oxidase and radical scavenging activities [24].
Most studies have been conducted on cell lines from the digestive system [26].For example, a hot-water chaga extract inhibited the proliferation of human colorectal cancer cells (HT-29) [27].Lee et al. (2015) showed that 95% ethanol extract of chaga is active in HT-29 human colon cancer cells [28].
Several studies of the chaga extracts showed excellent anticancer activities against colon cancers, but the bioactive compounds exhibiting those effects remain unknown [27].The water-soluble polysaccharide fraction of chaga is a promising group of substances associated with anticancer activity and insulin-improving sensitivity [29,30].Thus, the high content of polysaccharides seems to be responsible for the anticancer potential of chaga [31].Conversely, anticancer potential has been found in all types of extracts made by different solvents [32].Several studies [16,[33][34][35] have shown the cytotoxic activity of chaga triterpenoids against various cancer cells soluble in 80% ethanol [4].
The aim of the study was to compare the (1) chemical composition and (2) cytotoxic activity of chagas growing on Betula pendula and B. pubescens on various cancer cell lines.
To the best of our knowledge, except for cytotoxic activity against HepG2 cell lines, this is the first investigation of cytotoxicities against the tested cell lines of chaga parasitizing on B. pendula Roth and B. pubescens Ehrh.

Phytochemical Study of Chaga Extracts and Content of Biochemical Compounds
Freeze-dried ethanol extracts of chaga growing on two species, Betula pendula and B. pubescens, were prepared and analysed for triterpenoid composition and antioxidant activity (Table 1).
Although the ratio of dried raw material and solvent was the same for both samples, the yield of freeze-dried dry extract from samples differed slightly: 50 g of B. pendula conks resulted in 1.26 g of dry extract and the same amount of B. pubescens gave 1.45 g of dry extract.There were no significant differences in the content of sitosterol and betulinic acid.Also, antioxidant activity evaluated using the DPPH method did not reveal any differences between these extracts (Table 1).Significant differences were revealed between the content of lanosterol, inotodiol, and betulin in the extracts from conks from B. pubescens and B. pendula.The content of inotodiol in these samples was negatively correlated with the content of betulin and lanosterol, while betulin and lanosterol content had a strong positive correlation.

Cytotoxic Activity of Chaga Extracts
The cytotoxic activity of chaga (growing on B. pendula and B. pubescens) extract against thirty-one human cancer cell lines (Table 2) was evaluated by a sulforhodamine B assay [36,37].The results of the cytotoxic assays are summarised in Table 3.These data showed that the strongest inhibitions were observed with chaga (growing on B. pendula) extract on the HepG2 and CAL-62 cell lines and with chaga (growing on B. pubescens) extract on HepG2 cell line, with IC 50 values of 37.71, 43.30, and 49.99 µg/mL, respectively.Generally, chaga extracts demonstrated moderate activity against all cancer cell lines examined; high cytotoxicity (IC50 ≤ 20 µg/mL) was not detected.The extracts from B. pendula showed somewhat stronger experimental results than B. pubescens extracts on most cancer cell lines studied.

Discussion
The most abundant of the identified triterpenes was inotodiol, as has been reported in earlier studies [4,38].The content of lanosterol, inotodiol, and betulin significantly varied among the extracts from B. pubescens and B. pendula.Inotodiol was higher in B. pendula extract, and lanosterol and betulinic acid content in B. pubescens extracts.Correlation analysis revealed a positive correlation between inotodiol content in the extract and the effect on growth inhibition on most of the tested cell lines.A negative correlation was revealed between inotodiol and growth inhibition only on cell lines NTERA-2 and 8505c.
Also, in a previous study [4], the concentration of inotodiol was much higher than other triterpenes lanosterol, betulin, and betulinic acid.Upska et al. studied the extractability of chaga active ingredients using different methods and both polar and non-polar solvents.The best extractability of inotodiol was obtained by Soxhlet extraction using cyclohexane [39].Inotodiol and lanosterol are important compounds in chaga's anticancer effects [40,41].Inotodiol and other lanostanes were effective against A549 cancer cells [42,43].Inotodiol and lanosterol isolated from the conks of Betula spp.have immunological effects [17,44].Inotodiol and lanosterol may also have cosmetic importance as they activate tyrosinase and increase pigment synthesis in skin cells [45].Tian et al. showed that triterpenes of chaga may be an effective natural aid for treating and protecting various kidney diseases [46].
The antioxidant activity measurements performed using a DPPH assay showed the same results for chaga from both Betula spp.The level of antioxidant activity may depend on the solvent used to extract chaga and the origin of the raw material.The difference in the DPPH scavenging activity may be up to 40 times different in chagas from various countries [6].The scavenging activity of the water and ethanol extracts of chaga conks showed a similar level, but the methanol extract had maximum IC 50 values of 18.96, 16.25, and 24.90 mg/mL, respectively [47].
Here are some comparative data obtained using the same method in other natural products, which help to orient the strength of chaga's cytotoxic activity.The strongest anticancer effect of Matricaria chamomilla methanol extract against SK-MEL-2 cells was IC 50 40.7 µg/mL, but the extract of Calendula officinalis showed no remarkable cytotoxic activity against SK-MEL-2 and KB cells (IC 50 62.6 and 79.2 µg/mL, respectively) [48].The essential oils of Anthemis sylvestris roots and aerial parts had the strongest anticancer activity on KB cells (IC 50 19.7 µg/mL and 19.8 µg/mL, respectively), while the methanolic extract had no effect [37].The Pinus sylvestris needle methanol extract suppresses the viability of MDA-MB-231 cells on the level IC 50 35 µg/mL [49].In this context of anticancer activity, the effect of chaga's extract, having the strongest values between 37.7 and 43.3 µg/mL, showed remarkable but moderate effects.
Chaga has received considerable attention from many researchers due to its numerous biological attributes, particularly its potential anticancer effects.According to Ma et al., the petroleum ether and ethyl acetate extracts of chaga displayed significant cytotoxic activities against the human prostatic carcinoma cell PC 3 and breast carcinoma cell MDA-MB-231 [18].The inhibitory effects were mainly attributed to ergosterol peroxide and trametenolic acid [18].The aqueous extract of chaga in France containing a high content of betulin and betulinic acid and inotodiol showed an effect on human lung adenocarcinoma cells [21].Noticeably, chaga extract was reported to markedly reduce HepG2 cell viability due to G0/G1-phase arrest and apoptotic cell death, thus leading to downregulation of p53, pRb, p27, cyclins D1, D2, E, cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 expression [50].Through Matrigel-coated filters, the methanolic extract and its EtOAc-soluble fraction from Chaga collected in Japan showed the significant invasion inhibition of human fibrosarcoma HT 1080 cells.Importantly, compound 3β-hydroxylanosta-8,24-dien-21-al exhibited a strong inhibitory effect on HT 1080 cells.Furthermore, the methanolic extract of this sample significantly suppressed the formation of lung tumours in mice at 500 mg/kg/d [20].The methanolic extract of Russian chaga purchased in Korea showed inhibitory effects toward four human lung adenocarcinoma cell lines (including A549, H1264, H1299, and Calu-6) through induction of apoptosis accompanied by caspase-3 cleavage.Significantly, compounds 3β-hydroxylanosta-8,24-dien-21-al, trametenolic acid, and 3β-hydroxy-5αlanosta-8,25-dien-21-oic acid isolated from this extract showed cytotoxicities against these cell lines, with IC 50 values ranging from 75.1 to 227.4 µM in the same pattern [51].The aqueous extract of chaga (in Korea) displayed anti-proliferative activity on HCT116 and DLDl cell lines and reduced intestinal polyps in APC Min/+ and colon tumours in AOM/DSS-treated mice through downregulation of Wnt/β-catenin and NF-κB pathways [52].The extract of chaga collected from birch trees in Japan successfully reduced tumours in both tumour-bearing mice and metastatic mice by promoting energy metabolism [19].Additionally, the administration of chaga obtained in Japan could induce necrotic lesions, resulting in a decrease in the growth of the tumour and the weight of dog bladder cancer organoid-derived xenografts [22].
A limited number of fungi have been studied for bioactive compounds that can help treat various diseases including anticancer activity.Promising fungal species' anticancer activity on human cancer cell lines are many, but numerous studies, including clinical studies, are focused on Lentinula edodes, Coriolus versicolor, and Ganoderma lucidum [53].For example, Ganoderma species have demonstrated a wide range of health benefits, such as anticancer, anti-immunomodulatory, anti-inflammatory, antimicrobial, and antioxidant effects, which can be attributed to their bioactive compounds [54].Also, Lentinula edoides has shown antitumour, cytotoxic, and immunomodulating moiety in treating various cancers [55].Related to these named fungi, most in vitro cell studies use breast cancer cell lines (43.9%), followed by lung (14%) and colorectal cancer cell lines (13.1%) [53].
The current work and some others [4,15,56] indicate that fungal species origin and the fungus host or growth substrate affect the source of useful bioactive compounds.Inonotus obliquus grows naturally on Betula spp.and on Alnus spp. as well as some other broadleaved trees in northern Baltic conditions [2,3].In this area, the fungus grows on B. pendula and B. pubescens [2,3,57], and B. pendula is a widely known host to the chaga [4], but not B. pubescens.However, nothing is known about the bioactive compounds of the chaga growing on B. pubescens.The current work shows evidently that the bioactive compounds of chaga differ significantly according to the origin of quite close host species such as B. pendula and B. pubescens.This indicates that the fungus origin and background are important for further analyses including effects on different human cancer cell lines.
These findings highlight the potential of chaga as a source of bioactive compounds with selective anticancer properties only on the HepG2 cell line, encouraging further exploration of its therapeutic mechanisms and potential applications in cancer treatment.

Sample Sites, Fungal Isolation, Detection, and Preparation
The conks of I. obliquus were collected from B. pendula and B. pubescens, originating from Estonia between 21 December 2019 and 3 February 2022 (Table 4).The isolates were obtained from fresh conks to grow on 2% malt-extract agar (Biolife, Milano, Italy) plates.The DNA of I. obliquus isolates was extracted using a GeneJET Genomic DNA Purification Kit (Thermo Scientific, Vilnius, Lithuania), and the fungus was detected as described by Drenkhan et al. [58].ITS-PCR products from the isolates were sequenced at the Estonian Biocentre in Tartu.The ITS sequences were edited using the BioEdit program, Version 7.2.5 [59], and deposited in a GenBank (see Table 4).BLAST searches for the fungal taxa confirmation were performed in the GenBank database (NCBI).All the pure cultures were deposited into the Fungal Culture Collection (TFC) and GenBank (NCBI).
Before extractions and biochemical analysis, the conks were kept in a freezer at −20 • C.Then, the collected conks were dried in a laboratory oven (MMM Medcenter Einrichtungen GmbH, Planegg, Germany) at 50 • C, and all the material was ground into a coarse powder using a cutting mill Retch SM 300 with a 1 mm sieve (Retsch, Haan, Germany).Extracts were prepared from the mix of 10 individual conks samples per host species in equal proportions (see Table 4).For extract preparation, 500 mL of 80% ethanol was added to 50 g of dried and milled sample and agitated at room temperature for 12 h using Biosan ES20 orbital shaker.After vacuum filtration, the extracts were concentrated on rotary evaporator RV 10 control Flex (IKA-Werke GmbH & Co. KG., Staufen im Breisgau, Germany) to remove most of the ethanol and further dried in the VirTis AdVantage 2.0 EL freeze-drier (SP Scientific, Santa Cruz, CA, USA).Bioactive compound detection from I. obliquus was performed according to [4].We selected 80% ethanol for extraction based on the pre-trial from our previous research [4], where we optimised the sample preparation to determine free radical scavenging activity, polyphenols, and triterpenes content.In the pre-trial, we compared the following solvents: ethyl acetate, hexane, 80% EtOH, and a mixture of dichloromethane with MetOH and water in a proportion of 5:3:2.
Antioxidant activity measurements were performed in triplicate using a 2.2-diphenylpicrylhydrazyl (DPPH) assay [60].The absorbance values of the samples were measured at 515 nm using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan).The results were expressed in mg of gallic acid equivalent per g of dry weight (mg GA eq./g).
Qualitative and quantitative analyses were performed on a Shimadzu Nexera X2 UHPLC with mass spectrometer LCMS 8040 (Shimadzu Scientific Instruments, Kyoto, Japan).The UHPLC system had a binary solvent delivery pump LC-30AD, an autosampler Sil-30AC, a column oven CTO-20AC, and a diode array detector SPD-M20A.Five triterpenoids (betulinic acid, betulin, lanosterol, inotodiol, and sitosterol) were identified by comparing the retention times and parent and daughter ion masses with those of the standard compounds (Figure 1).Chromatographic separation was performed using the Ascentis Express column (C30 50 × 4.6 mm, Merck, Darmstadt, Germany) at 40 • C. The flow rate of the mobile phase was 1 mL/min, and the injected sample size was 1 µL.Mobile phases consisted of 1% formic acid in Milli-Q water (mobile phase A) and 1% formic acid in acetonitrile (mobile phase B).Separation was performed for 10 min at isocratic conditions with 92% of mobile phase B and 8% of mobile phase A. All samples were kept at 4 • C during the analysis.MS data acquisitions were performed on LCMS 8040 with the APCI source.All samples were analysed in triplicate, and the results were expressed as mg per g of dry weight (mg/g).
The standards betulin, betulinic acid, and lanosterol were purchased from Cayman Chemical Company (Ann Arbor, MI, USA), and inotodiol was from Aobious (Gloucester, MA, USA).All other standards (sitosterol, gallic acid) and chemicals (formic acid, methanol) used were of analytical grade and purchased from Sigma (Darmstadt, Germany).
The dry matter content in the samples was determined at 105 • C using the moisture analyser Precisa EM 120 HR (Precisa Gravimetrics AG, Dietikon, Switzerland).

Cytotoxic Assay
A total of 31 human cancer cell lines (Tables 1 and 2) were cultivated at 37 °C in a humidified atmosphere containing 5% carbon dioxide.An SRB cytotoxic assay for monolayer cells was analysed according to the method described in [61,62].
The 80% ethanolic dry extracts were first stocked in dimethyl sulfoxide (DMSO) 100% at the concentration of 20 mg/mL.The samples were then prepared in a range of diluted concentrations as 2000-400-80-16 µg/mL using basic RPMI medium (w/o FBS) in 96-well sample plates.Then, 10 µL from each well (with the diluted sample above) was added to the tested cell pre-seeded (190 µL) well to evaluate cytotoxic activities.Thus, the final concentration of the sample was 100-20-4-0.8 µg/mL.Ellipticine was tested with the final concentration of 20-4-0.8-0.16µg/mL.
The effects of Betula chaga hydroethanolic extracts on the viability of malignant cells were determined by sulforhodamine B cytotoxic assay [61,62].Briefly, cells were grown in 96-well microtiter plates, each containing 190 µL of medium.After 24 h, 10 µL of test samples dissolved in DMSO were added to each well.One plate with no samples served as a day 0 control.The cells were continuously cultured for an additional 48 h, fixed with trichloroacetic acid, and stained with sulforhodamine B, followed by determining optical densities at 515 nm using a Microplate Reader (BioRad, Hercules, CA, USA).The percentage of growth inhibition was calculated using the following equation: [OD (reagent)-OD (day 0)] ×100% Growth = [OD (negative control DMSO 10%)-OD (day 0)]

Cytotoxic Assay
A total of 31 human cancer cell lines (Tables 1 and 2) were cultivated at 37 • C in a humidified atmosphere containing 5% carbon dioxide.An SRB cytotoxic assay for monolayer cells was analysed according to the method described in [61,62].
The 80% ethanolic dry extracts were first stocked in dimethyl sulfoxide (DMSO) 100% at the concentration of 20 mg/mL.The samples were then prepared in a range of diluted concentrations as 2000-400-80-16 µg/mL using basic RPMI medium (w/o FBS) in 96-well sample plates.Then, 10 µL from each well (with the diluted sample above) was added to the tested cell pre-seeded (190 µL) well to evaluate cytotoxic activities.Thus, the final concentration of the sample was 100-20-4-0.8 µg/mL.Ellipticine was tested with the final concentration of 20-4-0.8-0.16µg/mL.
The effects of Betula chaga hydroethanolic extracts on the viability of malignant cells were determined by sulforhodamine B cytotoxic assay [61,62].Briefly, cells were grown in 96-well microtiter plates, each containing 190 µL of medium.After 24 h, 10 µL of test samples dissolved in DMSO were added to each well.One plate with no samples served as a day 0 control.The cells were continuously cultured for an additional 48 h, fixed with trichloroacetic acid, and stained with sulforhodamine B, followed by determining optical densities at 515 nm using a Microplate Reader (BioRad, Hercules, CA, USA).The percentage of growth inhibition was calculated using the following equation: [OD (reagent)-OD (day 0)] ×100% Growth = [OD (negative control DMSO 10%)-OD (day 0)] where OD is the optical density or absorbance value.The potent anticancer agent ellipticine was used as a positive control.

Statistics
Biochemical analysis data were analysed using Jamovi software [65].Metabolite concentrations were analysed by one-way analysis of variance (ANOVA).Differences between means were assessed with the post hoc Tukey test.The IC 50 values were analysed using TableCurve 2Dv4 software.

Conclusions
The strongest inhibitions were observed on the HepG2 cell line with chaga grown on both hosts (Betula pendula and B. pubescens).In general, chaga extract showed moderate activity against all 31 cancer cell lines studied.
The B. pendula origin chaga extracts exert somewhat stronger effects on most cancer cell lines studied than B. pubescens parasitizing chaga extracts.This effect can be attributed to a higher content of inotodiol in B. pendula origin extracts compared to B. pubescens.This is also the first comparative investigation of the chemical composition of I. obliquus parasitizing on B. pendula and B. pubescens.
The results of the study highlight the potential of chaga as a source of bioactive compounds with selective anticancer properties, encouraging further exploration of its therapeutic mechanisms and potential applications in cancer treatment.

Table 1 .
The average content of triterpenoids and the antioxidant activity of freeze-dried extracts of chaga conks growing on Betula pendula and B. pubescens.

Table 3 .
Cytotoxic activity of chaga extracts on various cancer cell lines.
a IC 50 (concentration that inhibits 50% of cell growth).b Positive control.

Table 4 .
Origin and hosts of Inonotus obliquus isolated from the conks and used in this study's fungal strains.