Chaga Mushroom Extract as a Dual-Action Agent against Microbial and Cancerous Cells: An In Vitro Study

The present study investigates the bioactive properties of the Ethyl Acetate Extract of Inonotus obliquus (EAEIO), sourced from the Chaga mushroom. Traditionally used in medicine, this mushroom is increasingly recognized for its potent antimicrobial and anticancer benefits. Our analysis sought to explore the impact of EAEIO on four diverse bacterial strains: Escherichia coli ATCC25922, Bacillus cereus EMCC1080, Pseudomonas aeruginosa ATCC10145, and Listeria monocytogenes NCTC7973, employing the disc diffusion method. We also investigated the potential cytotoxic effect of EAEIO on MCF7 breast cancer cells, HCT16 colon cells, and normal BHK cells using the MTT assay. Our results underscore the effective antimicrobial properties of EAEIO, evidenced by inhibition zones between 16 mm to 28 mm and minimum inhibitory concentrations (MICs) ranging from 6.25 µg/mL to 1.563 µg/mL. In addition, the EAEIO demonstrated remarkable anticancer activity against MCF7 and HCT16 cell lines, with IC50 values of 7.56 µg/mL and 11.2 µg/mL, respectively. To conclude, EAEIO - the Ethyl Acetate Extract of the Chaga mushroom, exhibited significant antimicrobial and anticancer properties while showing no toxic effect on BHK cells. These observations suggest EAEIO's potential as a valuable natural resource for antimicrobial and anticancer treatments. However, further research is essential to verify the safety and efficacy of EAEIO in cancer and infectious disease management.

The present study investigates the bioactive properties of the Ethyl Acetate Extract of Inonotus obliquus (EAEIO), sourced from the Chaga mushroom. Traditionally used in medicine, this mushroom is increasingly recognized for its potent antimicrobial and anticancer benefits. Our analysis sought to explore the impact of EAEIO on four diverse bacterial strains: Escherichia coli ATCC25922, Bacillus cereus EMCC1080, Pseudomonas aeruginosa ATCC10145, and Listeria monocytogenes NCTC7973, employing the disc diffusion method. We also investigated the potential cytotoxic effect of EAEIO on MCF7 breast cancer cells, HCT16 colon cells, and normal BHK cells using the MTT assay.
Our results underscore the effective antimicrobial properties of EAEIO, evidenced by inhibition zones between 16 mm to 28 mm and minimum inhibitory concentrations (MICs) ranging from 6.25 µg/mL to 1.563 µg/mL. In addition, the EAEIO demonstrated remarkable anticancer activity against MCF7 and HCT16 cell lines, with IC50 values of 7.56 µg/mL and 11.2 µg/mL, respectively.
To conclude, EAEIO -the Ethyl Acetate Extract of the Chaga mushroom, exhibited significant antimicrobial and anticancer properties while showing no toxic effect on BHK cells. These observations suggest EAEIO's potential as a valuable natural resource for antimicrobial and anticancer treatments. However, further research is essential to verify the safety and efficacy of EAEIO in cancer and infectious disease management.
Despite significant advancements in cancer treatments like chemotherapy and radiation therapy, cancer remains a prevalent global health issue. These conventional treatment modalities often present various side effects, leading to the need for alternative, less harmful, and more efficient treatment methods. As such, Gielecińska et al., (2023) and Soto et al., (2023) indicated a surge of interest in natural remedies, including medicinal mushrooms like the chaga mushroom, as also highlighted by Asma et al., (2022).
Previous research, both in vitro and in vivo, has explored the anticancer potential of chaga mushroom extracts, as per Abugomaa et al., (2023). Their findings indicated these extracts' ability to inhibit a variety of cancer cell lines Lee et al., (2021), induce apoptosis or programmed cell death in cancer cells Su et al., (2020), and suppress tumor expansion and metastasis in animal models Arata et al., (2016).
In addition to their anticancer capabilities, Glamočlija et al., (2015) reported that chaga mushroom extracts possess significant antimicrobial action against a diverse array of bacteria. This action is linked to the mushroom's abundant polysaccharides and triterpenes. İnci et al., (2022) carried out in vitro experiments showing that medicinal mushroom extracts exhibit broad-spectrum antimicrobial activity against bacteria such as Staphylococcus aureus, Escherichia coli, and Bacillus cereus. Cör et al., (2018) andZhou et al., (2019) attribute the antimicrobial potency of chaga mushroom extracts to the presence of polysaccharides and triterpenes.
Further research is required to fully understand the potential of chaga mushroom extracts as antimicrobial and anticancer agents, despite promising findings in previous studies. The extraction of bioactive compounds from natural sources holds interest due to their potential health benefits. As per Ma et al., (2013) and Xu et al., (2015), ethyl acetate, a commonly used solvent, can extract bioactive compounds from various sources, including chaga mushroom, which shows potent antioxidants.
This research examines the antimicrobial and anticancer capabilities of the ethyl acetate extract from chaga mushrooms. The study tests the extract's cytotoxic effects on breast (MCF7) and colon cancer (HCT16) cell lines, its antimicrobial strength against various bacteria, and identifies its bioactive compounds. It's the first study to explore chaga mushroom's ethyl acetate extract's properties against MCF7 and HCT16 cancer cell lines. The goal is to shed light on the potential use of this extract as an alternative cancer treatment and prevention method and as an antimicrobial agent.
The ethyl acetate organic solvent was purchased from Sigma-Aldrich.

Extraction of the Active Metabolites from Chaga Mushroom:
First, ten grams of chaga mushroom powder was mixed with 200 ml of ethyl acetate, and the mixture was incubated overnight at 50 o c with agitation at 200 rpm. Then the extracted solution was separated from chaga powder by centrifugation. The insoluble residue was treated twice again with the same method to increase the yield of the extracted compounds according to Nguyen et al., (2023). Finally, the collected supernatants were evaporated using a vacuum rotary evaporator to obtain the crude extract which was used in further experiments.

LC-MS/MS Analysis:
The sample analysis was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with an ExionLC AC system for separation and a SCIEX Triple Quad 5500+ MS/MS system equipped with electrospray ionization (ESI) for detection. The separation was carried out using an Ascentis® Express 90 Å C18 Column (2.1×150 mm, 2.7 µm) in both positive and negative ionization modes. The mobile phases consisted of two eluents A (5 mM ammonium formate at pH 3 for positive ionization mode and pH 8 for negative ionization mode) and B (LC grade acetonitrile). The mobile phase gradient was programmed as follows: 5% B at 0-1 min, 5-100% B from 1-20 min, 100% B from 20-25 min, 5% at 25.01, and 5% from 25.01-30 min, with a flow rate of 0.3 ml/min and an injection volume of 5 µl. For MS/MS analysis, negative ionization mode was applied with a scan (EMS-IDA-EPI) from 100 to 1000 Da for MS1 with the following parameters: curtain gas at 25 psi; IonSpray voltage at 5500 for positive ionization mode and -4500 for negative ionization mode; source temperature at 500°C; ion source gas 1 & 2 at 45 psi; and from 50 to 1000 Da for MS2 with a declustering potential of 80 for positive ionization mode and -80 for negative ionization mode, collision energy at 35 for positive ionization mode and -35 for negative ionization mode, and collision energy spread at 15. Compounds' identification was performed using MS-DIAL 4. Antibacterial Activity of The Extract: EAEIO was subjected to tests to evaluate their antibacterial activities. These were done by use of the disc-diffusion method. Reference strains of bacteria from the American Type Culture Collection (ATCC, LGC Standards, Teddington, UK) and Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA) were used in the study. These strains included two Grampositive bacteria (Bacillus cereus EMCC1080, Listeria monocytogenes NCTC7973) and two Gramnegative bacteria (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC10145). The purity of the bacteria was tested by culturing on nutrient agar and being maintained on nutrient agar slants.
The disc diffusion method was used. One mg of the chaga crude extract was dissolved in 5 ml DMSO, then the paper discs were impregnated with 5 µl of the dissolved extract. After that the discs were placed upon Müller-Hinton (MH) plates inoculated with 0.5 Mcfarland standard of the tested bacterial pathogens. Plates were incubated at 37 o c for 24 hr, then the inhibition zone diameters (mm) were measured. Negative control was only treated with DMSO. Positive control was treated with Chloramphenicol. Minimum inhibitory concentration (MIC) was carried out in Elisa plate with starting concentration of 100 µg/ml and diluted in bifold dilution, Chloramphenicol was used as a positive control.

Cell Viability Measured by MTT Assay:
Different concentrations of ethanol extract of chaga mushroom were tested on all cell lines to evaluate the toxicity by  5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide]-based colorimetric assay. Hela cells (1 x 10 4 ) cells were seeded wells of a 96-well microtiter plate and incubated for 24 h. Then, the cells were exposed to different concentrations of extract (10-500 lg/ml) at (37⁰C, 5% CO2 for 24 h incubation). After incubation, cells were washed with PBS, a concentration of (0.5 mg/ml) of MTT dye in each well and allowed the incubate dark at (37⁰C and 5% CO2 for 4 h). Finally, 100 ml of dimethyl sulfoxide (DMSO) was added to dissolve the purple formazan crystal in the reaction. The optical density (OD) was determined at 570 nm in an ELISA plate reader r (SpectraMax M5-Molecular Devices, USA).

Statistical Analysis
The present statistical analyses were executed using Statistical Package for Social Science (SPSS) software version 22. According to Kolmogorov-Smirnov test, data were normally distributed. An Independent ttest was applied to illustrate the statistical differences in the studied parameters in each of the experimental groups, as compared to the controls and nicotine-treated groups. P<0.05 represents significant differences. Data were displayed as mean ± standard error of the mean.

RESULTS 1. LC-ESI-MS/MS Analysis of EAEIO:
EAEIO was meticulously analyzed using LC-ESI-MS/MS under both negative and positive ionization conditions to elucidate the diverse spectrum of bioactive mycochemicals. a. Major Bioactive Mycochemicals Detected in EAEIO (Negative Ionization): Figure 1 illustrates the LC-ESI-MS/MS profile for EAEIO, obtained under negative ionization. This analysis uncovered a variety of bioactive compounds, each represented by a unique peak in the profile. Table 1 details the major compounds identified, including their retention times. For instance, 2,3-Dihydroxybenzoic acid, Shikimic acid, and Azelaic acid were detected early in the analysis, with a retention time of 0.969883 minutes. Other compounds such as Octanoate and Xanthine were detected later, with retention times of 1.672433 and 3.122583 minutes respectively. More complex compounds, such as Oxymetholone and Magnolol, had retention times of 7.854533 and 13.35777 minutes, respectively. The longest retention time recorded in our analysis was 28.29398 minutes, identified for both Allopurinol and 3-Methylbenzoic acid.

b. Major Bioactive Mycochemicals Detected in EAEIO (Positive Ionization)
When LC-ESI-MS/MS analysis of EAEIO was conducted under positive ionization conditions, as represented in Figure  2, a distinct set of bioactive mycochemicals was detected. The major compounds identified and their respective retention times are enumerated in Table  2. Lauryldiethanolamine was detected at 12.83568 minutes, and Berberine at the longest retention time of 22.58867 minutes.
The broad range of compounds revealed under both negative and positive ionization conditions illuminates the rich and complex mycochemical composition of I. obliquus, reinforcing its potential as a potent source of bioactive compounds with significant therapeutic implications.   Table 1 for reference.   Table 2 for reference.

Antibacterial Activity of EAEIO against Select Bacterial Strains:
The antibacterial efficacy of EAEIO was scrutinized against four different bacterial strains, namely E. coli, Bacillus, Listeria, and Pseudomonas. Chloramphenicol, a broad-spectrum antibiotic, served as the positive control. The comparative analyses of both the inhibition zone diameters and minimum inhibitory concentrations (MICs) are presented below.

a. Inhibition Zone Analysis:
The inhibition zones of EAEIO and Chloramphenicol against the tested bacterial strains were measured and compared. For EAEIO, the average inhibition zones were found to be 18.00 ± 0.58 mm for E. coli, 25.00 ± 0.29 mm for Bacillus, 18.00 ± 0.46 mm for Listeria, and 16.00 ± 0.58 mm for Pseudomonas (Table 3).
In contrast, Chloramphenicol demonstrated smaller inhibition zones: 15.00 ± 1.15 mm for E. coli, 18.00 ± 0.58 mm for Bacillus, 15.00 ± 0.58 mm for Listeria, and 12.00 ± 0.58 mm for Pseudomonas (Fig.3). It's noteworthy that the inhibition zones for EAEIO were significantly larger for Bacillus, Listeria, and Pseudomonas with p-values of less than 0.05. However, against E. coli, the difference in inhibition zones was not statistically significant (p=0.08), despite EAEIO demonstrating a larger average zone.

Fig.3: Bar Graph Illustrating the Antibacterial Activity of EAEIO Assessed via Inhibition
Zones. Each bar indicates the mean diameter of the inhibition zone (in mm) ± standard error of the mean (SEM). An asterisk (*) signifies a statistically significant difference (p<0.000) when compared with Chloramphenicol.
These findings underscore the potential of EAEIO as a promising source of antibacterial agents, demonstrating significant inhibitory activity against the tested bacterial strains, superior to that of the conventional antibiotic Chloramphenicol.

Cytotoxic Impact of EAEIO Assessed Through IC50 Values Across Varied Cell Lines:
A significant facet of the present investigation revolved around understanding the cytotoxic potential of EAEIO. This was quantified through the determination of the IC50 values across different cell lines (Fig. 5). The IC50 value denotes the concentration of a substance required to inhibit 50% of cell proliferation.
In the context of the MCF-7 breast cancer cell line, EAEIO demonstrated a potent cytotoxic effect. The IC50 value was recorded at 7.56 µg/ml. This low IC50 value points to a strong suppressive influence of EAEIO on MCF-7 cell proliferation.
The anticancer efficacy of EAEIO was also examined against the Hct-16 colon cancer cell line. Here, the IC50 value was slightly higher, registering at 11.2 µg/ml.
Despite the increase, this figure still denotes significant growth inhibition of the Hct-16 cells. Finally, the EAEIO's impact was assessed against a non-cancerous cell line, the BHK (Baby Hamster Kidney) cells. The IC50 value, in this case, was measured at 22.5 µg/ml, suggesting that a higher concentration of EAEIO was required to inhibit the proliferation of these normal cells.
These findings, graphically represented in the subsequent figure, highlight the promising cytotoxic potential of EAEIO. This is particularly true in the case of breast and colon cancer cells, where the extract displayed marked growth inhibitory effects. While these are preliminary results, they lay the groundwork for further, more detailed investigations into the therapeutic potential of EAEIO as an anticancer agent.  Newman and Cragg, (2016), making this line of inquiry an increasingly popular one. The bioactive chemicals produced by this species are well-known Zheng et al., (2010). Commonly known as Chaga, the medicinal mushroom I. obliquus has a high concentration of bioactive substances and has been studied extensively Glamočlija et al., (2015). Because of these characteristics, it has attracted a lot of interest in the field of oncology. This research aimed to investigate the presence of bioactive mycochemicals and therapeutic qualities in an ethyl acetate extract of Inonotus obliquus (EAEIO). To completely characterize the EAEIO, this work used a three-pronged strategy that included LC-ESI-MS/MS analysis, antibacterial efficacy analysis, and cytotoxic evaluation.
There is growing evidence that bioactive compounds found in fungi could have medicinal applications Ratnaweera et al., (2015). The bioactive components of fungal extracts have been the subject of extensive research, and the results have shown promising anticancer and antibacterial effects Keller, (2019;Liu et al., (2020). Chaga extract contains bioactive components with potential anticancer action, including polysaccharides, betulinic acid, and polyphenols Drenkhan et al., (2022). As a further advantage, chaga has been shown to have antibacterial activity against a wide range of harmful bacteria, providing new avenues in the fight against antibiotic resistance Garádi et al., (2021).
LC-ESI-MS/MS analysis was performed under both negative and positive ionization conditions, revealing a broad range of bioactive compounds (Figures 1 and 2). It is consistent with prior studies highlighting the diverse mycochemical composition of Inonotus species Y. O. Kim et al., (2005). Among the significant bioactive compounds identified under negative ionization (Table 1) were Shikimic acid and Azelaic acid. Shikimic acid is known for its antiinflammatory properties and plays a crucial role in the biosynthesis of the antiviral medication Tamiflu Sheng et al., (2023). On the other hand, Azelaic acid has demonstrated significant antibacterial activity against acnecausing bacteria Spaggiari et al., (2023). These findings underline the possible medical relevance of EAEIO. Other key compounds identified included Octanoate and Xanthine. Octanoate has been reported to modulate metabolic activities Zhao et al., (2023) while Xanthine, a purine base, is involved in the biosynthesis of caffeine and has a role as a bronchodilator Baraldi et al., (2007). Finally, the detection of complex compounds Magnolol suggests possible antiinflammatory and antioxidant activities Peng et al., (2023). These results imply the richness of EAEIO in potentially bioactive compounds. Under positive ionization (Table  2), a distinct set of mycochemicals was detected. Lauryldiethanolamine, identified at a retention time of 12.83568 minutes, is known for its potential role in lipid metabolism and signaling Hishikawa et al., (2014). Similarly, Berberine, identified as having the longest retention time, has a wide spectrum of biological effects, including antimicrobial, anti-inflammatory, and antineoplastic activitiesGasmi et al., (2023). The identification of such bioactive compounds implies the medicinal potential of EAEIO. The varying array of compounds identified under the different ionization conditions underscores the complexity of the mycochemical profile of EAEIO and the suitability of LC-ESI-MS/MS as a powerful tool for detecting and identifying metabolites.
The exploration of plants and fungibased bioactive compounds as potential antimicrobial agents is a rapidly growing field of study Hashem et al., (2023); Pastare et al., (2023). As the current investigation showed, EAEIO demonstrated considerable antibacterial efficacy against different bacterial strains, including E. coli, Bacillus, Listeria, and Pseudomonas (Table 3, & Fig.  3). Interestingly, the inhibition zones for EAEIO were significantly larger for Bacillus, Listeria, and Pseudomonas when compared to Chloramphenicol, a broad-spectrum antibiotic commonly used in microbial susceptibility tests. This result indicates a potential edge of EAEIO over conventional antibiotics in combatting these bacterial strains. Although the difference against E. coli was not statistically significant, EAEIO demonstrated a larger average zone, suggesting comparable efficacy. The minimum inhibitory concentrations (MICs) provide an additional metric to gauge the antibacterial potency of EAEIO (Table 4, Figure 4). The significance of this thread. Lower MIC values for EAEIO, compared to Chloramphenicol, particularly against Bacillus and Pseudomonas, suggest a higher potency of the extract. This result further underscores the potential application of EAEIO in combating bacterial infections. However, further studies are needed to unravel the specific mechanisms through which EAEIO exerts its antibacterial effects.
In the MTT assay, our study further explored the anticancer potential of EAEIO, particularly against MCF-7 breast cancer and Hct-16 colon cancer cell lines. The observed cytotoxicity, as indicated by IC50 values, supports previous findings on the antitumor properties of I. obliquus extractsMa et al., (2013;Youn et al., (2008). Interestingly, the higher IC50 against the BHK non-cancerous cell line might hint towards a selective cytotoxic action of EAEIO, a desirable attribute in cancer therapies to minimize damage to healthy cells Fulda, (2010).

CONCLUSION
This study's findings reveal the diverse mycochemical composition of the ethyl acetate extract of Inonotus obliquus (EAEIO), its considerable antibacterial activity, and its cytotoxicity against two human cancer cell lines. The potential therapeutic advantages of EAEIO, as suggested by these results, underline the importance of continued research into its bioactive compounds and mechanisms of action. Such studies could further clarify the potential applications of EAEIO in both antibacterial therapies and cancer treatment.
In summary, our findings substantiate the potent bioactivity of EAEIO, highlighting its potential as a source of novel antimicrobial and anticancer agents. Future investigations could help elucidate the mechanisms underlying these bioactivities and potentially contribute to the development of new therapeutic strategies based on I. obliquus bioactive compounds.