Chemical characterization and biological activity of Chaga ( Inonotus obliquus ), a medicinal “mushroom”

Chemical composition and biological properties of aqueous and ethanolic extracts of Inonotus obliquus (Pers.:Fr.) Pilat from different origins, i.e. of Finland, Russia, and Thailand, were studied. Concerning biological activity, antimicrobial, antiqourum, antioxidative, and antitumor and cytotoxic effects were tested. Oxalic acid was found as the main organic acid, with the highest amount in Russian aqueous extract. Gallic, protocatechuic and p-hydroxybenzoic acids were detected in all samples. I. obliquus extracts showed high antioxidant and antimicrobial activity. Extracts were tested at subMIC for anti-quorum sensing (AQS) activity in Pseudomonas aeruginosa and all of them showed definite AQS activity. The assays were done using twitching and swarming of bacterial cultures, as well as the amount of produced pyocyanin as QS parameters. All the extracts possessed good antitumor effect on four cells lines and did not show hepatotoxicity on porcine liver primary cells designed as PLP2. As the I. obliquus presence in Chaga conks is limited, further purification is necessary to draw quantitative conclusions. The presence of AQS activity in medicinal mushrooms suggests a broader anti-infectious disease protection than only immunomodulatory effects.


Introduction
In the present study we searched for comparable bioactive effects in extracts prepared from the well known medicinal mushroom Inonotus obliquus (Pers.:Fr.) Pilat, commonly known as Chaga. This mushroom is the hyperplastic conk consisting of wood and mycelium that occurs on Birch after infection and invasive growth of the fungus. Extracts of Chaga have been used in China, Korea, Russia and the Baltics for their presumed antibacterial, hepatoprotective, anti-inflammatory, antitumor, and antioxidant activities (Lemieszek et al. 2012). In vitro, the properties of Chaga were found to be non-toxic and mostly antiviral (Ahwad Ali et al. 2003), antioxidative (Nakajima et al. 2007) and antiinflammatory (Kim et al. 2007). Song et al (2007) found opposite results showing that its extract was both toxic and proinflammatory in macrophages in vitro. Artificially induced acute colitis in mice could be cured by oral administration of Chaga extract (Mishra et al. 2012), suggesting suppression of inflammatory cytokines. No clear antibiotic effects were reported in the scientific literature for Chaga until now.
Quorum sensing (QS) is a communication system between individuals or individual cells that allows recognizing and reacting to the size of the surrounding population. Among QS controlled activities of bacteria, coordination of gene expression will allow different forms of defense against competitors, and of adaptation to changing environments (Ward et al. 2012).
Bacteria as Pseudomonas aeruginosa use small diffusible signaling molecules that regulate expression of various genes including those for swarming motility, virulence, cell aggregation and production of polysaccharides that protect bacterial biofilms (Danchin et al. 2004, Rasmussen and Givskov, 2006, Fiqua et al. 2001. At least 65 % of all the infectious diseases are associated with bacterial biofilms (Fiqua et al. 2001). Bacteria apply QS based 4 mechanisms for their protection against antibiotics and are able to rapidly transfer antibiotic resistance throughout their own species as well as to different species. Each year in the United States, at least 2 million people become infected with bacteria that are resistant to antibiotics and at least 23000 people die each year as a direct result of these infections (CDC, 2013). Clostridium difficile, carbapenem-resistant Enterobacteriaceae (CRE) and drugresistant Neisseria gonorrhoeae were considered the most urgent threats, and multidrugresistant Acinetobacter, drug-resistant Campylobacter, extended spectrum β-lactamase producing Enterobacteriaceae (ESBLs), multidrug-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA) and drug-resistant tuberculosis were also among serious threats (CDC, 2013). The rapid emergence of multiple resistances against the present broad spectrum antibiotics used for infectious disease needs immediately search for alternatives. Non-toxic natural products that can inhibit microbial quorum sensing and thereby stop the appearance of new antibiotic resistant bacterial strains could be such an alternative.
Recently we have described direct antibiotic effects as well as anti-QS properties of extracts of the well known medicinal mushroom Agaricus blazei Murill (ABM) against various bacteria (Stojkovic et al. 2014). Based on its immunomodulatory effects, ABM was previously found to show protective activity against bacterial disease (Rumbaugh et al. 2009, Coates et al. 2002, CDC, 2013, Stojkovic et al. 2014. Antimicrobial effects against Mycobacterium tuberculosis, M. bovis and Streptococcus pneumoniae had been related with the immune system (Coates et al. 2002, Stojkovic et al. 2014, but this was contradicted by the study of Fantuzzi et al. (2011) who described that ABM extract did not promote immunostimulation and protection during experimental Salmonella enterica infection in 5 mice. The finding of Fantuzzi et al. (2011) was a good reason for us to look for possible anti-QS properties of ABM extracts. MIC's and MBC's of these extracts turned out equal to or better for inactivation of P. aeruginosa than those of ampicillin and streptomycin. The effect was caused by anti-QS compounds present in the extracts of Agaricus blazei (Sokovic et al., 2014).
In the present work, we report the chemical composition, antioxidant, antimicrobial, anti-QS and antitumor activities of extracts of I. obliquus wild samples obtained from Russia, Finland and Thailand.

Mushroom material
Wild Inonotus obliquus (Thailand) fruiting bodies were obtained from the Natural Medicinal trypsin-EDTA (ethylenediaminetetraacetic acid), nonessential amino acids solution (2 mM), penicillin/streptomycin solution (100 U/mL and 100 mg/mL, respectively), DMEM media was from Hyclone (Logan, Utah, USA). Methanol and all other chemicals and solvents were of analytical grade and purchased from common sources. Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA).

Extracts preparation
Hot aqueous extraction: The material of Inonotus obliquus conks was ground to a fine powder with a grinder. The dry powder (25 g/0.5 l water) was heated for 2 h at 80°C. The extract was centrifuged at 3000 rpm for 15 min and filtered through Whatman no. 4 filter paper; upon which the filtrate was concentrated in an evaporator at 40 °C (rotary evaporator Buchi R-210, Flawil, Switzerland) to dryness (Hu et al. 2009). Ethanolic extraction: The residue from hot aqueous extraction was extracted in 70 % ethanol at 70°C temperature overnight. The extract was then centrifuged at 3000 rpm for 15 min and filtered through the 7 Whatman no. 4 filter paper and dried by a rotary evaporator (Buchi R-210, Flawil, Switzerland) under vacuum at 40°C (Hu et al. 2009).

Chemical composition of the extracts
Organic acids. Organic acids were determined by ultra fast liquid chromatography (UFLC, Shimadzu 20A series) coupled with a photodiode array detector (PDA), after dissolving each extract in metaphosphoric acid (4%), at a known concentration (Barros et al., 2013). The organic acids were quantified by the comparison of the area of their peaks recorded at 215 nm with calibration curves obtained from commercial standards of each compound. The results were expressed in mg per g of extract.
Phenolic compounds. Phenolic acids and related compounds were determined using the UFLC mentioned above (Barros et al., 2009), after dissolving the aqueous extract in water and ethanolic extract in 20% aqueous ethanol, at a known concentration. Detection was carried out in a photodiode array detector (PDA), using 280 nm as the preferred wavelength.
The phenolic acids and related compounds were quantified by comparison of the area of their peaks with calibration curves obtained from commercial standards of each compound. The results were expressed in mg per g of extract.

Evaluation of the antioxidant potential of the extracts
General. Successive dilutions were made from the stock solution of aqueous and ethanolic extracts in water and ethanol, respectively and further submitted to different in vitro assays to evaluate the antioxidant activity of the samples (Reis et al. 2012). The sample concentrations providing 50% of antioxidant activity or 0.5 of absorbance (EC 50 ) were calculated from the 8 graphs of antioxidant activity percentages (DPPH, β-carotene/linoleate and TBARS assays) or absorbance at 690 nm (ferricyanide/Prussian blue assay) against sample concentrations.
Trolox was used as standard.
Folin-Ciocalteu assay. The extract solution (1 mL) was mixed with Folin-Ciocalteu reagent (2.5 mL, previously diluted with water 1:10, v/v) and sodium carbonate (75 g/L, 2 mL). The tubes were vortex mixed for 15 s and allowed to stand for 30 min at 40°C for colour development. Absorbance was then measured at 765 nm (Analytikjena spectrophotometer; Jena, Germany). Gallic acid was used to obtain the standard curve and the reduction of Folin-Ciocalteu reagent by the samples was expressed as mg of gallic acid equivalents (GAE) per g of extract.
Reducing power or ferricyanide/Prussian blue assay. The extract solutions with different concentrations (0.5 mL) were mixed with sodium phosphate buffer (200 mmol/L, pH 6.6, 0.5 mL) and potassium ferricyanide (1% w/v, 0.5 mL). The mixture was incubated at 50°C for 20 min, and trichloroacetic acid (10% w/v, 0.5 mL) was added. The mixture (0.8 mL) was poured in the 48 wells plate, the same with deionised water (0.8 mL) and ferric chloride (0.1% w/v, 0.16 mL), and the absorbance was measured at 690 nm in ELX800 Microplate Reader (Bio-Tek Instruments, Inc; Winooski, VT, USA).
DPPH radical-scavenging activity assay. The reaction mixture was made in a 96 wells plate and consisted of 30 µL of a concentration range of the extract and 270 µL methanol containing DPPH radicals (6×10 -5 mol/L). The mixture was left to stand for 1 h in the dark, and the absorption was measured at 515 nm in ELX800 Microplate Reader. The radical scavenging activity (RSA) was calculated as a percentage of DPPH discolouration using the 9 equation: % RSA = [(A DPPH -A S )/A DPPH ] × 100, where A S is the absorbance of the solution containing the sample and A DPPH is the absorbance of the DPPH solution.
Inhibition of β-carotene bleaching or β-carotene/linoleate assay. A solution of β-carotene was prepared by dissolving β-carotene (2 mg) in chloroform (10 mL). Two millilitres of this solution were pipetted into a round-bottom flask. The chloroform was removed at 40ºC under vacuum and linoleic acid (40 mg), Tween 80 emulsifier (400 mg), and distilled water (100 mL) were added to the flask with vigorous shaking. Aliquots (4.8 mL) of this emulsion were transferred into test tubes containing 0.2 mL of a concentration range of the extract. The tubes were shaken and incubated at 50ºC in a water bath. As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm. β-Carotene bleaching inhibition was calculated using the following equation: Absorbance after 2h of assay/initial absorbance) × 100.
Thiobarbituric acid reactive substances (TBARS) assay. Porcine (Sus scrofa) brains were obtained from official slaughtering animals, dissected, and homogenized with a Polytron in ice cold Tris-HCl buffer (20 mM, pH 7.4) to produce a 1:2 w/v brain tissue homogenate which was centrifuged at 3000g for10 min. An aliquot (100 µL) of the supernatant was incubated with 200 µL samples of a concentration range of the extract in the presence of FeSO 4 (10 mM; 100 µL) and ascorbic acid (0.1 mM; 100 µL) at 37ºC for 1 h. The reaction was stopped by the addition of trichloroacetic acid (28% w/v, 500 µL), followed by thiobarbituric acid (TBA, 2%, w/v, 380 µL), and the mixture was then heated at 80ºC for 20 min. After centrifugation at 3000 g for 10 min to remove the precipitated protein, the color intensity of the malondialdehyde (MDA)-TBA complex in the supernatant was measured by its absorbance at 532 nm. The inhibition ratio (%) was calculated using the following formula: Inhibition ratio (%) = [(A -B)/A] × 100%, where A and B were the absorbance of the control and the sample solution, respectively.
The cultures were stored at +4°C and subcultured once a month. (v/v) and spore suspension was adjusted with sterile saline to a concentration of 1.0×10 5 .
Ethanolic and aqueous extracts of I. obliquus from Russia, Finland and Thailand were dissolved in 5% DMSO solution containing 0.1% Tween 80 (v/v) (10 mg/mL) and immediately added in broth Malt medium with inoculum (0.2-6.0 mg/mL). The lowest concentrations without visible growth (at the binocular microscope) were defined as MICs.
The fungicidal concentrations (MFCs) were determined by serial subcultivation of 2 µL of tested compounds dissolved in medium and incubated for 72 h at 28°C. The lowest concentration with no visible growth was defined as MFC indicating 99.5% killing of the original inoculum. DMSO was used as a negative control, and commercial fungicides, bifonazole (Srbolek, Belgrade, Serbia) and ketoconazole (Zorkapharma, Šabac, Serbia), were used as positive controls (1-3000 µg/mL). Five percent DMSO was used as a negative control.

Antiquorum sensing (AQ) activity of the extracts
Bacterial strains, growth media and culture conditions. P. aeruginosa PA01 (ATCC 27853) used in this study is from the collection of the Mycoteca, Institute for Biological Research "Sinisa Stankovic", Belgrade, Serbia. Bacteria were routinely grown in Luria-Bertani (LB) medium (1% w/v NaCl, 1% w/v Tryptone, 0.5% w/v yeast extract) with shaking (220 rpm) and cultured at 37 °C.

Twitching and flagella motility
After growth in the presence or absence of ethanolic and aqueous extracts of I. obliquus

Inhibition of synthesis of Pseudomonas aeruginosa PA01 Pyocyanin
Overnight culture of P. aeruginosa PA01 was diluted to OD 600 nm 0.2. Then, ethanolic and aqueous extracts of I. obliquus were dissolved in 5% of DMSO (0.25-0.75 mg/mL), added to P. aeruginosa (5.00 mL) and incubated at 37 °C for 24 h. The treated culture was extracted with chloroform (3 mL), followed by mixing the chloroform layer with 0.2 M HCl (1 mL).
Absorbance of the extracted organic layer was measured at 520 nm using a Shimadzu UV1601 spectrophotometer (Kyoto, Japan) (Sandy and Foong-Yee, 2012). The experiment was done in triplicate and repeated two times. The values were expressed as ratio (OD 520 /OD 600 ) x 100.

Cytotoxicity of the extracts for tumor cell lines and non-tumor liver cells primary cultures
The aqueous and ethanolic extracts were re-dissolved in water at 8 mg/mL to obtain a more efficient dissolution. Successive dilutions were made from the stock solution and tested 14 against five human tumor cell lines: MCF-7 (breast carcinoma), NCI-H460 (non-small cell lung cancer), HeLa (cervical carcinoma) and HepG2 (hepatocellular carcinoma). Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium containing 10% heatinactivated FBS (MCF-7, NCI-H460, HeLa and HepG 2 ) and 2 mM glutamine (, at 37ºC, in a humidified air incubator containing 5% CO 2 . Each cell line was plated at an appropriate density (7.5×10 3 cells/well for MCF-7 and NCI-H460 or 1.0×10 4 cells/well for HeLa and HepG 2 ) in 96-well plates and allowed to attach for 24 h. Cells were then treated for 48h with the diluted sample solutions. Following this incubation period, the adherent cells were fixed by adding cold 10% trichloroacetic acid (TCA, 100 µL) and incubated for 60 min at 4 ºC.
Plates were then washed with deionized water and dried; sulforhodamine B solution (0.1% in 1% acetic acid, 100 µL) was then added to each plate well and incubated for 30 min at room temperature. Unbound SRB was removed by washing with 1% acetic acid. Plates were air dried, the bound SRB was solubilised with 10 mM Tris (200 µl, pH 7.4) and the absorbance was measured at 540 nm (Monks et al., 1991) in the microplate reader mentioned above. The results were expressed in GI 50 values (sample concentration that inhibited 50% of the net cell growth). Ellipticine was used as positive control.
For hepatotoxicity evaluation, a cell culture was prepared from a freshly harvested porcine liver obtained from a local slaughter house. It was designed as PLP2. Briefly, the liver tissues were rinsed in Hank's balanced salt solution containing 100 U/mL penicillin and 100 µg/mL streptomycin and divided into 1×1 mm 3 explants. Some of these explants were placed in 25 cm 2 tissue flasks in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM nonessential amino acids and 100 U/mL penicillin, 100 mg/mL streptomycin and incubated at 37ºC with a humidified atmosphere containing 5% CO 2 . The medium was changed every 2 days. Cultivation of the cells was continued with direct monitoring every 2-3 days using a phase contrast microscope. Before confluence, cells were sub-cultured and plated in 96-well plates at a density of 1.0×10 4 cells/well, and cultivated in DMEM medium with 10% FBS, 100 U/mL penicillin and 100 µg /mL streptomycin (Abreu et al., 2011). Cells were treated for 48h with the different diluted sample solutions and the same procedure described in the previous section for Sulforhodamine B colorimetric (SRB) assay was followed. The results were expressed in GI 50 values (sample concentration that inhibited 50% of the net cell growth). Ellipticine was used as positive control.

Statistical analysis
For each species, three samples were used and all the assays were carried out in triplicate.
The results were expressed as mean values and standard errors, and analyzed using one-way analysis of variance (ANOVA) followed by Tukey's HSD Test with α = 0.05. This analysis was carried out using SPSS v. 20.0 program.

Chemical characterization, antioxidant and antimicrobial effects I. obliquus extracts
The yields of ethanolic extracts were 1, 2.4 and 1.3 g for samples from Russia, Finland and Thailand, respectively, while of, aqueous extracts were 2, 5.5. and 0.5 g for the same samples, in all cases starting from 25 g of dry powder.
Oxalic acid was the only organic acid detected in the extracts (6.72-97.59 mg/g extract). The phenolic acids found were gallic, protocatechuic and p-hydroxybenzoic acids, as also the related compound cinnamic acid (Table 1). Gallic acid was only found in both, aqueous and ethanolic, extracts from Thailand (0.32 and 0.20 mg/g extract), and protocatechuic acid was detected in all samples except the aqueous extract from Finland (0.07-0.94 mg/g extract). p-Hydroxybenzoic acid was found in all samples (0.43-59.20 mg/g extract) and the related compound cinnamic acid was detected in all samples except the aqueous extract from Thailand (0.03-0.40 mg/g extract).
Regarding the antioxidant potential ( Table 2), it might be concluded that the ethanolic extract of I. obliquus from Thailand revealed the lowest EC 50 values for Ferrocyanide/Prussian blue (0.07 mg/mL), DPPH radical-scavenging activity (0.23 mg/mL), β-carotene bleaching inhibition (0.13 mg/mL) as well as for TBARS inhibition (0.08 mg/mL) assays. I. obliquus proved to have high potential for antioxidant purposes, since the obtained EC 50 values were lower than those reported for other wild edible species (Pereira et al., 2012). In previous study of antioxidant activity of mycelia of I. obliquus (Debnath et al., 2013), the scavenging activity of water, ethanol and methanol extracts on DPPH's were determined, and it was concentration dependent. The scavenging activities were ranged from 12.50-200 mg/mL, respectively. Therefore, the all extracts showed higher scavenging activity on DPPH• radical.
The IC50 (concentration at which 50% of DPPH•s are scavenged) were 18.96, 16.25 and 24.90 mg/mL for the water, ethanol and methanol extracts, respectively.
The results for antibacterial activity of ethanol and aqueous extracts of I. obliquus are presented in Table 3. The ethanolic extracts of I. obliquus from Russia, Finland and Thailand exhibited inhibitory effects at 0.30-3.00 mg/mL, 0.75-3.00 mg/mL, 0.50-2.25 mg/mL, respectively. A bactericidal effect was achieved at 1.50-6.00 mg/mL, 1.50-6.00 mg/mL and 0.75-3.00 mg/mL, respectively. The highest antibacterial effect was observed for ethanolic extract obtained from Thailand. The aqueous extracts showed inhibitory activity at 0.40-3.00 mg/mL, 0.75-3.75 mg/mL and 0.75-3.00 mg/mL from each one of the mentioned origins, and bactericidal activity at 1.50-6.00 mg/mL, 1.50-7.50 mg/mL and 1.50-6.00 mg/mL. The sample from Russia exhibited the highest antibacterial activity. The most sensitive bacteria to the extracts were S. aureus and B. cereus, while the most resistant ones were L.
monocytogenes, E. coli and E. cloacae. All tested extracts exhibited lower antibacterial activity than the control antibiotics, with exception of ethanolic extract from Thailand against P. aeruginosa (Table 3).
Regarding the antifungal activity (Table 4), the results were quite similar. All tested extracts showed again considerable antifungal effects on all tested fungi. Inhibitory effect of ethanolic extracts prepared with samples from Russia, Finland and Thailand was 0.40-1.50 mg/mL, 0.75-1.50 mg/mL and 0.40-1.50 mg/mL, respectively. A fungicidal effect was achieved at 0.75-3.00 mg/mL, 1.50-3.00 mg/mL and 0.75-.3.00 mg/mL, respectively. Aqueous extracts of the tested samples showed inhibition at 0.20-0.75 mg/mL, 0.20-0.75 mg/mL and 0.20-1.50 mg/mL, respectively. A fungicidal activity of these extracts was observed at 0.75-3.00 mg/mL, 0.40-1.50 mg/mL and 0.40-3.00 mg/mL, respectively. The most susceptible fungus was T. viride, while the most resistant were A. niger and A. fumigatus. The antifungal effect of tested extracts on P. ochrochloron and T. viride was higher than the one observed for ketoconazole ( Table 4).

Antiquorum sensing activity and cytotoxicity of I. obliquus extracts
Many mechanisms of actions have been proposed to interfere with the quorum sensing system such as inhibition of biosynthesis of autoinducer molecules, inactivation or degradation of the autoinducer, interference with the signal receptor, and inhibition of the genetic regulation system (Rasmussen and Givskov, 2006).
In addition to QS, the initiation of biofilm formation by P. aeruginosa depends on two cellassociated structures; the flagellum and type IV pili (O'Toole and Kolter, 1998a,b). The flagellum is responsible for swarming motility while the type IV pili are responsible for twitching motility (Henrichsen, 1972). Both types of motility are important in the initial stages of biofilm formation by P. aeruginosa (O'Toole and Kolter, 1998a,b). Therefore, we tried to determine if I. obliquus extracts can influence either one or both motilities. On swarming plates, the motile strain PAO1 was used as the 100% standard (control) for motility, while the Petri dishes with the same strain plus ethanol and aqueous extracts of I. obliquus were compared with the control. The normal colonies of P. aeruginosa, i.e. in the absence of extracts, were flat with a rough appearance displaying irregular colony edges (Fig.   1G) and a hazy zone surrounding the colony. The cells grew in a very thin layer. After 2 days of incubation at ambient temperature, colony expansion occurred very rapidly due to twitching motility; the control P. aeruginosa isolates produced swimming zones ( Table 5) of 12.6±1.0 mm. Statistically they all had the same colony size; the color of all treated samples was however different and went from white to light brown. Microscopically it was seen that bacteria grown with EIOR and AIOR extracts had reduced twitching motility. These colonies were incapable of producing such a twitching zone and had almost round, smooth, regular colony edges, the flagella were reduced both in size and in numbers (Fig. 1A, B) and the colony diameter was also reduced in comparison with control (10.7 mm). EIOR caused flatter protrusions than AIOR and the colony had a different color. EIOF extract influenced the colony color (orange) and diameter (10.3 mm), while also the protrusions were flatter. AIOF extract showed slightly lower antiquorum effect than EIOF. Colony color was light brown, diameter was reduced (10.5 mm) but the protrusions was only partly reduced (Fig. 1D). EIOT extract induced light yellow color of colony and diameter was reduced to 11.3 mm, and protrusions were flatter (Fig. 1E). AIOT extract showed lower AQ effect; colony color was white yellow, diameter was 11.6 mm and protrsions partly reduced (Fig. 1F). Streptomycin changed colony color in white, reduced diameter to 5.0 mm, and completely reduced protrusions (Fig. 1H), while Ampicillin changed color in white, and slightly reduced diameter to 12.0 mm and did not affect the formation of protrusions at all (Fig. 1I). It can be seen that extracts from Russia in general showed the highest reduction of protrusions. The best diameter reduction was shown by both Finland extracts, and the highest protrusion reduction was also shown by one of the Finland extracts, EIOF. Thailand extracts possessed the lowest antiqourum activity of the three different extract origins investigated; colony diameter was reduced in low percentage, protrusions were reduced but still wide and merged by EIOT, while AIOT reduced diameter in even lower percentage, protrusions were slightly reduced, still long and wider than AIOF but narrower than the untreated control ( Fig. 1A-I). I think we should do much more to explain what happens here, especially why this is important for the formation of biofiilms. Maybe there are inhibitors of flagellar growth. I can imagine that we start point inoculation and cover a sector of the plate with a new agarlayer after some hours and again later to see what happens. I think we have to make sure that the readers see the importance of preventing swarming. I am not experienced in bacteriology, but I will think of it. Let's discuss.

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The activity against pyocyanin production in a flask assay was used to quantify quorum sensing inhibitory activity of the ethanol and aqueous extracts of I. obliquus. The effect of extracts on production pyocyanin of P. aeruginosa (PAO1) was tested in subMIC concentration (Figure 2). The ethanolic and aqueous extracts of I. obliquus demonstrated concentration-dependent pyocyanin inhibitory activity. The pyocyanin assays revealed that subMIC amounts of extracts produced less (102-141%) pyocyanin than PAO1 (145.00%).
Streptomycin reduced the production of pyocyanin in 70.00%, while ampicillin stimulated production of extracts 1.5 times (215.00%). All tested extracts showed reduction of pyocyanin production but only in low amount. The highest reduction of pyocyanin production could be seen for extracts from Finland, especially for ethanolic extract ( Figure   2).
The results obtained for cytotoxic activity in human tumor cell lines are presented in Table 6.
The lowest GI 50 values were obtained for both Russian extracts in all cell lines except NCI-H460, which proved to be more sensitive to the extracts (80.93-91.20 µg/mL). In summary, our study indicated that aqueous and ethanolic extracts of I. obliquus obtained from three different localities possessed clear antioxidant, antimicrobial and anti-quorum 21 sensing activity, as well as antitumor effects without toxicity for non-tumor liver cells. It should be noted that extracts were prepared from Chaga conks, hyperplastic structures from Birch cortex that consist only partly of fungal material. One of us (LJLDVG) estimated the amount of mycelium in the conks on 10% only, based on microscopical observation. This indicates that further purification of the extracts is needed to draw definite conclusions on the possible anti-QS activity of Chaga and of I. obliquus extracts. Anti-quorum sensing property of this "mushroom" derived material may play an important role in antibacterial activity and could offer an additional strategy for fighting bacterial infection. Inhibition of different kind of bacteria and fungi, and bacterial quorum sensing, but also antitumor effect, offers a new strategy for the using of this mushroom as a medicinal mushroom.