Effect of bioactive extracts with high cytokinin content from micelial biomass of Hericium coralloides and Fomitopsis officinalis on tumor cells in vitro

Phytohormones cytokinins are known to promote cell division in plants. Contrary, in animal's and human's tissues they induce apoptosis and block the cell cycle of a wide spectrum of tumour cells. Therapeutic effects of cytokinins, specifically their anticancer and immunomodulatory actions are similar to those of medicinal mushrooms. We detected cytokinins in mycelial biomass of two species of medicinal mushrooms growing in vitro (Fomitopsis officinalis strain 5004 and Hericium coralloides strain 2332) using HPLC-MS. Trans-zeatin, zeatin riboside, zeatin-O-glucoside and isopentenyladenine were found. Crude extracts and purified cytokinin fractions from mycelial biomass were tested on the growth and development of cultures of tumor cells lines: Hela (MTT-assay), T24/83 (viability and level apoptotic cells) and HepG2 (consumption of glucose). The effect of cytokinin fraction from mycelial biomass of Fomitopsis officinalis on pathogenic cells was higher compared to Hericium coralloides one. The data obtained revealed a higher cytotoxic/cytostatic effect of the purified cytokinin fractions in comparison with crude methanolic extracts; also higher apoptotic index was found. Under the influence of the test agents the intensification of glucose uptake into cells was observed. This indicator was higher for crude mushroom mycelium extracts, whereas under the action of purified fractions the glucose uptake rate was lower, thus decreased glycolysis level was recorded. Also, the effect of both crude extract and purified fraction from H. coralloides mycelial biomass on glucose uptake in the conditioned medium was lower against F. officinalis.These results confirm the assumption that biologically active substances of medicinal mushrooms with high pharmacological potential include cytokinins.

Introduction. Deadly side effects of artificially synthesized drugs can be avoided only by means of natural preparations or those that are as close as possible to them in composition and structure of substances. Therefore, the urgent task of today is to find alternative therapies using natural biological raw materials. Macromycetes have enormous potential in this regard. Medicinal mushrooms have a wide range of medicinal properties. They exhibit more than 130 therapeutic effects due to the content of biologically active substances in fruiting bodies and cultured mycelium that enhance innate and acquired immune responses and demonstrate antitumor activity in animals and humans [1,2]. Polysaccharides and terpenoids are the most studied among them. However, since the systematic study of the biochemical composition of mushrooms, the physiological and medical action of its components has begun recently, the list of such compounds is not comprehensive. Most likely, the medicinal properties of mushrooms are determined by the presence of a complex of compounds that act jointly, enhancing the effect of each other [3]. The active substances of medicinal mushrooms probably include phytohormones, in particular, cytokinins, whose therapeutic effect has been detected in the last decade [4]. Cytokinins are polyfunctional hormones of plants that are involved in regulating of their growth and development in many aspects, in particular, positively regulating cell division [5]. At the same time, in animal cell culture the addition of cytokinins is known to have an opposite effect. Thus, cytokinin analogues have been found to block cell cycle patern and inhibit the growth of many types of human cancers [6,7]. The therapeutic properties of cytokinins were similar to those shown by medicinal mushrooms. The ability to produce cytokinins is inherent in both fruiting bodies [8] and mycelial culture [9] of many medicinal mushroom species. Therefore, it can be assumed that the medicinal properties of mushrooms depend on the cytokinins synthesized in their cells in combination with fungal specific metabolites. However, medical cytokinins testing has only recently begun, and detailed information is lacking today.
Species of basidiomycetes, which have been used in traditional Chinese and European medicine for many centuries, include the honeysuckle coral-like Hericium coralloides and the larch sponge Fomitopsis officinalis [10]. These are tree-destructive species with a large fruiting body that cause wood rot. In H. coralloides, they contain a complex of substances that are used as antidepressants, antioxidants, in the treatment of Alzheimer's and Parkinson's diseases and a number of cancers, to fight insomnia and impotence, to reduce blood cholesterol, etc. [11]. Extracts from F. officinalis fruiting body have a wide range of antimicrobial and antiviral activity due to the content of coumarins and triterpenoids, but the crude extract exhibits the highest activity, in particular against Mycobacterium tuberculosis and Yersinia pseudotuberculosis [12].
A necessary step to determine the nature of the biologically active substances of medicinal mushrooms is to investigate the effect of cytokinins produced by them on the growth and development of pathogenic cells.
Mycelial biomass was separated from the culture medium through filtration under vacuum followed by a double rinsing with 50 ml of potassium-phosphate buffered saline, pH 6.5.

Cytokinins purification
The sample (10 g of mycelial biomass) was homogenized during 3 min using an electrical homogenizer (Mechanika Precyzyjna, Poland) in 80 % methanol solution. Cytokinins were extracted with 80 % methanol (10 ml per 1 g) thrice during 24 h at +4 o C. The obtained extract was evaporated under vacuum using the rotor evaporator (Unipan, Poland) at +50 o C to a water phase state. Water residue was kept for 24 h in a freezer at -20 o C. After a fast defrosting, it was centrifuged (pH 2.5, 15000 g, +4 o C) for 30 min in the K 24 centrifuge (Janetzky, Germany). Supernatant was fractionated with n-butanol (1:1 by volume) at pH 8 and purified using the ion-exchange chromatography on column 20х2 cm (Bio-Rad, USA) with Dowex 50Wx8 (Serva, Germany) in H + form, elution with 0.1 N ammonia. Eluate was evaporated under vacuum to a dry residue, which was dissolved in 1 ml of 96 % ethanol and applied on thin layer chromatography plates Silicagel 60 F254 (Merk, Germany), run in solvent system isopropanol:ammonia:water (10:1:1 by volume).
3. HPLC/MS analysis Detection and quantification of cytokinins were performed using the HPLC-MS system (Agilent 1200, USA). Solid samples were dissolved in 200 µl of mobile phase and 5µl aliquot was injected into Agilent Zorbax Eclipse XDB-C18 column (4.6x250 mm, 5 μm). The column was eluted with an isocratic solvents system methanol:water:acetic acid (37:62.9:0.1 by volume) at a flow rate of 0.5 ml/min and column temperature of +30°C. The fractions eluted were directly passed through the mass spectrometer (Agilent 6120 Quadrupole LC/MS) in a combined regime "multi mode" (electrospray and chemical ionization at atmosphere pressure) of positive ionization. Data were analysed and processed using the software Agilent Chem-Station, version В.03.01 online. Concentrations were calculated based on the peak areas for the endogenous compounds relative to those determined for the internal standards. All experiments were carried out in three biological replicates. HPLC-MS analysis was done in five analytical replications. The data was processed by standard methods of variation statistics using Microsoft Excel 2007 program. Values of P < 0.05 were considered to be significant.

MTT-assay.
For in vitro screening of the epithelial line of Hela cells (human cervical carcinoma) has been employed. Cell lines were maintained in a RPMI medium (Sigma, USA) supplemented with 10% FBS, 2 mМ L-glutamine and Penicillin-Streptomycin solution at a final concentration of 50-100 I.U./ml penicillin and 50-100 µg/ml streptomycin. After cell inoculation into 96 well microtiter plates (Falcon, USA), the cells were incubated at 37 o C, 5% of CO2, 95% air and 100% relative humidity for 24 h prior to addition of test compounds. For a typical screening experiment, cells were cultured in medium containing H. coralloides and F. officinalis extracts with the growing concentration from 0.0156 to 500 mkg in 100 µL within 24 hrs. The initial plating density of cells was about 5 × 10 4 cells/mL in 100 µL plate volume. The test agents were added to cells in 100 μL of medium, and cells were cultured for 24 hrs. It was used cells at the about 70-80 % confluent growth. Proliferative parameters of the cultivated cells were defined as it was described elsewhere [14] with the MTT-colorimetric method. The biochemical essence of this method is based on the fact that mitochondrial dehydrogenases of living cells are capable to cleave tetrazolium rings with formation of insoluble purple crystals (formazan). MTT (20 µl) was added to the culture medium 4 h before the termination of the cells incubation in order to achieve the final concentration of 0.6 mM. Formazan crystals formed after the incubation with MTT were dissolved in 100 µl of dimethylsulfoxide. The plate was analyzed on the spectrophotometer at 540 nm. 5. Apoptotic and necrotic cells was determined in wells counts were performed using a trypan blue (necrotic cells) and by cytofluorimetry assay (apoptotic cells) dye after incubation with agents in concentration 0,08 mg/ml as described previously [15]. For them used cells line T24/83 (human bladder tumor).
6. Determination of glucose by glucose-oxidase method.
Determining the level of glucose in the incubation medium of HepG2 cells (human hepatocellular carcinoma) was performed using a standard set based on glucoseoxidase reaction which we modified for culture medium of cells. Initial cell concentration was about 1×10 5 cells/ml in the sample volume of 200 µl. Determination was performed according to the protocol of the manufacturer "Felicit-Diagnostics" (Ukraine) [16].

Results and Discussion.
Analysis of mycelial biomass of two species of medicinal macromycetes resulted in finding the main cytokinins, the presence of which is characteristic of most plantstrans-zeatin zeatinriboside, zeatin-O-glucoside and isopentenyladenine (Table 1). In H. coralloides 2332, the content of free cytokinin -trans-zeatin -was the highest. In F. officinalis 5004, mycelial biomass was characterized by a high concentration of the bound form, zeatin-O-glucoside. The total cytokinin hormone content in H. coralloides was almost four times higher than that of F. officinalis. Notes.*t-Z -trans-zeatin, ZR -zeatin riboside; iPa -isopentenyladenosine, iP -isopentenyladenine, ZOG -zeatin-О-glucoside Modern science considers mushrooms as producers of a wide range of compounds that can affect multiple processes in the human body synergistically, so it is important to study the combinations of molecules in fungal extracts [17]. We have established the ability of mycelial biomass of two species of fungi with a high pharmacological potential to produce cytokinins in large quantities. Comparison of the spectra of the pharmacological properties of medicinal mushrooms and cytokinins suggests that cytokinins are one of the components providing the therapeutic effect of macromycetes. In this regard, cytokinin-containing mycelial biomass fractions of H. coralloides and F. officinalis were tested on cultures tumor cells lines: Hela (MTT-assay), T24/83 (viability and level apoptotic cells) and HepG2 (consumption of glucose) The crude methanol extracts and cytokinin fractions, purified to the stage of ion exchange chromatography inclusive, were investigated.

Mushrooms species t-Z
The cytotoxic/cytostatic or proliferative effect (cell viability) was estimated as a percent of live cells in comparison against control and expressed in term of median growth inhibition (GI50, the compound's concentration that causes 50 % decrease in the net cell growth or mitogenic stimulation vs control). Higher activity was found for purified cytokinin fractions than for crude methanol extracts. The inhibitory effect for both mushroom species after cytokinin fractions treatment was in the range of 0,04-0,08 mg/ml, whereas IC50 was not determined for crude methanol extract due to large deviations in parallel measurements ( Fig. 2 and Fig. 3).  For metabolic pathways studies we used usually Hep G2 cells. As can be seen from the data under the influence of the test agents the intensification of glucose uptake into cells was observed. This indicator was higher for crude mushroom mycelium extracts, whereas under the action of purified fractions the glucose uptake rate was lower ( Table 2). The effect of both crude extract and purified fraction from H. coralloides mycelial biomass on glucose uptake in the conditioned medium was lower against F. officinalis.  Cytokinins are known to change morphology and disorganize actin cytoskeleton of bladder carcinoma T24 cells, block DNA synthesis and increase the level of cyclinedependent kinase inhibitor and induce genes involved in a negative regulation of the cell cycle in tumour cells of epithelium [18][19][20]. Cytokinins also inhibit the human enterovirus replication, show immunostimulating effects promoting proliferation of natural killer cells, provoke apoptosis in myeloid leukemia HL-60 cells [21][22][23]. Thus, сomparing the data obtained with the literature, we can reasonably assume that the inhibitory effect of fungal extracts on tumor cells is associated with the presence of cytokinins in them. Moreover, the cytokinin fraction from the F. officinalis mycelial biomass exhibited greater activity compared to H. coralloides one despite the higher cytokinin concentration in it. It has previously been found that aqueous and ethanol fungal extracts from F. officinalis exhibit growthinhibition effect on different cancer cell lines (mouse sarcoma, human hepatoma, lung cancer, colon cancer and breast cancer) [24], whereas H. coralloides has another medicinal properties. Perhaps, cytokinins act in complex with different compounds in these mushrooms species.
Conclusion. In the present study the influence of extracts from two species of medicinal Basidiomycetes mycelial biomass on tumor cells of different origin was examined. The data obtained revealed a higher cytotoxic/cytostatic effect of the purified cytokinin fractions in comparison with crude methanolic extracts; also higher apoptotic index and decreased glycolysis were recorded. These results confirm the assumption that biologically active substances of medicinal mushrooms with high pharmacological potential include cytokinins.