PaPer Ital. J. Food Sci., vol. 27 - 2015 1 - Keywords: anticancer activity, antifungal activity, antioxidant activity, Flammulina velutipes, Se-enriched mycelium - Se effect on biological activity of Flammulina velutipes i. Milovanović1, t. Stanojković2, M. Stajić1*, j. vukojević1 and a. knežević1 1University of Belgrade, Faculty of Biology, Takovska 43, 11000 Belgrade, Serbia 2Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia *Corresponding author: stajicm@bio.bg.ac.rs AbstrAct The goals of the study were evaluation of antioxidant, antifungal and anticancer potential of Flam- mulina velutipes mycelium ethanol extract and examination of Se effect on those activities. Both Se-amended and non-amended mycelium extracts exhibited significant antioxidant and antifungal potential. Se-enriched extract was more effective against Candida krusei and C. albicans and bet- ter DPPH• scavengers than non enriched one. Carriers of the antioxidant activity were phenol com- pounds. Contrary to antioxidant and antifungal potential, tested extracts were many times weaker cytotoxic agents against HeLa and LS174 cell lines than cis-DDP. Thus, Se-enriched mycelium could be supplement with antioxidant and antifungal capacity. 2 Ital. J. Food Sci., vol. 27 - 2015 INtrODUctION A need for new antimicrobial agents exist for a long time due to the emergence of microorgan- ism resistance as a result of the uncontrolled us- age of commercial antibiotics and antimycotics. Preference is given to natural compounds due to their health-beneficiary and environmentally- friendly effect. Besides diseases caused by mi- croorganisms, nowadays, cancer, diabetes, ath- erosclerosis, as well as neurodegenerative disor- ders occur frequently. One of the significant trig- gers for the mentioned diseases and disorders is oxidative stress (LIMÓN-PACHECO and GONSE- BATT, 2009). Since the capacity of cellular anti- oxidant defence is insufficient in some cases and synthetic antioxidants could have toxic and mu- tagenic effects, and as awareness about healthy lifestyle is permanently raised, search for nat- ural antioxidants presents current trend. Now- adays, natural remedies are also favoured for cancer treatment, because of the many side ef- fects of the chemotherapy, such as the same ef- fect on cancer and healthy cells, occurrence of mutations which could pass into future genera- tions, as well as more aggressive metastasis of returned disease. Based on traditional experience and scientific data, numerous mushroom species represent im- portant resources of antimicrobial, antioxidant, and anticancer agents. Although special attention is given to the species of the genera Ganoderma, Lentinus, Trametes, and Pleurotus, other mush- rooms also produce active compounds and their biological activities should be tested (ZHANG et al., 2007; CHEN et al., 2008; SHI et al., 2012). One of them is Flammulina velutipes (Curt. Fr.) Karst, known as Golden needle mushroom, Eno- ki, Enokidake and Enokitake. This species is a fa- mous edible and medicinal mushroom due to syn- thesis of numerous bioactive molecules, such as polysaccharides, proteins, sterols etc. Significant immunomodulating, antitumor, antioxidant, and antiviral potential of its extracts and compounds were reported (BAO et al., 2010; CHANG et al., 2010; YANG et al., 2012). CHANG et al. (2010) demonstrated cytotoxic effect of immunomodu- latory protein FVE against murine hepatocellu- lar carcinoma, and YANG et al. (2012) showed anti-proliferation activity of polysaccharide FVP against human gastric and lung cancer cells. How- ever, antibacterial and especially antifungal activ- ities of the extracts were poorly studied (RODRI- GUES de MELO et al., 2009; WANG et al., 2012). Among essential microelements, Se takes sig- nificant place in animal and human diet, due to its participation in biosynthesis of selenopro- teins and selenoenzymes involved in cell protec- tion against free radicals and indirectly in cell prevention from malignant transformation (RAY- MAN, 2005; BRIGELIUS-FLOHÉ, 2006). Organic Se compounds, depending on form and concentra- tion, also have antimicrobial activity based on ca- talysis of O 2 - production which causes oxidative damage of bacterial cell membrane and cell dis- function (XU et al., 2008). Considering that Se bioavailability from organic forms is better than from inorganic ones, and that mushrooms have ability to absorb inorganic Se and convert it to organic, their usage as food and dietary supple- ments could contribute the prevention of disease appearance (KALAĆ , 2010). However, there are just a few reports about biological activity of Se- enriched mushroom mycelia (MALINOWSKA et al., 2009; TURLO et al., 2010; SHI et al., 2010), and there are no available data for F. velutipes. Considering everything mentioned, the aims of the study were to evaluate antioxidant, antifun- gal and anticancer potential of F. velutipes my- celial extract and examine in which way myceli- um enrichment with Se affects those activities. MAtErIALs AND MEtHODs Organism and cultivation conditions The culture of Flammulina velutipes HAI 966 was obtained from Institute of Evolution, Uni- versity of Haifa, Israel (HAI), and maintained on malt agar medium in the culture collection of the Institute of Botany, Faculty of Biology, University of Belgrade. The inoculum preparation involved the follow- ing steps: (i) inoculation of 100.0 mL of synthet- ic medium (glucose, 10.0 g/L; NH 4 NO 3 , 2.0 g/L; K 2 HPO 4 , 1.0 g/L; NaH 2 PO 4 x H 2 O, 0.4 g/L; MgSO 4 x 7H 2 O, 0.5 g/L; yeast extract, 2.0 g/L; pH 6.5) with 25 mycelial discs (Ø 0.5 cm, from 7-day-old culture from malt agar); (ii) incubation at room temperature (22 ± 2ºC), on a rotary shaker (100 rpm), for 7 days; (iii) washing of obtained biomass (3 times) by sterile distilled water (dH 2 O); (iv) bi- omass homogenization with 100.0 mL of sterile dH 2 O in a laboratory blender. Homogenized inoculum (30.0 mL) was used for inoculation of 400.0 mL modified synthetic medium (with glucose in the amount of 65.0 g/L and peptone in the concentration of 2.0 g/L, pre- viously determined as the optimal carbon and ni- trogen sources and concentrations for biomass production) enriched with sodium selenite (Na- 2 SeO 3 ) in the initial Se concentration of 1.3 mg/L. The medium without Se was used as the control. Submerged cultivation was carried out at room temperature on rotary shaker for 21 days. The ob- tained biomass was filtered, washed 3 times with dH 2 O at magnetic stirrer and temperature of 30°C with the aim of removing the remaining Se from cell wall, and dried at 50°C to constant weight. Preparation of the fungal extracts Dry Se-amended and non-amended myce- lia (3.0 g) were extracted by stirring with 90.0 mL of 96% ethanol at the 30°C for 72 h. The Ital. J. Food Sci., vol. 27 - 2015 3 obtained extracts were filtered through What- man No. 4 filter paper, concentrated under re- duced pressure in a rotary evaporator (BÜCHI R-114, Switzerland) at 40°C to dryness, and re- dissolved in 96% ethanol (for the testing of an- tioxidant activity) or in 5% dimethylsulphoxide (DMSO) (for the analysis of antifungal and an- ticancer activity). Antioxidant activity DPPH assay Antioxidant activity was defined by measuring bleaching of the purple-coloured methanol solu- tion of stable 1,1-diphenyl-2-picryl-hydrazil rad- ical (DPPH•) (BLOIS, 1958). 1800.0 µL of 4% methanol solution of DPPH• and 200.0 µL of ex- tract of defined concentration (series of double dilutions from 32.0 mg/mL to 0.5 mg/mL) were mixed and shaken. After 30 min of incubation in the darkness, the absorbances of reactive mix- tures were measured at 517 nm against methanol as blank by spectrophotometer (CECIL CE2501, U.K.). The negative control contained all the re- action reagents except the extract. Scavenging ef- fects was calculated by equation: DPPH scavenging effect (%) = = [(A 0 - A sample )/A 0 ] x 100, A 0 - the absorbance of the negative control; A sam- ple - the absorbance of reaction mixture. The EC 50 value (mg extract/mL) is the effective concentration at which the DPPH• were scavenged by 50% and was obtained by interpolation from linear regression analysis. Commercial antioxi- dant, butylated hydroxyanisole (BHA) was used as a positive control. Determination of total phenolic content Total soluble phenolic compounds in the eth- anolic extracts of Se-amended and non-amend- ed mycelium were estimated with Folin-Ciocal- teu reagent according to the method of SINGLE- TON and ROSSI (1965), using galic acid as a standard. 1000.0 µL of 10% Folin-Ciocalteu rea- gent and 200.0 µL of the extract were reacted in the dark for 6 min before addition of 800.0 µL of 7,5% Na 2 CO 3 . The reaction mixture was vortexed vigorously and incubated on a rotary shaker (100 rpm) in the dark and at the room temperature for 2 h. The absorbance was measured at 760 nm by spectrophotometer against blank (mixture with- out extract). The total concentration of phenolic compounds in tested extracts determined as µg of galic acid equivalents (GAE) per mg of dry ex- tract, using an equation that was obtained from standard galic acid graph as: Absorbance = 0.012 x total phenols + + 0.041 (R2 = 0.999) Determination of total flavonoid content Total flavonoid content was determined using the methods of PARK et al. (1997). 1000.0 µL of the mycelium extract was diluted with 4300.0 µL mix- ture containing 4100.0 µL of 80% ethanol, 100.0 µL of 10% aluminium nitrate and 100.0 µL of 1 M aqueous potassium acetate. The mixture was in- cubated at room temperature for 40 min, and ab- sorbance was measured spectrophotometrically at 415 nm. The amount of total flavonoids was ex- pressed as µg of quercetine equivalents (QE) per mg of dry extract, using an equation that was ob- tained from standard quercetin hydrate graph as: Absorbance = 0.014 x total flavonoid - 0.072 (R2 = 0.989) Antifungal activity The tested micromycetes (Table 1) are main- tained on Malt agar at 4°C in the culture collec- tion of the Institute of Botany, Faculty of Biology, University of Belgrade. Tested micromycetes were cultivated on Sab- ouraud dextrose agar (SDA) at temperature of 25 ± 2°C for 21 days. Spore suspensions were prepared by washing of agar surface with sterile 0.9% saline containing 0.1% Tween 80 (v/v). Turbidity was deter- mined spectrophotometrically at 530 nm and spore number was adjusted to 106 CFU/mL (NCCLS, 1998). DMSO extracts of Se-amended and non-amend- ed mycelia were sterilized by filtration through Whatman No. 4 filter paper and 0.2 µm mem- brane filter. Antifungal potential of the tested ex- tracts was studied by microdilution method using 96-well microtiter plate (SARKER et al., 2007). Se- ries of double extract dilutions (from 32.0 mg/mL to 0.5 mg/mL) was analyzed. Each well contained SDA, spore suspension, resazurine, and crude eth- anol extract of defined concentration. The mixture without extract was used as the negative control, while positive control contained commercial anti- mycotic, ketoconazole, instead extract. Tested ke- toconazole concentrations ranged from 0.0313 mg/ mL to 0.0019 mg/mL (series of double dilutions). Effect of 5% DMSO on the spore germination was also analysed by its addition in the mixture instead SDA. Microtiter plates were incubated at 25 ± 2°C for 72 h. The lowest extract concentration without visible mycelium growth was defined as minimal inhibitory concentration (MIC). Minimal fungicidal concentration (MFC) was determined as the lowest extract concentration with no mycelial growth after reinoculation of 2 µL of the mixture on SDA. The experiments were repeated three times. Cytotoxic activity Cell lines Human cervix adenocarcinoma HeLa and hu- man colon carcinoma LS174 cell lines were ob- 4 Ital. J. Food Sci., vol. 27 - 2015 tained from the American Type Culture Collec- tion (ATCC) (Manassas, VA, USA). Both cancer cell lines were maintained in the recommended Roswell Park Memorial Institute (RPMI) 1640 me- dium supplemented with 100.0 g/L heat-inactivat- ed (56°C) fetal bovine serum (FBS), 3 mM L-glu- tamine, 100.0 mg/mL streptomycin, 100.0 IU/mL penicillin, and 25 mM 4-(2 hydroxyethyl)-1-pip- erazineethanesulfonic acid (HEPES) and adjust- ed to pH 7.2 with bicarbonate solution. Cells were grown in a humidified atmosphere of 95% air/5% CO 2 (v/v) at 37°C. Treatment of cell lines Stock solutions (100.0 mg/mL) of extracts, made in 50.0 g/L DMSO, were dissolved in en- riched RPMI 1640 medium to the required work- ing concentrations. Neoplastic HeLa cells (2000 cells per well) and neoplastic LS174 cells (7000 cells per well) were seeded into 96-well microtiter plates. 24 h later, after cell adherence, five differ- ent doubly diluted concentrations of the extracts were added to the wells. The final concentrations applied to target cells were 200.0, 100.0, 50.0, 25.0 and 12.5 µg/mL, except in the control wells where only the nutrient medium was added to the cells. The cultures were incubated for 72 h. Determination of cell survival (MTT test) The effect of extracts on cancer cell survival was determined by microculture tetrazolium test (MTT test), according to MOSMANN (1983) with modifi- cation by OHNO and ABE (1991). 20.0 µL of MTT solution [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- nyltetrazolium bromide in phosphate-buffering saline] of concentration of 5.0 mg/mL was added to each well. Samples were incubated for 4 h at 37°C in a humidified atmosphere of 95% air/5% CO 2 (v/v). Then, 100.0 µL of 10% sodium dodecyl sulfate was added to extract to dissolve the insol- uble product formazan resulting from the conver- sion of the MTT dye by viable cells. The number of viable cells in each well was proportional to the in- tensity of the light absorbance (A), which was read in an enzyme-linked immunosorbent assay (ELI- SA) plate reader at 570 nm 24 h later. The inhibi- tion rate was calculated according to the formula: Cell growth inhibition rate (%) = = (A control – A sample )/A control x 100 It was implied that the A of the blank was al- ways subtracted from the A of the corresponding sample with target cells. IC 50 was defined as the concentration of the extracts inhibiting cell surviv- al by 50%, compared with a vehicle-treated con- trol. Cis-diamminedichloroplatinum (cis-DDP) was used as a positive control. All experiments were done in triplicate. Statistical analysis The results were expressed as the mean ± stan- dard error of data obtained from three parallel me- asurements. One-way analysis of variance (ANO- VA) followed by LSD post-hoc determinations were performed using STATISTIKA software, version 5.0 (StatSoft, Inc) to test any significant differen- ces. P-values less then 0.01 were considered sta- tistically significant. Table 1 - Antifungal activity (MIC and MFC) of ethanol extracts of Se-non amended and Se-amended mycelium of Flammulina velutipes and commercial antimycotic. Tested organisms Flammulina velutipes Ketoconazole MIC (mg/mL) MFC (mg/mL) MIC MFC (mg/mL) (mg/mL) Se non-amended Se-amended Se non-amended Se-amended mycelium mycelium mycelium mycelium Acremonium strictum W. Gams 4.0 4.0 - - 0.0078 0.0156 Aspergillus flavus Link 8.0 4.0 - - 0.0078 0.0078 Aspergillus fumigatus Fresen. 32.0 16.0 - - 0.0078 0.0156 Aspergillus niger Teigh. 32.0 16.0 - - 0.0156 0.0313 Aspergillus terreus Thom 16.0 8.0 - - 0.0156 0.0313 Candida albicans (C.P. Robin) Berkhout 8.0 16.0 - - 0.0078 0.0156 Candida krusei (Castell.) Berkhout 8.0 2.0 - - 0.0078 0.0156 Candida parapsilosis (Ashford) Langeron & Talice 16.0 2.0 - - 0.0078 0.0156 Cladosporium sp. - - - - 0.0078 0.0039 Fusarium verticillioides (Sacc.) Nirenberg 8.0 4.0 - - 0.0156 0.0156 Microsporum gypseum (E.Bodin) Guiart & Grigoraki - - - - 0.0019 0.0039 Penicillium funiculosum Thom 8.0 4.0 - - 0.0039 0.0078 Trichoderma viride Pers. 8.0 8.0 - - 0.0039 0.0078 Trichophyton mentagrophytes (C.P. Robin) Sabour. - - - - 0.0019 0.0039 Ital. J. Food Sci., vol. 27 - 2015 5 Fig. 1 - DPPH radical scavenging capacity of ethanol extracts of Flammulina velutipes mycelium. Se non-amended (◻); Se- amended (◾). (Data represent mean value of activities of three different samples. Variations are given as standard errors). rEsULts Antioxidant activity Ethanol extracts of both Se-amended and non- amended F. velutipes mycelium showed good an- tioxidant potential that was dependent on the con- centration, at higher concentrations extracts were more effective in DPPH• scavenging (Fig. 1). How- ever, significant difference between the extracts, especially at higher concentrations, was noted (P<0.01). Se-enriched mycelium extract had high- er scavenging effect than the control, and it exhib- ited a progressive increase of the activity at con- centrations up to 8.0 mg/mL (from 4% to even 15% at concentration of 32.0 mg/mL). These re- sults were confirmed by EC 50 values, which were 30.5 ± 0.3 mg/mL in Se-enriched mycelium ex- tract and 43.8 ± 0.6 mg/mL in non-amended one. Commercial antioxidant BHA was more efficient comparing with F. velutipes mycelium extracts, with EC 50 value of 13.4 µg/mL. Total phenol compounds were detected in both tested extracts. Their content in the non-amend- ed extract was 9.5 ± 1.8 µg/mg, while the high- er concentration of 14.5 ± 1.1 µg/mg was noted in Se-amended extract. However, flavonoids were not detected in any of the extracts. Direct correla- tion between phenol content and DPPH• scaveng- ing effect existed, and linear relationship with R2 = 0.989 in non-amended mycelium extract and R2 = 0.957 in Se-amended one were noted. Antifungal activity The antifungal potential of ethanol extracts of Se-amended and non-amended mycelium was tested against 14 micromycetes including sap- robes as well as plant, animal and human patho- gens. In the most cases, MICs of Se-enriched my- celium extract were lower, except for Candida al- bicans where control extract had twice more ef- fect, and for Acremonium strictum and Trichoder- ma viride where MICs of Se-amended and non- amended extracts were the same (Table 1). The most sensitive species, with MIC of Se-enriched mycelium extract of 2.0 mg/mL, were C. krusei and C. parapsilosis. The most resistant species were Aspergillus fumigatus and A. niger, which growth was inhibited only with the maximum ex- tract concentration (32.0 mg/mL for non-amended and 16.0 mg/mL for Se-amended one). Tested ex- tract concentrations (from 0.5 mg/mL to 32.0 mg/ mL) have no inhibitory effect on Cladosporium sp. and both causal agents of dermatomycosis, Micro- sporium gypseum and Trichophyton mentagro- phytes. The maximum tested concentration of ex- tract (32.0 mg/mL) did not show fungicidal effect for any tested fungal species (Table 1). Sensitivity of the tested species to commercial antimycotic, ketoconazole, was more higher. Thus, the lowest tested concentration of 0.0019 mg/mL was MIC for Cladosporium sp., M. gypseum, and T. mentagrophytes. The concentration of 0.0039 mg/mL was MIC for Penicillium funiculosum and T. viride and MFC for Cladosporium sp., M. gyp- seum, and T. mentagrophytes. Mycelium growth of A. strictum, A. flavus, A. fumigatus, C, albi- cans, C. krusei, and C. parapsilosis was inhibit- ed at ketoconazole concentration of 0.0078 mg/ mL, which was also MFC for A. flavus, P. funicu- losum and T. viride. Concentration of 0.0156 mg/ mL was MIC for A. niger, A. terreus, and Fusar- ium verticillioides and MFC for most of the test- ed species, while the highest tested concentra- table 2 - cytotoxic activity (Ic 50 ) of se-non amended and se-amended Flammulina velutipes mycelium extracts and commer- cial cytostatic against HeLa and Ls 174 cell lines. Mycelial extract/cytostatic IC 50 (µg/mL) HeLa LS 174 Se-non Se-amended Se-non Se-amended amended extracts extracts amended extracts extracts F. velutipes 259.69 ± 0.70 331.91 ± 0.49 338.47 ± 0.97 348.46 ± 0.34 cis-DDP 0.72 ± 0.14 2.61 ± 0.11 6 Ital. J. Food Sci., vol. 27 - 2015 tion (0.0313 mg/mL) was MFC for A. niger and A. terreus (Table 1). 5% DMSO, used as a nega- tive control, had no inhibitory effect on the test- ed micromycetes. Cytotoxic activity Comparing with cis-DDP, which was used as the positive control, the tested extracts showed low cytotoxic activity against both HeLa and LS174 cell line (Table 2). IC 50 of non-amended myceli- um extract against HeLa cells was 360- and 130- fold, respectively, higher than values obtained for cis-DDP. Se-amended mycelium extract was weak- er cytotoxic agent than control one, especially for HeLa cell line. DIscUssION Enrichment of cultivation medium with Se in the initial concentration of 1.3 mg/L and ability of F. velutipes HAI 966 mycelium to absorb and in- corporate it in significant amount (10.0 µg/g) (MI- LOVANOVIĆ et al., 2013) led to enhancement of an- tioxidant and antifungal capacity, while cytotoxic activity was reduced and was not in correlation with antioxidant potential. DPPH• scavenging activity of F. velutipes eth- anol extracts was reported by BAO et al. (2010), who demonstrated higher efficiency of mycelium extract than fruiting body one in almost 4-fold. Ac- cording to SALTARELLI et al. (2009), the activity bases on redox properties of phenols, which ena- ble them to act as reducing agents and hydrogen donators. Direct dependence of antioxidant ca- pacity on phenol content was confirmed by CHEN et al. (2008) and BAO et al. (2010). Lower phenol content in F. velutipes HAI 966 mycelium extracts, compared with that in reported data, was respon- sible for negligible antioxidant potential that was significant only at the highest concentration. Lack of flavonoid in the ethanol extracts was in accord- ance with results of KARAMAN et al. (2009) who also have not found those compounds in myceli- um extracts of F. velutipes. However, other com- pounds from those extracts could also be carriers of antioxidant activity (SHI et al., 2012; WANG et al., 2012). Thus, SHI et al. (2012) demonstrated significant scavenging effects of polysaccharides, even 90% at concentration of 2.5 mg/mL, while WANG et al. (2012) have noted high efficiency of sesquiterpenoids. Enhanced antioxidant potential of Se-amend- ed F. velutipes HAI 966 mycelium extract is in accordance with results of TURLO et al. (2010) who noted higher level of antioxidant activity in Lentinus edodes after cultivation in Se-enriched medium. The better effect could be explained by the fact that selenomolecules react with free rad- icals (•OH, •H) and successfully neutralize them (SHEN et al., 2010). WANG et al. (2012) tested antifungal potential of sesquiterpenoids isolated from F. velutipes myce- lium against numerous species and reported weak activity against A. fumigatus and absence of any inhibitory effect on C. albicans. Similar, negligi- ble activity of ethanol extract of F. velutipes HAI 966 mycelium against A. fumigatus was also not- ed but it was enhanced with Se addition. Howev- er, tested Candida spp. were very sensitive to the extracts, especially in the Se presence, which was contrary to the results of WANG et al. (2012). Re- sults of this study showed that Se acts as an anti- fungal agent which is in accordance with results of SHAHVERDI et al. (2010) who clearly demon- strated antifungal activity of biogenic Se against selected clinical micromycetes. Mycelium enrich- ment with this element presents a way for getting biogenic Se nanoparticles that could be a potent ingredient for the preparation of antifungal for- mulations (SHAHVERDI et al. 2010). Contrary to Se stimulatory effect on antioxidant activity, this trace element caused regression of cy- totoxic activity. Numerous biologically active com- pounds, such as diverse types of sesquiterpenes, polysaccharides, glycoproteins, ribosome inacti- vating proteins, and sterols, isolated from myce- lium of F. velutipes, take important place as cyto- toxic agents against various cancer cell lines (LE- UNG et al., 1997; NG and WANG, 2004; WANG et al., 2012; YANG et al., 2012; YI et al., 2013). LE- UNG et al. (1997) showed that soluble homopol- ysaccharide, composed of glucose and isolated from F. velutipes fruiting bodies, were very effi- cient in Sarcoma-180 cells regression in vivo. The same effect was noted by YANG et al. (2012) for alkaline-soluble heteropolysaccharides with a glu- can as backbone chain and triple helix structure, which also have strong anti-proliferation activity against lung cancer cells (A549) and human gas- tric cancer cells (BGC-823) (inhibitory rate was 32.3% and 95%, respectively). The cytotoxic effect of polysaccharides was based on proliferation of B-cell, T-cell or both cells and not on tumor cell kill. Activation of immune system and in such a way production of interferon-gamma with anti- proliferate effect on tumor cells was also lied in the base of antitumor activity of F. velutipes gly- coprotein FVE (CHANG et al., 2010). However, ri- bosome inactivating proteins (flammulin, velutin, flammin, and velin) stopped cancer cell prolifera- tion by ribosome inactivation and translation in- hibition (NG and WANG, 2004). Efficiency of ses- quiterpenoids against human liver carcinoma cell line (HepG2), breast cancer cell line (MCF-7), hu- man gastric cancer cell line (SGC7901) and A549 was modest (IC 50 was in range between 20.0 and 100.0 µM) (WANG et al., 2012), while inhibition rate of human glioma cell line (U251) by sterols, at concentration of 20 µg/mL, was even 57% (YI et al., 2013). However, the sterol was not so effi- cient against HeLa cell line (IC 50 > 40.0 µg/mL), but more efficient comparing with tested Se-amended and non-amended mycelium extract. Although tested extracts showed lower biolog- Ital. J. Food Sci., vol. 27 - 2015 7 ically activities than commercial antimycotic, an- tioxidant and cytostatic, preference should be giv- en to natural products. 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