COPRINELLUS DISSEMINATUS (PERS.) J.E. LANGE 1938: in VITRO ANTIOXIDANT and ANTIPROLIFERATIVE EFFECTS Aleksandra R

DOI: 10.5937/FFR1602093N UDK 635.8:615.279+547.56 Original research paper

COPRINELLUS DISSEMINATUS (PERS.) J.E. LANGE 1938: IN VITRO ANTIOXIDANT AND ANTIPROLIFERATIVE EFFECTS Aleksandra R. Novaković1*, Maja A. Karaman 2, Sonja N. Kaišarević2 , Miona M. Belović1, Tanja I. Radusin1, Mirjana B. Beribaka3, Nebojša M. Ilić1

1University of Novi Sad, Institute for Food Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia 2University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia 3University of East Sarajevo, Faculty of Technology Zvornik, Karakaj bb, Bosnia and Herzegovina

*Corresponding author: Phone: +381214853770 Fax: +38121450725 E-mail address: [email protected]

ABSTRACT: Coprinellus disseminatus (Pers.) J.E. Lange 1938 is an edible saprotrophic fungal species with bioactive potential which is not extensively investigated, mostly due to its small size. Accordingly, the aim of this study was to determine chemical and biological properties of crude ethanol (CdEtOH) and water extracts (CdAq). The bioactivity screens included antioxidant and antiproliferative (human breast MCF-7 cancer cell-line; MTT and SRB assays) activity. In addition, CdAq and CdEtOH were primarily characterized by UV-VIS spectrophotometry, with an aim of determining its chemical composition (total phenol and flavonoid contents). The best antioxidant activities were obtained for • hydroxyl radicals (OH ) and superoxide anion radical (SOA) assays for both crude extracts (IC50 =4.02 µg/ml for CdAq and IC50 =1.40 µg/ml for CdEtOH) while total phenolic (TP) and total flavonoid (TF) content was higher in CdEtoH than in CdAq. Both crude extracts have shown activity against MCF-7 cell line although CdEtOH showed better effect than CdAq extract after 72h in MTT and SRB assay, reaching IC50 at 217.90 and 205.90 µg/ml, respectively. Based on obtained results, the saprotrophic fungal species Coprinellus disseminatus (Pers.) J.E. Lange 1938 could be considered as a good potential alternative source оf nutraceuticals and biologically active compounds.

Key words: Coprinellus disseminatus, antioxidant activities, antiproliferative activities, total phenolic content, total flavonoid content

INTRODUCTION Free radicals are classified as highly reac- of various diseases for which modern me- tive molecules that have one or more un- dicine still does not have appropriate for- paired electrons (Halliwell, 2006). Conse- mulations via standard preparations. Since quently, they can induce oxidative damage modern style of living continuously causes in biomolecules and play an important role disturbance of homeostasis through in aging process as well as in many di- stress, radiation or increased presence of seases, such as cardiovascular and in- xenobiotics in the environment (Kaloge- flammatory diseases, cancer, diabetes, ropoulos et al., 2013), there is an essential impaired immune system etc. (Valko et al., need for enhancement of human an- 2007; Ferreira et al., 2009). Nowadays, we tioxidant system in order to prevent oxi- are witnesses of challenges that humanity dative cell damage. The best approach is meets trying to resolve growing incidence to enhance the prevention, over curing, by

93 Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Food and Feed Research, 43 (2), 93-101, 2016 increasing consumption of foods rich in myco-pharmacological investigations of natural antioxidants, such as vegetables, medicinal mushrooms still play an im- fruits or mushrooms (Barros et al., 2007). portant role in the development of new biotech-products and bio-pharmaceuticals. Traditional medicinal use of fungi has been well-known in many Asian countries, Coprinellus disseminatus (Pers.) J.E. such as China, Russia, Japan and Korea, Lange 1938 is a small fungal species while the western medicine has recently known as "fairies bonnets" or "trooping taken an interest in their valuable bioactive crumble cap" which belongs to the phylum properties (Zaidman et al., 2005; Kalo- Basidiomycota and family Psathyrel- geropoulos et al., 2013; Karaman et al., laceae. Unlike other coprinoid species, its 2014). Since a growing interest in natural sporocarp does not deliquesce (dissolve resources with bioactive potentials, in- into black ink) with maturity. It is an edible cluding fungi, in the treatment and pre- species that can be found growing in sa- vention of many diseases during the protrophic clusters near the bases of recent decades (Espín et al., 2000), edible stumps, mostly in very high numbers. Only mushrooms are also analyzed as thera- one recently published study reported that peutic alternatives (Zaidman et al., 2005; methanol extracts of this species possess Kalogeropoulos et al., 2013; Karaman et antioxidant activities, while dichlorome- al., 2014). Mushrooms represent rich sour- thane, methanol and aqueous extracts ces of nutraceuticals (Benzie and Strain show antibacterial properties (Al-Fatimi et 1999; Ribeiro et al., 2009) and have al., 2013). Data on antiproliferative activity demonstrated to have a physiological be- of this species on human tumor cell lines, nefit or provide protection against certain breast cancer cells, are very modest (Gu chronic diseases (Zhou et al., 2009). and Leonard, 2006). Nowadays, both entire fungal sporophores Since the bioactivity of fungal species is and extracts are used for cancer treatment mostly influenced by geographical origin (Beattie et al., 2011), while medicinal (Puttaraju et al., 2006) or specificity of the mushroom preparations together with single fungal strain, the aim of this study isolated fractions and specific compounds was to evaluate antioxidant and antiprolife- are used as adjuvants to surgery, rative properties of both ethanol and water radiotherapy or chemotherapy (Lindequist crude extracts of autochthonous lignico- et al., 2005). Numerous bioactive pro- lous species C. disseminatus from the perties of mushrooms, such as antioxi- area of northern Serbia (Novi Sad). dant, antitumor (Wasser 2002; Zaidman, et al., 2005; Ferreira et al., 2010; Vaz et al. MATERIAL AND METHODS 2012), and antimicrobial (Barros et al., Chemicals 2007; Stojković et al., 2013) are being dis- covered during the last two decades. Folin-Ciocalteu (FC) reagent, anhydrous Many high molecular compounds (Wasser, sodium carbonate, gallic acid (GA), alu- 2002; Lindequist et al., 2005; Moradali et minium trichloride hexahydrate, sodium al., 2007; Zhang et al., 2007) and low acetate trihydrate, quercetin hydrate, 2,2- molecular compounds (Zaidman et al., diphenyl-1-picrylhydrazyl (DPPH•), anhy- 2005; Paterson, 2006; Barros et al., 2007; drous iron(III) chloride, 2,4,6-Tris(2-pyri- Ferreira et al., 2010; Stojković et al., 2013; dyl)-s-triazine (TPTZ), 2-thiobarbituric Karaman et al., 2014) have been proven acid, disodium hydrogen phosphate, thia- to possess antioxidant activities. These zolyl blue tetrazolium bromide, phenazine compounds have already been determined methosulfate (PMS), β-nicotinamide ade- as the main bioactive components in some nine dinucleotide (NADH), 3-(4,5-dimethyl- medicinal mushrooms (e.g. Ganoderma thiazole-2-yl)-2,5-diphenyltetrazolium bro- lucidum, Grifola frondosa, Coprinus coma- mide (MTT), dimethyl sulfoxide (DMSO), tus, Meripilus giganteus) which exhibit sulforhodamine B (SRB) and trichloro- both antioxidant and antimicrobial active- acetic acid (TCA) were purchased from ties (Barros et al., 2007; Ferreira et al., Sigma-Aldrich (Steinheim, Germany). Dul- 2010; Karaman et al., 2014). Therefore, becco's Modified Eagle's Medium with

94 Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Food and Feed Research, 43 (2), 93-101, 2016 Food and Feed Research, 43 (2), 93-101, 2016 increasing consumption of foods rich in myco-pharmacological investigations of 4.5% of glucose (DMEM) and fetal calf se- weight (d.w.). The experiments were per- natural antioxidants, such as vegetables, medicinal mushrooms still play an im- rum (FCS) were obtained from PAA Labo- formed in triplicate. fruits or mushrooms (Barros et al., 2007). portant role in the development of new ratories (Pasching, Austria). biotech-products and bio-pharmaceuticals. Total flavonoid content Traditional medicinal use of fungi has Mushroom samples Total flavonoid (TF) content of of CdEtOH been well-known in many Asian countries, Coprinellus disseminatus (Pers.) J.E. The species C. disseminatus was collec- and CdAq respectively was measured such as China, Russia, Japan and Korea, Lange 1938 is a small fungal species ted from urban area (street) in Novi Sad spectrophotometrically, in a 96-well plate while the western medicine has recently known as "fairies bonnets" or "trooping during 2012. Identification of the species reader, using a modified method by Chang taken an interest in their valuable bioactive crumble cap" which belongs to the phylum was based on classical determination ba- et al. (2002). The relevant sample (30 μl) properties (Zaidman et al., 2005; Kalo- Basidiomycota and family Psathyrel- sed on determinative keys (Phillips, 1983) was mixed with 90 μl of methanol, 6 μl of geropoulos et al., 2013; Karaman et al., laceae. Unlike other coprinoid species, its and a voucher specimen (12-00662) was 0.75 M aluminium-trichloride, 6 μl of 1 M 2014). Since a growing interest in natural sporocarp does not deliquesce (dissolve deposited at the Herbarium BUNS (Faculty sodium acetate and 170 μl of distilled resources with bioactive potentials, in- into black ink) with maturity. It is an edible of Sciences Department of Biology and water. Absorbance was measured at 414 cluding fungi, in the treatment and pre- species that can be found growing in sa- Ecology, University in Novi Sad, Serbia). nm, after incubation of 30 min. The vention of many diseases during the protrophic clusters near the bases of The samples were frozen at -20 °C and experiments were performed in triplicate, recent decades (Espín et al., 2000), edible stumps, mostly in very high numbers. Only freeze-dried during 48 h (Bio alpha, Martin while results were expressed as mg mushrooms are also analyzed as thera- one recently published study reported that Christ GmbH, Switzerland). Lyophilized quercetin equivalents (QE)/g of dry weight peutic alternatives (Zaidman et al., 2005; methanol extracts of this species possess samples were ground to a fine powder, (d.w.). Kalogeropoulos et al., 2013; Karaman et antioxidant activities, while dichlorome- and after that packed in plastic tubes and al., 2014). Mushrooms represent rich sour- thane, methanol and aqueous extracts • stored in a dark place at room temperature DPPH scavenging activity ces of nutraceuticals (Benzie and Strain show antibacterial properties (Al-Fatimi et prior to analysis. • 1999; Ribeiro et al., 2009) and have al., 2013). Data on antiproliferative activity DPPH scavenging activity was evaluated of this species on human tumor cell lines, demonstrated to have a physiological be- Extraction according to Espin et al. (2000). The nefit or provide protection against certain breast cancer cells, are very modest (Gu reaction mixture consisted of 10 μl of • chronic diseases (Zhou et al., 2009). and Leonard, 2006). The whole freeze dried and powdered sample, 60 μl of DPPH solution and 180 Nowadays, both entire fungal sporophores Since the bioactivity of fungal species is sporocarps of C. disseminatus (10 g) were μl of methanol. After incubation of 60 min and extracts are used for cancer treatment mostly influenced by geographical origin extracted with two solvents separately: (dark place, at room temperature), absor- (Beattie et al., 2011), while medicinal (Puttaraju et al., 2006) or specificity of the ethanol (EtOH) and distilled water (Aq) for bance was measured at 540 nm. Each mushroom preparations together with single fungal strain, the aim of this study 24 h on a shaker (Thermofisher Scientific, sample was tested at five double diluted isolated fractions and specific compounds was to evaluate antioxidant and antiprolife- USA) at 120 rpm at room temperature (25 concentrations (100-1600 µg/ml) to obtain are used as adjuvants to surgery, rative properties of both ethanol and water °C). The extracts were filtered through IC50, and experiments were performed in radiotherapy or chemotherapy (Lindequist crude extracts of autochthonous lignico- Whatman No.4 filter paper and the sol- triplicate. et al., 2005). Numerous bioactive pro- lous species C. disseminatus from the vents were removed by rotary evaporation in vacuum at 40 °C (Büchi, Switzerland). Nitric oxide radical scavenging capa- perties of mushrooms, such as antioxi- area of northern Serbia (Novi Sad). • dant, antitumor (Wasser 2002; Zaidman, The obtained ethanolic (CdEtOH) and city - NO assay aqueous (CdAq) extracts were stored at - et al., 2005; Ferreira et al., 2010; Vaz et al. MATERIAL AND METHODS Nitric oxide scavenging capacity was 20 °C prior to analysis. Dry residues were 2012), and antimicrobial (Barros et al., determined according to the procedure of Chemicals redissolved in DMSO for determination of 2007; Stojković et al., 2013) are being dis- (Green et al. 1982). The reaction mixtures antioxidant and antiproliferative activities covered during the last two decades. Folin-Ciocalteu (FC) reagent, anhydrous in test tubes consisted of 30 µl extract, 500 (5 mg/ml, w/v). Many high molecular compounds (Wasser, sodium carbonate, gallic acid (GA), alu- µl SNP, and 500 µl of phosphate buffer, 2002; Lindequist et al., 2005; Moradali et minium trichloride hexahydrate, sodium (pH=7.4.) The control contained equivalent al., 2007; Zhang et al., 2007) and low acetate trihydrate, quercetin hydrate, 2,2- Total phenolic content • volume of ethanol, while reagents were re- molecular compounds (Zaidman et al., diphenyl-1-picrylhydrazyl (DPPH ), anhy- Total phenol content (TP) of CdEtOH and placed with phosphate buffer in the 2005; Paterson, 2006; Barros et al., 2007; drous iron(III) chloride, 2,4,6-Tris(2-pyri- CdAq respectively was determined accor- correction. Test tubes were incubated at Ferreira et al., 2010; Stojković et al., 2013; dyl)-s-triazine (TPTZ), 2-thiobarbituric ding to method by Singleton et al. (1999), room temperature for 90 min, under light Karaman et al., 2014) have been proven acid, disodium hydrogen phosphate, thia- adapted for a 96-well plate reader (Multi- exposure. After incubation, 1 ml of Griess to possess antioxidant activities. These zolyl blue tetrazolium bromide, phenazine skan Ascent, Thermo Electron Corpo- reagent (0.2% solution of NEDA and 2% compounds have already been determined methosulfate (PMS), β-nicotinamide ade- ration). Folin-Ciocalteu reagent (125 μl, solution of SA in 4% phosphoric acid in the as the main bioactive components in some nine dinucleotide (NADH), 3-(4,5-dimethyl- 0.1 M) was added to 25 μl of diluted ex- ratio 1:1 (v:v)) was added equally to medicinal mushrooms (e.g. Ganoderma thiazole-2-yl)-2,5-diphenyltetrazolium bro- tracts. After 10 min, 100 μl of 7.5% w/v so- samples, corrections and control. Aliquots lucidum, Grifola frondosa, Coprinus coma- mide (MTT), dimethyl sulfoxide (DMSO), dium carbonate was added and reaction of 250 µl were transferred to the plate, and tus, Meripilus giganteus) which exhibit sulforhodamine B (SRB) and trichloro- mixture was incubated for 2 h. Absorbance their absorbance was measured using pla- both antioxidant and antimicrobial active- acetic acid (TCA) were purchased from was read at 690 nm. TP was expressed as te reader at 540 nm. Samples were tested ties (Barros et al., 2007; Ferreira et al., Sigma-Aldrich (Steinheim, Germany). Dul- mg gallic acid equivalents (GAE)/g of dry at five different concentrations (70-600 2010; Karaman et al., 2014). Therefore, becco's Modified Eagle's Medium with

95 Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Food and Feed Research, 43 (2), 93-101, 2016 Food and Feed Research, 43 (2), 93-101, 2016

µg/ml) to obtain IC50, and experiments Antiproliferative activity while absorbance was measured at 492 RESULTS AND DISCUSSION were performed in triplicate. Cells nm and 690 nm, using the microplate reader. DMSO was used as a positive Antioxidant activity and total phenol Estrogen dependent MCF-7 cells were Ferric reducing antioxidant power control. The results were expressed in IC and flavonoid contents grown in DMEM (PAA Laboratories) sup- 50 (FRAP) values (sample concentration that inhibited plemented with 10% FCS. The cells were Radical scavenging capacity (RSC) and FRAP assay was performed according to 50% of the net cell growth). These seeded in a 96-well microplate (5000 cells total phenolic and flavonoid content of a modified procedure of Benzie and Strain experiments were performed in triplicate. per well). After incubation of 24 h, the analyzed extracts are presented in Table (1999). The FRAP reagent consisted of growth medium was replaced with 100 μl The absorbance was calculated from the 1. According to the obtained results, both 300 mM acetate buffer (pH=3.6), 10 mM of medium containing the examined sam- difference of two absorbances: A=A540- crude extracts possessed good RSC TPTZ in 40 mM HCl and 20 mM FeCl3, in ples (extracts) at four different concen- A690 for MTT assay and A=A492-A690 for activity. Based on statistical analysеs the the ratio 10:1:1 (v:v:v). The sample (10 µl), trations (33.3 μg/ml, 100.0 μg/ml, 300.0 SRB assay. Percentage of cytotoxicity was water extract exhibited significantly higher 225 µl of FRAP reagent and 22.5 µl of μg/ml, and 900.0 μg/ml). Untreated cells calculated as the ratio of treated group ab- activity than ethanol extract in NO• assay distilled water were added in a 96-well served as the control, while pure DMSO sorbance and the control group absor- (thirteen times lower IC value), while the plate. Absorbance was measured after 6 25 was used as a positive control. The effects bance, multiplied by 100. ethanol extract showed better activity in min at 620 nm. The results were ex-pres- of the extracts on the growth of MCF-7 SOA assay (IC50 = 1.40 µg/ml). sed as mg ascorbic acid equivalents cells were evaluated by standard colori- Experiments were performed twice in (AAE)/g of dry weight (d.w.). Each analysis metric assays – MTT and SRB. triplicate, and the obtained results were Moreover, both analyzed extracts showed was performed three times. expressed as IC values (sample con- MTT Assay 50 very good scavenging activity towards centration that inhibited 50% of the net cell Superoxide anion radical scavenging DPPH and OH radical although IC50 capacity – SOA assay After 24 h and 72 h respectively, the cell growth). IC50 values were calculated from values did not differ significantly. In viability was determined by the prolife- the cytotoxicity (%) - extract concentration general, both analysed extracts showed Superoxide anion radical scavenging ration MTT assay (Mosmann, 1983). This (μg/mL) plot using the Origin v. 6.0 gra- higher activities in DPPH assay than capacity of extracts was determined by assay is based on the colour reaction of phing and data analysis software (1999). methanol extract of C. disseminatus from measuring their ability to neutralize super- mitochondrial dehydrogenase in living Yemen with RSC 42.8% at 1000 µg/ml, oxide anion radicals generated during Statistical analysis cells with MTT reagent. After incubation, (Al-Fatimi et al., 2013) as well as from the aerobic reduction of nitro blue tetrazolium MTT reagent was added to each well (50 species C. comatus originated from Serbia by NADH mediated by PMS (Nishikimi et Results were expressed as mean ± μg/100 μl/well; at 37 °C, in 5% CO2 for 3 standard deviation (SD). Statistical ana- (Stojković et al., 2013). al., 1972). 100 µl of 677 µM NADH, 100 µl h). The crystals of produced formazan of 60 µM PMS, 200 µl of 144 µM NBT and lysis was performed using Statistica soft- were dissolved in 100 μl acidified iso- These differences between our research 1.1 ml of phosphate buffer (pH = 8.3) were ware system (STATISTICA, 2013). Signifi- propanol (0.04 M HCl in isopropanol). results and other published work on this mixed with 10 µl of extract in the test tube. cant differences between two groups were Absorbance was measured at 540 nm and determined by Student’s t-test. topic, could be assigned to different geo- The control contained ethanol instead of 690 nm on a 96 well plate reader graphical origin of the fungal species, extract, and correction contained 10 µl of (Multiskan Ascent, Thermo Electron Cor- Finally, Pearson correlation coefficients content of secondary metabolites, different 2 extract and 1.5 ml of phosphate buffer. poration, USA). These experiments were (r ) were calculated for TP and TF, DPPH, strains as well as different solvents used After 5 min, aliquots of 250 µl were trans- performed in triplicate. The results were FRAP and IC50 values for antiproliferative for extraction. ferred to the well plate and their absor- expressed in IC50 values (sample concen- activity. bance was measured at 540 nm using pla- tration that inhibited 50% of the net cell te reader. Five different concentrations of growth). Table 1. Antioxidant activity of C. disseminatus extracts and TP and TF content each sample (0.4-6.6 µg/ml) were tested SRB Assay to obtain IC50, and experiments were per- a Antiradical assays IC50 (µg/ml) CdEtOH CdAq formed in triplicate. This assay estimates cell number indi- rectly, by staining cellular protein with the DPPH• 397.28 ± 64.17* 250.37 ± 15.74* Hydroxyl radical scavenging capacity - protein-binding dye SRB (adapted pro- SOA 1.40 ± 0.66** 24.84 ± 2.38* • * * OH assay cedure, by Skehan et al. (1990)). After in- OH• 7.37 ± 1.46 4.02 ± 0.29 a Antiradical assays IC25 (µg/ml) CdEtOH CdAq The content of OH radicals was deter- cubation, the cells were fixed adding cold • * ** mined from the degradation reaction of 2- 50% TCA and incubated for 1 h at 4 °C. NO 272.30 ± 21.53 21.28 ± 6.08 Antioxidant assayb deoxy-D-ribose into fragments (Cheesman Wells were washed with deionized water * ** et al., 1988), while the malondialdehyde and dried; SRB solution (0.4% in 1% FRAP 9.74 ±0 .79 4.02±0.60 Total contentc MDA reaction with 2-tiobarbituric acid TBA acetic acid) was then added to each plate * ** TP 59.94 ± 0 30.74 ± 0.36 well and incubated for 30 min at room * ** reagent was determined spectrophoto- TF 11.37 ± 0 0.59 ± 0.1 4 temperature. Unbound SRB was removed a b metrically at 532 nm. Each reaction was Results are expressed as IC50 and IC25 (µg/ml). Ferric reducing antioxidant power (FRAP) is expressed as mg performed using 10 μL of fungal extract by washing with 1% acetic acid. Plates ascorbic acid equivalents/g extract dry weight (mg AAE/g d.w.).cTotal phenol content (TP) was expressed as mg were air dried, the bound SRB was gallic acid equivalents/g extract dry weight (mg GAE /g d.w.) and total flavonoid content (TF) was expressed in (1.9–38.38 μg/ml) to obtain IC50, and expe- *,** riments were performed in triplicate. solubilised with 10 mM Tris (pH 10.5), mg quercetine equivalents /g extract dry weight (mg QE/g d.w.). Values with different asterisk in superscript within a column are statistically different t-test p<0.001

96 Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Food and Feed Research, 43 (2), 93-101, 2016 Food and Feed Research, 43 (2), 93-101, 2016

97 Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Food and Feed Research, 43 (2), 93-101, 2016 Food and Feed Research, 43 (2), 93-101, 2016

Furthermore, ethanolic extract showed they measure distinct biological parame- The highest correlation was obtained 4. Benzie, I. F., Strain, J.J. (1999). [2] Ferric higher activity than water extract according ters in living cells. While SRB assay does between antiproliferative effects with TF reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological to reducing power capacity (FRAP) assay not depend on enzymatic activity but on and TP content especially in MTT assay fluids and modified version for simultaneous which is in accordance with literature data protein content of the cells, in MTT assay (Table 3), indicating that these compounds measurement of total antioxidant power and (Vaz et al. 2011), which can be attributed the results reflect the activity of mitochon- strongly influenced the expressed cyto- ascorbic acid concentration. Methods in Enzy- to higher TP and TF content in ethanolic drial dehydrogenase which is more sen- toxic activities. By comparing two cytotoxic mology, 299, 15-27. extract compared to water extract (Table sitive parameter, and therefore in many assays, MTT assay showed to be more 5. Chang, C.C., Yang, M.H., Wen, H.M., Chern, 1) according to correlation analysis. cases with changes detectable at lower suitable assay for determination of the J.C. (2002). Estimation of total flavonoid con- concentrations. However, the effects ob- tent in propolis by two complementary colori- Taking into account both parameters (anti- antiproliferative activity of mushroom ex- metric methods. Journal of Food and Drug Ana- served for each sample also strongly de- oxidant activity and chemical composition), tracts. lysis, 10 (3), 178-182. pend on the specific combination of com- it may be assumed that phenol and fla- 6. Cheeseman, K.H., Beavis, A., Esterbauer, H. pounds present in the sample, their inte- CONCLUSIONS vonoid compounds are not the only com- (1988). Hydroxyl-radical-induced iron-catalysed raction and action of their metabolites that degradation of 2-deoxyribose. Quantitative de- ponents responsible for the observed acti- Both crude extracts of C. disseminatus induce specific and often unexpected cel- termination of malondialdehyde. Biochemical vities. possessed high antiradical and antioxidant Journal, 252 (3), 649-653. lular responses. The obtained results were properties and antiproliferative activity 7. Espin, J.C., Soler-Rivas, C., Wichers, H.J. Antiproliferative activity not consistent with previously published against MCF-7 human breast cancer cell data for water extract of Coprinellus sp. (2000). Characterization of the total free radical The cytotoxic effect against MCF-7 human line. Very high correlation between cyto- scavenger capacity of vegetable oils and oil breast cancer cell line was evaluated by that reached IC50 at 120 µg/ml at 24 h (Gu toxic effects and TP and TF content indi- fractions using 2, 2-diphenyl-1-picrylhydrazyl MTT and SRB assays (Table 2). Under the and Leonard, 2006) because the analyzed cates a significant effect of phenolic com- radical. Journal of Agricultural and Food Che- mistry, 48 (3), 648-656. experimental conditions, both extracts in extracts showed lower activity (IC50 at pounds on this activity. Based on mani- 625.26 µg/ml). fested bioactivities, the saprotrophic fungal 8. Ferreira, I.C., Barros, L., Abreu, R. (2009). Anti- two different assays showed antipro- oxidants in wild mushrooms. Current Medicinal liferative activity. The ethanol extract de- species Coprinus disseminatus (Pers.) In addition, correlation analyses between Chemistry, 16 (12), 1543-1560. monstrated significantly higher cytotoxic Gray 1821 could be considered as a good radical scavenging activity and antipro- 9. Ferreira, I.C., Vaz, J.A., Vasconcelos, M.H., activity (lower IC ) than water extract, ex- potential alternative source for nutraceu- 50 liferative activities based on IC values Martins, A. (2010). Compounds from wild mu- cept for SRB assay at 72 h. 50 ticals and biologically active compounds. shrooms with antitumor potential.Anti-Cancer (Table 3) showed very strong correlation Additionally, further biochemical studies Agents in Medicinal Chemistry (Formerly Cur- Differences in IC values obtained for the for MTT assay with NO, SOA, and FRAP 50 regarding the chemical profile of bioactive rent Medicinal Chemistry-Anti-Cancer Agents), same samples by the two assays can assays at both incubation times, while no 10 (5), 424-436. compounds, as well as possible molecular probably be attributed to the difference in significant correlations were observed with mechanisms responsible for the demon- 10. Green, L.C., Wagner, D.A., Glogowski, J., sensitivity of targets they reflect, since DPPH and OH assays. Skipper, P.L., Wishnok, J.S., Tannenbaum, S. strated cytotoxic effects should be conduc- R. (1982). Analysis of nitrate, nitrite, and [15 N] ted in the future. nitrate in biological fluids. Analytical Bioche- Table 2. mistry, 126 (1), 131-138. Cytotoxic activities of C. disseminatus extracts on MCF-7 (IC50 µg/ml) АCKNOWLEDGEMENTS 11. Gu, Y.H., Leonard, J. (2006). In vitro effects on MTT assay SRB assay proliferation, apoptosis and colony inhibition in Extracts 24h 72h 24h 72h This study was supported by the Ministry ER-dependent and ER-independent human CDAq ˃900** 718.07±37.36** 625.26±26.80** 211.01±25.07* of Education, Science and Technological breast cancer cells by selected mushroom CDEtOH 249.47±11.53* 217.90±24.79* 511.37±6.46* 205.90±35.98* Development of the Republic of Serbia species. Oncology Reports, 15 (2), 417-424. All values are represented as mean values and standard deviations obtained from three measurements (mean ± (Research Grant No. III 46001). 12. Halliwell, B. (2006). Reactive species and anti- *,** SD; n=3). Values with different asterisk in superscript within a column are statistically different (p<0.05), t-test oxidants. Redox biology is a fundamental the- p<0.001 me of aerobic life. Plant Physiology, 141 (2), REFERENCES 312-322. Table 3. 1. Al-Fatimi, M., Schröder, G., Kreisel, H., Linde- 13. Kalogeropoulos, N., Yanni, A.E., Koutrotsios, Correlation (r2) between antiproliferative and antioxidant assays and TF, TP content in crude extracts quist, U. (2013). Biological activities of selected G., Aloupi, M. (2013). Bioactive microconsti- tuents and antioxidant properties of wild edible IC (μg/ml) MTT assay SRB assay basidiomycetes from Yemen. Die Pharmazie - 50 An International Journal of Pharmaceutical mushrooms from the island of Lesvos, Greece. 24h 72h 24h 72h Sciences, 68 (3), 221-226. Food and Chemical Toxicology, 55, 378-385. 0.55 0.54 0.6 0.16 DPPH 2. Barros, L., Baptista, P., Estevinho, L.M., & 14. Karaman M., Stahl M., Vulić J., Vesić M., FRAP 0.89** 0.87** 0.67* 0.00 • *** ** * Ferreira, I.C. (2007). Bioactive properties of the Čanadanović Brunet, J. (2014). Wild growing NO 0.97 0.92 0.82 0.00 medicinal mushroom Leucopaxillus giganteus • lignicolous mushroom species as sources of OH 0.54 0.53 0.58 0.26 mycelium obtained in the presence of different novel agents with antioxidative and antibacterial *** ** SOA 0.95 0.90 0.63 0.00 nitrogen sources. Food Chemistry, 105 (1), potentials. International Journal of Food 179-186. Sciences and Nutrition, 65 (3), 311-319 *** *** * TF content (mg AAE/100 g) 0.99 0.97 0.82 0.00 3. Beattie, K.D., Ulrich, R., Grice, I.D., Uddin, S. 15. Lindequist, U., Niedermeyer, T.H., Jülich, W.D. *** *** * TP content (mg GAE/100 g) 0.99 0.95 0.80 0.00 J., Blake, T.B., Wood, K.A., Tiralongo, E. (2005). The pharmacological potential of mush- *Correlations are significant at p<0.05, **correlations are significant at p<0.01,***correlations are significant at (2011). Ethanolic and aqueous extracts derived rooms. Evidence-Based Complementary and p<0.001 from Australian fungi inhibit cancer cell growth Alternative Medicine, 2 (3), 285-299. in vitro. Mycologia, 103 (3), 458-465.

98 Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Food and Feed Research, 43 (2), 93-101, 2016

The highest correlation was obtained 4. Benzie, I. F., Strain, J.J. (1999). [2] Ferric between antiproliferative effects with TF reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological and TP content especially in MTT assay fluids and modified version for simultaneous (Table 3), indicating that these compounds measurement of total antioxidant power and strongly influenced the expressed cyto- ascorbic acid concentration. Methods in Enzy- toxic activities. By comparing two cytotoxic mology, 299, 15-27. assays, MTT assay showed to be more 5. Chang, C.C., Yang, M.H., Wen, H.M., Chern, suitable assay for determination of the J.C. (2002). Estimation of total flavonoid content in propolis by two complementary colori- antiproliferative activity of mushroom ex- metric methods. Journal of Food and Drug Ana- tracts. lysis, 10 (3), 178-182. CONCLUSIONS 6. Cheeseman, K.H., Beavis, A., Esterbauer, H. (1988). Hydroxyl-radical-induced iron-catalysed Both crude extracts of C. disseminatus degradation of 2-deoxyribose. Quantitative determination of malondialdehyde. Biochemical possessed high antiradical and antioxidant Journal, 252 (3), 649-653. properties and antiproliferative activity 7. Espin, J.C., Soler-Rivas, C., Wichers, H.J. against MCF-7 human breast cancer cell (2000). Characterization of the total free radical line. Very high correlation between cyto- scavenger capacity of vegetable oils and oil toxic effects and TP and TF content indi- fractions using 2, 2-diphenyl-1-picrylhydrazyl cates a significant effect of phenolic com- radical. Journal of Agricultural and Food Che- mistry, 48 (3), 648-656. pounds on this activity. Based on mani- fested bioactivities, the saprotrophic fungal 8. Ferreira, I.C., Barros, L., Abreu, R. (2009). Anti- oxidants in wild mushrooms. Current Medicinal species Coprinus disseminatus (Pers.) Chemistry, 16 (12), 1543-1560. Gray 1821 could be considered as a good 9. Ferreira, I.C., Vaz, J.A., Vasconcelos, M.H., potential alternative source for nutraceu- Martins, A. (2010). Compounds from wild mu- ticals and biologically active compounds. shrooms with antitumor potential.Anti-Cancer Additionally, further biochemical studies Agents in Medicinal Chemistry (Formerly Cur- regarding the chemical profile of bioactive rent Medicinal Chemistry-Anti-Cancer Agents), 10 (5), 424-436. compounds, as well as possible molecular mechanisms responsible for the demon- 10. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S. strated cytotoxic effects should be conduc- R. (1982). Analysis of nitrate, nitrite, and [15 N] ted in the future. nitrate in biological fluids. Analytical Bioche- mistry, 126 (1), 131-138. АCKNOWLEDGEMENTS 11. Gu, Y.H., Leonard, J. (2006). In vitro effects on proliferation, apoptosis and colony inhibition in This study was supported by the Ministry ER-dependent and ER-independent human of Education, Science and Technological breast cancer cells by selected mushroom Development of the Republic of Serbia species. Oncology Reports, 15 (2), 417-424. (Research Grant No. III 46001). 12. Halliwell, B. (2006). Reactive species and antioxidants. Redox biology is a fundamental the- me of aerobic life. Plant Physiology, 141 (2), REFERENCES 312-322. 1. Al-Fatimi, M., Schröder, G., Kreisel, H., Linde- 13. Kalogeropoulos, N., Yanni, A.E., Koutrotsios, quist, U. (2013). Biological activities of selected G., Aloupi, M. (2013). Bioactive microconsti- basidiomycetes from Yemen. Die Pharmazie - tuents and antioxidant properties of wild edible An International Journal of Pharmaceutical mushrooms from the island of Lesvos, Greece. Sciences, 68 (3), 221-226. Food and Chemical Toxicology, 55, 378-385. 2. Barros, L., Baptista, P., Estevinho, L.M., & 14. Karaman M., Stahl M., Vulić J., Vesić M., Ferreira, I.C. (2007). Bioactive properties of the Čanadanović Brunet, J. (2014). Wild growing medicinal mushroom Leucopaxillus giganteus lignicolous mushroom species as sources of mycelium obtained in the presence of different novel agents with antioxidative and antibacterial nitrogen sources. Food Chemistry, 105 (1), potentials. International Journal of Food 179-186. Sciences and Nutrition, 65 (3), 311-319 3. Beattie, K.D., Ulrich, R., Grice, I.D., Uddin, S. 15. Lindequist, U., Niedermeyer, T.H., Jülich, W.D. J., Blake, T.B., Wood, K.A., Tiralongo, E. (2005). The pharmacological potential of mush- (2011). Ethanolic and aqueous extracts derived rooms. Evidence-Based Complementary and from Australian fungi inhibit cancer cell growth Alternative Medicine, 2 (3), 285-299. in vitro. Mycologia, 103 (3), 458-465.

99 Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Food and Feed Research, 43 (2), 93-101, 2016

16. Moradali, M.F., Mostafavi, H., Ghods, S., 26. Stojković, D., Reis, F.S., Barros, L., Glamočlija, Hedjaroude, G.A. (2007). Immunomodulating J., Ćirić, A., van Griensven, L.J., Ferreira, I.C. and anticancer agents in the realm of macro- (2013). Nutrients and non-nutrients composition mycetes fungi (macrofungi). International Im- and bioactivity of wild and cultivated Coprinus munopharmacology, 7 (6), 701-724. comatus (OF Müll.) Pers. Food and Chemical 17. Mosmann, T. (1983). Rapid colorimetric assay Toxicology, 59, 289-296. for cellular growth and survival: application to 27. Valko, M., Leibfritz, D., Moncol, J., Cronin, proliferation and cytotoxicity assays. Journal of M.T., Mazur, M., Telser, J. (2007). Free radicals Immunological Methods, 65 (1), 55-63. and antioxidants in normal physiological fun- 18. Nishikimi, M., Rao, N. A., Yagi, K. (1972). The ctions and human disease. The International occurrence of superoxide anion in the reaction Journal of Biochemistry & Cell Biology, 39 (1), of reduced phenazine methosulfate and mo- 44-84. lecular oxygen. Biochemical and Biophysical 28. Vaz, J.A., Almeida, G.M., Ferreira, I.C., Martins, Research Communications, 46 (2), 849-854. A., Vasconcelos, M.H. (2012). Clitocybe ale- xandri extract induces cell cycle arrest and 19. Paterson, R.R.M. (2006). Ganoderma–a thera- apoptosis in a lung cancer cell line: iden- peutic fungal biofactory. Phytochemistry, 67 tification of phenolic acids with cytotoxic po- (18), 1985-2001. tential. Food Chemistry, 132 (1), 482-486. 20. Puttaraju, N.G., Venkateshaiah, S.U., Dhar- 29. Vaz, J.A., Barros, L., Martins, A., Santos- mesh, S.M., Urs, S.M. N., Somasundaram, R. Buelga, C., Vasconcelos, M.H., Ferreira, I.C. (2006). Antioxidant activity of indigenous edible (2011). Chemical composition of wild edible mushrooms. Journal of Agricultural and Food mushrooms and antioxidant properties of their Chemistry, 54 (26), 9764-9772. water soluble polysaccharidic and ethanolic 21. Phillips, R. (1981). Mushrooms and Other Fungi fractions. Food Chemistry, 126 (2), 610-616. of Great Britain and Europe, Pan Books Ltd., 30. Wasser, S. (2002). Medicinal mushrooms as a London. source of antitumor and immunomodulating 22. Ribeiro, B., de Pinho, P.G., Andrade, P.B., Bap- polysaccharides. Applied Microbiology and Bio- tista, P., Valentão, P. (2009). Fatty acid compo- technology, 60 (3), 258-274. sition of wild edible mushrooms species: A 31. Zaidman, B.Z., Yassin, M., Mahajna, J., Was- comparative study. Microchemical Journal, 93 ser, S.P. (2005). Medicinal mushroom modu- (1), 29-35. lators of molecular targets as cancer thera- 23. Singleton, V.L., Orthofer, R., Lamuela-Raven- peutics. Applied Microbiology and Biotech- tos, R.M. (1999). Analysis of total phenols and nology, 67 (4), 453-468. other oxidation substrates and antioxidants by 32. Zhang, M., Cui, S.W., Cheung, P.C.K., Wang, means of Folin-Ciocalteu reagent. Methods in Q. (2007). Antitumor polysaccharides from Enzymology, 299, 152-178. mushrooms: a review on their isolation process, 24. Skehan, P., Storeng, R., Scudiero, D., Monks, structural characteristics and antitumor activity. A., McMahon, J., Vistica, D., Boyd, M.R. Trends in Food Science & Technology, 18 (1), (1990). New colorimetric cytotoxicity assay for 4-19. anti-cancer-drug screening. Journal of the Na- 33. Zhou, X., Gong, Z., Su, Y., Lin, J., Tang, K. tional Cancer Institute, 82 (13), 1107-1112. (2009). Cordyceps fungi: natural products, 25. STATISTICA (Data Analysis Software System) pharmacological functions and developmental (2013). v.12.0., Stat-Soft Inc., Tulsa, OK, USA products. Journal of Pharmacy and Pharma- (www.statsoft.com) cology, 61 (3), 279-291.

100 Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Aleksandra Novaković et al., Coprinellus disseminatus (Pers.) J.E. Lange 1938: in vitro antioxidant and antiproliferative effects, Food and Feed Research, 43 (2), 93-101, 2016 Food and Feed Research, 43 (2), 93-101, 2016

16. Moradali, M.F., Mostafavi, H., Ghods, S., 26. Stojković, D., Reis, F.S., Barros, L., Glamočlija, COPRINELLUS DISSEMINATUS (PERS.) J.E. LANGE 1938: IN VITRO Hedjaroude, G.A. (2007). Immunomodulating J., Ćirić, A., van Griensven, L.J., Ferreira, I.C. and anticancer agents in the realm of macro- (2013). Nutrients and non-nutrients composition АНТИОКСИДАТИВНИ И АНТИПРОЛИФЕРАТИВНИ ЕФЕКАТ mycetes fungi (macrofungi). International Im- and bioactivity of wild and cultivated Coprinus 1 2 2 munopharmacology, 7 (6), 701-724. comatus (OF Müll.) Pers. Food and Chemical Александра P. Новаковић *, Маја A. Караман , Соња Н. Каишаревић , Миона M. Беловић1, Тања И. Радусин1, Мирјана Б. Берибака3, Небојша M. Илић1 17. Mosmann, T. (1983). Rapid colorimetric assay Toxicology, 59, 289-296. for cellular growth and survival: application to 27. Valko, M., Leibfritz, D., Moncol, J., Cronin, 1Универзитет у Новом Саду, Научни институт за прехрамбене технологије у Новом proliferation and cytotoxicity assays. Journal of M.T., Mazur, M., Telser, J. (2007). Free radicals Immunological Methods, 65 (1), 55-63. and antioxidants in normal physiological fun- Саду, Булевар цара Лазара 1, 21000 Нови Сад, Србија 2Универзитет у Новом Саду, Природно-математички факултет, Департман за биологију 18. Nishikimi, M., Rao, N. A., Yagi, K. (1972). The ctions and human disease. The International Journal of Biochemistry & Cell Biology, 39 (1), и екологију, Трг Доситеја Обрадовића 2, 21000 Нови Сад, Србија occurrence of superoxide anion in the reaction 3 Универзитет у Источном Сарајеву,Технолошки факултет Зворник, Каракај бб, Босна и of reduced phenazine methosulfate and mo- 44-84. lecular oxygen. Biochemical and Biophysical 28. Vaz, J.A., Almeida, G.M., Ferreira, I.C., Martins, Херцеговина Research Communications, 46 (2), 849-854. A., Vasconcelos, M.H. (2012). Clitocybe ale- xandri extract induces cell cycle arrest and Сажетак: Coprinellus disseminatus (Pers.) J.E. Lange 1938 је јестива сапротрофна врста 19. Paterson, R.R.M. (2006). Ganoderma–a thera- apoptosis in a lung cancer cell line: iden- peutic fungal biofactory. Phytochemistry, 67 гљиве, чији биопотенцијал није много истраживан, углавном због мале величине. Сходно томе, tification of phenolic acids with cytotoxic po- (18), 1985-2001. циљ овог истраживања био је да се утврди хемијски и биолошки потенцијал сирових етанолних tential. Food Chemistry, 132 (1), 482-486. CdEtOH и водених CdAq екстраката. Скрининг биоактивности обухватио је антиоксидативну и 20. Puttaraju, N.G., Venkateshaiah, S.U., Dhar- 29. Vaz, J.A., Barros, L., Martins, A., Santos- антипролиферативну активност (ћелијска линија хуманог аденокарцинома дојке, MCF-7; тестови mesh, S.M., Urs, S.M. N., Somasundaram, R. Buelga, C., Vasconcelos, M.H., Ferreira, I.C. (2006). Antioxidant activity of indigenous edible МТТ и SRB). Поред тога CdEtOH и CdAq хемијски су прелиминарно окарактерисани (садржај (2011). Chemical composition of wild edible укупних фенола и флавоноида) помоћу UV-VIS спектрофотометрије. Највећа антирадикалска mushrooms. Journal of Agricultural and Food mushrooms and antioxidant properties of their Chemistry, 54 (26), 9764-9772. активност уочена је за OH and SOА радикал за оба сирова екстракта (IC50 =4,02 µg/ml for CdAq water soluble polysaccharidic and ethanolic 21. Phillips, R. (1981). Mushrooms and Other Fungi fractions. Food Chemistry, 126 (2), 610-616. and IC50 =1,40 µg/ml for CdEtOH), док је садржај укупних фенола (TP) и укупних флавоноида био већи код CdEtOH у односу на CdAq. Оба сирова екстракта су показала активност у односу на of Great Britain and Europe, Pan Books Ltd., 30. Wasser, S. (2002). Medicinal mushrooms as a London. MCF 7 ћелијску линију, иако је CdEtOH показао бољи ефекат у односу на CdAq екстракт након source of antitumor and immunomodulating 72h на MTT и SRB тесту постужући IC 217,90 и 205,90 µg/ml. На основу испољене 22. Ribeiro, B., de Pinho, P.G., Andrade, P.B., Bap- polysaccharides. Applied Microbiology and Bio- 50 tista, P., Valentão, P. (2009). Fatty acid compo- technology, 60 (3), 258-274. биоактивности сапротрофна врста C. disseminatus може се сматрати добрим ресурсом алтернативних извора нутрацеутика и биолошки активних једињења. sition of wild edible mushrooms species: A 31. Zaidman, B.Z., Yassin, M., Mahajna, J., Was- comparative study. Microchemical Journal, 93 ser, S.P. (2005). Medicinal mushroom modu- Coprinellus disseminatus, антиоксидативна, антипролиферативна (1), 29-35. Кључне речи: lators of molecular targets as cancer thera- активност, укупни феноли, флавоноиди 23. Singleton, V.L., Orthofer, R., Lamuela-Raven- peutics. Applied Microbiology and Biotech- tos, R.M. (1999). Analysis of total phenols and nology, 67 (4), 453-468. Received: 21 November 2016 other oxidation substrates and antioxidants by 32. Zhang, M., Cui, S.W., Cheung, P.C.K., Wang, Accepted: 19 December 2016 means of Folin-Ciocalteu reagent. Methods in Q. (2007). Antitumor polysaccharides from Enzymology, 299, 152-178. mushrooms: a review on their isolation process, 24. Skehan, P., Storeng, R., Scudiero, D., Monks, structural characteristics and antitumor activity. A., McMahon, J., Vistica, D., Boyd, M.R. Trends in Food Science & Technology, 18 (1), (1990). New colorimetric cytotoxicity assay for 4-19. anti-cancer-drug screening. Journal of the Na- 33. Zhou, X., Gong, Z., Su, Y., Lin, J., Tang, K. tional Cancer Institute, 82 (13), 1107-1112. (2009). Cordyceps fungi: natural products, 25. STATISTICA (Data Analysis Software System) pharmacological functions and developmental (2013). v.12.0., Stat-Soft Inc., Tulsa, OK, USA products. Journal of Pharmacy and Pharma- (www.statsoft.com) cology, 61 (3), 279-291.

101