AN INSIGHT INTO IN VITRO BIOACTIVITY OF WILD-GROWING PUFFBALL SPECIES LYCOPERDON PERLATUM ( PERS ) 1796

Lycoperdon perlatum (Pers) 1796 is saprobic puffball species with a global distribution. It is edible if young, when the gleba is still homogeneous and white. Since this species has a pleasant texture and taste, it has been used in soups as a substitute for dumplings. The aim of this work was to study bioactivity of crude extracts prepared from wild-growing sporocarps of L. perlatum collected from Eastern Serbia during 2012. The bioactivity screens included antioxidant (DPPH • and FRAP assays), antiproliferative (human breast MCF-7 cancer cell-line; MTT and SRB assays) and antibacterial (three referent ATCC strains; microdilution assay) effects. Polar extracts (aqueous LycAq and ethanol – LycEtOH) and a nonpolar extract (hexane LycHex) of the examined mushroom species were screened. In addition, LycAq and LycEtOH were primarily characterized by UV-VIS spectrophotometry, due to determination of chemical composition (total phenol and flavonoid contents). The highest anti-DPPH radical activity was observed for LycAq (IC50 = 46.56 μg/ml). In comparison with LycAq, less polar LycEtOH showed slightly better ferric reducing antioxidant power (FRAP) (IC50 = 21.87 μg/ml and IC50 = 19.28 μg/ml, respectively). However, total phenol contents of both extracts were similar (≈ 2.0 mg GAE/g d.w.). Conversely, modest activities were found against Staphylococcus aureus ATCC 25922 (LycHex, MIC = 3.12 mg/ml) and MCF-7 cells (with the highest one obtained for LycEtOH after 72 h, IC50 = 367.54 μg/ml and IC50 = 390.03 μg/ml, MTT and SRB assays, respectively). According to the obtained experimental data, L. perlatum can be considered as a good source of novel and potent natural antioxidants for use in regular diet.


INTRODUCTION
Since the ancient times, mushrooms have been valued by humankind as a culinary wonder and traditional medicine.Recently, it has been discovered that many fungal species, including puffballs, represent miniature pharmaceutical factories producing hundreds of natural products with potent and broad range bioactivities.Furthermore, these organisms have a long history of use in Oriental medicine, supported by contemporary studies (Miles and Chang, 2004).Production of reactive oxygen and nitrogen species occurs during normal cell metabolism.The excess of free radicals leads to oxidative stress, resulting in oxidative DNA damage, implicated in the pathogenesis of numerous disorders, such as cardiovascular, atherosclerosis, rheumatoid arthritis and cancer (De Silva et al, 2013).On the other hand, antibiotic resistance has become a global concern (Westh et al., 2004).The clinical efficacy of many existing antibiotics is being threatened by the emergence of multidrug resistant pathogens (Bandow et al., 2003).The increasing failure of chemotherapeutics has led to the screening of a number of medicinal mushrooms for their antimicrobial potential (Karaman et (Dickinson and Lucas, 1982).Several chemicals have been identified in its fruit bodies such as sterol derivatives ((S)-23-hydroxylanostrol, ergosterol α-endoperoxide, ergosterol 9,11dehydroendoperoxide and (23E)-lanosta-8,23-dien-3β,25-diol), volatile compounds (3-octanone, 1-octen-3-ol and (Z)-3-octen-1-ol) and an unusual amino acid such as lycoperdic acid (Lamotte et al., 1978;Szumny et al., 2010).The puffball extracts are known for exhibiting antibacterial activity against human pathogens such as Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, with efficiency comparable to that of the antibiotic ampicillin (Ramesh and Pattar, 2010).Moreover, antifungal activities against Candida albicans, C. tropicalis, Aspergillus fumigates and Alternaria solani have been also reported (Pujolet et al., 1990).
This study, focusing on in vitro bioactivity evaluation of autochthonous L. perlatum, included antioxidant, antiproliferative and antibacterial screens.To the best of our knowledge, this is the first report on bioactive properties of L. perlatum originating from Serbia.

Puffball samples
Lycoperdon perlatum (Pers) 1796 was collected in Sikole area (a village near the Negotin town, Serbia) during 2012.After its identification, a voucher specimen (MYC12-00664) was deposited at the Herbarium BUNS, University of Novi Sad, Serbia.The puffball samples were frozen at -20 °C, prior to freeze-drying procedure (Bio alpha, Martin Christ GmbH, Germany).Freeze dried samples were ground to a fine powder, wrapped in plastic bags and stored in a dark place at room temperature, until further use.

Total phenol content
Total phenol (TP) content of LycEtOH and LycAq was determined according to method by Singleton et al. (1999), adapted for a 96-well plate reader (Multiskan Ascent, Thermo Electron Corporation).Folin-Ciocalteu reagent (125 μl, 0.1 M) was added to diluted extracts (25 μl).After 10 min, 100 μl of 7.5% w/v sodium carbonate was added and the reaction mixture was incubated for 2 h.Absorbance was read at 690 nm.TP is expressed as mg gallic acid equivalents (GAE)/g of dry weight (d.w.).
Absorbance was measured at 620 nm, after incubation of 6 min.The results are expressed as mg ascorbic acid equivalents (AAE)/g of dry weight (d.w.).

Antibacterial activity
LycHex was the only extract screened for antibacterial activity at in vitro conditions, after dissolving in 5% DMSO, to reach a final concentration of 0.5% (w/v) (CLSI procedure, 2008; slightly modified by Karaman et al., 2009b).Standard American Type Culture Collection (ATCC) strains of two Gram-positive bacteria, namely S. aureus ATCC 25922 and B. subtilis ATCC 6633, and one Gram-negative bacterium, namely E. coli ATCC 25923, were used.Two-fold microdilution assay in 96-well microplates (Spektar, Ĉaĉak, Serbia) was applied for determination of minimal inhibitory and bactericidal concentrations (MIC and MBC, respectively).Pure bacterial strains were subcultured on nutrient agar slants at 37 ºC for 24 h; their suspensions corresponding to McFarland 0.5 optical density, ≈ 1.5×10 8 CFU/ml.The extract concentration ranged from 0.78 to 25.00 mg/ml.After incubation at 35 ºC for 18-24 h, MIC and MBC were determined.Last two wells served as a growth control (positive control) and negative control (5% DMSO), respectively.The reference antibiotics (gentamicin and ampicillin) were applied as control standards.

Antiproliferative activity Cells
Estrogen dependent MCF-7 cells were grown in DMEM (PAA Laboratories) supplemented with 10% FCS.The cells were seeded in a 96-well microplate (5000 cells per well).After incubation of 24 h, the growth medium was replaced with 100 μl of medium containing the examined samples (extracts) at four different concentrations (33.3 μg/ml, 100.0 μg/ml, 300.0 μg/ml and 900.0 μg/ml).The untreated cells served as the control, while pure DMSO was used as a positive control.The growth of MCF-7 cells was evaluated by standard colorimetric assays, MTT and SRB.

MTT Assay
After incubation of 24 h and 72 h respecttively, the cell viability was determined by the proliferation MTT assay (Mosmann, 1983).This assay is based on the color reaction of mitochondrial dehydrogenase in living cells with MTT reagent.Upon the incubation, MTT reagent was added to each well (50 μg/100 μl/well; at 37 °C, in 5% CO 2 , for 3 h).The crystals of produced formazan were dissolved in 100 μl acidified isopropanol (0.04 M HCl in isopropanol).Absorbance was measured at 540 nm and 690 nm on a 96 well plate reader (Multiskan Ascent, Thermo Electron Corporation, USA).The results are expressed as IC 50 values (sample concentration which inhibited 50% of the net cell growth).

SRB Assay
This assay estimates cell number indirectly, by staining cellular protein with the protein-binding dye SRB (adapted procedure, by Skehan et al., 1990).After incubation, the cells were fixed adding cold 50% TCA and incubated for 1 h at 4 °C.The wells were washed with deionised water and dried; SRB solution (0.4% in 1% acetic acid) was then added to each plate well and incubated for 30 min at room temperature.The unbound SRB was removed by washing with 1% acetic acid.The plates were air dried, the bound SRB was solubilized with 10 mM Tris (pH = 10.5), while absorbance was measured at 492 nm and 690 nm, using the microplate reader.DMSO was applied as a positive control.The results are expressed as IC 50 values.

Data analysis
The absorbance was calculated from the difference of two absorbances: A = A 540 -A 690 and = A 492 -A 690 for MTT and SRB assays, respectively.Percentage of cytotoxicity was calculated as the ratio of the treated and the control group absorbance respectively, multiplied by 100.The obtained results are expressed as IC 50 .These values were calculated from the cytotoxicity (%) -extract concentration plot (μg/ml) using the Origin v. 6.0 graphing and data analysis software (1999).

Statistical analysis
All the assays were carried out in triplicate.The data were statistically analyzed using the software Statistica (2013).The results of antioxidant and antiproliferative activities as well as total phenol (TP) and flavonoid (TF) contents are expressed as a mean value ± standard deviation (SD).The statistical significance was determined by analysis of variance (ANOVA), with Duncan's multiple range test as post hoc test.The Pearson correlation coefficients (r 2 ) were calculated for TP, TF, FRAP and IC 50 values for anti-DPPH • and antiproliferative activities.

Antioxidant activity and total phenol and flavonoid contents
Among the examined extracts, IC 50 value of LycAq stood out (46.56 µg/ml) in DPPH• scavenging assay (Table 1).Interestingly, in both assays these extracts showed higher activities compared with the experimental data obtained for L. perlatum originating from Portugal and Turkey (Barros et al., 2008b;Sarikurkcu et al., 2015).While there was no significant difference in their TP contents, TF content of LycAq was more than three times higher (Table 1).

Table 1.
Antioxidant activity and total phenol and flavonoid contents of Lycoperdon perlatum extracts *Expressed as concentration of the extracts that caused 50% activity -IC50 (µg/ml).**Expressed as mg ascorbic acid equivalents/g extract of dry weight (mg AAE/g d.w.).***Total phenol (TP) content is expressed as mg gallic acid equivalents/g extract of dry weight (mg GAE/g d.w.), while total flavonoid (TF) content is expressed as mg quercetin equivalents/g extract of dry weight (mg QE/g d.w.) Taking into account both parameters (antioxidant activity and chemical composition), it may be assumed that phenolic and flavonoid compounds are not the only ones responsible for the observed activities.

Antiproliferative activity
The antiproliferative activity against human breast MCF-7 cancer cell-line was evaluated by MTT and SRB assays.The examined extracts (LycAq and LycEtOH) exhibited different activity in the aforementioned bioassays (Table 2).Generally, it was modest with LycAq (IC 50 = 327.86µg/ml) being the most potent one (SRB assay, 24 h).As other bioactivities, this one also directly depends on the chemical composition (

Antibacterial activity
LycHex practically displayed a modest antibacterial activity only against the strain S. aureus ATCC 25922 (MIC = 3.12 mg/ml, MBC = 6.25 mg/ml) (Table 4).These results are in good accordance with recently published data for the same puffball species collected in India (Ramesh and Pattar, 2010).

CONCLUSIONS
L. perlatum, the examined fungal species in this study, may be considered as a promising source of novel natural antioxidants with potential significance for regular diet.If its autochthonous origin is taken into account, this fact is even more important.Puffball species in Serbia have not been examined enough so far.To our knowledge, this is the first report on bioactivity of the autochthonous species L. perlatum.The future research work should be primarily directed towards elucidation of chemical profiles of LycAq and LycEtOH and their mechanism(s) of action.

Table 2 .
a,bThe results are expressed as a mean value ± SD.The means with different superscript within the same row are statistically different (p<0.05)Antiproliferative activity of Lycoperdon perlatum extracts on MCF-7 cells a

Table 4 .
Antibacterial activity of Lycoperdon perlatum hexane extract **The obtained values against the strain Staphylococcus aureus ATCC 25922