Cent. Eur. J. Biol.• 6(4) • 2011 • 575-582 DOI: 10.2478/s11535-011-0029-5

Central European Journal of Biology

The antioxidant properties of polypore mushroom Daedaleopsis confragosa

Research Article

Senka Vidović1,*, Zoran Zeković1, Ibrahim Mujić2, Žika Lepojević1, Marija Radojković1, Jelena Živković3

1Faculty of Technology, Department of Biotechnology and Pharmaceutical Engineering, The University of Novi Sad, 21000 Novi Sad, Serbia 2Collegium Fluminense Polytechnic of Rijeka, 52441 Poreč, Croatia 3Faculty of Medicine, The University of Niš, The Department of Pharmacy, 18000 Niš, Serbia Received 17 November 2010; Accepted 15 February 2011

Abstract: Daedaleopsis confragosa belong to a large and remarkable group of mushrooms called polypores. This type of mushroom could be easily said to be quite unexplored and unused when it comes to its antioxidant properties. Thus, in order to evaluate its antioxidant activity, the investigation had to include the total phenolics and flavonoide content, the content of Selenium, the content of Zinc, the scavenging capacity on DPPH˙and OH˙ radicals, reducing power and capacity to remove lipid peroxidation. The investigated mushroom extract contained 54.17 mg GAE/g of total phenols and 48.46 mg CE/g of total flavonoides. Zinc and Selenium were detected and quantified by using inductively coupled plasma mass spectroscopy. The scavenging activity of the radicalswas

found to exhibit 50% of the inhibition value (IC50 value) at the extract concentration of 0.015±0.007 mg/ml for the investigated D. confragosa extract. By using electron paramagnetic resonant spectroscopy it was found that the investigated extract does not have a significant role in the prevention of lipid peroxidation. It was effective in scavenging on ˙OH radical, RI was 56±2%.

Keywords: Mushrooms • Daedaleopsis confragosa • Antioxidant activity • Antioxidant compounds • Zinc • Selenium © Versita Sp. z o.o.

1. Introduction is believed that one species was used for its antibacterial properties and the other one for starting fire [3]. There Daedaleopsis confragosa belong to the order of the are two famous medicinal polypore mushrooms that Polyporales, the family of Polyporaceae and genus are in use today: Ganoderma lucidum, well known as Daedaleopsis. It is also known by its common name reishi, and Trametes versicolor. They have been used “the blushing bracket”. The polyporales are among in traditional medicine and in many scientific medicinal the most common and easily identifiable group of wild researches related to immune system recovery and mushrooms. They are usually inedible, because of its cancer [4]. As D. confragosa belong to this large and hardness and woody structure, except for a few special significant group of mushrooms, the further research of edible species [1,2]. Almost all of these mushrooms the latter could be of tremendous importance. grow on wooden bases such as trees, logs and stumps. Considering the growing interest for the natural Some of the polypore mushrooms have been used in sources of antioxidants on one hand, and the unexplored rituals many centuries ago. Famous Otzi the Iceman, the and unused field of polypore mushrooms on the other, the mummy of a man from about 53 centuries ago, who was main objective of this research was the investigation of the found in 1991 in a glacier on the border between Austria antioxidant properties and some antioxidant compounds and Italy, was carrying two different polypore species. It of the woody D. confragosa mushroom, collected from

* E-mail: [email protected] 575 The antioxidant properties of polypore mushroom Daedaleopsis confragosa

the Istrian peninsula in Croatia during late summer 2008. total phenolics and flavonoide content, the content of The powerful antioxidant properties of these mushrooms Selenium, the content of Zinc, the scavenging capacity refer to different antioxidant compounds. Mushrooms are on DPPH˙ and OH˙ radicals, the reducing power and generally considered to be a good source of protein and capacity to remove or prevent lipid peroxidation. phenolic antioxidants, which is the case with variegatic acid and diboviquinone [5]. Bearing in mind the presence of the conjugated ring structures and the number and 2. Experimental Procedures arrangement of the polar hydroxyl groups [6], many phenolic compounds can fuction as antioxidants by 2.1 Chemicals and Reagents scavenging superoxide anion [7], singlet oxygen [8], and 1,1-Diphenyl-2-picryl-hydrazyl-hydrate (DPPH), lipid peroxyl radicals [9], and by stabilizing free radicals that Folin-Ciocalteu reagent, L-α-phosphatidylcholine, are involved in oxidative processes through hydrogenation (±)-catechin, hypoxanthine and xanthine oxidase or complexing with the oxidizing species [10]. ˙OH radical was purchased from Sigma (Sigma-Aldrich GmbH, is one of the most reactive radicals in biological systems Sternheim, Germany). The gallic acid was purchased and is marked as a highly damaging species in free radical from Sigma (Sigma, St. Luis, MO, USA). Potassium

pathology, capable of damaging bio-molecules of living ferricyanide, FeSO4 was purchased from Merck cells [11]. Thus, the inhibition of this destructive activity can (Darmstadt, Germany). 7-DS (2-(5-carboxypentyl)-2- be seen as crucial. Now, most active phenolic antioxidants, undecyl-4,4-dimethyloxazolidine-3-oxylstearate) was in view of their chemical structure, are flavonoides. purchased from Molecular Probes (Junction City, OR, Selenium and Zinc act as integral parts of the USA). DEPMPO (5-diethoxyphosphoryl-5-methyl- antioxidant systems. Alone, these micronutrients cannot 1-pyrroline-N-oxide) was purchased from Alexis be considered as antioxidant compounds. Selenium is Biochemical (Laursen, Switzerland). All other chemicals important as constituents of protective enzymes via special and reagents were of the analytical reagent grade. amino acids (selenocysteine, selenomethionine), or structures like selenium-proteins. Selenium-proteins have 2.2 Sample Preparation a key role in a variety of biological processes, including Voucher specimens (Daedaleopsis confragosa (Bolton) antioxidant defence [12]. For example, we may consider J. Schröt., Croatia, Istria, Ćićarija Mune, 08.2008. (det.: four glutathione peroxidases, among them cytosolic Maja Karaman) were confirmed and deposited at the glutathione peroxidase, the first Selenium-protein that Herbarium of the Department of Biology and Ecology was identified, which protects cells against peroxidative (BUNS Herbarium), Faculty of Natural Sciences, damage by reducing hydrogen peroxide, free fatty acid University of Novi Sad [18]. The mushrooms fruiting hydroperoxides, and phospholipids hydroperoxides [13]. bodies were collected in their natural environment, after This trace element is established as an essential element they were gently cleansed of any residual compost. of fundamental importance to human health. It is known The fruiting bodies of the fresh mushrooms were air- primarily for its antioxidant activity and its therapeutic dried and then stored in an air-tight plastic bag at room aspects, for its chemo-preventive, anti-inflammatory and temperature. The dried mushroom samples were milled antiviral properties [14]. Zinc antioxidant properties belong in a blender before the extraction. The mushroom to some other structures, for example Zn-metallothionein samples (10.0 g) were extracted by using 50% ethanol or antioxidant enzyme Cu-Zn superoxide dismutase. Zinc (50.0 ml). The extraction process was carried out using deficiency leads to oxidative damage of lipids, proteins, the ultrasonic bath (B-220, Branson and SmithKline DNA oxidation [15], or diseases such as cancer and Company, USA) at 45°C for 40 minutes. After the atherosclerosis. This second essential mineral could well filtration, the extraction solvent was removed bya perform its antioxidant actions using different mechanisms rotary evaporator (Devarot, Elektromedicina, Ljubljana, including sulfhydryl protection against oxidation, the Slovenia) under a vacuum. The obtained extract was induction of metallothionein, a cystein rich protein with the then dried at 60°C to the constant mass. In the end, capacity to scavenge oxidants and bind redox active metals, the dry extract was placed in glass bottles and stored and as a constituent of the intracellular and extracellular at -4°C to prevent oxidative damage until the analysis. antioxidant enzyme Cu-Zn superoxide dismutase [16]. It can replace the redox active metals, Copper and Iron, in 2.3 The Determination of Antioxidant membrane binding sites and thus inhibit the oxidation of Compounds and trace elements Selenium liposomes [17]. and Zinc For evaluating D. confragosa antioxidant activity, The content of total phenolic compounds in a dry the following elements have been investigated: the mushroom extract was determined by Folin-Ciocalteu

576 S. Vidović et al.

procedure [19] using gallic acid as a standard. The for 20 min at 50°C. After the incubation, 2.5 ml of absorbance was measured at 765 nm. The content of 10% trichloroacetic acid solution was added to the total phenolic compounds was expressed as mg of gallic mixture, and the mixture was centrifuged for 10 minutes acid equivalent (GAE) per g of dry mushroom extract. (1509 x g). The obtained supernatant (2.5 ml) was mixed

The total flavonoids content was determined by with bidestilated water (2.5 ml) and 0.1% FeCl3 solution aluminium chloride colorimetric assay [20] using catehin (0.5 ml). The absorbance was measured at 700 nm. The as a standard. It was expressed as mg of catehin higher absorbance indicates a higher reducing power of equivalents (CE) per g of dry mushroom extract. the investigated extract. For the analysis of the trace elements, Selenium and Zinc, the mushroom extract was digested in concentrated 2.6 Scavenging Activity on ˙OH radical

HNO3 and afterwards the elements were quantified The ability of the investigated mushroom extract to by inductively coupled plasma mass spectroscopy remove radicals was evaluated by the difference (ICP/MS, PerkinElmer 9000, USA). between the amplitudes of the EPR signals of trapped radicals in radical-generating systems, with and without 2.4 DPPH Assay the addition of the investigated extract. The results are The free radical scavenging activity of the mushroom here presented as Relative Inhibition (%), which presents extracts was determined as described by Espin [21]. This the relative decrease of radical production (Eq. 2). In assay was used for a preliminary free radical scavenging equation (2) PArgsis the second peak amplitude (radical– evaluation. It seemed to be the simplest and fastest generating system), PArgs+e is the second peak amplitude method than any other method in this case. DPPH˙ is a (radical-/-generating system + extract) and PIrgs is the stable free radical and it allows an electron or hydrogen second peak intensity (radical-generating system). radical to become a stable diamagnetic molecule [18].

The decrease in the absorbance of DPPH˙ radical was RI = ( (PArgs – PArgs+e)/ PIrgs)×100 (Eq. 2) caused by the reaction between antioxidant molecules and the radical, which resulted in the scavenging of the The ability of the mushroom extracts to scavenge radical by the hydrogen donation, which was visually ˙OH radicals was tested using the Fenton reaction noticeable as discoloration from purple to yellow. Briefly, as a generating system. The reaction for ˙OH radical the mushroom extract was mixed with methanol (96%) production was performed by combining 0.5 mM H2O2 and 90 mM 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and 0.075 mM FeSO4. DEPMPO (5-diethoxyphosphoryl- to give a different final concentration of dry extract. 5-methyl-1-pyrroline-N-oxide), in the final concentration After 60 min at room temperature, the absorbance of 28 mM, was used to form DEPMPO/OH adduct. The was measured at 517 nm and expressed as radical final concentration of the extract (previously dissolved in scavenging capacity. Radical scavenging capacity water) was 0.2 mg/ml. The sample with no extract added (%RSC) was calculated using the equation (Eq. 1), was used as a blank sample. The antioxidant activity of where A sample is the absorbance of sample solution and ascorbate at the final concentration of 0.2 mg/ml was

A blank is the absorbance of a blank sample. estimated so as to compare the effects of the extracts with the effects of this frequently used antioxidant. In

%RSC = 100-(A sample×100)/A blank (Eq. 1) all of these experiments ultra-pure water (18 ΜΩ) was used. This activity was also expressed as the inhibition Electron Paramagnetic Resonant Spectroscopy concentration at 50% (IC50), the concentration of the (EPR) was the only employed technique used for solution tested was required to obtain 50% of radical direct detection and identification of free radicals [23], scavenging capacity. with sensitivity as high as 10-10 M. EPR spectra were recorded at room temperature using a Varian E104-A 2.5 The Determination of Reducing Power EPR spectrometer operating at X-band (9.51 GHz) with The reducing power of the mushroom extracts the following settings: the modulation amplitude, 2 G; and ascorbic acid, the standard antioxidant, were the modulation frequency, 100 KHz; the microwave determined by the Oyaizu method [22]. Various power, 10 mW; the time constant, 0.032 s; the field concentration of the mushroom extracts (0.1, 0.2 and centre, 3,410 G; the scan range, 200 G. The spectra 0.5 mg/ml) and ascorbate (0.01, 0.02, 0.05, 0.1, 0.2 were recorded using EW software (Scientific Software, and 0.5 mg/ml) were mixed with the phosphate buffer Bloomington, IL, USA). The samples were drawn into (2.5 ml, 0.2M, pH 6.6) and 2.5 ml of 1% potassium 10 cm long gas-permeable Teflon tubes (wall thickness ferricyanide [K3Fe(CN)6]. The mixture was incubated 0.025 mm and internal diameter 0.6 mm; Zeus

577 The antioxidant properties of polypore mushroom Daedaleopsis confragosa

industries, Raritan, USA). The measurements were buffer (Na2HPO4 8.45 mM, NaH2PO4 3.58 mM, performed by using quartz capillaries, in which Teflon pH 7.4) was added to obtain 125 mM lipids concentration. tubes were placed. The recording started 2 minutes The dry fungal extract was dissolved in the phosphate after the beginning of the reaction, and lasted for buffer to the final concentration of 0.2 mg/ml. The

4 minutes [24,25]. liposomes were exposed to 0.5mM H2O2 and 0.75 mM

FeSO4 in the presence or absence of the fungal extract 2.7 Lipid Peroxidation for 20 minutes at room temperature. After that, the The ability of the mushroom extracts to inhibit lipid liposome solution was dissolved 15 times to stop the peroxidation was tested by using the Fenton reaction reaction. The samples were centrifuged for 10 minutes as the generating system for OH˙ radical. This radical is (10600 x g) and the supernatant was discarded. The a major cause of lipid peroxidation . EPR spin probing liposomes were again dissolved in the buffer, and technique and membrane spin probe 7-Doxyl Stearate the solution was applied to dried 7-DS, redissolved (7-DS), shown in Figure 1, were used to determine the in methanol. The control samples were treated in the membrane fluidity that correlates with the increased lipid same manner, but without the exposure to the radical- peroxidation. The order parameter (S), which correlates generating system [24,25]. The spin probe/membrane with the membrane fluidity, was calculated from the lipid ratio used was approximately 1:200 [26]. spectral parameters (Figure 2). EPR spectra were recorded at room temperature

In the Figure 2, 2TII is outer hyperfine splitting using a the Varian E104-A EPR spectrometer operating

and 2T┴ - inner hyperfine splitting. Parameter a at X-band (9.51 GHz) using the following settings: the is isotropic hyperfine coupling constant in crystal modulation amplitude, 2 G; the modulation frequency,

[a = 1/3(Txx + Tyy + Tzz)] and a’ is isotropic hyperfine 100 kHz; the microwave power, 10 mW; the time

coupling constant in membrane [a’ = 1/3(TII + 2T┴)] and constant, 0.25 s; the field centre, 3390 G; the scan

Txx, Tyy, Tzz - hyperfine constants (for 7-DS, they were range, 100 G; the scan time, 8 minutes. The spectra

taken to be Txx = Tyy = 6.1 G, Tzz = 32.4 G) (Eq. 3). were recorded using the EW software. The samples were drawn into 10 cm long gas-permeable Teflon

S = (TII -T┴)/(TZZ-TXX) x (a/a’) (Eq. 3) tubes (the wall thickness 0.025 mm and internal diameter 0.6 mm, Zeus industries, Raritan, USA). The The Chloroform solution of L-α-phosphatidylcholine, measurements were performed using quartz capillaries, that evaporated under a vacuum to dryness, was used in which the Teflon tubes were placed. for the preparation of the liposomes. The phosphate 2.8 Statistical Analysis The statistical analysis was performed using Statistica 6.0. (StatSoft Inc, Tulsa, OK, US). All the experiments were done at least in triplicate unless specified otherwise. The results are here presented as mean values ± SD. The significant levels were defined at P<0.05. Figure 1. Chemical structure of 7-DS used to determine membrane fluidity. 3. Results and Discussion 3.1 Antioxidant Compounds and trace elements Selenium and Zinc As the chemical structure of phenolic compounds is mainly responsible for their antioxidant activity, the measurement of the total phenolic content may be related to the antioxidant properties of an investigated material. The higher extraction yields of phenolic compounds can be obtained by the increase of a solvent polarity [27]. That being so, the 50% ethanol polar solvent was used to obtain the dry mushroom extract. The total phenolics Figure 2. EPR spectra of liposomes labeled with 7-DS (black: content, the total flavonoids content, the content of liposomes treated with ˙OH radicals; gray: untreated liposomes). Two narrow lines originate from 7-DS in the Selenium and Zinc were investigated and presented solution (arrows). (Table 1). The extraction yield was determined based on

578 S. Vidović et al.

the weight of the mushroom sample that had been used achieved a slightly higher capacity, 75.5%, but also in the extraction process and the mass of the obtained at a much higher concentration, 5mg/ml. At the same dry mushroom extract. The obtained extraction yield concentration radical scavenging capacity of this fruity was 8.34%. This low extraction yield had been expected body mushroom water extract was 36.8% [33]. since D. confragosa is a woody mushroom with a low The radical scavenging activity was found to exhibit moisture content. The total flavonoide/ total phenolic 50% of the inhibition value (IC50 value) at the extract ratio is very high, around 90%. This could contribute concentration of 0.015±0.007 mg/ml for the investigated to a higher extract antioxidant activity, as flavonoides, D. confragosa extract. The calculated IC50 value was considering their chemical structure, are referred to slightly lower than the reported IC50 value for Boletus as phenolic compounds with the highest antioxidant edulis (0.016±0.003 mg/ml) and lower than the reported properties. These compounds can play a double role value for Boletus aurantiacus (0.024±0.004 mg/ml) dry in reducing the rate of oxidation, as they participate extract [30], or methanolic extract of Ganoderma tsugae, in Iron chelation and trapping radicals [28]. The total known as Ling chic (0.25±0.0016 mg/ml) [34], which phenolic content of D. confragosa extract is higher than indicated a higher antioxidant activity in comparison to it was reported for some other mushrooms extracts the reported values. (39.09±0.17 mg GAE/g for Boletus edulis, 38.53±0.03 mg Ferrous ion, which commonly exists in food systems, GAE/g for Boletus aurantiacus, 26.89 mg GAE/g for is well-known as an effective pro-oxidant component Armillaria mellea and 24.48 mg GAE/g for Lycoperdon [35]. Ferrous iron has the capacity to reduce oxygen to saccatum) [29,30]. It was also higher than the reported superoxide radicals, it can catalyze the decomposition content of the methanolic extracts for Grifola frondosa of peroxide and yielding hydroxyl radical from hydrogen (12.31 mg/g), Hericium erinaceus (12.05 mg/g) and peroxide [36]. The ability of certain components, Trichloma giganteum (7.61 mg/g) [31]. Both essential ascorbic acids and polyphenols to reduce Fe3+ to Fe2+, trace elements, Selenium and Zinc, were detected in is in fact the basis of theirs prooxidative activity. This the investigated extract, but the content of the essential activity plays the important role in some cases (cancers, trace element Zinc was much higher in comparison to virus and bacterial infections), where the generation of that of essential Selenium. certain radical and products like H2O2 are needed. For example as a result of prooxidative action, generated

3.2 Antioxidant activity H2O2 is required and is important as it causes certain The antioxidant activity of the investigated mushroom cancer cell death [37]. For the measurements of extract was checked through different radical systems. the reductive ability Fe3+→Fe2+ transformation in the The first method applied was a quick and simple DPPH presence of the mushroom extract was investigated. assay. It was noticed that the scavenging effect of the Figure 3 indicates the reductive capabilities of the mushroom extracts on DPPH˙ radicals increased with analyzed mushroom extract and the standard antioxidant the increase of the concentrations. At concentration compound ascorbic acid. of 0.01mg/ml, the radical scavenging capacity of the A characteristic feature of the investigated extract investigated extract was 34.22%, while at the extract is its antioxidant activity. Confirmation of this is a much concentration of 0.02 mg/ml radical the scavenging lower reducing power in comparison to that of ascorbic capacity was 67.55%. Scavenging ability similar to the acid. It was clear, as we expected, that ascorbic acid ability of investigated extract at 0.02 mg/ml showed a has more reductive capabilities than D. confragosa methanolic extract of medicinal mushroom Ganoderma extract. For example at the concentration of 0.2 mg/ml lucidum but at higher concentration, 0.64 mg/ml [32]. measured absorbance of ascorbic acid was 1.192±0.03, Lee et al. (2008) found that the scavenging capacity while for D. confragosa was 0.442±0.06. However, the on DPPH radicals, of ethanolic extracts of Hypsizigus reducing power of the investigated mushroom extract marmoreus mushroom, also known as hon-shimeji, was higher than the previously reported reducing power

Mushroom TP TF (TF/TP) x 100 Zinc content Selenium content sample mg GAE/g of extract mg CE/g of extract (%) mg/kg of extract mg/kg of extract

D. confragosa 54.17±0.002 48.46±0.004 89.46±0.003 32.64±0.001 0.217±0.003

Table 1. Total phenolics (TP), total flavonoids (TF), total flavonoids and total phenolics ratio (TF/TP), Zinc and Selenium content in dryD. confragosa extract.

579 The antioxidant properties of polypore mushroom Daedaleopsis confragosa

for mushroom Boletus edulis and Boletus aurantiacus starts with the peroxidation of polyunsaturated fatty acid (0.223 and 0.192, respectively) [30] and also for the components in low density lipids (LDL) of the membrane. reported reducing powers of the methanolic extracts The oxidative modification of LDLs may play a significant of Dictyophora indusiata, Grifola frondosa, Hericium role in the development of atherosclerosis. Several erinaceus and Tricholoma giganteus [31]. This property toxic byproducts can be derived from the peroxidation suggests that D. confragosa extracts may act as free process and they can also damage bio-molecules [38]. radical chain terminators, transforming reactive free Such membrane modification and free radical activity radical species into more stable non-radical products. can be inhibited by antioxidants. The application of an For further analysis, an extract antioxidant activity antioxidant should prevent or remove lipid peroxides, EPR method was used. The ability of the mushroom so that the membrane fluidity remains unchanged after

extract to scavenge ˙OH radicals was also investigated H2O2 treatment [25]. by this method. The scavenging of ˙OH radical existed for For the investigation of the lipid peroxidation order, the investigated mushroom extract and it was the inhibitor the parameters S: for pure liposomes, liposomes for ˙OH radical. The determined relative inhibition of exposed to the Fenthon system, liposomes mixed with ˙OH radical was 56±2% at the extract concentration of mushroom extract and liposomes mixed with mushroom 0.2 mg/ml. This activity was slightly lower than the activity extract exposed to the Fenton system - were measured against ˙OH radical which was reported for dry ethanolic and are presented in Table 2. extracts of the mushroom extracts of Boletus edulis The lipid peroxidation leads to the decrease of the (82±3%) [30], Armillaria mellea (85±4%) and Lycoperdon membrane fluidity which was evaluated by the EPR saccatum (73±4%) [29] at the same concentration. spin-probes-doxyl stearates. The measurement of the S Relative inhibition of the investigated mushroom extract parameter for pure liposomes and the liposomes exposed was compared to relative inhibition of the well known to the Fenton system was performed so as to evaluate the antioxidant compound ascorbate. The relative inhibition of ascorbate, as a standard antioxidant compound, was Ascorbic acid D. confragosa extract 100±3%. As we expected this activity was higher than that of the investigated mushroom extract, but it still 1.4 could be considered as an effective scavenger of this 1.2 radical species. Lee et al. (2008) reported that ethanolic extracts of H. marmoreus, at the extract concentration 1.0 of 20 mg/ml, achieved 12.2% relative inhibition of this 0.8 radical, while for a water extract it was slightly higher, 27.67% [33]. Also, Mau et al. (2002) reported that 0.6

medicinal mushrooms Coriolus versicolor, G. lucidum Absorbance 0.4 and G. tsuage showed scavenging abilities of hydroxyl radical from 38.0% to 52.6% for the extract concentration 0.2 of 16 mg/ml [32]. Compared to the previously reported 0.0 results this investigated extract could be considered to be 0.0 0.1 0.2 0.3 0.4 0.5 more effective in inhibition of hydroxyl radicals. Concentration (mg/ml)

3.3 Lipid Peroxidation The fluidity and permeability of the cell membrane can Figure 3. Reducing power of D. confragosa dry extract and ascorbic acid, measured by spectrophotometric detection of be seriously affected by free radicals. The free radical Fe3+→ Fe2+ transformation at different concentration, species can induce lipid peroxidation, which can break showing better reductive capabilities of standard down the membrane integrity. This oxidative modification antioxidant compound ascorbic acid.

Samples Treatment S±S.D.

Untreated liposomes 0.583±0.002 Control Treated liposomes 0.609±0.009

Untreated liposomes/extract 0.597±0.002 D. confragosa Treated liposomes/extract 0.604±0.007

Table 2. The values of the order parameter S for pure liposomes and liposomes/extract mixture and liposomes and liposomes/extract mixture exposed to Fenton system for evaluation of the decrease of the membrane fluidity provoked by the Fenton system.

580 S. Vidović et al.

level of the decrease of the membrane fluidity provoked in today’s food industry. As we used different methods by the Fenton system. S of the untreated liposome/ and techniques for obtaining more details about extract mixtures was acquired for determining whether antioxidative properties of D. confragosa mushroom the components of the extracts provoked changes in and its extract, we successfully provided a lot of various the membrane fluidity, which may imply the existence and important information. The results of this study of the mechanisms of defense from peroxidation and clearly show that D. confragosa mushroom extract has some other possible applications of the extracts. The antioxidant activity against various radical producing S parameter value of the untreated liposome/extract systems. The mushroom is a source of networked mixture was slightly higher than the value of the untreated antioxidant compounds. The high contribution of total liposome, what indicated certain influence on membrane flavonoide compounds can contribute to its higher fluidity. In the case of pure liposome exposed to oxidative antioxidant activity, as flavonoids are considered as stress and liposome exposed to oxidative stress in the the most active antioxidant phenolic compounds. The presence of extract the order parameter S were almost extract contains the trace mineral elements, Selenium the same, 0.609±0.009 and 0.604±0.007, respectively. and Zinc, which are rather relevant considering their There was no significant decrease of order parameter vital role in human health. Many European countries S in the presence of mushroom extract exposed to still have dietary Selenium and Zinc intake below the the radical generated Fenthon system, in comparison recommended amount so, an additional intake of these to that of pure liposome exposed to radical system, elements is of importance. Further evaluation of the which indicates that D. confragosa extract could not be investigated extract antioxidant activity proves that it is considered as an efficient antioxidant in lipid peroxidation an effective scavenger of different radical species and prevention. Though, some other mushrooms extracts, for has the ability to chelate ferrous ion, but it is not efficient example Boletus edulis, Boletus aurantiacus, Lycoperdon in lipid peroxidation prevention. Still, from all the saccatum etc., have been reported as efficient obtained facts this polypore mushroom and investigated components in the prevention of the lipid peroxidation extract could be considered as an antioxidant source [29,30]. that could be used for different applications in food and Intensive research of new unexplored natural pharmaceutical industries. sources of antioxidants is very significant and can help bring new natural products into the pharmaceutical and food industries referring to their everyday battle Acknowledgments with reactive oxygen species. Therefore, finding a new antioxidant source could be important for health benefits Specimens’ determination was done in Herbarium of the considering the many diseases that reactive oxygen Department of Biology and Ecology (BUNS Herbarium), species induce in biological systems. Discovering a Faculty of Natural Sciences, University of Novi Sad. natural source of antioxidants could be significant also Authors would like to thank to Goran Ačkov from this for the replacement of the artificial toxic antioxidants Department.

References [1] Shiono Y., Tamesada Y., Muravayev Y.D., Murayama [5] Kasuga A., Aoyagi Y., Sugahara T., Antioxidant T., Ikeda M., N-Phenethylhexadecanamide from activity of fungus Suillus bovines, J. Food. Sci., edible mushroom Laetiporus Sulphareus, Nat. 1995, 60, 1113-1115 Prod. Res., 2005, 4, 363-366 [6] Cotelle N., Bernier J.L., Catteau J.P., Pommery J., Wallet [2] Yu L., Zhang B., Geza R., Szalvay K., Sun R., Janis J.C., Gaydou E.M., Antioxidant properties of hydroxyl J., et al., Protein HGF1 from edible mushroom flavones, Free Radical Biol. Med., 1996, 20, 35-43 Grifola frondosa is a novel 8kda class I hydrophobin [7] Robak J., Gryglewski R.J., Flavonoids are that forms rodlets in compressed monolayers, scavengers of superoxide anion, Biochem. Microbiology, 2008, 154, 1677-1685 Pharmacol., 1998, 37, 83-88 [3] Peintner U., Poder R., Pumpel T., The iceman’s [8] Husain S.R., Cillard, J., Cillard P., Hydroxyl radical fungi, Mycol. Res., 2005, 102, 1153-1162 scavenging activity of flavonoids, Phytochemistry, [4] Lindequist U., Niedermayer T., Wolf D.J., The 1987, 26, 2489-2491 Pharmacological potential of mushrooms, eCAM [9] Torel J., Chillard J., Chillard P., Antioxidant activity (Evidence-based complementary and alternative of flavonoids and reactivity with peroxy radical, medicine), 2005, 3, 285-299, 2005, 3, 285-299 Phytochemistry, 1986, 25, 383-385

581 The antioxidant properties of polypore mushroom Daedaleopsis confragosa

[10] Shahidi F., Janitha P.K., Wanasundara P.D., [25] Živković J., Zeković Z., Mujić I., Gođevac D., Phenolic antioxidants, Crit. Rev. Food Sci. Nut., Mojović M., Mujić A., et al., EPR Spin trapping and 1992, 32, 67-103 Spin Probing Spectroscopy in Assesing Antioxidant [11] Halliwell B., Antioxidants and Human Disease: A Properties: Example on Extract of Catkin, Leaves General Introduction, Nutr. Rev., 1997, 55, s44-s52 and Spiny Burs of Castanea Sativa, Food Biophys., [12] Brenneisen P., Steinbrenner H., Seis H., Selenium, 2009, 4, 126-133 oxidative stress, and health aspects, Mol. Aspect [26] Cooper R.A., Abnormalities of cell-membrane Med., 2005, 26, 256-267 fluidity in the pathogenesis of disease, New Engl. J. [13] Brigelius-Flohe R., Flohe L., Is there a role of Med., 1977, 297, 371-377 gluthatione peroxidases in signaling differenatiation, [27] Cheung L.M., Cheung P.C.K., Oi V.E.C., Antioxidant Biofactors, 2003, 17, 93-102 activity and total phenolics of edible mushroom [14] Rayman M.P., The importance of selenium to extracts, Food Chem., 2003, 81, 249-255 human health, Lancet, 2000, 356, 233-241 [28] Van Acker S.W.A., Van Balen G.P., Van den Berg [15] Ho E., Courtemanche C., Ames B.N., Zinc deficiency D.J., Bast A., Van der Vijgh W.J., Influence of iron induces oxidative DNA damage and increases p53 chelation on the antioxidant activity of flavonoids, expression in human lung fibroblasts, J. Nutr., Biochem. Pharmacol., 1998, 56, 935-943 2003, 133, 2543-2548 [29] Mujic I., Zekovic Z., Lepojevic Z., Vidovic S., [16] Oteiza P., Mackenzie G., Zinc, oxidant-triggered Milosevic S., Mujic A., In: Wasser S.P., Li Y., Buswell cell signaling, and human health, Mol. Aspect Med., J.A., Chen M., Yang X., (Eds.), Proceedings of the 2005, 26, 245-255 5th International Medicinal Mushroom Conference [17] Powell S.R., The antioxidant properties of zinc, J. (5-8 September 2009, Nantong, China), Nantong, Nutr., 2000, 130, 1447S-1454S 2009, 205-211 [18] Holmgren P.K., Holmgren N.H., Additions to Index [30] Vidović S., Mujić I., Zeković Z., Lepojević Ž., Herbariorum (Herbaria), 8th Ed., Taxon, 2003, 52, Tumbas V., Mujić A., Antioxidant properties of 385-389 selected Boletus mushrooms, Food Biophys., [19] Singleton V.L., Rossi J.A., Colorimetry of total 2009, 1, 49-58 phenolics with phosphomolybdic-phosphotungstic [31] Mau J.L., Lin H.C., Song S.F., Antioxidant properties acid reagents, Am. J. Enol. Viticult., 1965, 16, of several specialty mushrooms, Food Res. Int., 144-158 2002, 35, 519-526 [20] Markham K. R., Plant Phenolics, In: Harborne J.D., [32] Mau J. L., Chao G. R., Chen, C. C., Antioxidant (Ed.), Methods in Plant Biochemistry, Academic properties of several medicinal mushrooms, J. Press, San Diego, 1989 Agric. Food Chem., 2002, 50, 6072-6077 [21] Espin J.C., Soler-Rivas C., Wichers H.J., [33] Lee Y.L., Jian S.Y., Lian P.Y., Mau J.L., Antioxidant Characterization of the Total Free Radical properties of extracts from a white mutant of Scavenger Capacity of Vegetable Oils and Oils mushroom Hypsizigus marmoreus, J. Food Comp. Fractions using 2, 2-Diphenyl-1-pycrilhydrazyl Anal., 2008, 21, 116-124 Radical, J. Agric. Food Chem., 2000, 48, 648- [34] Mau J.L., Tsai S.Y., Tseng Y.H., Huang S.J., 656 Antioxidant properties of methanolic extracts from [22] Oyaizu M., Studies on product of browning reaction Ganoderma tsugae, Food Chem., 2005, 93, 641-649 prepared from glucose amine, Jpn. J. Nutr., 1986, [35] Hsu C.L., Chen W., Weng Y.M., Tseng C.Y., 44, 307-315 Chemical composition, physical properties and [23] Lundqvist H., Danmark S., Johansson U., antioxidant activity of yam as affected by different Gustafsson H., Ollinger K., Evaluation of electron drying methods, Food Chem., 2003, 83, 85-92. spin resonance for studies of superoxide anion [36] Fraga C., Oteiza P., Iron toxicity and antioxidant production by human neutrophilis interacting nutritiens, Toxicology, 2002, 180, 23-32 with Staphylococcus aureus and Staphylococcus [37] Chen Q., Espay M.G., Sun A.Y., Lee J.H., Krisha epidermis, J. Biochem. Biophys. Methods, 2008, M.C., Shacter E., et al., ascorbate in pharmacologic 70, 1059-1065 concentrations selectively generates ascorbat [24] Spasojević I., Mojović M., Blagojević D., Spasić S., radical and hydrogen peroxide in extractellular Jones D., Nikolić-Kokić A., et al., Relevance of the fluid in vivo, Proc. Natl. Acad. Sci. USA, 2007, 21, capacity of phosphorylated fructose to scavenge 8750-8754 the hydroxyl radical, Carbohydr. Res., 2009, 344, [38] Box H.C., Maccubbin A.E., Lipid peroxidation and 80-84 DNA damage, Nutriton, 1997, 13, 920-921

582