ANTIOXIDATIVE ACTIVITY of Lenzites Warnieri BASIDIOCARPS

Зборник Матице српске за природне науке / Matica Srpska J. Nat. Sci. Novi Sad, № 133, 163—171, 2017 UDC 615.322:582.28 https://doi.org/10.2298/ZMSPN1733163K Aleksandar Z. KNEŽEVIĆ1 * , Ivan N. MILOVANOVIĆ1 , Jelena B. VUKOJEVIĆ1 1 University of Belgrade, Faculty of Biology, Takovska 43, 11000 Belgrade, Republic of Serbia ANTIOXIDATIVE ACTIVITY OF Lenzites warnieri BASIDIOCARPS ABSTRACT: Considering that mushrooms synthesize different kinds of compounds with antioxidative activity and that search for natural antioxidants is a topical study area, testing of unstudied species is fully justified. The aim of the study was to evaluate antioxidative capacity of Lenzites warnieri basidiocarps using different solvents. Antioxidative potential of 96% ethanolic, 70% ethanolic and methanolic extracts was evaluated by 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) bleaching assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay. Additionally, total content of phenols and flavonoids in extracts was determined as galic acid equivalent (GAE) and quercetin equivalent (QE), respectively. Presented as EC50, 70% ethanolic extracts showed the highest antioxidative capacity by DPPH assay (3.08 ± 0.49 mg/mL) and 96% ethanolic extract by ABTS assay (3.08 ± 0.24 mg/mL). Methanolic extract exhibited the lowest antioxidative activity in both assays (6.02 ± 0.99 mg/mL and 4.92 ± 0.38 mg/mL, respectively). Results showed that antioxidative capacity of extracts depended on solvents and assay used, indicating that ethanolic extracts were with higher capacity in free radicals neutralization. The highest content of total phenols was detected in 70% ethanolic extract (37.45 ± 0.36 µg GAE/mg of dried extract) while the lowest amount was noted in methanolic extract (22.73 ± 0.05 µg GAE/mg of dried extract). Total flavonoid contents were negligible and ranged between 1.91 ± 0.10 and 2.24 ± 0.13 µg QE/mg of dried extract. The obtained results indicate that Lenzites warnieri possess significant antioxidative capacity which is mainly correlated to phenols present in the extracts. KEYWORDS: Lenzites warnieri, antioxidative activity, basidiocarps, extracts INTRODUCTION Mushrooms have been used for thousands of years as food but also in traditional medicine as a reach source of biologically active compounds (Was- ser, 2010; Roupas et al. 2012). However, this great pharmacological potential is still underutilized since, approximately, only 5% of the species are well studied and comprehensive studies are needed for both unstudied and already examined species (Wasser, 2002). * Corresponding author. E-mail: [email protected] 163 Species of the genus Lenzites has been used in traditional Chinese medicine for treatment of several diseases and disorders such as haunch and femoral pain, acropathy, apoplexy and cold (Ren et al. 2006). Medicinal effects have been also demonstrated in conventional medicine and until nowadays, there have been several studies on the various properties of Lenzites spp. basidiocarps and mycelium extracts as scavengers of free radicals, antimicrobials, anti-oxidant, anti-viral and immunosuppressant (Liu et al. 2014; Milovanović et al. 2015; Hussin et al. 2016). The contemporary life style brings human and animal populations to face with abundant presence of free radicals, endogenously or in the environment, which further leads to appearance of oxidative stress which is a basis of aging and the initiation of various diseases and disorders (Limón- Pacheco and Gonsebatt, 2009). The capacity of organisms to defend against free radicals can be improved by various synthetic or natural antioxidants. From this aspect, macromycetes can be of a great interest due to the synthesis of different antioxidative compounds such as phenols, tocopherols, carotenoids, ascorbic acid, etc. (Stajić et al. 2013). Lenzites warnieri Mont. & Durieu (Polyporaceae) is a white rot plant pathogen which can be found on living wood of several deciduous trees such as alder, cottonwood, elm and willow, prefering wet habitats such as riparian woodland or fens and warm temperatures (Ryvarden and Gilbertson, 1993). Species primarily inhabit Central and Southern Europe but also parts of Asia and Northern Africa (Tortić, 1972; Winterhoff, 1986). Formation of resupinate fruiting bodies occurs in autumn while sporulation is expected in following spring (Ryvarden and Gilbertson, 1993). Since described activities might have been responsible for medicinal effects of Lenzites spp. in traditional practice, the goal of the study was to evaluate possible antioxidative potential of basidiocarp extracts of unstudied Lenzites warnieri. MATERIALS AND METHODS Organism and extraction The basidiocarps were collected in Gornje Podunavlje Special Nature Reserve, Serbia, and identified as Lenzites warnieri according to the macroscopic features and the micro morphology of the reproductive structures (Ryvarden and Gilbertson, 1993; Vukojević and Hadžić, 2013). Fruiting bodies (3.0 g) were air-dried, grinded to powder in laboratory blender (Waring Commercial 8010S, USA). Extraction was carried out with 96% ethanol, 70% ethanol and methanol (90.0 mL) on a magnetic stirrer (150 rpm, 30 °C) for 72 hours. The resultant extracts were then centrifuged (20 °C, 3,000 rpm, 10 min), and supernatants filtrated through Whatmann No. 4 filter paper. The filtrates were concentrated under reduced pressure in a rotary evaporator (Büchi R-114, Germany) at 40 °C to dryness and redissolved in 96% ethanol 164 to an initial concentration of 16.0 mg/mL. The extraction yield was determined as the ratio of dry fungal biomass before extraction and dry extract weight after the evaporation process. Determination of antioxidative activity Antioxidative activities of basidiocarp extracts were determined spectrophotometrically (CECIL CE2501) by DPPH and ABTS tests. DPPH assay The free radical scavenging activity of extracts was based on the reduction of a methanol solution of 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH●) (Blois, 1958). The reaction mixtures composed of 1,800.0 µL of 4% methanol solution of DPPH● and 200 µL of extract at defined concentration (series of double dilutions with ethanol from 16.0 mg/mL to 0.125 mg/mL) were mixed and shaken vigorously. After 30 min of incubation in the dark, absorbance was measured at 517 nm against methanol as a blank. The negative control contained all the reaction reagents except the extract. Scavenging effect was calculated using the equation: DPPH scavenging effect (%) = ((Ac-As)/Ac) × 100 where Ac – absorbance of the negative control; As – absorbance of the reaction mixture (with samples at different concentrations). EC50 values (mg extract/mL) represent the concentration of test samples or L-ascorbic acid (standard antioxidant) providing 50% inhibition of DPPH radicals were calculated by interpolation of DPPH● absorbance curve at 517 nm from linear regression analysis. ABTS assay This test was based on measuring the level of ABTS+● stock solution color change in presence of antioxidants according to the procedure of Miller et al. (1993). The initial solution of ABTS cation radicals was prepared by dissolution of 9.0 µg ABTS in 2.5 mL dH2O and addition of 44.0 µL 140 mM potassium persulphate (K2S2O8) solution, 12 to 16 hours before use, while the stock solution was prepared immediately prior to measurement by dilution of the initial solution with dH2O and adjustment of solution absorbance on 0.700 ± 0.020 at 734 nm. The reaction mixture (1,500.0 µL of the ABTS stock solution and 15.0 µL of extract of concentration of 1.0 mg/mL) was incubated at room temperature for 4 min and absorbance change was measured at 734 nm. Distilled water was used as a blank. Extract concentration required for ABTS cation radicals reduction, equivalent to reduction of 1.0 mg/mL ascorbic acid (AAEC) was determined using equation of calibration curve for ascorbic acid. EC50 value (mg extract/mL) presents effective extract concentration that scavenges 50% of ABTS cation radicals and it is obtained by linear regression analysis. 165 Determination of phenol and flavonoid contents The total contents of soluble phenolic compounds in the extracts of fruiting bodies were estimated using Folin-Ciocalteu reagent and gallic acid as stand- ards according to the method described by Singleton and Rossi (1965). The reaction mixture (1,000.0 µL of 10% Folin-Ciocalteu reagent and 200.0 µL of sample in concentration of 1.0 mg/mL) was incubated for 6 min in dark and thereafter 800.0 µL of 7.5% Na2CO3 was added. The mixture was then vortexed and incubated on a rotary shaker (100 rpm) in dark at room temperature for 2 hours. The absorbance was measured spectrophotometrically at 740 nm against a blank (mixture without extract). The total concentration of phenols was presented as µg of gallic acid equivalent (GAE) per mg of dried extract using equation of calibration curve for GAE. Total flavonoid contents in extracts were determined using quercetin as standard according to the method of Park et al. (1997). The reaction mixture (1,000.0 µL of extract in concentration of 1.0 mg/mL, 4,100.0 µL of 80% ethanol, 100.0 µL of 10% Al (NO3)3 × 9H2O and 100.0 µL of 1.0 M water solution of potassium acetate) was incubated in dark on a rotary shaker (100 rpm) and room temperature for 40 min and thereafter absorbance was measured at 415 nm against blank (mixture which contained ethanol instead extract). Total flavonoid content was expressed as µg of quercetin equivalent (QE) per mg of dried extract using equation of calibration curve for QE. Statistical analysis All measurements were carried out in triplicate and the results are expressed as mean ± standard error. One-way analysis of variance (ANOVA) and Tukey`s HSD post-hoc test were performed to test any significant differences among means. Statistical significance was declared at P < 0.01. All statistical analyses were done using software STATISTICA, version 6.0 (StatSoft, Inc., Tulsa, USA). RESULTS AND DISCUSSION Extraction yields of Lenzites warnieri basidiocarps differed statistically depending on solvent used for the extraction (P < 0.01).

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