|Year : 2015 | Volume
| Issue : 42 | Page : 6-18
Cytotoxicity of some edible mushrooms extracts over liver hepatocellular carcinoma cells in conjunction with their antioxidant and antibacterial properties
Gökhan Sadi1, Bugrahan Emsen1, Abdullah Kaya1, Aytaç Kocabas1, Seval Çinar1, Deniz Irtem Kartal2
1 Department of Biology, Kamil Özdağ Faculty of Science, Karamanoğlu Mehmetbey University, Karaman, Turkey
2 Department of Biology, Biochemistry Graduate Programme, Middle East Technical University, Ankara, Turkey
|Date of Submission||30-Sep-2014|
|Date of Acceptance||01-Dec-2014|
|Date of Web Publication||27-May-2015|
Dr. Gökhan Sadi
Department of Biology, Kamil Özdağ Faculty of Science, Karamanoğlu Mehmetbey University, 70100, Karaman
Source of Support: The authors are grateful to Karamanoglu Mehmetbey
University Scientific Research Fund for financial support (37-M-12), Conflict of Interest: None
| Abstract|| |
Background: Mushrooms have been valued for their nutritive content and as traditional medicines; several important medicinal properties of mushrooms have been recognized worldwide. Objective: The purpose of this study was to elucidate the cell growth inhibitory potential of four edible mushrooms; Coprinus comatus (O.F. Mull.) Pers. (Agaricaceae), Tricholoma fracticum (Britzelm.) Kreisel (Tricholomataceae), Rhizopogon luteolus Fr. and Nordholm (Rhizopogonaceae), Lentinus tigrinus (Bull.) Fr. (Polyporaceae) on hepatocellular carcinoma (HepG2) cells in conjunction with their antioxidant and antibacterial capacities. Materials and Methods: Five different extracts of edible mushrooms were obtained using water, methanol, acetone, n-hexane and chloroform as solvent systems for cytotoxic, antioxidant and antibacterial properties. Results: C. comatus showed substantial in vitro cytotoxic activity against HepG2 cell lines with all extracts especially with chloroform 50% inhibition (IC 50 value of 0.086 mg/ml) and acetone (IC 50 value of 0.420 mg/ml). Chloroform extract of C. comatus had maximum amount of β-carotene (25.94 μg/mg), total phenolic content (76.32 μg/mg) and lycopene (12.00 μg/mg), and n-hexane extract of L. tigrinus had maximum amount of flavonoid (3.67 μg/mg). While chloroform extract of C. comatus showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) capturing activity (1.579 mg/ml), the best result for metal chelating activity was obtained from methanolic extract (0.842 mg/ml). Moreover, all tested mushrooms demonstrated antibacterial activity and n-hexane extract of L. tigrinus and acetone extracts of T. fracticum were the most active against tested microorganism. Conclusion: These results indicate that different extracts of investigated mushroom have considerable cytotoxic, antioxidant and antibacterial properties and may be utilized as a promising source of therapeutics.
Keywords: Cancer cells, cell toxicity, edible macrofungi, radical scavenging
|How to cite this article:|
Sadi G, Emsen B, Kaya A, Kocabas A, Çinar S, Kartal DI. Cytotoxicity of some edible mushrooms extracts over liver hepatocellular carcinoma cells in conjunction with their antioxidant and antibacterial properties. Phcog Mag 2015;11, Suppl S1:6-18
|How to cite this URL:|
Sadi G, Emsen B, Kaya A, Kocabas A, Çinar S, Kartal DI. Cytotoxicity of some edible mushrooms extracts over liver hepatocellular carcinoma cells in conjunction with their antioxidant and antibacterial properties. Phcog Mag [serial online] 2015 [cited 2019 Nov 22];11, Suppl S1:6-18. Available from: http://www.phcog.com/text.asp?2015/11/42/6/157665
| Introduction|| |
Wild edible mushrooms have been consumed in fresh or dried forms by human for a long period due to its organoleptic properties such as flavor and texture. ,, Although thousands of mushrooms are present in nature, few of them are accepted as edible and commercial. , Studies on nutritional values of mushrooms showed richness in protein, dietary fibers, vitamins and minerals along with low fat and energy ,,, enables to use as edible. Local consumption of wild mushrooms are also increasing because of their medicinal properties , due to the presence of secondary metabolites having pharmaceutical importance. ,
Functional foods are new emerging area of food science and defined as food, which exerts health improving effects other than its nutritional value. Whereas, nutraceuticals are defined as concentrated phytochemicals derived from food and applied as a supplement.  Mushrooms are rich in nutraceuticals having antioxidant, antitumor and antimicrobial features. ,,, There has been also a continuous search for new antimicrobial substances for decades and mushrooms are of interest for several researchers. Albeit nearly 60 antimicrobial compounds have been isolated from mushrooms; only the compounds from microscopic fungi have been present in the market as antibiotics until now. 
Antioxidants protect the living systems from free radicals, which results in oxidative damage of protein and nucleic acids. Even though humans are well protected against free radicals, many diseases such as cancer may result from the uncontrolled production of reactive oxygen species.  Therefore, antioxidant supplementation either as foods or drugs helps people to reduce the risk resulted from oxidative stress. Drugs of herbal origin have been used in traditional systems of medicine since ancient times, and many researchers have benefited from medicinal plants to prevent oxidative stress. ,,,,
Although, antioxidant capacities of mushrooms are well-established, anticancerogenic properties of wild edible mushrooms in clinical studies  is of immense important due to very few reports. Therefore, this study focused on analysis of bioactive feature of some wild edible mushrooms. In this concern, we elucidated the cell growth inhibitory potential of four edible mushrooms; Coprinus comatus (O.F. Mull.) Pers. (Agaricaceae), Tricholoma fracticum (Britzelm.) Kreisel (Tricholomataceae), Rhizopogon luteolus Fr. and Nordholm (Rhizopogonaceae), Lentinus tigrinus (Bull.) Fr. (Polyporaceae) on liver hepatocellular carcinoma (HepG2) cells in conjunction with their antioxidant and antibacterial capacities.
| Materials and Methods|| |
Collection and identification of mushroom samples
Samples were collected in 2012 from different localities within Karaman province of Central Anatolian Region of Turkey. Necessary morphological and ecological characteristics of the samples were recorded, and they were photographed in their natural habitats. Then they were brought to the fungarium and microstructural properties were investigated. Comparing the obtained macroscopic and microscopic data with literature,  they were identified as T. fracticum (Kaya 7482), L. tigrinus (Kaya 7454), C. comatus (Kaya 7483) and R. luteolus (Kaya 7455). The samples are kept at Karamanoğlu Mehmetbey University, Science Faculty, Department of Biology.
Extraction of bioactive ingredients
After drying under mild heat evaporator, entire mushroom samples (cap and stripe) were powdered with liquid nitrogen, mortar and paste. Then bioactive ingredients were extracted by 250 ml of different solvent systems such as, water, methanol, chloroform, acetone and n-hexane using Soxhlet extraction apparatus throughout 2 days. After extraction, solvents were evaporated with rotary evaporator (IKA, Staufen Germany) under vacuum to dryness and lyophilized to get ultra dry powders that were solubilized with minimum amount of sterile distilled water. They were diluted to different concentrations according to experiments before the experimental setup.
Determination of cell growth inhibitory potential of mushrooms on hepatocellular carcinoma cells
Cell viability was quantified using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)- 2H-tetrazolium-5-carboxanilide (XTT) in vitro cellular toxicity assay.  Accordingly, HepG2 cells were cultured in RPMI 1640 medium with L-glutamine and 25 mM HEPES (Lonza, USA) supplemented with 10% fetal bovine serum and 0.2% gentamycin sulfate. They were grown in a humidified incubator at 37°C with 5% CO 2 . After growing to expected confluency (90%), they were harvested by 2 ml 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA), complete detachment was obtained by incubation at 37°C for 5 min. Later, trypsin activity was brought to end by the addition of 5 ml growth medium. Cell suspension was counted with trypan blue dye and cell concentration was adjusted to 100,000 cells/ml. In sterile microtiter culture disc, 50 μl HepG2 cell in fresh complete medium and 50 μl extracts in different concentration (0.5-10 mg/ml) were mixed and incubated for 48 h or 72 h at 37°C in 5% CO 2 .
Cell growth inhibitory potential of the mushrooms on HepG2 cells was studied using commercial XTT cell proliferation kit (Biological Industries, Israel). 100 μl of activation solution (PMS-N methyl dibenzopyrazine methyl sulfate) was added to 5 ml XTT reagent (supplied with kit) and mixed. Then, 50 μl of this solution was added to each well containing cells and extracts and incubated for 10 h with activated XTT under 5% CO 2 at 37°C in a humidified incubator. Finally, the intensity of the formazan was measured at 415 nm with Multiscan Go microplate reader (Thermo Scientific, USA), and 50% inhibition (IC 50 ) values were calculated.
Determination of antioxidant potentials
Possible antioxidant potential of different extracts of tested mushrooms were evaluated by measuring their total flavanoids, β-carotene and lycopene amount and phenolic contents as well as reducing power, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and metal chelating activity. The methods used to evaluate these indicators of antioxidative capacity are given in the following parts.
Determination of total phenolic contents
Concentration of total phenolic compounds in different extracts of T. fracticum, L. tigrinus, C. comatus and R. luteolus was determined spectrophotometrically according to the method described previously by  with slight modifications. In this method, gallic acid with various concentrations (0.01-1.0 mM) was used as a standard phenolic compound. This method was adapted to microtiter plate measurements in triplicate. A volume of 20 μl of standards or different extracts (10 mg/ml) were mixed with the same amount of Folin and Ciocalteu's phenol reagent (2N) and kept in dark for 3 min. Afterward, 20 μl of 35% sodium carbonate (w/v) and 140 μl dH 2 O were added to start incubation period for 10 min. After that, the absorbance was measured at 725 nm and the results were calculated as mean ± standard error of mean (SEM) from gallic acid calibration curve and expressed as mg of gallic acid equivalents/mg of extracts.
Determination of total flavonoid contents
Total flavonoids of water, methanol, chloroform, acetone and n-hexane extracts obtained from edible mushrooms were determined using protocols reported by  with slight modifications. A volume of 50 μl of extracts (10 mg/ml) were mixed with 215 μl of ethyl alcohol (80% v/v), 5 μl of aluminum nitrate (10% w/v) and 5 μl potassium acetate (1 M) in microtiter plates and incubated for 40 min at room temperature. After reading at 415 nm, total flavonoid contents were calculated according to following equation:
Total flavonoid contents (μg/mg extract) = (A 415 + 0.01089)/0.002108
Determination of β-carotene and lycopene contents
Different extracts with water, methanol, n-hexane, acetone and chloroform that were obtained from four edible mushroom was re-extracted with 10 ml of acetone: hexane (4:6) mixture and filtered through Whatman No. 4 filter paper in order to determine β-carotene and lycopene contents. After filtration, absorbance of the filtrates was measured at 453, 505 and 663 nm. β-carotene and lycopene contents were determined according to following equations. 
β-carotene content (mg/100 mg) =0.216 A 663 - 0.304 A 505 + 0.452 A 453
Lycopene content (mg/100 mg) = −0.0458 A 663 + 0.372 A 505 - 0.0806 A 453
DPPH radical scavenging activity
Free radical scavenging activities (RSA) of extracts obtained from T. fracticum, L. tigrinus, C. comatus and R. luteolus were determined by monitoring DPPH reduction. Gallic acid (0.01-0.5 mM) was used as standard antioxidant molecule and to determine RSA, 20 μl of various concentrations of standard or extracts were mixed with 180 μl of DPPH solution (0.06 mM in methanol) and incubated for 1 h at dark in microtiter plates. Blank measurements without standard or extracts were also performed. After reading absorbance at 517 nm, reduction of the DPPH radical was determined as percent discoloration of DPPH which was calculated according to following formula;
The extract concentrations providing the IC 50 was calculated from the graph of RSA versus extract amount that was used for the comparison of different extracts of tested mushrooms. 
Determination of metal chelating activity
Chelating abilities of various extracts from edible mushrooms were determined using EDTA as a standard chelating agent.  Different concentrations (50 μl) of extracts (2, 4, 6, 8, 10 mg/ml) and standard (0.1-5 mM) were added to microplate wells and mixed with 10 μl ferrozine (5 mM), 5 μl iron (II) chloride (2 mM) and 185 μl absolute methanol. After incubation for 10 min at room temperature, absorbances were read at 562 nm, and IC 50 values were calculated.
Determination of reducing power
Reducing powers of different extracts were determined according to the method  with an adaptation to microplate measurement. Gallic acid (0.01-0.1 mM) was used as a standard antioxidant. In this method, various concentrations of 50 μl mushroom extracts (2, 4, 6, 8, 10 mg/ml) were mixed with 75 μl phosphate buffer (0.2 M pH: 6.6) and 75 μl potassium ferricyanide (1% w/v) in a total volume of 200 μl and incubated at 50°C for 20 min. After adding 75 μl trichloroacetic acid (10% w/v), samples were centrifuged for 10 min at 1000 × g. Supernatant (75 μl) were transferred to another microtiter plate and mixed with 75 μl distilled water and 15 μl iron (III) chloride (0.1% w/v). After reading the absorbance at 700 nm, effective concentrations (EC 50 ) at which the absorbance was 0.5 for reducing power was calculated.
Determination of antibacterial activities
The antibacterial activities of different extracts of four edible mushrooms (40 μg/μl) were determined against Bacillus subtilis (Ehrenberg) Cohn, Bacillus licheniformis, Staphylococcus aureus Rosenbach, Escherichia More Details coli T. Escherich and Agrobacterium tumefaciens Smith and Townsend by the disc diffusion susceptibility method.  Penicillin, gentamicin and tetracycline were used as the standard antibiotic discs. Microorganisms were grown in Müller Hilton broth at 35°C and 28°C for 18 h and then cell density were adjusted according to 0.5 McFarland standards. After that, 100 μl of adjusted microorganism suspension was inoculated onto Müller Hilton Agar by spread plate technique. A volume of 40 μl of extract (40 μg/μL) loaded discs and standard antibiotic discs were placed on inoculated Petri dish More Detailses. Negative controls were prepared with water alone. Inhibition zones (IZ) around the discs after overnight incubation were measured to determine antibacterial activities of the extracts. The study was performed in triplicates of each sample.
All the assays were carried out at least in triplicate measurements. The results are expressed as mean values and SEM. Antioxidant, antibacterial and cytotoxicity activities of mushrooms were compared with Analysis of Variance (ANOVA) test followed by the appropriate post-hoc test (Duncan Test) and values with P < 0.05 were considered as significantly different. IC 50 and EC 50 values were calculated with Probit Regression Analysis and associated 95% confidence limits for each treatment. Relations among the variables were tested by Bivariate Correlation Analysis. These calculations were carried out using Statistical Package for Social Sciences (SPSS ® , version 21.0, IBM Corporation, Armonk, NY, USA).
| Results and Discussion|| |
Naturally occurring substances present in edible mushrooms have beneficial effects on health. Several species of mushroom show free RSA and high growth inhibitory potential against cancer cells and microorganisms ,,,,,,,, that's why, mushrooms have recently become attractive as nutritionally beneficial foods and as a source material for the development of some drugs. This study presents the comparison of four different edible mushrooms; T. fracticum, L. tigrinus, C. comatus and R. luteolus which are consumed locally in different regions of Turkey in terms of their important medicinal properties such as cytotoxic, antioxidant and antibacterial effects.
Cell growth inhibitory potential of mushrooms on liver hepatocellular carcinoma cells
Liver tissue is involved in the metabolism of various nutrients including edible mushrooms and their digestive products in the form of glucose, amino acids and fatty acids. Furthermore, it contains thousands of enzymes essential to perform vital metabolic functions to metabolize both beneficial and harmful substances and store some of the compounds having antioxidant properties. ,,
Cancer diseases are one of the main causes of death worldwide and hepatocellular carcinoma (also called malignant hepatoma) is the most common type of liver cancer.  Liver HepG2 cells are the model cell lines to study liver functions in vitro since they maintain several liver properties. Our study reports on the investigation into the cytotoxic activity of different extracts of four edible mushrooms on HepG2 cells that have not been reported previously.
To study the growth inhibitory activity of the four edible mushrooms in vitro, HepG2 cell lines were incubated with various concentrations of four edible mushrooms obtained from different solvent extraction. The potentially cytotoxic effect of the mushroom extracts was assessed using in vitro XTT assay. Findings from our study clearly demonstrated that elevated concentrations of mushroom extracts are cytotoxic to human liver carcinoma (HepG2) cells. In particular, C. comatus displayed the strongest growth inhibitory potential on HepG2 cells. However, all extracts of C. comatus are more effective anticancer agent than other mushrooms tested with very low IC 50 values (0.086 mg/ml for chloroform and 0.42 mg/ml for acetone extracts). In addition to C. comatus, L. tigrinus also possess high degree of growth inhibitory potential over HepG2 cells for all extracts (IC 50 value of 0.376 mg/ml for acetone, 0.434 mg/ml for n-hexane and 2.854 mg/ml for chloroform). In contrast, T. fracticum was the least effective cytotoxic material among the all tested mushroom since nearly tenfold higher amount of extract was needed to obtain IC 50 values as compared with the most effective C. comatus. IC 50 value of acetone extract was 3.851 mg/ml, 5.164 mg/ml for chloroform and 51.982 mg/ml for water extract of T. fracticum that was significantly higher than those of other mushrooms. Cytotoxic activities of all tested extracts were summarized in [Table 1].
|Table 1: 48 and 72 h IC50 values (mg/ml) of different extracts obtained from four mushrooms for cytotoxicity test |
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Substances that can inhibit or retard oxidation of substances are called antioxidants and recently there has been increasing interest in discovering natural antioxidants, especially those of macrofungal origin. Various macrofungi species, plants, lichens and algae have antioxidant constituents which are important materials due to the fact that these components have anti-inflammatory, antiallergic, antiviral, anti-aging, anticarcinogenic properties and behave like biological response modifiers. ,,, Therefore, they are used in medicinal and pharmaceutical fields by many researchers. The present study was undertaken with the aim to carry out a systematic comparative antioxidant study on different extracts gained from four edible mushrooms using different in vitro methods, and to find any correlation between the antioxidant activity and bioactive ingredients such as total phenolic, total flavonoid, lycopene and β-carotene contents of the extracts. To evaluate the total antioxidant properties of four edible mushrooms, three different assays were carried out namely; scavenging activity on DPPH radicals, reducing power and metal chelating activity.
Scavenging ability on DPPH radicals
The model of scavenging DPPH radical is a widely used method to evaluate the free RSA of antioxidants. DPPH is a stable free radical, and when antioxidants react with DPPH, unpaired electron is paired off and the DPPH solution is decolorized. Therefore, decolorization degree demonstrates the radical scavenging activity of an antioxidant containing materials.
Previous studies showed that extracts or chemical constituents obtained from various mushrooms possess DPPH scavenging activities due to their antioxidant compounds. Studies carried out with Laetiporus sulphureus;  Pleurotus ostreatus; , Lentinus edodes, Sparassis crispa and Mycoleptodonoides aitchisonii; , Stereum hirsutum;  Russula virescens;  Pleurotus eryngii;  Macrolepiota procera; , Pleurotus flabellatus;  Russula laurocerasi;  Suillus bovinus;  Phellinus fastuosus, Phellinus grenadensis, Phellinus merrillii and Phellinus badius;  Lenzites betulina;  Lactarius piperatus;  Cordyceps militaris;  Tricholoma portentosum and Lactarius deliciosus;  Pholiota nameko revealed the high DPPH scavenging activities of these fungi correlated with their antioxidant compounds.
In the present study, all the mushroom species showed DPPH free RSA that were found to rise with increasing concentration. Maximum concentrations (10 mg/ml) of methanol extracts from R. luteolus and T. fracticum and chloroform extract from C. comatus showed DPPH scavenging activities over 70% (71.38, 71.19 and 70.28%, respectively). Among all the mushroom extracts, highest activity was obtained from the lowest concentration (2 mg/ml) of chloroform extract from C. comatus (53.56%). Comparing activity rates of the extracts of four mushrooms, chloroform extracts gave best results and all activity rates were over 60%. The scavenging effects of four mushrooms (in chloroform extract) on DPPH radical were in the descending order of C. comatus >T. fracticum >L. tigrinus >R. luteolus. The lowest activity values belonged to acetone extract. In acetone extract, DPPH scavenging activity rates of four mushrooms ranged from 13.67% to 40.08% for maximum concentration (10 mg/ml) and was in the order of L. tigrinus <T. fracticum <C. comatus <R. luteolus [Figure 1]. The chloroform extract from C. comatus showed strong DPPH radical scavenger that was reflected by its low IC 50 value (1.579 mg/ml). Other extracts that had low IC 50 values (under 3 mg/ml) are methanol and chloroform extract from R. luteolus (2.398 and 2.518 mg/ml) and methanol extract from C. comatus (2.566 mg/ml). IC 50 value of standard (gallic acid) was found as 0.037 mg/ml and there was statistically (P < 0.05) significant difference between this value and the values belonging to all mushroom extracts [Table 2].
|Figure 1: Scavenging abilities of extracts in different concentrations from four mushrooms on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals|
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|Table 2: IC50 values (mg/ml) of different extracts from four mushrooms and standard for scavenging activity on DPPH radicals |
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Chelating abilities of metal ions
The presence of metal ions; especially Fe 2+ lead the production of reactive species such as hydroxyl radicals and metal-oxygen complex in biological systems and cellular damage eventually occurs with metal catalyzed reactions.  To prevent oxidative stress caused by metal ions, edible mushrooms that have metal chelating abilities are very important in foods. In the determination of metal chelating activities, extracts of the mushroom species was used to suppress the formation of ferrous and ferrozine complex, indicating their chelating activity and capture ferrous ion before the production of ferrozine in the system.
Many researchers have previously examined chelating activities of different extracts of some mushroom species and chemical constituents of different fungi in their studies and achieved positive results. ,,,,,,,,,
The present study has also been focused on metal chelating activities of different extracts from T. fracticum, L. tigrinus, C. comatus and R. luteolus. Correlations between chelating capacities of various extracts of four mushrooms and their concentrations were observed. Methanol extracts at concentrations of 8 and 10 mg/ml and chloroform extract at concentration of 10 mg/ml of of C. comatus displayed over 70% metal chelating activity (70.17, 78.01 and 70.52%, respectively). Methanol extract gave higher results compared to other extracts. For the chelating effect of ferrous ion, four mushrooms in methanol extract at concentration of 10 mg/ml were effective in the following order: C. comatus >R. luteolus >T. fracticum >L. tigrinus [Figure 2].
|Figure 2: Chelating abilities of extracts in different concentrations from four mushrooms on ferrous ions|
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As shown in [Table 3], for chelating on ferrous ions, highly potent inhibitions were found by methanol extract from C. comatus (IC 50 = 0.842 mg/ml), methanol extract from R. luteolus, (IC 50 = 2.397 mg/ml) and chloroform extract from C. comatus (IC 50 = 5.650 mg/ml) when compared with standard (EDTA) inhibitor (IC 50 = 0.158 mg/ml) [Table 3].
|Table 3: IC50 values (mg/ml) of different extracts from four mushrooms and standard for chelating on ferrous ions |
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Determination of reducing power
The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity.  In the used protocol, an increase in the color of the test solution depends on the reducing power of each compound. The presence of reducers (antioxidants) causes the conversion of the Fe 3+ /ferricyanide complex used in this method to the ferrous form. Higher absorbance indicated higher reducing power of the selected extracts.
Previously, researchers reported that many mushroom species have reducing power. ,, They also reported that methanolic extracts of some mushroom species had high reducing power activity.
In the present study, reducing power of the various extracts of edible mushrooms increased with concentration. Based on the reducing power, among the mushroom extracts predominant one was acetone extract from R. luteolus (EC 50 = 1.754 mg/ml). While the highest reducing powers in chloroform and methanol extracts were belonged to C. comatus (EC 50 = 4.045 and 2.541 mg/ml, respectively), in n-hexane and water extracts R. luteolus showed the highest reducing capacities (EC 50 = 4.869 and 2.468 mg/ml, respectively) [Table 4].
|Table 4: EC50 values (mg/ml) of different extracts from four mushrooms and standard for reducing power |
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However, as shown in [Figure 3], L. tigrinus demonstrably had the lowest reducing activity in acetone and chloroform extracts. Whereas in n-hexane and water extracts, T. fracticum had minimum reducing power. Similarly, in methanol extract, lower reducing power were determined in L. tigrinus and T. fracticum.
Amount of bioactive components working as antioxidants
Significant differences between the chemical composition of wild and commercial edible mushrooms have been reported.  Natural antioxidants derived from macrofungi are of considerable interest since these compounds have important constituents with free radical scavenging ability and hence may contribute directly to the antioxidative action. Among them, phenolic compounds, flavonoids, β-carotene and lycopene contents correlates well with the antioxidant potential of the edible mushrooms. Numerous studies carried out by scientists with some mushrooms demonstrated that these materials had antioxidant components such as β-carotene, flavonoid, lycopene and phenol. ,,,,,,,,, Previous studies also revealed that many other edible mushrooms such as Agaricus bisporus, , Amanita vaginata,  Grifola frondosa,  P. eryngii,  P. ostreatus, , Russula delica and S. bovinus had high rate of phenol, flavonoids, ascorbic acid, β-carotene and lycopene. For that reason, various extracts from T. fracticum, L. tigrinus, C. comatus and R. luteolus were analyzed for their antioxidant components including β-carotene, flavonoids, lycopene and total phenols.
As for the total phenolic contents, highest quantities were found in chloroform and acetone extract of C. comatus (76.32 and 61.09 μg/mg, respectively). At the same time, considerable phenolic content was determined in water extract of L. tigrinus (46.76 μg/mg). The total phenolic content in other mushroom extracts was lower than 37.00 μg/mg.
Considering flavonoid content, n-hexane and water extract of L. tigrinus showed the maximum amounts (3.67 and 3.22 μg/mg respectively). The flavonoid content in other mushroom extracts was lower than 3.00 μg/mg.
Among the mushroom extracts, chloroform extract of C. comatus had the highest β-carotene content (25.94 μg/mg). Furthermore acetone and methanolic extracts of this mushroom had higher β-carotene level than the other edible mushrooms (23.32 and 6.55 μg/mg, respectively), n-hexane and water extracts of L. tigrinus (17.28 and 13.13 μg/mg, respectively) had also considerable amount.
Chloroform and acetone extract of C. comatus revealed higher lycopene content (12.00 and 9.16 μg/mg, respectively) as compared with other mushroom extracts. However, lycopene contents in other mushroom extracts were found in lower amounts (0.96-6.25 μg/mg) [Table 5].
|Table 5: Antioxidant contents of different extracts from four edible mushrooms |
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In this study, it was also determined that there is a positive correlation between antioxidant activity and bioactive constituents of tested edible mushrooms [Table 6]. Our results suggested that phenolic acids and flavonoids may be the second major contributors for the antioxidant activity as the IC 50 values of radical scavenging activity of various soluble fractions of edible mushrooms and also the contents of phenolics or flavonoids exhibited significant correlation. Present study also revealed that DPPH radical scavenging activity was in significant correlation with the amount of phenolics, lycopene, flavonodis and β-carotene. Furthermore, reducing capacity of edible mushrooms was in positive correlation with the amount of flavonoids. Therefore, many foods that have a high rate of phenolics or flavonoids can be used for the purpose of medical therapies.
|Table 6: Correlations between amounts of bioactive components and antioxidant activities |
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Antibacterial activities of different extracts
Mushrooms are rich sources of natural antibiotics, and many of the secondary metabolites may combat with many bacteria and viruses.  With an increasing number of bacteria developing resistance to commercial antibiotics, extracts and derivatives from mushrooms hold great promise for novel medicines. Therefore, antibacterial activity of tested edible mushrooms were analyzed by disc diffusion method on two Gram-negative (E. coli and A. tumefaciens) and three Gram-positive (B. subtilis, B. licheniformis, S. aureus) bacteria. All four tested mushrooms showed antibacterial activity at least on one microorganism. On the other hand, 10 out of 20 extracts (five different extracts of each four mushrooms, total 20 extracts) had stronger antimicrobial potential. [Table 7] summarizes the antibacterial effects of edible mushroom extracts. Accordingly, the largest IZ was measured as 13 mm with chloroform extract of R. luteolus on B. subtilis. Besides, wide range of activity was observed with n-hexane extract of L. tigrinus. It inhibited the growth of entire microorganism used in this survey, including E. coli, because, none of the other extracts did inhibit the growth of E. coli. Strong antibacterial activity of L. tigrinus was followed by T. fracticum in such that its acetone extract showed antimicrobial activity against all microorganisms other than E. coli. The results revealed that for all tested extracts Bacillus sp. were more susceptible, since at least one extract from this mushroom showed antibacterial activity. Moreover, water extract of C. comatus has antibacterial activity against B. licheniformis. Indeed it is well known that Gram-negative microorganism more resistant to antibacterial than Gram-positives.
|Table 7: Antibacterial activities of different extracts obtained from four mushrooms |
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This study provided consistent result with earlier reports that mushrooms produce wide variety of antimicrobial compounds. , It was previously reported that C. comatus had antimicrobial acitivity against S. aureus and B. subtilis whereas in this study it showed its activity against B. licheniformis. This discrepancy could result from cultivation from the different environment because climate, environment and the soil type can effect chemical composition of the mushrooms. On the other hand, similar result was obtained with L. tigrinus that it showed a wide range of antimicrobial activity.
| Conclusion|| |
Edible macrofungi have gained increasing reputation in recent decades, not only due to their nutritive value, but also for their possible health benefits and therapeutic uses. Therefore, wild beneficial sources bring new natural products into the pharmaceutical industry. Our results on four edible mushrooms elucidates that, C. comatus showed substantial in vitro cytotoxic activity against HepG2 cell lines that was in parallel with its high β-carotene, total phenolic and lycopene contents. High antioxidant potential of C. comatus obtained from highest DPPH capturing, and metal chelating activity was in significant positive correlation with the abovementioned bioactive ingredients. As for antibacterial activity, L. tigrinus and T. fracticum were the most active against tested microorganism. These results indicate that different extracts of investigated edible mushrooms have considerable cytotoxic, antioxidant and antibacterial properties and may be utilized as a promising source of therapeutics. Therefore, edible mushrooms might provide an appropriate source of antioxidant and cytotoxic natural compounds and could also be searched as a potent antibacterial drug against infectious diseases. C. comatus deserves further studies due to its incredible cytotoxic and antioxidant potential, and it can be a promising mushroom that would be benefitted in the field of pharmacology with safer and better effectiveness.
| Acknowledgment|| |
The authors are grateful to Karamanoğlu Mehmetbey University Scientific Research Fund for financial support (37-M-12).
| References|| |
Cheung LM, Cheung PC, Ooi VE. Antioxidant activity and total phenolics of edible mushroom extracts. Food Chem 2003;81:249-55.
Cheung PC. The nutritional and health benefits of mushrooms. Nutr Bull 2010;35:292-9.
Beluhan S, Ranogajec A. Chemical composition and non-volatile components of croatian wild edible mushrooms. Food Chem 2011;124:1076-82.
Barros L, Baptista P, Correia D, Casal S, Oliveira B, Ferreira I. Fatty acid and sugar compositions, and nutritional value of five wild edible mushrooms from Northeast Portugal. Food Chem 2007;105:140-5.
Reis FS, Barros L, Martins A, Ferreira IC. Chemical composition and nutritional value of the most widely appreciated cultivated mushrooms: An inter-species comparative study. Food Chem Toxicol 2012;50:191-7.
Barros L, Cruz T, Baptista P, Estevinho LM, Ferreira IC. Wild and commercial mushrooms as source of nutrients and nutraceuticals. Food Chem Toxicol 2008;46:2742-7.
Kalač P. Chemical composition and nutritional value of European species of wild growing mushrooms: A review. Food Chem 2009;113:9-16.
Espín JC, García-Conesa MT, Tomás-Barberán FA. Nutraceuticals: Facts and fiction. Phytochemistry 2007;68:2986-3008.
Fan L, Pan H, Soccol AT, Pandey A, Soccol CR. Advances in mushroom research in the last decade. Food Technol Biotechnol 2006;44:303-11.
Palacios I, Lozano M, Moro C, D′Arrigo M, Rostagno MA, Martínez JA, et
. Antioxidant properties of phenolic compounds occurring in edible mushrooms. Food Chem 2011;128:674-8.
Ramesh Ch, Pattar MG. Antimicrobial properties, antioxidant activity and bioactive compounds from six wild edible mushrooms of western ghats of Karnataka, India. Pharmacognosy Res 2010;2:107-12.
Wong JY, Chye FY. Antioxidant properties of selected tropical wild edible mushrooms. J Food Compost Anal 2009;22:269-77.
Kotan E, Alpsoy L, Anar M, Aslan A, Agar G. Protective role of methanol extract of Cetraria islandica
(L.) against oxidative stress and genotoxic effects of AFB1 in human lymphocytes in vitro
. Toxicol Ind Health 2011;27:599-605.
Ceker S, Agar G, Nardemir G, Anar M, Kizil HE, Alpsoy L. Investigation of anti-oxidative and anti-genotoxic effects of Origanum vulgare
L. essential oil on human lymphocytes in vitro
. J Essent Oil Bear Plants 2012;15:997-1005.
Türkez H, Togar B. Olive (Olea europaea
L.) leaf extract counteracts genotoxicity and oxidative stress of permethrin in human lymphocytes. J Toxicol Sci 2011;36:531-7.
Turkez H, Togar B, Polat E. Olive leaf extract modulates permethrin induced genetic and oxidative damage in rats. Cytotechnology 2012;64:459-64.
Türkez H, Togar B. Aluminium phosphide-induced genetic and oxidative damages in vitro
: Attenuation by Laurus nobilis
L. leaf extract. Indian J Pharmacol 2013;45:71-5.
Breitenbach J, Kränzlin F. Fungi of Switzerland. Vol. 1-5. Lucerne: Verlag Mykologia;1984-2000.
Abe K, Matsuki N. Measurement of cellular 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase release using MTT. Neurosci Res 2000;38:325-9.
Taga MS, Miller EE, Pratt DE. Chia seeds as a source of natural lipid antioxidants. J Am Oil Chem Soc 1984;61:928-31.
Pal J, Ganguly S, Tahsin KS, Acharya K. In vitro
free radical scavenging activity of wild edible mushroom, Pleurotus squarrosulus
(Mont.) Singer. Indian J Exp Biol 2010;48:1210-8.
Turkoglu A, Duru ME, Mercan N, Kivrak I, Gezer K. Antioxidant and antimicrobial activities of Laetiporus sulphureus
0 (Bull.) Murrill. Food Chem 2007;101:267-73.
Oyetayo VO, Dong CH, Yao YJ. Antioxidant and antimicrobial properties of aqueous extract from Dictyophora indusiata
. Open Mycol J 2009;3:20-6.
Karamac M, Amarowicz R, Weidner S, Abe S, Shahidi F. Antioxidant activity of rye caryopses and embryos extracts. Czech J Food Sci 2000;20:209-14.
Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-6.
Costantini S, Colonna G, Castello G. A holistic approach to study the effects of natural antioxidants on inflammation and liver cancer. Cancer Treat Res 2014;159:311-23.
Fernandes Â, Barreira JC, Antonio AL, Oliveira MB, Martins A, Ferreira IC. Effects of gamma irradiation on chemical composition and antioxidant potential of processed samples of the wild mushroom Macrolepiota procera
. Food Chem 2014;149:91-8.
Ito T, Urushima H, Sakaue M, Yukawa S, Honda H, Hirai K, et al.
Reduction of adverse effects by a mushroom product, active hexose correlated compound (AHCC) in patients with advanced cancer during chemotherapy - The significance of the levels of HHV-6 DNA in saliva as a surrogate biomarker during chemotherapy. Nutr Cancer 2014;66:377-82.
Kim SP, Nam SH, Friedman M. Hericium erinaceus
(Lion′s Mane) mushroom extracts inhibit metastasis of cancer cells to the lung in CT-26 colon cancer-tansplanted mice. J Agric Food Chem 2013;61:4898-904.
Wang S, Zhu F, Meckling KA, Marcone MF. Antioxidant capacity of food mixtures is not correlated with their antiproliferative activity against MCF-7 breast cancer cells. J Med Food 2013;16:1138-45.
Fontes Vieira PA, Gontijo DC, Vieira BC, Fontes EA, de Assunção LS, Leite JP, et
. Antioxidant activities, total phenolics and metal contents in Pleurotus ostreatus
mushrooms enriched with iron, zinc or lithium. LWT Food Sci Technol 2013;54:421-5.
Alves MJ, Froufe HJ, Costa AF, Santos AF, Oliveira LG, Osório SR, et al.
Docking studies in target proteins involved in antibacterial action mechanisms: Extending the knowledge on standard antibiotics to antimicrobial mushroom compounds. Molecules 2014;19:1672-84.
Petrovic J, Glamoclija J, Stojkovic DS, Ciric A, Nikolic M, Bukvicki D, et al. Laetiporus sulphureus
, edible mushroom from Serbia: Investigation on volatile compounds, in vitro
antimicrobial activity and in situ
control of Aspergillus flavus
in tomato paste. Food Chem Toxicol 2013;59:297-302.
Vázquez-Velasco M, González-Torres L, López-Gasco P, Bastida S, Benedí J, Sánchez-Reus MI, et al.
Liver oxidation and inflammation in Fa/Fa rats fed glucomannan/spirulina-surimi. Food Chem 2014;159:215-21.
Tavakol HS, Farzad K, Fariba M, Abdolkarim C, Hassan G, Seyed-Mostafa HZ, et al.
Hepatoprotective effect of Matricaria chamomilla
.L in paraquat induced rat liver injury. Drug Res (Stuttg) 2015;65:61-4.
Nichols JA, Katiyar SK. Skin photoprotection by natural polyphenols: Anti-inflammatory, antioxidant and DNA repair mechanisms. Arch Dermatol Res 2010;302:71-83.
Christhudas IV, Kumar PP, Sunil C, Vajravijayan S, Sundaram RL, Siril SJ, et al
. In vitro
studies on a-glucosidase inhibition, antioxidant and free radical scavenging activities of Hedyotis biflora
L. Food Chem 2013;138:1689-95.
Dasgupta A, Rai M, Acharya K. Chemical composition and antioxidant activity of a wild edible mushroom Pleurotus flabellatus
. Int J PharmTech Res 2013;5:1655-63.
Dulay RM, Arenas MC, Kalaw SP, Reyes RG, Cabrera EC. Proximate composition and functionality of the culinary-medicinal tiger sawgill mushroom, Lentinus tigrinus
(higher basidiomycetes), from the Philippines. Int J Med Mushrooms 2014;16:85-94.
Lung MY, Huang WZ. Antioxidant potential and antioxidant compounds of extracts from the medicinal sulphur polypore, Laetiporus sulphureus
(Higher basidiomycetes) in submerged cultures. Int J Med Mushrooms 2013;15:569-82.
Ejelonu OC, Akinmoladun AC, Elekofehinti OO, Olaleye MT. Antioxidant profile of four selected wild edible mushrooms in Nigeria. J Chem Pharm Res 2013;5:286-95.
Lee MR, Hou JG, Begum S, Xue JJ, Wang YB, Sung CK. Comparison of constituents, antioxidant potency, and acetylcholinesterase inhibition in Lentinus edodes
, Sparassis crispa
, and Mycoleptodonoides aitchisonii
. Food Sci Biotechnol 2013;22:1747-51.
Kim MJ, Chu WM, Park EJ. Antioxidant and antigenotoxic effects of shiitake mushrooms affected by different drying methods. J Korean Soc Food Sci Nutr 2012;41:1041-8.
Lee J, Hong JH, Kim JD, Ahn BJ, Kim BS, Kim GH, et al.
The antioxidant properties of solid-culture extracts of basidiomycetous fungi. J Gen Appl Microbiol 2013;59:279-85.
Hasnat MA, Pervin M, Debnath T, Lim BO. DNA protection, Total phenolics and antioxidant potential of the mushroom Russula virescens
. J Food Biochem 2014;38:6-17.
Yildirim NC, Turkoglu S, Yildirim N, Ince OK. Antioxidant properties of wild edible mushroom Pleurotus eryngii
collected from Tunceli province of Turkey. Dig J Nanomater Biostruct 2012;7:1647-54.
Fernandes Â, Barros L, Barreira JC, Antonio AL, Oliveira MB, Martins A, et
. Effects of different processing technologies on chemical and antioxidant parameters of Macrolepiota procera
wild mushroom. LWT Food Sci Technol 2013;54:493-9.
Khatua S, Roy T, Acharya K. Antioxidant and free radical scavenging capacity of phenolic extract from Russula laurocerasi
. Asian J Pharm Clin Res 2013;6:156-60.
Pereira E, Oliveira I, Baptista P. Guttation droplets of the edible mushroom Suillus bovinus
as a new source of natural antioxidants. Sci Hortic (Amsterdam) 2012;148:89-92.
Ayala-Zavala JF, Perez-Carlon JJ, Esqueda M, Gonzalez-Aguilar GA, Leyva JM, Cruz-Valenzuela MR, et al.
Polar fractionation affects the antioxidant properties of methanolic extracts from species of genus Phellinus
quel.(higher Basidiomycetes). Int J Med Mushrooms 2012;14:563-73.
Liu K, Wang JL, Gong WZ, Xiao X, Wang Q. Antioxidant activities in vitro
of ethanol extract and fractions from mushroom, Lenzites betulina
. J Food Biochem 2013;37:687-93.
Barros L, Baptista P, Ferreira IC. Effect of Lactarius piperatus
fruiting body maturity stage on antioxidant activity measured by several biochemical assays. Food Chem Toxicol 2007;45:1731-7.
Reis FS, Barros L, Calhelha RC, Ciric A, van Griensven LJ, Sokovic M, et al.
The methanolic extract of Cordyceps militaris
(L.) Link fruiting body shows antioxidant, antibacterial, antifungal and antihuman tumor cell lines properties. Food Chem Toxicol 2013;62:91-8.
Ferreira IC, Baptista P, Vilas-boas M, Barros L. Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal: Individual cap and stipe activity. Food Chem 2007;100:1511-6.
Zhang Y, Liu Z, Ng TB, Chen Z, Qiao W, Liu F. Purification and characterization of a novel antitumor protein with antioxidant and deoxyribonuclease activity from edible mushroom Pholiota nameko.
Soltaninejad K, Kebriaeezadeh A, Minaiee B, Ostad SN, Hosseini R, Azizi E, et al. Biochemical and ultrastructural evidences for toxicity of lead through free radicals in rat brain. Hum Exp Toxicol 2003;22:417-23.
Wong SP, Leong LP, Hoe J, Koh W. Antioxidant activities of aqueous extracts of selected plants. Food Chem 2006;99:775-83.
Liu Y, Du YQ, Wang JH, Zha XQ, Zhang JB. Structural analysis and antioxidant activities of polysaccharide isolated from Jinqian mushroom. Int J Biol Macromol 2014;64:63-8.
Nandi AK, Samanta S, Maity S, Sen IK, Khatua S, Devi KS, et al. Antioxidant and immunostimulant ß-glucan from edible mushroom Russula albonigra (Krombh.) Fr. Carbohydr Polym 2014;99:774-82.
Elmastas M, Isildak O, Turkekul I, Temur N. Determination of antioxidant activity and antioxidant compounds in wild edible mushrooms. J Food Compost Anal 2014;20:337-45.
Yeh JY, Hsieh LH, Wu KT, Tsai CF. Antioxidant properties and antioxidant compounds of various extracts from the edible basidiomycete Grifola frondosa (Maitake). Molecules 2011;16:3197-211.
Ji H, Du A, Zhang L, Li S, Yang M, Li B. Effects of drying methods on antioxidant properties and phenolic content in white button mushroom. Int J Food Eng 2012;8.
Meir S, Kanner J, Akiri B, Philosoph-Hadas S. Determination and involvement of aqueous reducing compounds in oxidative defense systems of various senescing leaves. J Agric Food Chem 1995;43:1813-9.
Barros L, Falcão S, Baptista P, Freire C, Vilas-boas M, Ferreira IC. Antioxidant activity of Agaricus sp. mushrooms by chemical, biochemical and electrochemical assays. Food Chem 2008;111:61-6.
Sulkowska-Ziaja K, Muszynska B, Motyl P, Pasko P, Ekiert H. Phenolic compounds and antioxidant activity in some species of polyporoid mushrooms from Poland. Int J Med Mushrooms 2012;14:385-93.
Paloi S, Acharya K. Antioxidant activities and bioactive compounds of polyphenol rich extract from Amanita vaginata (Bull.) Lam. Int J PharmTech Res 2013;5:1645-54.
Barros L, Calhelha RC, Vaz JA, Ferreira IC, Baptista P, Estevinho LM. Antimicrobial activity and bioactive compounds of Portuguese wild edible mushrooms methanolic extracts. Eur Food Res Technol 2006;225:151-6.
Yamaç M, Bilgili F. Antimicrobial activities of fruit bodies and/or mycelial cultures of some mushroom isolates. Pharm Biol 2006;44:660-7.
Ranadive KR, Belsare MH, Deokule SS, Jagtap NV, Jadhav HK, Vaidya JG. Glimpses of antimicrobial activity of fungi from World. J New Biol Rep 2013;2:142-62.
Dyakov MY, Kamzolkina OV, Shtaer OV, Bisko NA, Poedinok NL, Mikhailova OB, et
al. Morphological characteristics of natural strains of certain species of basidiomycetes and biological analysis of antimicrobial activity under submerged cultural conditions. Microbiology 2011;80:274-85.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]