Bioactivity of Edible Mushroom Agaricus Campestris

“XXII SAVETOVANJE O BIOTEHNOLOGIJI” Zbornik radova, Knjiga 2, 2017. BIOACTIVITY OF EDIBLE MUSHROOM AGARICUS CAMPESTRIS  Marijana Kosanić1, Branislav Ranković1, Tatjana Stanojković2 Abstract: Here we determined antioxidant, antimicrobial and anticancer properties of edible mushroom Agaricus campestris. Antioxidant activity was evaluated by free radical scavenging, superoxide anion scavenging and reducing power. A. campestris extract had moderate free radical scavenging activity (IC50=179.65 µg/mL) and potent superoxide anion scavenging potential (IC50=35.204 µg/mL). Measured values of absorbance for reducing power varied from 0.3701 to 0.141. Further, the antimicrobial potential was determined by a microdilution method on 12 microorganisms. Extract of A. campestris showed relatively strong antimicrobial activity with minimum inhibitory concentration values ranging from 0.08 to 7.5 mg/mL. Finally, the cytotoxic activity was tested using MTT method on the human epithelial carcinoma Hela cells, human lung carcinoma A549 cells and human colon carcinoma LS174 cells. A. campestris extract expressed cytotoxic activity with IC50 values ranging from 9.54 to 16.87 μg/mL. Key words: anticancer activity, antimicrobial activity, antioxidant activity, mushrooms. Introduction In many parts of the world, wild mushrooms are regularly collected and used directly as a main source of food or as a condiment. Today mushrooms are valuable not only because of the appealing texture and unique taste, but because of their important chemical and nutritional characteristics (Kalac, 2012). Besides the important nutritional values, mushrooms also have a valuable health benefits. Their consumption has consistently been shown to have beneficial effects on human health. One of the noticeable features of many mushrooms is their ability to function as immunomodulators and thus to improve the resistance to disease. While much attention in recent years has focused on various immunological and anti-cancer properties of certain mushrooms, they also offer other potentially important health benefits, including antioxidants, anti-hypertensive and cholesterol-lowering properties, liver protection, as well as anti-inflammatory, anti-diabetic, anti-viral and anti-microbial properties (Mishra et al., 2015; Pandimeena et al., 2015; Kosanić et al., 2017). These properties have attracted the interest of many pharmaceutical companies, which are viewing the medicinal mushroom as a rich source of innovative biomedical molecules. 1 Department of Biology, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, Kragujevac, Serbia ([email protected]); 2 Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia 579 “XXII SAVETOVANJE O BIOTEHNOLOGIJI” Zbornik radova, Knjiga 2, 2017. Material and methods Fungal materials Fungal sample of A. campestris L.:Fr., was collected from Šumarice, Kragujevac, Serbia, in September of 2016. The demonstration samples are preserved in facilities of the Department of Biology and Ecology of Kragujevac, Faculty of Science. The determination of mushrooms was done using standard keys (Uzelac, 2009). Finely dry ground thalli of the examined mushrooms (100 g) were extracted using methanol (500 mL) in a Soxhlet extractor. The extracts were filtered and then concentrated under reduced pressure in a rotary evaporator. The dry extracts were stored at −180C until they were used in the tests. The extracts were dissolved in 5% dimethyl sulphoxide (DMSO) for the experiments (Kosanić et al., 2017). DMSO was dissolved in sterile distilled water to the desired concentration Antioxidant activity Antioxidant activity was evaluated by free radical scavenging, superoxide anion radical scavenging and reducing power. The free radical scavenging activity of lichen extracts was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH) according to the Kosanić et al. method (2017). Oyaizu method (1986) was used to determine the reducing power. The superoxide anion radical scavenging activity was detected according to the Nishimiki et al. method (1972). The amount of total phenols in the lichen extracts was determined as pyrocatechol equivalent using Folin-Ciocalteu reagent according to Slinkard and Singleton’s method (1997). Antimicrobial activity The sensitivity to extracts of the investigated species of microorganisms was tested by determining the minimal inhibitory concentration (MIC) by the broth microdilution method with using 96-well micro-titer plates (Sarker et al., 2007). Cytotoxic activity Human epithelial carcinoma Hela cells, human lung carcinoma A549 cells and human colon carcinoma LS174 cells were obtained from American Type Culture Collection (Manassas, VA, USA). All cancer cell lines were cultured as a monolayer in the RPMI 1640 nutrient medium, with 10% (inactivated at 56ºC) FBS, 3 mM of Lglutamine, and antibiotics, at 37ºC in humidified air atmosphere with 5% CO2. Stock solutions (50 mg ml-1) of the extracts, made in DMSO, were dissolved in a corresponding medium to the required working concentrations. The final concentrations applied to the cells were 200, 100, 50, 25 and 12.5 μg ml-1. In the control wells only the nutrient medium was added. The effect on cancer cell survival was determined 72 h after the addition of the extract, by the MTT test (Mosmann, 1983). 580 “XXII SAVETOVANJE O BIOTEHNOLOGIJI” Zbornik radova, Knjiga 2, 2017. Results and discussion The scavenging DPPH radicals, superoxide anion radical scavenging, reducing power and phenolics content of the studied extract are represented in Table 1 and Table 2. The IC50 values were 179.65 and 35.204 μg ml-1 for DPPH radicals and superoxide anion radicals scavenging activity, respectively. Measured values of absorbance for reducing power varied from 0.3701 to 0.141. The total phenolic compounds was determined as the pyrocatechol equivalent using an equation obtained from a standard pyrocatechol graph (y = 0.0057x - 0.1646, R2 = 0.9934). Table 1. DPPH radical scavenging activity, superoxide anion scavenging activity and phenolics content of extract of Agaricus campestris Mushroom species Agaricus campestris Ascorbic acid DPPH radical scavenging IC50 (µg ml-1) 179.65 ± 1.11 6.42 ± 0.18 Superoxide anion scavenging IC50 (µg ml-1) 35.204 ± 3.08 115.61 ± 1.16 Phenolics content (μg PE mg-1 of extract) 50.57 + 1.024 Table 2. Reducing power of extract of Agaricus campestris Mushroom species Agaricus campestris Ascorbic acid 1000 µg ml-1 0.3701 ± .031 3.862 ± .992 Absorbanca (700 nm) 500 µg ml-1 0.195 ± .025 2.113 ± .032 250 µg ml-1 0.141 ± .008 1.654 ± .021 Extract from A. campestris acted on all the microorganisms tested. The MIC fluctuated in a range of 0.08–0.16 mg ml-1 for bacteria and 0.4–7.5 mg ml-1 for fungi (Table 3). Table 3. Minimum inhibitory concentration (MIC) of extract Agaricus campestris Mushroom species Bacillus cereus Bacillus subtilis Eschrichia coli Proteus mirabilis Staphylococcus aureus Trichophyton mentagrophytes Geotrichum candidum Paecilomyces variotii Fusarium solani Candida albicans Aspergillus flavus Penicillium italicum A. campestris (mg ml-1) 0.08 0.08 0.16 0.16 0.16 0.81 1.62 0.81 1.62 0.4 1.62 7.5 581 Streptomycin (mg ml-1) 0.02 0.02 0.06 0.03 0.03 - Ketoconazole (mg ml-1) 0.16 0.08 0.16 0.16 0.04 0.31 0.16 “XXII SAVETOVANJE O BIOTEHNOLOGIJI” Zbornik radova, Knjiga 2, 2017. The data obtained for anticancer effect of A. campestris extract are shown in Table 4. The IC50 against Hela, A549 and LS174 cell lines was 9.54, 16.87 and 12.84 μg ml-1 respectively. Table 4. Growth inhibitory effect of Agaricus campestris on Hela, A549, and LS174 cell survival Cell lines Mushroom species Agaricus campestris Cis-DDP Hela A549 IC50 (μg ml-1 ) 16.87 ± 1.57 4.91 ± 0.42 9.54 ± 0.63 0.86 ± 0.33 LS174 12.84 ± 2.23 3.18 ± 0.29 The extract tested had strong antioxidant activity against various oxidative systems in vitro. In this study, we used DPPH test just to verify that extracts exhibit antioxidant activity, and in future complex research we would use some other tests to explain the mechanisms of antioxidant activity. Several researchers explored antioxidant activity of A. campestris (Kosanić et al., 2017; Woldegiorgis et al., 2014) by using different extraction solvents. In this study, the antioxidant activity of selected mushroom was confirmed by acetone extracts. Depending on the polarity, different extraction solvents can extract different metabolites which contribute to the great antioxidant potential, which means that between antioxidative components in the extracts there is a synergistic effect, which leads to prominent antioxidant activity of mushrooms. Similar to our results, numerous researchers found relatively strong antimicrobial activity for A. campestris (Giri et al., 2012; Kosanić et al., 2017). The probable mechanisms of antimicrobial action of tested mushroom are inhibition of cell wall synthesis, protein synthesis, or nucleid acid synthesis, like antibiotics, but less effective. In our experiments, the examined mushroom in the same concentrations showed a stronger antibacterial than antifungal activity. This different sensitivity between the fungi and bacteria is probably due to the different permeability of their cell walls. The cell wall of the gram-positive bacteria consists of mureins and teichoic acids, the gramnegative bacteria consist of lipopolysaccharides and lipopoliproteins, while the cell wall of fungi consists of hitchin and glucan (Kosanić et al., 2017). Numerous findings suggest that some mushrooms in combination with commercial anti-cancer drugs work in synergy as an effective tool for treating drug-resistant cancers. Mushrooms also are known to complement chemotherapy and radiation therapy by countering the side-effects of cancer, such as nausea, bone marrow suppression, anemia, and lowered resistance (Patel and Goyal, 2012). The evidences from various researchers across the globe, regarding anti-tumor application of mushroom extracts unarguably make it a fast-track research area worth mass attention. Results of our study promise that the A. campestris extract can be very effective therapy for malignant cancer cells. A. campestris was previously tested on cytotoxic activity by several researchers. For instance, Kosanić et al. (2017) shown that methanol extract from A. campestris has anticancer activity. Also, Li et al. (2005) found antitumor activities of different solvent extracts of A. campestris. The mechanism of action of the tested extracts is yet to be tested. For further research is necessary to isolate active anticancer compounds from this extract. 582 “XXII SAVETOVANJE O BIOTEHNOLOGIJI” Zbornik radova, Knjiga 2, 2017. Conclusion The present investigation can be concluded that the tested mushroom appear to be good natural antioxidant, antimicrobial and anticancer agent. The identification of active antioxidant, antimicrobial and anticancer compounds of this mushroom species can lead to their potential commercial usage in food production, medicine, and the cosmetic industry. 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