Investigation of Nutritional Content, Antioxidant Anticancer and Antimicrobial Activities of Pleurotus eryngii (DC. Ex Fr.) Quel, Extract Obtained by Different Solvents

: cumalikeskin@artuklu.edu.tr ABSTRACT The present study related the nutritional contents, fatty acid compositions, in vitro antioxidant, anticancer and antimicrobial activities of different solvents extracts of Pleurotus eryngii. The nutritional contents of the P. eryngii were investigated in detail. Crude protein (g 100 kg-1), carbohydrate (g 100 kg-1), lipid (g 100 kg-1), ash (g 100 kg-1), dietary fiber (g 100 kg-1) and energy value (cal 100 g-1) of fruiting bodies were analysed. Fatty acid compositions of total lipid, phospholipid and triacylglycerol fractions of Pleurotus eryngii were investigated by Gas chromatography. Totally 13 different fatty acids constituents were identified. Antioxidant properties including total antioxidant activity, free radical scavenging activity, reducing power activity, metal chelating activity, and superoxide anion radical scavenging activity were evaluated using different tests. The highest total antioxidant, DPPH scavenging, metal chelating, and superoxide anion radical scavenging activities were determined in ethyl acetate extracts. The potential of cytotoxic activity on PC-3 (prostate cancer) cell lines was found to depend on the concentration and the type of the tested extracts fractions. It was determined that hexane, ethyl acetate, and methanol extracts of Pleurotus eryngii have inhibition effect on the growth of tested microorganisms. The present results suggested that used as a daily nutrient, the Pleurotus eryngii could be a novel source for new drug developments and promising in some Investigation of Nutritional Content, Antioxidant Anticancer and Antimicrobial Activities of Pleurotus eryngii (DC. Ex Fr.) Quel, Extract Obtained by Different Solvents.


INTRODUCTION
People have consumed wild mushrooms since ancient times as part of their nutrients due to their unique taste, nutritional value, and rich mineral contents. Wild edible mushrooms have been identified as a source of many chemical and nutritional components. They are quite rich in terms of some minerals, water, protein, fibre, carbohydrates and vitamins such as Ca, Mg, K, selenium, choline, thiamine, riboflavin, ascorbic acid and ergocalciferol. On the other hand fatty acids, mono-, di-and triglycerides, sterols, and phospholipids contents are very low. Therefore, they are attractant and considered as low-calorie foods by the people for daily dietary consumption (Heleno et al. 2009;Kalac 2009;Kalac 2012). Various medicinal and nutritional properties of fungi have been notified in much research. They are including many bioactive compounds, such as phenolics. These compounds have antioxidant, antitumor, anti-inflammatory, antibacterial and antifungal activities (Dundar et al. 2013;Ferreira et al. 2010;Ren et al. 2016;Taofiq et al. 2015;Alves et al. 2013). Pleurotus eryngii is known as king oyster mushroom, compared to other species of Pleurotus, it has a unique flavour, different aromatic structure, high nutrient content, important medicinal properties, economic importance, longer shelf life, fleshy structure and other characteristics related to cookery (Rodriguez and Royse, 2007;Moonmoon et al. 2010). Recently it has become an extremely preferred species among North African, Asian and European consumers (Jeong et al. 2010). Nutritional contents of Pleurotus species such as chemical composition, macro and micronutrients, amino acids, proteins have been investigated (Ahmed et al. 2013;Oyetayo and Ariyo 2013;Musieba et al. 2013). But, the nutrient contents of the Pleurotus species may vary according to the physical and chemical differences in the habitat, substrate composition, pileus size and harvest seasons (Khan and Tania 2012). Genus Pleurotus contains a lot of substances that are biologically active including polysaccharides, proteoglycans, lectins, β-and glucans lipids, peptide, sterols, dietary fibre and lowmolecular-weight compounds such as terpenoids, fatty acid esters and polyphenols (Liu et al. 2010;Iwona et al. 2018). Hwang et al. (2003) reported that the water extract of Pleurotus eryngii showed inhibitory activity on the human colon cell lines  and the human colorectal adenocarcinoma (Caco-2) cell lines. Dündar et al. (2013) reported that cultured P. eryngii mycelium ethanol extract showed 60.68% antioxidant activity in the β-carotene-linoleic acid test system at a concentration of 10 mg/ml. P. ostreatus, a selenium accumulator, has been reported to show antitumor activity by inhibiting DNA cytosine methyltransferase (Ferreira et al. 2010). Llaurado et al. (2012 reported that the Pleurotus sp. water, and methanol extract had an immunomodulatory effect (on murine spleen cells) in mice, and the stimulation index was 1.90 and 1.28 respectively in 72 hours. In a study, researchers reported that the water-soluble polysaccharide extract (6.4 mg/mL) of P.eryngii increases the activities of antioxidant enzymes and effectively removes the DPPH free radicals (90.46%) in the CCl4-induced hepatotoxicity (Chen et al. 2012). In another study, researchers reported that the HepG-2 cells which were treated with 100 mg/mL P. eryngii polysaccharide extract showed antiproliferative effect (20% of early apoptotic cells) at 6 h (Ren et al. 2016). Also β-glucans and -glucans have anticancer properties (Jedinak et al. 2010;Wu et al. 2011). Furthermore, Pleurotus eryngii has phenolic and flavonoid compounds with antioxidant properties such as p-coumaric acid, gentisic acid, chlorogenic acid, syringic acid, caffeic acid, quercetin, rutin, kaempferol, catechin, protocatechuic acid, ferulic acid, t-cinnamic acid, vanillic acid, gallic acid, luteolin, naringenin, benzoic acid, 4-hydroxybenzoic acid, 2,5-Dihydroxybenzoic acid Nagendra 2013, Gasecka et al. 2016). However, the cytotoxic activity of the P. eryngii extracts on PC-3 cell lines has not been reported yet. These studies are very important in terms of better evaluation of P. eryngii as an alternative nutrient source and in obtaining new comparative data for the investigation of new therapeutic alternatives. Herein, the antioxidant properties, fatty acid compositions, nutritional content, the antimicrobial and cytotoxic activity of different solvents extracts of Pleurotus eryngii were investigated in detail.

Material
Pleurotus eryngii samples were collected from the field study in the province of Hakkari/Türkiye between April to May 2016. Taxonomic identification of material was according to Phillips (1994), using ecological, macroscopic data.
Determination of nutritional contents of Pleurotus eryngii Energy value, crude protein (nitrogen), ash, lipids and carbohydrate amounts of mushroom samples (of dry material%) were investigated according to the methods specified in the literature (Kjeldahl 1883;AOAC, 1990;Dundar et al. 2009). Khejdahl system was used for nitrogen determination. Energy value was determined by using the calorimeter (IKA C 200). Cauterization at 600±15 degrees C was used for the determination of ash. The dietary fibre amount was determined according to AOAC 993.21 method (AOAC 1990). Amino acid concentrations in the fungus sample were determined by using the HPLC accoutered with a combined system with an Agilent 1260 Infinity HPLC-DAD (Das et al. 2014). Each experiment was repeated 3 times and the results were given with mean and standard deviations. The isolation and transmethylation of fatty acids were carried out as described by Kaçar et al. (2016). Shimadzu GC 2010 PLUS model Gas Chromatography device, flame ionization detector (FID) and DB-23 (Bonded 50% cyanopropyl) (J & W Scientific, Folsom, CA, USA) capillary column (30m x 0.25mm inside diameter x 0.25 μm film thickness) were used for the analysis of fatty acids methyl esters. Firstly, 5 g of ground mushrooms were extracted into a mixture of chloroform-methanol (2: 1 v/v). Secondly, the total lipids in the sample were separated by thin-layer chromatography into phospholipids and triacylglycerol fractions. Total lipids; 80: 20: 1 ratio was carried out in a mixture of petroleum ether-diethyl ether-acetic acid. Air-drying plates were sprayed with 2.7dichlorofluorescein to make lipid fractions visible under the UV lamp. The bands of phospholipids and triacylglycerol were determined using the standards (Sigma-AldrichChemicals of fatty acids). Finally, the per cent content of the fatty acids of the hexane-extracted material was analyzed by gas chromatography, with the conversion of fatty acids to fatty acid methyl esters. Total amounts of fatty acids were obtained by the GC Solution (Version 2.4) computer program. SPSS 15 computer program was applied to compare the percentages of fatty acids of fungi. The t-test was used to compare the fatty acid percentages of the two groups. Duncan's multiple range tests were used to determine the difference between means (Duncan 1955).

Preparation of macrofungi extracts for in vitro assays
The fruiting bodies of the P. eryngii, dried at room temperature, were made ready for analysis by milling. The 50 g powdered mushroom samples were taken in a flask and extracted with series of organic solvents (200 ml hexane, ethyl acetate, and methanol) with different polarities in the constant stirring rate at 250 rpm for 24 hours and then filtered. This procedure was repeating three times for each solvent. Finally, solvent of hexane, ethyl acetate, and methanol extracts has been evaporated in the rotary evaporator at 40 °C under vacuum. Crude extracts were re-dissolved in methanol/DMSO (8:2) for preparing stock solution (7 mg/ml) and stored at -20°C for later use. The polarity index of hexane, ethyl acetate, and methanol were reported as 0, 4.3, and 6.6 respectively (Keskin et al. 2018).

In vitro antioxidant assays
The total antioxidant activity of mushroom extracts was determined by the β-carotene-linoleic acid method (Dapkevicius et al. 1998). DPPH radical scavenging activities of mushroom extracts were performed spectrophotometrically according to Cu et al. (2000). Superoxide anion radical scavenging activity was performed according to the riboflavin-methioninelight-NBT method (Zhishen et al. 1999). The metal chelating activity of the mushroom extract was detected by the method of Dinis et al. (1994). The reducing power activity of the mushroom extracts was determined according to Oyaizu, (1986).

Cytotoxic activity of mushroom extracts
Cytotoxic activity of P. eryngii extracts was evaluated according to the method of Alley et al. (1998). MTT test was used to evaluate the PC-3 (prostate cancer) cell line. MTT solution: 5 mg MTT was dissolved in phosphate buffer solution (CMFPBS) (pH = 7.0) without 1 ml divalent cations (Ca ++ and Mg ++ ). The solution was stored in the dark at 4 °C. Serum media for PC-3 (CRL-1435, ATCC) cell culture: 10 ml inactivated FBS (Fetal Bovine Serum) (10%), 1 ml penicillin (100 U)/streptomycin (100 mg/ml) solution (1%) DMEM-F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12). 24-h culture phases were applied in each well for PC-3 cells in 200 µl medium Research Article with 105 cells. The 96-well microplate was incubated for 24 hours in a humidified incubator containing 5% CO2 at 37 °C. After 24 hours, the culture medium was removed from the wells using an 8-channel automatic pipette. 50 µl of PBS and 90 µl of fresh media was added to each well. Subsequently, 10 µl of the test substance was applied to the wells at a concentration of ½ per cent dilution to the MTT test. Cytotoxicity tests were performed 7 times, 4 replicates for each concentration and 8 repetitions in the same order for a second day. 20 µl of MTT solution was added to each well of the 96well microplate containing the cell line incubated for 24 hours at 37 °C 5% CO2 with P. eryngii extracts.
After shaking at 150 rpm for 5 min, it was incubated at 37 °C for 2-3 hours. The top liquid in the wells was discarded. 100 µl of DMSO was added to the wells. Shaken at 150 rpm for 5 min. The intensity of the resulting colour was measured at 590 nm (versus 670 nm reference wavelength). By comparing the absorbance value of the tested compounds and the solvent control group, the number of cells killed % (Inhibition concentration, IC) was calculated. The absorbance values of the wells (solvent controls) containing the solution of the test substance instead of the test sample showed 100% viability. It was determined that DMSO used as 1% in the experiment had no cytotoxic effect on the cells. The absorbances at 590 nm for the MTT test were measured against the 690 nm reference wavelength. Corrected absorbance values were obtained by subtracting the blind absorbance from each solvent control and sample absorbance. Calculations were made by taking the average of absorbance values for repeats in a microplate. The relative inhibition activity (IC) was calculated according to the following formula as a percentage of the solvent control;

Inhibition%=
(corrected mean Absorbance sample/corrected mean Absorbance solvent control) x100 Viability % was calculated using solvent control for MTT testing. The inhibition % curve against the exposure concentration was plotted. The concentration corresponding to 50% inhibition from the curve was determined as IC50. Studies on cytotoxic activity were performed at the laboratories of Istanbul University Faculty of Pharmacy.
Determining antimicrobial activity of mushroom extracts Antimicrobial activity of obtained extracts was determined by the disk diffusion test taking into consideration the National Committee for Clinical Laboratory Standards rules. The disk diffusion test was performed using Nutrient Agar and Sabouraud Dextrose Agar medium. Before the microorganisms were inoculated, the solid media were incubated at 35-36 ºC for 30 minutes. It was sterilized and cooled to 45-50 °C, Nutrient Agar and Sabouraud Dextrose Agar (SDA Merck) were poured into 25 ml sterile petri dishes with a diameter of 9 cm. It was sterilized and cooled to 45-50 °C, Nutrient Agar and Sabouraud Dextrose Agar (SDA Merck) were poured into 25 ml sterile petri dishes with a diameter of 9 cm. The mediums were incubated at 37 degrees for overnight for contamination control. Bacteria (108 pieces / mL) and yeast (107 pieces/mL) were added to the water bath and 150 μL of them were collected and distributed to the sterile petri dishes with sterile cotton swabs. 30 mg/L (210 μg/disc) of mushroom (7 mg/mL) solutions were impregnated on 6 mm sterile blank paper discs. Discs impregnated with fungal solutions were placed on the solidified agar by lightly pressing. Petri dishes prepared in this way were incubated in the oven at 37 °C for 24/48 hours, after which the inhibition zone diameters on the medium were measured as mm using a ruler.

RESULT and DISCUSSIONS
In this study, nutritional contents, fatty acid composition, antioxidant, anticancer and antimicrobial activities of extracts obtained with solvents that vary from non-polar to polar (hexane to methanol) were investigated.

Determination of nutritional contents
Macrofungi have the essential nutrients for human health because of their structures. Mushrooms are an edible fungus that can ensure diverse major nutrients. The many varieties of mushroom have different compositions and nutritional profiles. Mushrooms are in rich with protein, vitamins, minerals, and antioxidants. These natural compounds have various health benefits. For this purpose, data on nutrients (fibre, protein, fat, carbohydrate, ash and energy amounts) of P. eryngii were analyzed in detail. Crude protein (Nx4.38), carbohydrate, lipids, ash, dietary fibre and energy value of P. eryngii fruiting bodies (% of dry substance) were determined as 15.930.06 g 100 kg-1, 68.40.59 g 100 g-1, 3.340.02 g 100 g-1, 9.90.01 g 100 g-1, 24.60.05 g 100 g-1 and 38880.23 cal 100 g-1 respectively. There are many studies on the different nutritional contents of Pleurotus species. Ahmet et al. (2013) investigated the nutritional content of P. high-king (PHK), P. ostreatus (PO3), P. geesteranus (PG1), P. geesteranus (PG3), and P. ostreatus (PO2) strains also they reported that the highest Ca (3381.0), Mg (1222.0), and K (140.0) amount was found in PG3 and the highest amount of Fe (46811) and Cu (3.60.0) was in PHK type. However, they reported that the highest protein and ash content was found in strain PG1, while the highest lipid and crude fiber content was found in strains PO2 and PO3, respectively. Oyetayo et al. (2013) reported that the KSU J. Agric Nat 24 (2) were higher in those grown in Pycnanthus ongoleubis. Musieba et al. (2013) reported that ash, protein, fat and fiber contents of P. citrinopileatus were 9.121.57, 22.102.0, 1.322.03 and 20.782.72 g/100 g dry matter, respectively. Mazi et al. (1999) reported that ash content was between 6.9% and 10.5% and potassium and magnesium content was higher than other elements in a study they conducted on different types of edible mushrooms (P. ostreatus, P. eryngii, P. pulmunarius and Lentinula edodes). Also, they reported the most abundant amino acids in edible mushrooms, expressed as percentages of total amino acids, were glutamic acid (12.8±20.9%), aspartic acid (9.1±12.1%), and arginine (

P. eryngii has rich carbohydrate and protein content
and has a small amount of ash and oil compared to other edible fungi. This rich nutritional composition ensures that this fungus is the perfect food for use in low-calorie diets. Some researchers have suggested that the essential amino acid content of mushrooms can be compared to animal proteins (Longvah and Deosthale 1998;Mattila et al. 2001). Figure 1 shows the essential amino acid content of P. eryngii. It was detected eight essential amino acids for humans, and the total contents of essential amino acids varied between 0.05 mg 100g-1 to 9.83 mg 100g-1. The major components of the essential amino acid were found as threonine, methionine and leucine. Edible mushroom, Pleurotus contains 40% of the essential amino acids which humans need to intake, and thus it is considered as the potential diet for individuals having malnutrition problems (Pushpa and Purushothoma 2010).  Table 1). The following main fatty acids were identified in total lipid, triacylglycerol and phospholipid fractions: C8:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, and C20:0 as saturated fatty acids (SFAs); C16:1n-7, C18:1n-9, and C20:1n-9 as monounsaturated fatty acids (MUFAs); and C18:2n-6, C18:3n-3, as polyunsaturated fatty acids (PUFAs). C 8:0 was identified only in the TG fraction at a trace amount. The results show that MUFA percentages (44.5%) were higher than SFA (18.25%) and PUFA (37.2%) in total lipid fraction. In the phospholipid fraction, the PUFA ratio (50.14%) was the significantly highest when compared with SFA (11.85) and MUFA (30.54). The MUFA (52.95%) percentage was found higher than SFA (11.17%) and PUFA (29.71%) in triacylglycerol fraction. Linoleic (C18:2n-6), oleic (C18:1n-9) and palmitic (C16:0) acids were found as major fatty acids in all fractions (Table 1). Oleic acid is the most abundant fatty acid in the human body and makes up almost half of the fatty acids. Fats with high oleic acid in its composition have been reported to have many advantages for human well-being. It is stated that oleic acid, a bioactive compound, is an effective glucosyltransferase inhibitor and inhibits high levels of human telomerase activity in a cell-free enzymatic assay (Oda et al. 2002;Won et al. 2007). Linoleic acid is an essential fatty acid because it cannot be synthesized in the human cells and it must be taken from the diet. In its insufficiency reported that retardation in growth, reproductive disorders and dermatoses in the skin, pathological changes in the liver and kidney (Dimou et al. 2002). We conclude that the presence of linoleic (C18: 2n-6) and oleic acids (C18: 1n-9) in P. eryngii and the abundance of essential fatty acids may create a new perspective in formulating new mushroom-based dietary supplements in further studies. * Each data is the average of 3 replicates. Three injections were performed per repeat. § The data determined by the same letters in each line are not different from each other at the level of p> 0.05 probability. SEM.: Standard error mean, SFA: Saturated Fatty Acids, MUFA: Monounsaturated Fatty Acids, PUFA: Unsaturated Fatty Acids. The percentages of fatty acids in total, triacylglycerol and phospholipid fusion were evaluated in their own right.

Determination of antioxidant properties
In living organisms, free radicals are formed in different ways. They react with membrane lipids, nucleic acids, proteins, enzymes and other small molecules to cause cellular damage. Low doses of synthetic antioxidants were able to inhibit cancer but some studies with experimental animals, indicate that they caused carcinogenesis and liver damage when used in high doses. This has led to increased interest in natural antioxidant sources. Mushrooms contain antioxidant substances to reduce oxidative damage in humans' cell (Kosanić et al. 2012;Khatua et al. 2013;Adebayo et al. 2014). Radicals scavenging activity, reducing power activity, metal chelating activity, total antioxidant activity and superoxide anion radical scavenging activity methods were applied to determine antioxidant properties. The highest results (DPPH, KSU J. Agric Nat 24 (2): 267-277, 2021 Araştırma Makalesi Research Article metal chelating, total antioxidant and superoxide radical extinguishing activity) were determined in ethyl acetate extract, except for reduction power activity. It was determined that the activities increased with the concentration and the results changed with the used solvents. The data obtained from ethyl acetate extract was quite close to the standards used as the positive control (Table 2). P. eryngii methanol extract had the highest reducing power activity, but it was found that they had moderate activity in comparison with positive controls (BHT, BHA, Ascorbic acid, Trolox). These differences might be due to chemical alteration in the active compounds present in this mushroom caused by using the organic solvent extraction process. Similar results have been reported in different research (Le et al. 2007;Tan et al. 2015).

Determination of cytotoxic activity
The result of the cytotoxic activity of P. eryngii extracts on the PC-3 cell lines and 50 % growth inhibition (IC50) shown in Table 3. In recent studies reported that different types of the extract obtained from fruit bodies or mycelium of Pleurotus mushrooms may have been as potential anticancer agents on different cancer cell lines (Hwang et al. 2003;Sun et al. 2017). However, no studies have been set against the PC-3 cell line.
Cytotoxic activity (against PC-3 cell lines) of P. eryngii was investigated by using the various solvent extract. Ethyl acetate extract of P. eryngii showed significant inhibitory activity at the concentration of 530 μg ml-1 (86.203 %) against PC-3 cell lines with IC50; 109.89±2.01 μg mL-1 ( Figure 2). Methanol extracts did not show any cytotoxic activity. It has been found that the potential of cytotoxic activity is depended on the concentration and solvent type of extracts. There has been increasing attention regarding the minimal toxicity fungal metabolites and potent bio pharmacological activities in cancer therapy. In particular, it can be said that naturally occurring anticancer compounds with low toxicity and high potency to normal cells should be in human diets. The positive results obtained from the ethyl acetate extract may have positive consequences for other cancer cells.

Determination of antimicrobial activity
There are several reports in the literature regarding the antimicrobial activity of extracts from the genus of Pleurotus (Mondal et al. 2013;Neelam and Singh 2013;Rahman et al. 2013). P. eryngii extracts have inhibitory activity against a group of bacterial reference strains were stated in some studies (Shang et al. 2013;Schillaci et al. 2013; Akyüz and Kırbağ  ). In the current study, the highest antimicrobial activity was obtained against Pseudomonas aeruginosa ATCC 27853 (11 mm) in ethyl acetate extract. P. eryngii hexane extract showed weak growth inhibition on P. aeruginosa, B. subtilis, S. aureus bacterial strains and C. albicans yeast. The methanol extract showed moderate growth inhibition against P. aeruginosa, S. aureus, S. pyogenes bacterial strains and C. albicans yeast. The ethyl acetate extracts only inhibited P. aeruginosa growth. However, none of the tested extracts showed an inhibitory effect on the growth of E. coli bacteria strain (Figure 3). In this regard, the Pleurotus eryngii extracts were not showed strong antimicrobial activities when compared with standard antibiotics but we believe that the values may change when solvent differences considered.

CONCLUSIONS
Mushrooms are very rich in classes of phenolic compounds such as coumaric acid, chlorogenic acid, syringic acid, caffeic acid, quercetin, rutin, catechin, protocatechuic acid, ferulic acid, gallic acid, which are considered as secondary metabolites. These compounds are very important in terms of antioxidant metabolism. They eliminate the effects of free radicals caused by cellular activities. Also, by directing the cells to apoptosis, they prevent cancer cell proliferation and tumour generation. Furthermore, Pleurotus mushrooms are noted as one of the richest and wellbalanced sources in terms of human nutrition and commonly human food and nutraceutical industries. Moreover, due to high nutritional values, these mushrooms can help significantly against malnutrition disease and can have positive effects on general health. In this respect, the further chromatographic and structural study needs to be conducted to know which compounds are in charge of from which activity. Also, in vivo studies are needed to understand how mushrooms are efficacious in living creatures. Following these studies, we suggest that usage of these potentials of mushrooms in the pharmaceutical and food industry may be beneficial to reduce the utilization of synthetic compounds. The believe that results will provide new baseline information for further study on Pleurotus eryngii.