2.1. Source of Strains and Tissue Culture

Wild fruiting bodies of P. cystidiosus were collected from the forest area of Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines and brought to the laboratory to rescue the mycelia. Internal tissues from the premature basidiocarp were obtained and inoculated onto potato dextrose agar plates. Culture plates were labeled properly as WS218-1, WS218-2, CST01, and CST02, and incubated at room temperature to allow mycelial ramification for 7 days. Mycelial disks in the pure culture were prepared using a flame sterile 10 mm-diameter cork borer and these were used as an inoculant in the evaluation of mycelial biomass production in liquid culture.

2.2. Mycelial Biomass Production in Submerged Culture

In the evaluation of mycelia biomass production of P. cytidiosus, coconut water from mature coconut was served as the medium in liquid culture. Fifty ml was dispensed into each glass bottle, plugged with cotton, and covered with paper. Ten replicates per strain were done. Culture media were sterilized in an autoclave at 121°C, 15 psi for 30 minutes. After cooling, 10 mm-diameter mycelial disk was inoculated into each medium. Cultures were incubated at 30°C for 15 days to allow mycelia growth. The mycelia were harvested, air-dried, and weighed.

2.3. Ethanol Extraction

Five grams of powdered air-dried mycelia of each strain were extracted in 500 ml of 80% ethanol for 48 hours. Extracts were filtered using Whatman filter No. 2 and concentrated to dryness using rotary evaporator. The extracts were harvested and subjected to assay.

2.4. 2,2’diphenyl-1-1picrylhydrazyl (DPPH) Radical Scavenging Activity Assay

The 2,2’diphenyl-1-1picrylhydrazyl (DPPH) radical scavenging assay following the standard method of Kolak et al. [3] with minor modifications was used to determine the antioxidant activities of the extracts. One ml of extract at 1,000 μg/ml (dilution in ethanol) concentration was mixed with 4 ml of 0.1 mM DPPH solution in the separate plastic cuvette. A 1,000 μg/ml concentration of catechin was also prepared and this served as the positive control. Triplicate test was done. The prepared mixtures were incubated in the dark at 37°C for 30 minutes. The absorbance readings were monitored at 517 nm using a UV–VIS spectrophotometer (Spectrumlab 752S, Hinotek Instrument Co., LTD, China). The ability to scavenge the DPPH radical was calculated using the formula:

$\%\mathrm{Radical}\mathrm{Scavenging}\mathrm{Effect}=\mathrm{[(}{\mathrm{A}}_{\mathrm{control}}–{\mathrm{A}}_{\mathrm{sample}}\mathrm{)/}{\mathrm{A}}_{\mathrm{control}}]\times 100.$

2.5. Estimation of Phenolic Content

The total phenolic contents of the extracts were estimated using the Folin–Ciocalteu method of Sunita and Dhananjay [4] with modifications. Gallic acid was used as a standard and the total phenolics were expressed as mg gallic acid equivalents (GAE)/g of the sample. The different concentrations of gallic acid and the extract at 1 mg/ml (dilution in methanol) concentration were prepared. Each sample (0.5 ml) was introduced into test tubes and mixed with 2.5 ml of a 10-fold dilute Folin–Ciocalteu reagent and 2 ml of 7.5% sodium carbonate. All tests were performed in triplicate. The tubes were covered with parafilm and allowed to stand at 30°C for 30 minutes prior to absorbance reading at 760 nm using a UV–VIS spectrophotometer (Spectrumlab 752S, Hinotek Instrument Co., LTD, China).

2.6. Mycochemical Analysis

The mycochemical compositions of the fruiting bodies were detected following the procedures described by Guevara et al. [5].

2.7. Statistical Analysis

Data were analyzed using analysis of variance and the least significant difference at 5% level of significance was used to compare treatment means.

3.1. Mycelial Biomass Production

Mycelial growth and biomass production of mushrooms are species- and strain-dependent. In our intention to determine and to select the best strain, we evaluated the mycelial biomass production of four strains of P. cystidiosus in liquid culture using coconut water as a medium. Table 1 presents the mean yield of mycelial biomass of the four strains of P. cystidiosus after 15 days of incubation. Apparently, the different strains respond differently in the coconut water medium. WS218-2 strain recorded the highest mycelial biomass (6.57 g fresh wt. and 0.39 g dry wt.), whereas CSC02 strain had the lowest fresh weight and WS218-01 had the lowest dry weight. These results strongly indicate that the mycelial biomass production of P. cystidiosus could vary and it is dependent on the type of strain. This finding substantiates the observation of Kalaw et al. [6] who reported that the two strains of each mushroom, namely, Ganoderma lucidum, Lentinus tigrinus, Volvariella volvacea, Coprinopsis cinerea, and Schizophyllum commune performed differently on the nutritional and physical factors for mycelial growth on solid medium. Moreover, the biomass production performances of the different strains of Agaricus bisporus significantly varied as affected by temperature and method of composting of the substrate [7]. Black (AMRL 63) Morchella strains had a higher growth rate on wheat grains, potato peels, and a mixture than yellow (AMRL 52) strain in solid-state fermentation [8].

3.2. Radical Scavenging Activity and Total Phenolic Content

The DPPH free radical scavenging activity assay is one of the most common methods in determining the antioxidant activity of the extracts or compounds being tested. The antioxidant activity is definitely attributed to the antioxidative agents including total phenolics. In this study, the radical scavenging activities and total phenolic contents of ethanolic extracts of mycelia of four strains of P. cystidiosus were analyzed (Table 2). Apparently, the four extracts exhibited different radical scavenging activities and total phenolic contents. WS218-2 mycelia extract significantly recorded the highest radical scavenging activity of 79.01% and phenolic content 95.67 mg GAE/g of the sample. Accordingly, the results strongly suggest that phenolics were the major antioxidant components in the mushroom extract, which contribute to the antioxidative action via radical scavenging ability. This conforms with the report of Phan et al. [9] that the total phenolics present in the extracts were positively correlated to the free radical scavenging activities of two strains of Pleurotus giganteus. Among strains of P. cystidiosus evaluated, the WS218-2 strain could be a promising source of protective agents to help us reduce oxidative damage and the risk of cardiovascular diseases.

Table 1: Yield of mycelial biomass of the four strains of P. cystidiosus grown in liquid culture using coconut water medium after 15 days of incubation. [Click here to view]

Table 2: Radical scavenging activity and total phenolic content of the four strains P. cystidiosus mycelia. [Click here to view]

In contrast, CST02 had the lowest radical scavenging activity (57.41%), whereas CST01 contained the lowest phenolic content 81.50 mg GAE/g of sample). Although these values were found lower in the present work, these values are still higher when compared to the scavenging activities of methanolic extracts of specialty mushrooms (63.3%–67.8%) and commercial mushrooms (42.9%–69.9%) and the total phenolic content of methanolic extracts of specialty mushrooms (7.61–16.28 mg/g) and commercial mushrooms (6.27–15.65 mg/g) [10]. Moreover, DPPH free radical scavenging activities of Pleurotus djamour var. djamour (5.5 mg/ml), Pleurotus djamour var. roseus (7.5 mg/ml), P. ostreatus (12.0 mg/ml), P. pulmonarius (6.0 mg/ml), and S. commune (3.0 mg/ml) are far lower compared to the obtained values in the present study [11]. The maximum radical scavenging activity (18.94%) and total phenolics (26.59 mg AAE/g sample) of mycelial extract of L. tigrinus and the highest scavenging activity (16.94%) and total phenolics (25.60 mg AAE/g sample) of mycelial extract of Lentinus sajor-caju in liquid culture using rice bran decoction are also lower [12]. Based on the present results and findings of the previous works, it is noteworthy to say that the different strains of P. cystidiosus could exhibit superior antioxidant activity than among other mushrooms.

3.3. Mycochemical Composition

Mycochemicals are fungal-derived chemicals, which play major roles in several biological activities. The chemical components of some of the Philippine wild mushrooms have been elucidated including Trichaleurina celebica, Trametes elegans, Polyporus grammocephalus, Lentinus strigosus, L. tigrinus, C. cinerea, and Paneaolus antillarium [13–19]. In our intention to elucidate the bioactive compositions of the best wild strain, WS218-2, mycochemical detection in thin-layer chromatography was also carried out. Table 3 presents the results of the mycochemical screening. Eleven mycochemicals, namely, essential oil, triterpenes, anthraquinones, tannins, flavonoids, phenols, fatty acids, alkaloids, steroids, sugars, and coumarins were detected in P. cystidiosus WS218-2. In the elucidation of bioactive compounds of P. ostreatus conducted by Mohamed and Farghaly [20], nine chemical groups including 2 metabolites related to acids, 5 alcohols, 27 alkane, 3 amides, 27 esters, 8 fatty acids, 4 terpenoids, 29 heterocyclic, and 2 phenols were detected and these were related to anti-cholesterol and anti-cancer activity of the mentioned mushroom.

Table 3: Mycochemical composition of mycelia of WS218-2 strain of P. cystidiosus. [Click here to view]

Several research studies have focused on the elucidation of bioactive compounds from Pleurotus species (i.e., [20–23]) that can be isolated for nutraceutical and pharmacological purposes. Bioactive compounds from the mushrooms could possess antioxidant, antifungal, antibacterial, and antiviral activities [24]. Terpenoids, phenols, steroids, polysaccharides, fatty acids, and amino acid were reported to act as anticancer agents [25,26]. Phenolic compounds, especially flavonoid and organic acids, were known antioxidant metabolites [20]. The chemical constituents of P. cystidiosus obtained in the present work are very useful mycochemicals that revealed significant antioxidant properties and could provide interesting biological activities that need to be investigated in future studies.