BIOLOGICAL ACTIVITY OF ß-GLUCANS FROM EDIBLE MUSHROOM, SCHIZOPHYLLUM COMMUNE IN THAILAND

Objective: The objective of this research was to determine the antioxidant of ß-glucans from an edible mushroom, Schizophyllum commune in Thailand. Methods: The antioxidant activity of ß-glucans was measured in terms of hydrogen donating or radical scavenging ability by 2, 2-diphenyl-1picrylhydrazyl (DPPH) method,2,2 ́-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity analysis, and total phenolic compounds. The ß-glucans structure was analyzed using Fourier-transform infrared spectrophotometer. Results: The ß-glucans were extracted from S. commune and tested biological activities. Using the Folin–Ciocalteu Reactive method, we found that the phenolic compound contents of ethanol extracts of the mushroom sample were 284.41±1.22 mgGAE/g extract. The IC50 radical scavenging ability (DPPH) and IC50 radical scavenging activity (ABTS) of ß-glucans were 0.829±0.006 and 0.724±0.021 mg/mL, respectively. Conclusion: The ß-glucans from S. commune in Thailand showed a potent antioxidant activity and it will be able to apply in pharmaceutical cosmetics.


INTRODUCTION
The edible mushrooms are a natural product of the forest environment that grows on the most abundant nutrients such as cellulose [1]. They are a macro-fungus that has a distinctive fruiting body, have been mostly used as human food for centuries and have been famous for texture and flavor as well as having various medicinal properties [1,2]. However, in recently emerged, the edible mushrooms, as being an important source of biologically active material, have medicinal value. Mushrooms are rich in different biologically active compounds such as phenolics, tocopherol, lycopene, ß-carotene, vitamins, polysaccharide, and secondary metabolites in their fruiting bodies [3].
The edible mushrooms are the sources of bioactive substances such as secondary metabolites, have become attractive as a functional food which related to the beneficial health effects, antioxidants in biological systems [4]. It has been reported to interfere with the initiation and progression of tumor cell [5], antiaging [6], anti-inflammatory [7], brainprotective factors, and to protect against cardiovascular disease [8].
Hence, the present study primarily focused on the selection of solvents for extraction and analysis of antioxidant activities of Schizophyllum commune from Thailand. The results will be beneficial for pharmaceutical application and drug developments as well as the cosmetic industry. Consequently, it may be part of alternative antioxidant resources instead of synthetic antioxidant.

Fungal strain
S. commune-producing ß-glucans strain used in this study was S. commune, which obtained from Thai Traditional Medicine College, Rajamangala University of Technology Thanybauri, Pathum Thani, Thailand.

Inoculum preparation
S. commune was initially grown on Potato dextrose agar (PDA) in a Petri dish and then incubated at 30°C for 7 days.

Culture conditions
The flask culture experiments were performed in 250 ml Erlenmeyer flask that contained 50 ml of media. The five plugs of the seed culture were transferred into the culture medium and then inoculated at 30°C for 7 days.
Production of polysaccharide S. commune was initially grown on optimal condition in a 500 ml flask at 30°C on a rotary shaker incubator at 150 rpm for 7 days.

Extraction of polysaccharide
The fermentation broth was centrifuged at 8000 g for 20 min, and the resulting supernatant was filtered through Whatman filter paper No. 2 and mixed with four volumes of absolute ethanol, stirred vigorously, and left overnight at 4°C. The precipitate polysaccharide was collected by centrifugation at 8000 g for 10 min. The residue was reprecipitated with the same volume of ethanol, and the precipitate of pure polysaccharide was freeze-dried in a lyophilizer.

Preparation of β-glucan extracts solution to determine the antioxidant activity
Accurate weight of 0.1 g of the β-glucan extracts was dissolved in 5 ml dimethyl sulfoxide (Merck, Germany) and dilute to 250 mL of 99% methanol. This solution is to determine the total phenolic content, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ABTS radical scavenging activity.

Determination of total phenolic compounds
The total amount of phenolic compounds was determined with a modified standard Folin-Ciocalteu reagent [9]. Briefly, the 0.1 mL of the sample solution, 2 mL of the Folin-Ciocalteu reagent (Merck, Germany), and 1.5 ml of 2% sodium carbonate were mixed on a vortex mixer. After 30 min reaction, the absorbance at 765 nm was determined and used to estimate the phenolic content using the calibration curve made with gallic acid (Merck, Germany). The total amount of phenolic compounds was expressed in μg gallic acid equivalent per g dry weight of extractions.

ABTS radical scavenging activity analysis
The antioxidant activity was measured using a modified version [10]. To prepare ABTS cation radical solution, 2.45 mM of potassium persulphate (Merck, Germany) aqueous solution was added to 7 mM of ABTS (Sigma-Aldrich, Germany) in equal quantities and allowing them to react for 24 h at room temperature in the dark. One mL of the final dark-green radical solution was then diluted with 60 ml methanol and used in ABTS tests. 0.1 mL of different concentrations of the β-glucan extracts solution were added to 2 mL of ABTS+• solution. The mixtures were incubated at room temperature, in the dark for 6 min before reading the absorbance against a blank at 734 nm using UV-Vis GENESYS 10S spectrometer (Thermo Fisher Scientific, USA). Inhibition of free radical by ABTS in percent (I%) was calculated in the following way: where, A blank is the absorbance of the control reaction (containing all reagents except the test compound) and A sample is the absorbance of the test compound. The value of 50% inhibition (IC 50 ) was calculated from the graph plotted inhibition percentage against extract concentrations. Tests were carried out in triplicate. In the ABTS assay, Trolox was used as a positive control.

DPPH radical scavenging activity analysis
The radical scavenging activity of β-glucans was conducted using the modified method [10]. Different concentration of extract solutions (0.1 mL) was added to 3.0 mL of 0.1 mM methanol solutions of DPPH (Sigma-Aldrich, Germany). The samples were first kept in the dark place at room temperature, and after 30 min, the absorbance was measured at 517 nm using a spectrophotometer. The percent of inhibition and the value of 50% inhibition (IC 50 ) were calculated the same as ABTS radical scavenging activity test.

Chemical analysis of β-glucans
Infrared spectroscopy was performed with equipment from Perkin Elmer, model spectrum RX. The method used was an FT-IR in KBr solid. The KBr was pulverized in an agate mortar and pestle, and the sample, which was also sprayed together with KBr, was added. With the resulting powder, a tablet was made by compression with a pressure of 10 tons.

Total phenolic content and antioxidant activity of β-glucan
Phenolic compounds as a large group of biologically active molecule present in mushroom species [11]. Besides, their antioxidant activities, phenolics exhibit anti-inflammatory, and antimicrobial activity. Total phenolic content, antioxidant activity of β-glucans from S. commune, is shown in Table 1. This result showed a total phenolic of 284.41±1.22 mgGAE/g extract. The results indicating that ethanol was the suitable solvent for phenolic form S. commune. The antioxidant activity of methanolic extract of S. commune was subjected to DPPH and ABTS effect of the extract. ABTS activity was quantified in terms of percentage inhibition of the ABTS•+ radical cation by antioxidants in plant sample, as an oxidizing agent to the evaluated antioxidant activity of biological samples. Table 1 showed the IC 50 of ABTS scavenging activity and DPPH scavenging activity of 0.829±0.006 and 0.724±0.021 mg/ml, while Trolox showed the IC 50 of ABTS scavenging activity and DPPH scavenging activity of 0.128±0.009 and 0.116±0.009, respectively. This experimental result was similar to the previous reported [12].

Chemical analysis of β-glucans from S. commune
Infrared spectroscopy allows the measurement of molecular vibrations of covalent bonds. The infrared region 3900-400cm -1 provides information on the fundamental vibrations. The infrared spectra of extracted β-glucan are shown in Fig. 1.
In the region of 3700-3000 cm -1 , the extracted β-glucans spectra showed a wide band with maximum absorption at 3327/cm. This can be attributed to normal vibrational modes of asymmetric and symmetric stretching of OH groups because polysaccharides contain a significant number of OH groups, which exhibit an absorption band above 3000/cm [13,14].
The absorption peaks occurring at 2935/cm in the region of 2900-2400 cm -1 could be attributed to the relative values of the vibrational modes of asymmetric and symmetric stretches of CH groups [15].
The absorption at 1655/cm and 1636/cm was due to the stretching of CN groups and NH groups of the proteins indicating the presence of amide linkages and the presence of protein in the sample [15,16]. These results are in accordance with the chemical evaluation, indicating the presence of protein in the sample.
The region 1400-400 cm -1 , which showed peaks with maximum absorption at 1059/cm -1 , corresponds to COC and CO bonds of a ring of D-glucose [16]. It is also important to note that the spectra showed absorption peaks at 889/cm, which is indicative of β-glycosidic bonds [17,18].

CONCLUSION
In this study, we determined the antioxidant activities and β-glucan content of edible mushroom species, S. commune. Based on the results, the ß-glucans extracts from S. commune showed a potent antioxidant activity, and it will be able to apply in pharmaceutical cosmetics.

ACKNOWLEDGMENT
We are grateful to Thai Traditional Medicine College, Rajamanagala University of Thanyaburi (Rangsit center) for all facilities and we are also thankful to our advisor, Dr. Surachai Techaoei for providing necessary facilities during the study.