Antioxidant and Antimicrobial Activities of Polycephalomyces nipponicus

1department of biology, faculty of Science, Mahasarakham university, Kantarawichai district, Maha Sarakham Province, 44150, Thailand. 2department of chemistry, faculty of Science, Mahasarakham university, Kantarawichai district, Maha Sarakham Province, 44150, Thailand. 3Microbiology and applied Microbiology research unit, faculty of Science, Mahasarakham university, Kantarawichai district, Maha Sarakham Province, 44150, Thailand.

Antioxidants are compounds that can eliminate free radicals and protect cells from damage caused by oxidative stress 8 .Nowadays, natural antioxidants from mushrooms have attracted much interest because they are safer than synthetic antioxidants in the pharmaceutical and food industries 9 .Furthermore, pathogenic microorganisms are increasingly resistant to commercial drugs so medicinal agents from natural sources with efficacy against pathogenic microorganisms are being sought as alternative medicines.There have many benefits for human health because they are non-toxic and there are no side effects 10 .Natural antioxidant substances found in fruiting bodies, mycelia and broth of mushrooms include phenolics, flavonoids, and polysaccharides which have been shown to have the capacity to scavenge free radicals and inhibit pathogenic bacterial and fungal growth 11,12,13 .Several studies have demonstrated that various extracts of insect pathogenic fungi had potent activity as scavengers of free radicals in-vitro 2,14,15,16 .
The antimicrobial activities of insect pathogenic fungal species have been reported 15,17, 18,19 .Methanolic extract from the fruiting body of C. militaris contains antibacterial and antifungal compounds 20 .An aqueous extract from O. sinensis possed antibacterial activity 21 .Zhang et al . 22demonstrated that a polysaccharide from C. cicadae showed strong antibacterial activity against gram positive and gram negative bacteria.However, there have been few studies of biological activities from P. nipponicus.In this study, we investigated the antioxidant and antimicrobial activities in methanol, 50% methanol and water extracts of P. nipponicus TBRC 6537 mycelia.

Materials and Methods
All chemicals and reagents were commercially available and used without purification.

Fungal strain
Polycephalomyces nipponicus TBRC 6537 was purchased from Thailand Bioresource Research Center (TBRC).The stock culture was grown on potato dextrose agar (PDA) slant at 28 o C for 7 days and then kept at 4 o C.

submerged culture
The mycelia of P. nipponicus TBRC 6537 was cultured on PDA agar plate at 28 °C for 14 days and punched out with sterile cork borer (6 mm in diameter).This 20 mycelia plugs were grown on 400 ml of synthetic medium (dextrose 20 g/L, peptone 10 g/L, yeast extract 5 g/L, MgSO 4 •7H 2 O 0.5 g/L, K 2 HPO 4 1 g/L, thiamin 0.002 g/L and glycine 4 g/L, pH 6.8) in a 1,000 mL Erlenmeyer flasks .The culture was incubated at 28 °C for 30 days under static culture and filtered through Watman No.4 filter paper.Collected mycelia were freeze dried.extraction Samples of dried mycelium of P. nipponicus TBRC 6537 were ground before extraction.Approximately 2 g of dried and powdered samples was sequentially extracted using methanol, 50 % methanol and water, at 60 °C for 30 min using an ultrasonic cleaning bath (Bandelin Electronic, Ultrasonic Bath DT 255 H).The extract was filtered through Whatman No.1 filter paper and the filtrate evaporated to dryness under reduced pressure in a rotatory vacuum evaporator.The dried sample of mycelial extract was weighed and the yield was determined.The dried mycelial extracts were kept in darkness at 4 °C until further analyses.

total phenolic content
The total phenolic compound (TPC) of mycelial extracts was determined by the Folin-Ciocalteu method 23 with some modifications.500 µl of mycelia extract was mixed with 1,500 µl of Folin-Ciocalteu reagen (diluted 10 times in water) and held in the dark for 3 min before 1,500 µl of 7.5% Na 2 CO 3 was added.The absorbance of the mixed reaction was measured at 765 nm after incubation in the dark for 30 min.TPC was expressed as mg gallic acid equivalents (GAE) per gram extract.Values presented are the average of three measurements.

total flavonoid content
The total flavonoid content of mycelia extracts was determined by the method described by Chang et al . 24with some modifications.200 µl of mycelia extract was mixed with 75 µl of 5% NaNO 2 .After incubation in the dark at room temperature for 5 min, 150 µl of 10%AlCl 3 was added.The mixed reaction was incubated for 6 min and 500 µl of 1M NaOH added, followed by 1,075 µl of distilled water to a final volume of 2 ml.The absorbance of the mixed reaction was measured at 415 nm after incubation at room temperature for 30 min.Flavonoid content was expressed as mg quercetin equivalent (QE) per gram dried extract.Values presented are the average of three measurements.

antioxidant property dPPh assay
The DPPH radical-scavenging activity of mycelial extracts was determined using the method based on Reis et al. 20 using ascorbic acid as a standard.100 µl of mycelia extract at various concentrations was mixed with 900 µl of 0.1 mM radical DPPH solution.The absorbance of the mixed reaction was measured at 517 nm after incubation in the dark for 30 min.The DPPH scavenging percentage was calculated as follows: Where A 0 is the absorbance of a negative control and A s is the absorbance of the reaction mixture.The antioxidant activity is expressed as IC 50 .IC 50 was calculated by interpolation from a graph plotting concentration against scavenging activity.Tests were conducted in triplicate.

aBts assay
The 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid free radical (ABTS•+) neutralization was determined using a spectrophotometric method described by Re et al . 25with some modifications.100 µl of mycelia extract at various concentrations was mixed with 900 µl of ABTS cation radical solution.The absorbance of the mixed reaction was measured at 734 nm after incubation in the dark for 20 min.The ABTS scavenging percentage was calculated as follows: Scavenging ABTS radical (%) = [(A 0 -A s ) / A 0 ] x 100 Where A 0 is the absorbance of a negative control and A s is the absorbance of the reaction mixture.ABTS radical cation scavenging activity was express as equivalents of ascorbic acid.C. albicans TISTR 5957 were obtained from a culture collection at Thailand Institute of Scientific and Technological Research.These bacterial strains were used for assessing antibacterial activity of the extracts.The yeast strain was used for assessing the antifungal activity of the extracts.

agar well diffusion method
Antimicrobial activities of the different extracts were determined by the agar well diffusion method with some modifications 26 .The five tested bacteria were cultured at 37 o C for 16-18 hours on nutrient agar and C. albicans were cultured at 28 o C for 48 hours on Sabouraud dextrose agar.The cell suspensions were adjusted to 1.5 ×10 8 CFU/ml for bacteria and 1.5 ×10 6 CFU/ml for yeast in 0.85% NaCl which compared to 0.5 McFarland standard.Bacterial and yeast suspensions were then swabbed with sterilized cotton swab on to Mueller Hinton agar and Sabouraud dextrose agar, respectively.Agar wells were punched out with a sterile cork borer (6 mm in diameter).100 µl (200 mg/mL) of extracts was added to each well and then incubated at 37 o C for 18-24 hours (bacteria) and at 28 o C for 24 hours (yeast).Gentamycin (antibiotic) and ketoconazole (antifungal) were used as positive control.5% DMSO was used as negative control.

determination of the minimal inhibitory concentration (MiCs), minimum bactericidal concentration (MBC) and determination of minimum fungicidal concentration (MFC)
The antibacterial activity of each mycelia extract was quantified by determining minimum inhibitory concentrations (MICs) using the broth microdilution method 27,28 with some modification against the five bacterial strains.90 µl of various concentrations of the mycelia extract (2-fold dilutions of mycelia extract in Mueller Hinton broth starting from 400 mg/ml) were added to 96well microtiter plates .Ten microliters of bacterial suspension (ca.1.5×10 6 CFU/ml) was applied to each well.After incubation at 37 o C for 24 hours, 30 µl of resazurin (0.01%) indicator solution was added in to each well.The plates were further incubated for 2-4 hours.Color change of the indicator from blue to pink indicated viable cell growth.The lowest concentration at which no color change occurred was defined as the MIC value.Samples of the highest dilution with remaining blue color from the MIC were inoculated (10 µl) onto nutrient agar plates to determine the minimal bactericidal concentration (MBC) with no bacterial growth.
For antifungal activity, the MIC was determined by using the macrodilution method 29 against C. albicans.The mycelia extracts were diluted two fold in Sabouraud dextrose broth at concentrations ranging from 0.39 mg/ml to 200 mg/ml.Aliquots of 0.9 ml of yeast suspensions (ca.2.5×10 3 CFU/ml) were transferred to each tube containing 0.1 mL mycelia extract.MICs were determined after incubation at 28 o C for 24-48 hours as the lowest concentration of mycelial extract which could inhibit growth.In order to obtain the MFC, turbidity free media from the MIC assay was streaked onto Sabouraud dextrose agar plates and incubated at 28 o C for 48 hours.The MFC was defined as the lowest concentration of mycelia extract which could kill yeast cells.

statistical analyses
Neither log-transformed nor original size of clear zone conformed to normal distribution at 95% confidence level, according to Shapiro-Wilk tests (p<0.05).Independent-Samples Kruskal-Wallis Tests were therefore performed as nonparametric one-way analyses of variance for these two variables under the null hypothesis that the distributions of these variables are the same across categories of either extraction methods or test microbes.Pairwise comparisons were used as a post hoc analyses should the null hypothesis be rejected.

results extraction yield, total phenolic and flavonoid contents
Extraction yield, total phenolic and flavonoid contents of different extracts from P. nipponicus TBRC 6537 mycelia are reported in Table 1 antioxidant activity Free radical scavenging activity was investigated with the DPPH and ABTS assays.The water extract showed antibacterial activity against three bacteria tested (E. coli DMST 4212, S. epidermidis TISTR 518 and B. cereus DMST 5040) with the inhibition zones from 13.17±0.29 to 25.00±00 mm, and it did not inhibit P. aeruginosa DMST 4739 or S. aureus DMST 2933.The water extract exhibited the highest antibacterial activity against E. coli DMST 4212 (25.00±00) and S. epidermidis TISTR 518 (19.17±2.02).The 50% aqueous methanol and water extracts had efficient antifungal activities against C. albicans TISTR 5957 with the inhibition zones of 13.33±1.15and 22.33±1.53mm, while methanol extract had no antifungal activity.(Table 3 and Figure 1.) The MICs assay and MBCs/MFC of methanol, 50% aqueous methanol and water extracts from P. nipponicus TBRC 6537 mycelia against five bacterial and one yeast species are shown in Table 4 .The MIC values of methanol extract for five bacteria tested was ranged from 25 to 100 mg/ml and MBC values rang was 25 to >200 mg/ml.The MIC values of 50% aqueous methanol extract for four bacteria tested was ranged from 1.56-12.5 mg/ml and MBC values was ranged from 3.13 to 50 mg/ml.The MIC values of water extract for three bacteria tested ranged from 0.05 to 3.13 mg/ml and MBC values ranged from 0.16 to 6.25 mg/ml.The MIC values of 50% aqueous methanol and water extracts for C. albicans TISTR 5957 were 2.5 and 0.156 mg/ml, respectively.The MFC values of 50% aqueous methanol and water extracts were 10 and 0.313 mg/ml, respectively.The result of these study indicated that the water extract was found to be more effective for microorganisms tested than 50% aqueous methanol and methanol extracts, respectively.

disCussion
The yield of extraction depends on the solvent and its varying polarity, temperature, extraction time, and composition of the sample.Under constant extraction time and temperature, solvent and composition of sample are known as the most important parameters.In this work, P. nipponicus TBRC 6537 mycelia extracts were obtained by sequential extraction in an ultrasonic cleaning bath using methanol, 50 % aqueous methanol and water, respectively.Extraction yields ranged from 7.39% for water extract to 12.00% for methanol extract (Table 1).The yields of extraction by various solvents decreased in the following order: methanol > 50% aqueous methanol > water.It can be seen that the extraction yield of pure methanol (12.00%) is higher than that of 50% aqueous methanol (8.08%) and water (7.39%).This shows that the extraction yield decreases with increasing polarity of the solvent used in extraction.The total phenolic content values of the extracts range from 16.774 mg GAE/g for water extract to 28.249 mg GAE/g for 50% aqueous methanol extract (Table 1) and they decrease in the following order: 50% aqueous methanol > methanol > water.It was found that the total phenolic content of the extracts decreased with increasing water content in the aqueous methanol.The total phenolic contents of the 50% aqueous methanol extract (28.249 mg GAE/) is higher than that of the methanol extract (21.624 mg GAE/g).This may be attributable to the content of more nonphenolic compounds such as carbohydrates and terpene in methanol extracts than in 50% aqueous methanol extract.It may also be caused by the possible complex formation of some phenolic compounds in the extract that are soluble in 50% aqueous methanol.
The flavonoid contents of the extracts are reported in Table 1.It was observed that the extraction efficiency of different solvents on the flavonoid contents was different to that on the phenolic contents.The total flavonoid contents was highest in methanol extract (67.139 mg QE/g), followed by water extract (39.478 mg QE/g) and 50% aqueous methanol extract (28.350 mg QE/g).This indicates that flavonoids are not the dominating phenolic group in P. nipponicus TBRC 6537.
The antioxidant activity assay is based on the reduction of DPPH and ABTS.The ability of the extracts and standard ascorbic acid to scavenge free radicals and pair of the odd electron was shown in this assay (Table 2).It was observed that water extract (IC 50 0.228±0.001)and methanol extract (IC 50 0.697±0.000)were good as (p=0.015)ascorbic acid (IC 50 0.029±0.001) in DPPH radical scavenging activity.The water and methanol extracts exhibited the highest DPPH scavenging activities because of the polar compounds that they contains 31 .Additionally, water extract (IC 50 0.049±0.000)and 50% aqueous methanol extract (IC 50 0.157±0.002)were as good as (p=0.015)ascorbic acid (IC 50 0.012±0.001) in ABTS radical scavenging activity.Reis etal . 20reported that methanol extract from the fruiting body of C. militaris showed the highest DPPH radical scavenging activity with the lowest EC 50 value of 12.17 mg/ml.Furthermore, a hot water extract from cultured mycelia of O. sinensis had stronger DPPH radical scavenging effect than that of natural mycelium 32 .In addition, exo-polysaccharide (EPS) and intracellular polysaccharide (IPS) extracted from mycelium of C. cicadae showed high DPPH scavenging activity 15 which were similar to C. gracilis 33 .The study demonstrates that the extracts of Cordyceps sinensis (CSE) and Cordyceps militaris (CME) in aqueous systems showed strong ABTS radical scavenging activities 34 .The water extract contains polar compounds like polysaccharide that there is correlation to their antioxidant 32,35 .

antimicrobial activity
The results of antimicrobial activity of the different extracts tested by the agar well diffusion method, MIC and MBC/MFC against P. aeruginosa DMST 4739, E. coli DMST 4212, S. aureus DMST 2933, S. epidermidis TISTR 518, B. cereus DMST 5040 and C. albicans TISTR 5957 are shown in Table 3 and 4. The methanol extract exhibited antibacterial effects against all tested microorganisms except C. albicans TISTR 5957.The methanol extract was found to have antibacterial activity against P. aeruginosa DMST 4739 while 50% aqueous methanol and water extracts showed no antibacterial activities.Moreover, it exhibited the highest antibacterial activity against B. cereus DMST 5040 (MIC and MBC: 25 mg/ml), followed by S. aureus DMST 2933 and S. epidermidis TISTR 518 (MIC and MBC: 25 and 50 mg/ml), P. aeruginosa DMST 4739 (MIC and MBC: 100 and 200 mg/ml) and E. coli DMST 4212 (MIC and MBC: 100 and >200 mg/ml).In contrast, the methanol extract of P. nipponicus isolate Cod-MK1201 mycelia did not inhibit the growth of any of the gram positive and gram negative bacteria tested 6 .Reis et al. 20 who reported that a methanol extract of fruiting C. militaris revealed the highest antibacterial activity against B. cereus and P. aeruginosa with MIC values of 0.015 mg/ml and MBC values of 0.03 mg/ml.Dong et al. 14 reported that the methanol

table 1 .
Extraction yield, total phenolic content and total flavonoid content of different extracts of P. nipponicus TBRC 6537 mycelia Remark: Values are mean inhibition zone (mm) ± S.D of three replicates

table 2 .
IC 50 values of different extracts of P.

table 3 .
Antimicrobial activity of different extracts from P. nipponicus TBRC 6537 mycelia against microorganisms tested by the agar well diffusion method Remarks: Same letter in inhibition zone diameters indicate that values in same column are not statistically significantly different at 95 % confidence level.Values are mean inhibition zone (mm) ± S.D of three replicates.