Abstract: Crude extracts of mushroom Phellinus linteus were investigated for their antioxidant, cytotoxic, antimalarial and antibacterial activities. The crude EtOAc fraction from large scale extraction showed the strongest antioxidant activity with IC50 value of 17.73±0.27 μg mL-1 and the highest total phenolic value of 76.39±0.07 EGA. This activity was as good as (p>0.05) L(+)ascorbic acid (IC50 value of 16.56±0.50 μg mL-1). From small scale extraction, IC50 values of crude extracts were in the range of 24.15±0.50 to 207.02±1.95 μg mL-1 and the values of total phenolic content ranged from 68.11±0.06 to 5.96±0.18 EGA. Crude MeOH, CH2Cl2 and EtOAc extracts from large scale extraction exhibited cytotoxicity against MFC7 and NCI-H187 cancer cells. Crude MeOH and CH2Cl2 extracts also displayed antimalarial activity against Plasmodium falciparum with IC50 values of 3.15 and 3.08 μg mL-1, respectively. None of the extracts were found to have antibacterial activity against five different species of pathogenic bacteria.
INTRODUCTION
Mushroom Phellinus linteus, commonly referred as Sanghwang in Korea, Meshimakobu in Japan and Songgen in China, is the most well known species of the genus Phellinus. This mushroom has long been used as a traditional medicine in oriental countries for the treatment of stomachaches, inflammation, arthritis of the knee, gastroenteric disorders, inflammation, tumors and lymphatic disorders (Cho et al., 2002; Kang et at., 2004; Kim et al., 2004a). The active polysaccharides, especially β-glucan, produced by P. linteus have been known to exhibit potent biological activities. There were reports of polysaccharides extracted from P. linteus stimulating cell-mediated and humoral immunity and inhibiting tumor growth and metastasis (Han et al., 1999; Kim et al., 1996). The inhibitory activity of the polysaccharide isolated from P. lintues toward melanoma cell metastasis (Han et al., 2006) and the role of polysaccharides from P. linteus as effective immunomodulator and enhancing the antitumoral activity of peritoneal macrophages (Kim et al., 2004b) have also been reported. P. linteus was found to act as a natural anticancer agent by preventing the inhibition of gap junctional intercellular communication through the inactivation of ERK1/2 and p38 MAP kinases (Cho et al., 2002). Polysaccharides from P. linteus have also been reported to exhibit the hypoglycemic effect and they also decreased total cholesterol, triacylglycerol and aspartate aminotransferase activity (Kim et al., 2001). Its polysaccharide had an effect on the immunomodulatory activity of the murine splenic lymphocytes (Kim et al., 2003a) and also inhibited tumor growth through a mechanism leading to a Th-1 dominant immune state and the activation of CD11c+CD8+ DC (Kim et al., 2004c).
An active polysaccharide from P. linteus was reported to have been involved with the prevention and treatment of autoimmune joint inflammation (Kim et al., 2003b). The antiinflammatory, antinociceptive and antiangiogenic activities from an n-BuOH subfraction from a mycelia culture of P. linteus (Kim et al., 2004a) and the antiinflammatory activity from its fruiting body via mediation of heme oxygenase-1 (Kim et al., 2006) have been investigated. There were reports of P. linteus inhibition of inflammatory mediators by suppressing redox-based NF-kB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophage (Kim et al., 2007a). The anti-inflammatory activity mediated through the PKCδ/Nrf2/ARE signaling to up-regulation of heme oxygenase-1 (Kim et al., 2007b) was documented.
Crude extracts of P. linteus have also been studied for their antioxidant activity. There was a report that the 70% ethanol extract from fruiting body of P. linteus showed antiangiogenic and antioxidant activities when compared with standard vitamin C (Song et al., 2003). Recently, polysaccharide from hot water extracts from various medicinal mushrooms, including P. linteus, were found to be highly active to Reactive Oxygen Species (ROS) which could play an important role in the prevention of infection by destroying potential intracellular pathogens (Wei and Van Griensven, 2008).
In this study, we aim to investigate antioxidant property of crude extracts obtained from various solvent extractions of P. linteus, to evaluate these extracts for their cytotoxicity against NCI-H187, MFC7 and KB cancer cells and to characterize antimalarial and antibacterial activities which have never been investigated before.
MATERIALS AND METHODS
Mushroom sample: The fruiting body of mushroom P. linteus was kindly provided by Mr. Frankie Chan. The mushroom was identified by Dr. Usa Klinhom based on morphology and deposited at MSUT, Faculty of Science, Mahasarakham University. The specimen was dried at 60°C under a UV lamp, then powdered.
Extraction
Small scale extraction: Ten gram of mushroom powder was extracted with 100
mL (24 h, 3 times) of five different solvents; water, ethanol (EtOH), 50% EtOH,
80% EtOH and ethyl acetate (EtOAc), under two conditions; heated at 60°C
and room temperature extractions. The collected EtOH and EtOAc layers (cal.
300 mL each) were each evaporated under reduced pressure at 40°C to give
crude EtOH and EtOAc extracts, respectively. The collected water, 50% EtOH,
80% EtOH layers (cal. 300 mL each) were evaporated under reduced pressure and
residues from evaporation were subjected to freeze-drying to give crude extracts
from water, 50% EtOH and 80% EtOH.
Large scale extraction: Five hundred gram of mushroom powder was extracted with methanol (MeOH) (3 L, 60°C, 3 day, 2 times). The collected MeOH layer (cal. 6 L) was concentrated under reduced pressure to obtain a brownish-black crude MeOH extract. Three hundred milligram of this crude extract was taken for the antioxidant and biological assays. The remaining extract was suspended in water (150 mL) and then partitioned with hexane, dichoromethane (CH2Cl2) and EtOAc, respectively. The hexane (cal. 1 L), CH2Cl2 (cal. 700 mL) and EtOAc (cal. 1.2 L) layers were evaporated under reduced pressure to give crude hexane (1.67 g), CH2Cl2 (1.54 g) and EtOAc (3.39 g) fractions from MeOH, respectively.
Antioxidant assay
Radical scavenging activity toward DPPH radical (DPPH method): Free radical
scavenging activity of the crude extracts was measured according to the method
of (Chu et al., 2000) with the modifications in the concentration and
the ratio of mixed solutions. A 2 mL of 0.2 mM DPPH solution in MeOH was added
to 1 mL of sample solution (5-100 μg mL-1). The mixture was
kept at room temperature for 30 min in the dark. After that, the absorbance
values of the solutions were measured at λmax 517. The percentages
of radical scavenging activity (%RSA) of each concentration were calculated
and then converted to the value of the inhibition concentration at 50% (IC50
value). Quercetin, L(+)-ascorbic acid (vitamin C) and butylated hydroxytoluene
(BHT) were used as standard antioxidants.
RSA (%) = [1 - (Asample/Ablank)
x 100] |
Blank: One milliliter of methanol mixed with 2 mL of 0.2 mM DPPH solution.
Determination of total phenolic content (Folin- Ciocalteu method): Total phenolic content was determined by using the Folin-Ciocalteu method described by Miliauskas at al. (2004) with the modifications in the concentration and the ratio of mixed solutions. One milliliter of a 50 μg mL-1 of sample solution in MeOH was added with 2 mL of Folin-Ciocalteu’s reagent solution (10-fold dilution in H2O) and 4 mL of 7.5% (w/v) sodium carbonate solution. The absorbance values of the mixed solution were measured at λmax 761 nm after being left to stand for 1 h at room temperature in the dark. The phenolic content was expressed as equivalent of gallic acid (EGA); mg EGA: 100 mg of the extract.
Cytotoxicity and antimalarial assay: Crude MeOH extract and its crude CH2Cl2 and EtOAc extracts from large scale extraction were sent to BIOTEC for their cytotoxicity (Skehan et al., 1990) and antimalarial (Desjardins et al., 1979) activity.
Antibacterial assay: Antibacterial activity was evaluated using paper disc method. Crude MeOH extract its crude hexane, CH2Cl2 and EtOAc fractions from large scale extraction were test against five food borne pathogenic bacteria; Escherichia coli ATCC25922, Salmonella typhi DMST5784, Shigella flexneri DMST4423, Shigella dysenteriae DMST15111 and Vibrio cholerae ATCC14035. Bacterial concentrations of 107 cfu mL-1 were incubated before being placed on paper discs containing crude extracts at a concentration of 5,000 μg mL-1 on MHA medium. The zone of inhibition was observed and measured at 24 and 48 h.
The extraction, antioxidant and antibacterial activities testing were carried out at the Faculty of Science, Mahasarakham University while the bioassay for cytotoxic and antimalarial activities were carried out at BIOTEC (National Center for Genetic Engineering and Biotechnology), Bangkok, Thailand, during August to December, 2008.
Statistical analysis: The tests for antioxidant activity and total phenolic content were carried out in triplicate. The data was recorded as Mean±SD. The means of all parameters were examined for significance by Analysis of Variance (ANOVA) with Duncan’s significant difference post-hoc test (SPSS software). The p-values less than 0.05 were considered significant.
RESULTS
Antioxidant activity
DPPH radical scavenging activity: The strongest DPPH radical scavenging
capacity of the extracts was found in crude EtOAc fraction from large scale
extraction with IC50 value of 17.73±0.27 μg mL-1
(Table 1). This activity was as good as (p>0.05) L(+) ascorbic
acid (vitamin C) (IC50 value of 16.56±0.50 μg mL-1),
significantly (p<0.05) higher than BHT (IC50 value of 18.84±0.27
μg mL-1) but lower than quercetin (IC50 value of
10.92±0.13 μg mL-1). This activity was three and four
times stronger than the scavenging activity of crude EtOAc extracts from heated
extraction, IC50 value of 52.90±0.30 μg mL-1
and room temperature, IC50 value of 77.97±1.26 μg mL-1,
from small scale extractions. Comparing activity between crude MeOH and EtOH
extracts showed that crude MeOH extract from large scale extraction, IC50
value of 25.58±0.22 μg mL-1, displayed radical
scavenging activity significantly higher (p<0.05) than crude EtOH extracts
from heated, IC50 value 29.18±0.20 μg mL-1
and much better than room temp., IC50 value 43.06±0.52 μg
mL-1, from small scale extractions. Crude CH2Cl2
fraction from large scale extraction was found to have low antioxidant property
when compared with crude MeOH extract and EtOAc fraction. However, this activity
was significant (p<0.05) higher than the activity of crude water extracts
from small scale extractions. Hexane was used for large scale extraction in
order to remove the lipid from crude MeOH extract before fractionation. No significant
antioxidant activity (IC50 value>1,000 μg mL-1)
was observed for crude hexane fraction.
| Table 1: | Antioxidant activity from crude extracts of mushroom P. linteus |
| aHeated at the temperature of 60°C; bRoom temperature | |
For small scale extraction, scavenging activity on DPPH radical from crude extracts from heated extraction exhibited stronger activity than room temperature extraction for all kind of extracting solvents. In this group, crude 50% EtOH extract showed the highest antioxidant activity with IC50 values of 24.15±0.50 and 25.54±0.17 μg mL-1, while crude water extracts exhibited the lowest DPPH scavenging ability with IC50 values of 147.24±2.26 and 207.02±1.95 μg mL-1.
Total phenolic content: Crude EtOAc fraction from large scale extraction presented the highest amount of phenolic compounds with a value of 76.39±0.07 EGA (Table 1). For small scale extraction, crude extract from 80% EtOH showed the highest total phenolic content for both heated and room temperature extraction conditions with values of 68.11±0.06 (heat) and 51.64±1.47 (room temp.) EGA, respectively. The lowest total phenolic content was found in crude water extracts, 14.19±0.17 (heat) and 5.96±0.18 (room temp.) EGA. High amount of total phenolics relate to good antioxidant property.
Cytotoxic and antimalarial activities: Crude MeOH, crude CH2Cl2 and EtOAc fractions from large scale extraction of P. linteus showed cytotoxicity against MFC7 (IC50 values of 17.36, 25.78 and 27.26 μg mL-1, respectively), NCI-H187 (IC50 values of 19.14, 17.88 and 40.15 μg mL-1, respectively) and vero cells (IC50 values of 48.42 and 42.58 and non cytotoxic μg mL-1, respectively) while displayed inactive result to KB cell (Table 2). Also, crude extracts from MeOH and CH2Cl2 exhibited potent antimalarial activity against the multidrug-resistant strain of Plasmodium falciparum with IC50 values of 3.15 and 3.08 μg mL-1, respectively.
| Table 2: | Biological activities from crude extracts of P. linteus |
| dAgainst Plasmodium falciparum (K1, multidrug-resistant strain), KB: Oral cavity cancer cell, MCF7: Breast cancer call, NCI-H187: Small cell lung cancer cell, vero use African green monkey kidney fibroblasts | |
Antimicrobial activity: From our results, no significant antibacterial activity was observed from crude extracts of P. linteus against five species of pathogenic bacteria tested in this study at maximum concentration of 100 mg mL-1.
DISCUSSION
In our present work, medicinal mushroom P. linteus has been investigated for its antioxidant and biological activities. The strongest antioxidant capacity as comparable as (p>0.05) vitamin C was observed in crude EtOAc fraction. These results were in good agreement with the study of Kang et al. (2004) in the report of the antioxidant activity of L-tyrosine catalyzed by tyrosinase from crude fractions of P. linteus. The results indicated that successively fractionation of crude MeOH extract with CH2Cl2, EtOAc and n-BuOH, the highest active antioxidants came in crude EtOAc fraction. Slightly lower antioxidant property of crude EtOH-water (50% EtOH and 80% EtOH) extracts than the standard BHT, a synthetic antioxidant compound, was investigated. This result were similar to the antioxidant activity of mushroom Chaga extracts from sclerotium (ST) and Fruiting Body (FB) parts reported by Nakajima et al. (2007). Crude 80% MeOH extract from both of ST and FB parts showed relatively high activity than 50% MeOH, 20% MeOH and aqueous extracts using DPPH assay.
It has been reported that the antioxidant capacity of edible mushroom extracts was positively correlated with their content of phenolic compounds (Lim et al., 2007). Present results showed a good correlation between the amount of total phenolics and antioxidant activity of the P. linteus extracts. Yellow pigments that composed of hispidin derivatives and polyphenols which are a major contribution to their antioxidant property are commonly produced by mushrooms Phellinus (Lee and Yun, 2007) and pure polyphenol compounds isolated from mushroom Phellinus have been reported by Nagatsu et al. (2004), Min et al. (2006), Mo et al. (2004) and Wang et al. (2007) The significantly high amount of phenolic compounds in crude EtOAc fraction followed by crude 80% EtOH and EtOH extracts could be explained that the phenolics of P. linteus are high in polarity and likely easier soluble in polar organic and alcohol-aqueous solvents.
Crude MeOH extract and its crude hexane, CH2Cl2 and EtOAc fractions of P. linteus had no antibacterial activity against five species of pathogenic bacteria tested. However, Hur et al. (2004) reported antibacterial activity from n-BuOH fraction of crude MeOH extract of P. linteus against Staphylococcus aureus strains at MIC values ranging from 63-125 μg mL-1 while antibacterial activity from aqueous extracts of P. gilvus and P. igniarius against S. aureus, E. coli and Klebsiella pneumonia have been reported by Sittiwet and Puangpronpitag (2008a, b). Interestingly, antibacterial activity from crude water extract of P. linteus against pathogenic bacteria should be further studied.
The cytotoxicity against NCI-H187 and antimalarial activity of P. linteus are the first time reported. Recently, the activity of P. linteus suppressed phosphorylation of AKT at Thr308 and Ser473 in breast cancer cells has been investigated by Sliva et al. (2008).
In conclusion, our research suggested that P. linteus collected from Thailand has high potential would be used as natural antioxidants. Due to toxicological concerns associated with the used of synthetic substances in food and increasing awareness about natural foods, there has been increased interest in the use of natural substances as food preservatives and antioxidants (Peschel et al., 2006). Its antioxidant property, cytotoxicity and antimalarial activity could also have correlations with other pharmacological actions for its use in folk medicine.
Isolation and structure elucidation of pure compound from crude CH2Cl2 and EtOAc fractions of P. linteus are under the process.
ACKNOWLEDGMENTS
This research was financially supported by Faculty of Science, Mahasarakham University and Center of Excellence of Innovation in Chemistry (PERCH-CIC). The authors would like to thank Mr. Frankie Chan and Prof. Dr. Leo J.L.D. Van Griensven for kindly supplying the mushroom and their valuable guidance. The author acknowledges the BIOTEC for the biological assay of cytotoxic and antimalarial activities. Dr. Jolyon Dodgson, Department of Biology, Faculty of Science, Mahasarakham University, is appreciated for his generosity of editing the manuscript.