Botanical Studies (2007) 48: 407-417.
a
These authors contributed equally to this work.
*
Corresponding author: E-mail: yschang@mail.cmu.edu.tw;
Tel: +886-4-22030380; Fax: +886-4-22083362.
INTRODUCTION
It is commonly accepted that, in a situation of oxidative
stress, reactive oxygen species, such as superoxide (O
.
2
),
hydroxyl (OH
.
) and peroxyl (
.
OOH, ROO
.
) radicals, are
generated. The reactive oxygen species play an important
role related to the degenerative or pathological processes
of various serious diseases, such as aging (Burns et al.,
2001), cancer, coronary heart disease, Alzheimer¡¦s disease
(Smith et al., 1996; Diaz et al., 1997), neurodegenerative
disorders, atherosclerosis, cataracts, and inflammation
(Aruoma, 1998). Traditional medicine is widespread and
plants still presents a large source of natural antioxidants
that might serve as leads for the development of novel
drugs. Several anti-inflammatory, digestive, anti-necrotic,
neuroprotective, and hepatoprotective drugs have recently
Antioxidant and free radical scavenging activities of
Phellinus merrillii extracts
Heng-Yuan CHANG
1
, Yu-Ling HO
2
, Ming-Jyh SHEU
3
, Yaw-Huei LIN
4
, Mu-Chuan TSENG
5
,
Sheng-Hua WU
6
, Guan-Jhong HUANG
1, a
, and Yuan-Shiun CHANG
1,7,a,
*
1
Institute of Chinese Pharmaceutical Sciences, College of Pharmacy, China Medical University, Taichung 404, Taiwan
2
Department of Nursing, Hung Kuang University, Sha Lu, Taichung 433, Taiwan
3
Department of Physiology, School of Medicine, College of Medicine, China Medical University, Taichung 404, Taiwan
4
Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan
5
Bureau of Food and Drug Analysis, Department of Health, Executive Yuan,161-2 Kun-yang St., Nangang District, Taipei
115, Taiwan
6
Department of Botany, National Museum of Natural Science, Taichung 404, Taiwan
7
Chinese Crude Drug Pharmacy, China Medical University Hospital, Taichung 404, Taiwan
(Received May 22, 2007; Accepted July 26, 2007)
ABSTRACT.
This study aimed to investigate possible antioxidant activity of various extracts of Phellinus
merrillii (PM). The explored items include: ABTS free radical scavenging assay, determination of total
phenolics contents (TPC), ferric reducing antioxidant power assay (FRAP), rapid screening of antioxidant by
dot-blot DPPH (1, 1-diphenyl-2-picrylhydrazyl) staining, DPPH radical-scavenging activities and reducing
power measurement. In the ABTS free radical scavenging assay, the n-BuOH fraction displayed the highest
total antioxidant activity (17.13 ¡Ó 0.04 mM). In the determination of total phenolics contents (TPC) and ferric
reducing antioxidant power assay (FRAP), the EtOAc fraction had the highest phenolics contents (46.21 ¡Ó
0.02 mM) and reducing antioxidant power (19.09 ¡Ó 0.03 mM). In the rapid screening of antioxidant by dot-
blot DPPH staining, the n-BuOH fraction showed the highest strong dot-blot staining. In the reducing power
measurement, the crude extract had the highest reducing power at 2 mg/ml concentration. In the DPPH
radical-scavenging activities, the EtOAc fraction had the highest antioxidant activity (IC
50
= 0.66 ¡Ó 0.01 mg/
ml). As regard the correlation coefficients among ABTS assay, FRAP assay, and total phenolics contents, it
can be seen that correlation coefficients in each case were significant. Among all extracts, the highest amount
of total phenolics contents were found in the EtOAc fractiont. It is suggested that the PM might contribute
its antioxidant activities on EtOAc and n-BuOH fraction. In high-performance liquid chromatography tandem
mass (LC/MS/MS) analysis for hispolon, the daughter ion scanned chromatograms of PM was established.
Both hispolon and PM showed similar daughter ion spectrum at the retention time of 4.7 min and had more
lobes in m/z 219 and m/z 135. This indicated that PM did contain the active ingredient hispolon. Both the IC
50
of DPPH radical scavenging activity for hispolon and BHT were 42.4 ¡Ó 2.9 and 81.2 ¡Ó 3.2 £gM, respectively.
These findings mean that hispolon was most important in antiradical activities. It was suggested that hispolon
might contribute to its antioxidant activities in PM.
Keywords: 1,1-diphenyl-2-picrylhydrazyl (DPPH); Ferric reducing antioxidant power assay (FRAP);
Free radicals; Glutathione reduced form (GSH); High-performance liquid chromatography tandem mass
(LC/MS/MS); Phellinus merrillii (PM); Scavenging effect; Total antioxidant capacity; Total phenolics contents
(TPC); Trolox equivalent antioxidant capacity (TEAC).
BIOChemISTRy
pg_0002
408
Botanical Studies, Vol. 48, 2007
been shown to have an antioxidant and/or anti-radical
scavenging mechanism as part of their activity (Lin and
Huang, 2002; Repetto and Llesuy, 2002). In the search
for sources of natural antioxidants and compounds with
radical scavenging activity during recent years, some have
been found, such as echinacoside in Echinaceae root (Hu
and Kitts, 2000), anthocyanin (Espin et al., 2000), phenolic
compounds (Rice-Evans et al., 1997), water extracts
of roasted Cassia tora (Yen and Chuang, 2000), whey
proteins (Tong et al., 2000), and thioredoxin h protein
from sweet potato (Huang et al., 2004).
The antioxidants present in dietary mushrooms are
of great interest as possible protective agents to help
the human body reduces oxidative damage without any
interference (Adams and Wermuth, 1999). Now they
are recognized as functional foods and as a source of
physiologically beneficial components (Wasser and Weis,
1999). Mushrooms have been shown to boost heart health;
lower the risk of cancer; promote immune function; ward
off viruses, bacteria, and fungi; reduce inflammation;
combat allergies; and help balance blood sugar levels
and support the body¡¦s detoxification mechanism (Ada
et al., 2005). Mushrooms have also been shown to
accumulate a variety of secondary metabolites including
phenolic compounds, polypeptides, terpenes, steroids, etc.
Mushroom phenolics have been found to be an excellent
antioxidant and synergist (Li et al., 2005). Furthermore,
several companies are developing capsules from
combinations of mushrooms, and these capsules, although
expensive, have been shown to be health beneficial,
including fighting against cancer (Mau et al., 2005).
Macrofungi was commonly used as a nutrition
supplements to a variety of diseases in Asia (Jong and
Birmingham, 1992; Chen et al., 2006a). In Taiwan, several
different species of Phellinus were widely applied for
anticancer, antioxidant purposes and hepatoprotective
effects. Phellinus linteus demonstrated anti-tumor activity
in several studies (Lin et al., 2003; Kim et al., 2003; Li
et al., 2004; Bae et al., 2005). Also, there were several
reports about the antioxidant effects from Phellinus. The
methanolic extract of the basidiocarps of Phellinus linteus
demonstrated antioxidative effect (Chung et al., 1998) and
antimutagenic activities (Sohn and Nam, 2001). Studies
indicated that Phellinus linteus could protect primary
cultured rat hepatocytes against hepatotoxins (Kim et
al., 2004). Also Phellinus rimosus (Berk) Pilat possess
antioxidant and antihepatotoxic activities (Ajith and
Janardhanan, 2002).
Phytochemicals, especially phenolics in fruits and
vegetables, are suggested to be the major bioactive
compounds for health benefits. Phenolics are one of the
groups of nonessential dietary components that have
been associated with the inhibition of atherosclerosis and
cancer. The bioactivity of phenolics may be related to
their ability to chelate metals, inhibit lipoxygenase, and
scavenge free radicals (Mallavadhani et al., 2006; Lin et
al., 2005).
Hispolon and hispolon derivatives were isolated from
the fungus Phellinus igniarius (Mo et al., 2004). Hispolon,
a yellow pigment was first found in Inonotus hispidus in
1996 (Ali et al., 1996b). Hispolon has been reported to
exhibit apoptosis effect on human epidermoid KB cells
(Chen et al., 2006b) and antivirus activities (Awadh et al.,
2003). Hispolon also inhibit chemiluminescence response
of human mononuclear cells and suppress mitogen-
induced proliferation of spleen lymphocytes of mice (Ali
et al., 1996a).
No report on the antioxidant activities of PM was
presently available. In this work, we reported that PM
displayed antioxidant activities in a series of in vitro
tests such as total antioxidant activity, determination
of total phenolics contents (TPC), ferric reducing
antioxidant power assay (FRAP), DPPH (1, 1-diphenyl-2-
picrylhydrazyl) staining, DPPH radical-scavenging activity
and reducing power method.
mATeRIALS AND meThODS
materials
1, 1-Diphenyl-2-picrylhydrazyl(DPPH), potassium
peroxodisulfate (K
2
S
2
O
8
), tris (hydroxylmethyl)
aminomethane, glutathione reduced form (GSH),
potassium ferricyanide (K
3
Fe(CN)
6
), trichloroacetic
acid (TCA), ferric chloride (FeCl
3
), (+)-catechin,
aluminum chloride hexahydrate (AlCl
3
¡P6H
2
O), rutin,
sodium bicarbonate (Na
2
CO
3
), sodium phosphate
dibasic (Na
2
HPO
4
), sodium phosphate monobasic
(NaH
2
PO
4
), 2, 4, 6-tris(2-pyridyl)-s-triazine (TPTZ) were
purchased from Sigma Chemical Co. (St. Louis, MO
USA). Folin-Ciocalteu solution, methanol and ethanol
were purchased from Merck. Trolox (6-hydroxy-2, 5,
7, 8-tetramethychroman-2-carboxylic acid), ABTS [2,
2¡¦-azinobis (3-ethylbenzothiazoline)-6-sulfonic acid]
diammonium salt were purchased from Roche. Hispolon
was purchased from BJYM Pharm. & Chem. Co. Ltd.
(Beijing, China). Phellinus merrillii was purchased from
the Ji Pin mushroom store (Nantou, Taiwan) and identified
by Dr. Yu-Cheng Dai (Institute of Applied Ecology,
Chinese Academy of Science, China).
extraction of Phellinus merrillii
1.5 kg of PM was soaked with 5 l of 70% ethanol at
room temperature. The samples were filtered with filter
paper (Advantec No. 1, Japan) while the residue was
further extracted under the same conditions three times.
The filtrates collected from these separate extratcions
were combined and evaporated to dryness under vacuum.
The crude extract (60 g) was dissolved into water and
formed as suspension. Hexane 250 ml was added into the
suspension and mixed thoroughly. Then, hexane and water
layers were separated. EtOAc added into the water layer
was mixed well and were separated. Finally, n-BuOH was
mixed with water layer and formed n-BuOH, water soluble
and water insoluble fractions (Figure 1).
pg_0003
CHANG et al. ¡X Free radical scavenging activity of
Phellinus merrillii
409
ABTS free radical scavenging assay
Total antioxidant status of the PM was measured using
2, 2¡¦-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid
(ABTS) assay (Re et al., 1999). ABTS was dissolved in
deionized water to 7 mM concentration, and potassium
persulphate added to a concentration of 2.45 mM. The
reaction mixture was left to stand at room temperature
overnight (12~16 h) in the dark before use. The resultant
intensely-coloured ABTS
¡E+
radical cation was diluted
with 0.01 M PBS (phosphate buffered saline), pH 7.4, to
give an absorbance value of ~0.70 at 734 nm. The test
compound was diluted 100 ¡Ñ with the ABTS solution
to a total volume of 1 ml. Absorbance was measured
spectrophotometrically at time intervals of 1 min after
addition of each extract. The assay was performed at least
in triplicate. Controls containing 990 £gl of PBS, to replace
ABTS, were used to measure absorbance of the extract
themselves. The assay relies on the antioxidant capability
of the samples to inhibit the oxidation of ABTS to
ABTS
¡E+
radical cation. The total antioxidant activities were
expressed as mM trolox equivalent antioxidant capacity
(TEAC).
Determination of total phenolics contents (TPC)
Total phenolics contents were determined using the
Folin-Ciocalteu method (Ragazzi and Veronese, 1973).
One mL of the extract was added to 10.0 ml distilled water
and 2.0 ml of Folin-Ciocalteu phenol reagent (Merck-
Schuchardt, Hohenbrun, Germany). The mixture was
allowed to stand at room temperature for 5 min and then
2.0 ml sodium carbonate was added to the mixture. The
resulting blue complex was then measured at 680 nm.
The contents of phenolics contents were expressed as mM
trolox equivalent antioxidant capacity (TEAC) (Hou et al.,
2004).
Ferric reducing antioxidant power assay (FRAP)
The ferric reducing antioxidant power assay (FRAP)
of each standard solution was measured according to a
modified protocol developed by Benzie and Strain, 1996.
To prepare the FRAP reagent, a mixture of 0.1 M acetate
buffer (pH 3.6), 10 mM TPTZ, and 20 mM ferric chloride
(10:1:1, v/v/v) was made. To 1.9 ml of reagent was added
0.1 ml of extract. Readings at the absorption maximum
(593 nm) were taken every 15 s using a Shimadzu UV-
visible 2501 spectrophotometer, and the reaction was
monitored for up to 10 min. Trolox solution was used to
perform the calibration curves. The reducing antioxidant
power were expressed by the method of Benzie and
Strain, 1996, as mM trolox equivalent antioxidant capacity
(TEAC).
Rapid screening of antioxidant by dot-blot and
DPPh staining
Each diluted sample of the PM was carefully loaded
onto a 20 cm ¡Ñ 20 cm TLC layer (silica gel 60 F254;
Merck) and allowed to dry (3 min). Drops of each sample
were loaded, in order of decreasing concentration (2, 1,
0.5, 0.25, and 0.125 mg/ml), along the row. The staining
of the silica plate was based on the procedure of Soler-
Rivas et al., 2000. The sheet bearing the dry spots was
placed upside down for 10 s in a 0.4 mM DPPH solution.
Then the excess of solution was removed with a tissue
paper and the layer was dried with a hair-dryer blowing
cold air. Stained silica layer revealed a purple background
with white spots at the location where radical-scavenger
capacity presented. The intensity of the white color
depends upon the amount and nature of radical scavenger
present in the sample (Huang et al., 2004).
Determination of antioxidant activity by
reducing power measurement
The reducing powers of the PM and glutathione were
determined according to the method of Yen and Chen,
1995. The PM (0, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/ml)
and glutathione (0, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/ml)
were mixed with an equal volume of 0.2 M phosphate
buffer, pH 6.6, and 1% potassium ferricyanide. The
mixture was incubated at 50¢XC for 20 min, during which
period ferricyanide was reduced to ferrocyanide. Then
an equal volume of 1% trichloroacetic acid was added to
the mixture, which was then centrifuged at 5,000 g for
10 min. The upper layer of the solution was mixed with
distilled water and 0.1% FeCl
3
at a ratio of 1:1:2, and the
absorbance at 700 nm was measured to determine the
amount of ferric ferrocyanide (prussian blue) formed.
Increased absorbance of the reaction mixture indicated
increased reducing power of the sample (Huang et al.,
2005).
Scavenging activity against DPPh radical
The effect of crude extracts on the DPPH radical
was estimated according to the method of Yamaguchi
et al., 1998. An aliquot of crude extract (30 £gl) and
Figu re 1. Extraction and fractionation scheme of Phellinus
merrillii.
pg_0004
410
Botanical Studies, Vol. 48, 2007
glutathione (GSH) (0.5 mg/ml, 30 £gl) were mixed with
100 mM Tris-HCl buffer (120 £gl, pH 7.4) and then with
150 £gl of the DPPH in ethanol to a final concentration
of 250 £gM. The mixture was shaken vigorously and left
to stand at room temperature for 20 min in the dark.
The absorbance at 517 nm of the reaction solution was
measured spectrophometrically. The percentage of DPPH
decolourization of the sample was calculated according to
the following equation as: % decolourization = [1 - ABS
sample / ABS control] ¡Ñ 100. The 50% inhibition (IC
50
) of
antioxidant activity was calculated as the concentrations
of samples that inhibited 50% of scavenging activity of
DPPH radicals activity under these conditions (Huang et
al., 2007).
Analysis of hispolon and Pm by LC/mS/mS
LC/MS/MS was conducted to analyze both the standard
(hispolon) and the 70% ethanol crude extract of PM
according to Chen et al., 2006a, b; Lai et al., 2006. Its
purity was more than 95% based on reversed phase LC/
MS/MS analysis (Instrument: Micromass Quattro Ultima
tandem mass; HPLC: Waters 2695 Alliance LC & 996
PDA with Automatic Liquid Sampler and Injector; Data
processor: MassLynx NT Quattro Data Acquisition).
Analyses were carried out to using negative mode
electrospray ionization of LC/MS/MS analysis.
Chromatographic separation was performed on a C
18
column (Cosmosil 5-C
18
, 150 ¡Ñ 4.6 mm, i.d., 5 £gm)
under an isocratic elution of a mixed solvent system
in a composition of each 70% of methanol and 30% of
water at flow rate of 0.5 ml/min. A full UV spectrum was
scanned from 200 to 400 nm. The source and desolvation
temperature was set at 120¢XC and 350¢XC, respectively.
All processes of the capillary voltage setting 3kV, cone
voltage setting 100V and collision energy setting 15eV for
hispolon [M-H]
-
fragment (m/z=219.8) daughter ion scan.
The daughter ion spectrum obtained will be compared with
the LC/MS/MS library database.
Statistical analysis
Data are expressed as mean ¡Ó S.E.M. Means of
triplicate analyses were calculated. The Student¡¦s t test was
used for comparison between two treatments. A difference
was considered to be statistically significant when p <
0.05.
ReSULTS
extraction and fractionation of Phellinus
merrillii
From 1.5 kg of PM, 60 g of 70% ethanol extract was
obtained. The yield was 4%. The crude extract (60 g) was
suspended in water and partitioned with hexane, EtOAc,
n-BuOH sequentially to yield 3 g hexane fraction (5%),
20 g EtOAc fraction (33%), 23 g n-BuOH fraction (39%),
13 g water soluble fraction (21%) and 1 g water insoluble
fraction (2%) (Figure 1).
effect of the Phellinus merrillii on the total
antioxidant capacity
The antioxidant capacity of crude extract, hexane,
EtOAc, n-BuOH, soluble water and water insoluble
fractions of PM were evaluated according to the ABTS
decoloration method and the FRAP assay. Their total
phenoplics contents were also determined. The results
were shown in Figure 2. Inhibition of generation
of the ABTS£»
+
radical cation was the basis of the
spectrophotometric methods that had been applied to the
measurement of the total antioxidant activities of solutions
of pure substances, aqueous mixtures and beverages. Total
antioxidant activities of several different fractions of PM
(0.1 mg/ml) were measured using the ABTS£»
+
method.
Trolox was used as positive control. The total antioxidant
activity was expressed as Trolox mM by reference to the
Trolox standard calibration curve. The n-BuOH fraction
displayed the highest total antioxidant activity (17.13 ¡Ó
0.04 mM TEAC) (Figure 2). Total antioxidant capacity of
crude and subfractions of PM were evaluated as 16.29 ¡Ó
0.35 mM for crude extract, 16.10 ¡Ó 0.31 mM for EtOAc
fraction, 13.88 ¡Ó 0.19 mM for water insoluble fraction and
13.78 ¡Ó 0.39 mM for water soluble fraction. The hexane
fraction had the lowest total antioxidant capacity of 12.36
¡Ó 0.11 mM.
Total Phenolics contents of fractions of
Phellinus merrillii
The total phenolics contents of crude extract and
hexane, EtOAc, n-BuOH, soluble water and water
insoluble fractions were expressed as mM of trolox
equivalent. The EtOAc fraction had the highest phenolics
contents of 46.21 ¡Ó 0.02 mM, crude extract and n-BuOH
fractions had the phenolics contents of 36.32 ¡Ó 0.01 mM
and 44.95 ¡Ó 0.01 mM, water insoluble fraction and hexane
fraction had the phenolics contents of 8.19 ¡Ó 0.01 mM
and 9.71 ¡Ó 0.01 mM. The water fraction had the lowest
phenolics contents of 2.26 ¡Ó 0.01 mM (Figure 2).
Figure 2. TPC, ABTS, and FRAP assays of various fractions
including crude extrat, hexane, EtOAc, n-BuOH, water soluble
and water insoluble fractions. Each absorbance value represented
the average of triplicates of different samples analyzed.
ABTS assay
FRAP assay
Total phenolics
Crude hexane etOAC n-BuOh h
2
O h
2
O insoluble
pg_0005
CHANG et al. ¡X Free radical scavenging activity of
Phellinus merrillii
411
Ferric reducing antioxidant power assay (FRAP)
The ferric reducing antioxidant power of crude extract
and hexane, EtOAc, n-BuOH, water soluble and water
insoluble fractions were expressed as mM of trolox
equivalent. The EtOAc fraction had the highest reducing
antioxidant power of 19.09 ¡Ó 0.02 mM, crude extract and
n-BuOH fraction had the reducing antioxidant power of
12.00 ¡Ó 0.01 mM and 13.98 ¡Ó 0.02 mM, water insoluble
fraction and water fraction had the reducing antioxidant
power of 3.37 ¡Ó 0.01 mM and 1.01 ¡Ó 0.01 mM. The
hexane fraction had the lowest reducing antioxidant power
of 0.68 ¡Ó 0.01 mM (Figure 2).
Relationship between total antioxidant activity
(TeAC), total phenolics contents (TPC) and
FRAP assay
The Correlation cofficients (R
2
) of antioxidant capacity
(TEAC), total phenolics contents, and FRAP assay of
crude extract, hexane, EtOAc, n-BuOH, water soluble and
water insoluble fractions of PM were shown in Figure 3A
and 3B.
Relationship between ABTS assay (TEAC) and TPC
in crude extract, hexane, EtOAc, n-BuOH, soluble water
and water insoluble fractions were shown in Figure 3A (R
2
= 0.8145, p < 0.05). Relationship between ABTS assay
(TEAC) and FRAP assay in crude extract, hexane, EtOAc,
n-BuOH, soluble water and insoluble water fractions were
shown in Figure 3B (R
2
= 0.7878, p < 0.05).
The correlation coefficients (R
2
) values of TEAC
and total phenolics contents showed higher correlation.
The higher the total phenolics contents, the higher the
antioxidant activity the fractions exhibited.
Rapid screening of antioxidant by dot-blot and
DPPh staining
Antioxidant capacity of the PM was eye-detected semi-
quantitatively by a rapid DPPH staining TLC method.
Each diluted crude extract and hexane, EtOAc, n-BuOH,
water soluble and water insoluble fractions were applied
as a dot on a TLC layer that was then stained with DPPH
solution (Figure 4). This method was typically based on
the inhibition of the accumulation of oxidized products.
The generation of free radicals was inhibited by the
addition of antioxidants and scavenging of the free radicals
shifted the end point. The reduced glutathione was used
as a positive control. Initial faint spots appeared, and 1 h
later weak spots could be observed in sample row. This
white spots with strong intensity appeared quickly at the
concentration of 0.25 mg/ml of PM per application, and
down to the dilution at 0.125 mg/ml of the PM (Figure
4). In the DPPH staining, the n-BuOH fracton showed the
highest strong dot-blot staining.
Figure 4. Dot blot assay of the crude and various fractions of
Phellinus merrillii on a silica sheet stained with a DPPH solution
in methanol. The crude and fractions of PM (2, 1, 0.5, 0.25, and
0.125 mg/ml) were applied from left to right in sample row;
GSH (2, 1, 0.5, 0.25, and 0.125 mg/ml) were applied from left to
right in control row.
Figure 3. The antioxidant capacity of various extracts including
c rude extract, hexane, E tOAc, n-BuOH, water soluble and
water insoluble fractions of Phellinus merrillii were evaluated
acc ording to the ABTS decoloration method and the FRAP
assay. (A) correlation coefficient between TPC and ABTS assay;
(B) correlation coefficient between FRAP assay and ABTS.
Total phenolic contents (Trolox mm)
Ferric reducing ability (Trolox mm)
R
2
= 0.8145 (p < 0.05)
R
2
= 0.7878 (p < 0.05)
Crude extract
Hexane
EtOAc
n-BuOH
H
2
O soluble
H
2
O insoluble
GSH
2 1 0.5 0.25 0.125 (mg/ml)
pg_0006
412
Botanical Studies, Vol. 48, 2007
measurement of reducing power
We investigated the Fe
3+
-Fe
2+
transition to measure
the PM¡¦s reducing capacity. The reducing capacity of
a compound may serve as a significant indicator of its
potential antioxidant activity (Meir et al., 1995). The
antioxidant activities of putative antioxidants have been
attributed to various mechanisms, among which are
prevention of chain initiation, binding of transition metal
ion catalysts, decomposition of peroxides, prevention of
continued hydrogen abstraction, and radical scavenging
(Diplock, 1997). The reducing power of PM was shown in
Figure 5, with GSH as a positive control. PM exhibited a
dose dependent reducing power activity within the applied
concentrations (0, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/ml).
The reducing power of crude extract and hexane, EtOAc,
n-BuOH, water soluble, water insoluble fraction and GSH
were compared. The crude extract had the highest reducing
power at 2 mg/ml concentration. However, hexane fraction
showed lowest reducing power (Figure 5).
Scavenging activity against DPPh radical
The DPPH radical had been used widely in the
model system to investigate the scavenging activities of
several natural compounds such as phenolic compounds,
anthocyanins, or crude extracts of plants. DPPH radical
was scavenged by antioxidants through the donation
of hydrogen, forming the reduced DPPH-H
¡E
. The color
changed from purple to yellow after reduction, which can
be quantified by its decrease of absorbance at wavelength
517 nm. Table 1 showed the radical-scavenging activity of
the different fractions of PM and GSH, using the DPPH
coloring method. It was found in 0.5 mg/ml of EtOAc
fraction of PM had the highest radical-scavenging activity
(IC
50
= 0.66 ¡Ó 0.01 mg/ml), followed by n-BuOH fraction
(0.78 ¡Ó 0.01 mg/ml), crude extract (0.81 ¡Ó 0.02 mg/ml),
water fraction (0.83 ¡Ó 0.05 mg/ml) and water insoluble
fraction (0.93 ¡Ó 0.06 mg/ml). Hexane fraction had the
lowest radical-scavenging activity (IC
50
= 3.79 ¡Ó 0.01 mg/
ml).
Compositional analysis of hispolon and Pm by
LC/mS/mS
In high-performance liquid chromatography tandem
mass (LC/MS/MS) analysis for hispolon, the daughter
ion scanned chromatograms of PM was established. Both
hispolon and PM showed similar daughter ion spectrum
at the retention time of 4.7 min and had more lobes in m/z
219 and m/z 135 (Figure 6). This indicated that PM did
contain the active ingredient of hispolon.
Determination of the antioxidative activity of
hispolon
We used hispolon to measure antioxidant activity and
BHT was used as positive control. Both IC
50
values of
hispolon and BHT were 42.4 ¡Ó 2.9 and 81.2 ¡Ó 3.2 mM,
respectively, when scavenging activity of DPPH radicals
(%) was measured. These results demonstrated that
hispolon exhibited better antioxidative activity than BHT
(Table 2).
DISCUSSION
Mushrooms are nutritionally functional foods and
important sources of physiologically beneficial medicines.
They produce various classes of secondary metabolites
with interesting biological activities and, thus, have the
potential to be used as valuable chemical resources for
drug discovery (Zjawiony, 2004). Several mushrooms
belonging to the genera Inonotus and Phellinus, such
Table 1. DPPH radical scavenging activity of various fractions
of Phellinus merrillii.
DPPH radical scavenging activity IC
50
(mg/ml)
GSH
0.42 ¡Ó 0.04
H
2
O
0.83 ¡Ó 0.05
Crude
0.81 ¡Ó 0.02
Hexane
3.79 ¡Ó 0.01
EtOAc
0.66 ¡Ó 0.01
n-BuOH
0.78 ¡Ó 0.01
H
2
O Insoluble
0.93 ¡Ó 0.06
Table 2. Antioxidant activity of hispolon.
Scavenging activity of DPPH radicals (%),
IC
50
(£gM)
BHT (control)
81.2 ¡Ó 3.2
Hispolon
42.4 ¡Ó 2.9
Figure 5. Antioxidative activities of the crude extract and
various fractions of Phellinus merrillii (0, 0.125, 0.25, 0.5,
1.0, and 2.0 mg/ml) we re m easured by the reducing power
method. GSH was used as a positive control. Each absorbance
value represented the average of triplicates of different samples
analyzed.
GSH
Crude
Hexane
Et0Ac
n-BuOH
H
2
O
H
2
O Insoluble
Concentration (mg/ml)
pg_0007
CHANG et al. ¡X Free radical scavenging activity of
Phellinus merrillii
413
as Inonotus obliquua, Phellinus linteus, Phellinus ribis
and Phellinus igniarius have been used as traditional
medicines for the treatment of gastrointestinal cancer,
cardiovascular disease, tuberculosis, liver or heart diseases,
fester, bellyache, bloody gonorrhea, stomach ailments,
and diabetes (Nakamura et al., 2004). Polysaccharides,
especially £]-glucan, are considered to be responsible for
their biological activity. The isolation of polysaccharides
derived from medicinal mushrooms and their biological
activity had been reported (Vinogradov and Wasser, 2005).
Interestingly, these mushrooms often contain a bundle
of yellow antioxidant pigments which are composed of
hispidin derivatives and polyphenols.
The antioxidant activity of mushroom extracts with
stronger inhibition of lipid peroxidation occurring at high
concentrations of the extracts in most cases (Cheung and
Peter, 2005). The possible mechanism of antioxidant
activity of mushroom extracts includes scavenging of free
radicals possibly through hydrogen-holding capacity and
oxidation by peroxy radicals (Ali et al., 1996b).
In this paper, the results from in vitro experiments
demonstrated that crude extract, n-BuOH and EtOAc
fractions had better antioxidant activity in reducing power
method. The crude extract had the highest reducing power
at 2 mg/ml concentration. In the DPPH staining, the
EtOAc and n-BuOH fractions shared the same patterns
with the crude extract and appeared as white spots when
they were diluted from 2.0 to 0.125 mg/ml. In the DPPH
staining, the n-BuOH fraction showed the highest strong
dot-blot staining. In the DPPH colorimetric method, it was
found that EtOAc fraction of PM had the highest radical-
scavenging activities. In the ABTS free radical scavenging
assay, the n-BuOH fraction displayed the highest total
antioxidant activity. Total phenolics contents demonstrated
that the EtOAc fraction had the highest amounts of total
phenolic contents than other fractions. In ferric reducing
antioxidant power assay (FRAP), the EtOAc fraction
exhibited the highest reducing power. ABTS with high
correlation were established between total phenolics
contents and total antioxidant activity (TEAC) assay. This
data indicated that the correlation coefficients (R
2
) values
of TEAC and total phenolics contents showed higher
correlation. The higher the total phenolics contents the
higher the antioxidant activity of the fractions. The ex vivo
or in vivo antioxidant activity of PM should be studied in
the near future.
In LC/MS/MS analysis, the finger print chromatogram
of PM was established. Both PM and hispolon showed
similar peak at the retention time of 4.7 min and had more
lobes in m/z 219 and m/z 135. This implied that PM did
contain the active ingredient hispolon. Hispolon was better
than BHT in the IC
50
of DPPH radical scavenging activity.
These findings imply that hispolon was most important in
antiradical activities. It was suggested that hispolon might
contribute to the antioxidant activities of PM.
The relatively higher contents of phenolics in PM might
explain the high antioxidant property (Bimla and Punita,
2006). Positive correlations were established between
total phenolics contents in the mushroom extract and their
antioxidant activities, similar to those observed in Lentinus
edodes and Volvariella volvacea (Cheung et al., 2003).
Polyphenolic compounds have an important role
in stabilizing lipid oxidation and are associated with
antioxidant activity (Yen et al., 1993). The phenolic
compounds may contribute directly to antioxidative
action (Duh et al., 1999). It is suggested that polyphenolic
compounds may exhibit inhibitory effects on mutagenesis
and carcinogenesis in humans, when up to 1.0 g is
ingested daily from a diet rich in fruits and vegetables
sources (Tanaka et al., 1998). The antioxidative activities
could be ascribed to the different mechanisms exerted by
different phenolic compounds and due to the synergistic
effects of different compounds. The antioxidants present
in six different fractions may have different functional
properties, such as reactive oxygen species scavenging
(quercetin and catechin) (Hatano et al., 1989), inhibition of
the generation of free radicals and chain-breaking activity,
Figure 6. Comparison of daughter ion scanned (ES-D219.8)
chromatograms (A & B) of hispolon standard vs. sample and
each of daughter ion spectrum (C & D), it indicated that sample
contains hispolon.
pg_0008
414
Botanical Studies, Vol. 48, 2007
e.g. p-coumaric acids (Laranjinha et al., 1995) and metal
chelation (Van-Acker et al., 1998). These compounds
were normally phenolic compounds, which were effective
hydrogen donors, such as tocopherols, flavonoids, and
derivatives of cinnamic acid, phosphatidic and other
organic acids.
Although ethanol extract of Phellinus linteus was
shown to scavenge directly the stable DPPH radical
over a concentration range of 10 £gg/ml (30.1 ¡Ó 2.72%
inhibition) to 300 £gg/ml (85.5 ¡Ó 4.2% inhibition). I t
scavenged the stable radical DPPH in a concentraction-
dependent manner. The EC
50
value of Phellinus linteus
was calculated to be 22.07 £gg/ml, whereas that of Vitamin
C, used as a positive control, was 5.11 £gg/ml (Song et al.,
2003). They demonstrated that ethyl acetate extract of P.
rimosus exhibited significant in vitro antioxidant activity.
The ethyl acetate extract of P. rimosus also showed potent
anthepatotoxic activity against CCl
4
-induced acute toxicity
in rat liver (Ajith and Janardhanan, 2002). Our previous
studies also showed the similar results.
In conclusion, PM possessed antioxidant and free
radical scavenging activities. Above all, there are many
total phenolic compounds in PM including hispolon.
PM might be an agent with anti-cancer, hepatoprotective
and ant-inflammatory potentials and should be further
investigated in the future.
Acknowledgement. The authors gratefully appreciated
the support by Dr. Yu-Cheng Dai from Institute of
Applied Ecology, Chinese Academy of Science, China.
Dr. Dai helped us identifying the authenticity of PM.
The authors also like to thank China Medical University
and Committee of Chinese Medicine and Pharmacy,
Department of Health, Executive Yuan, TAIWAN for the
grant support (CMU94-020; CCMP93-RD-037).
LITeRATURe CITeD
Ada, M.A., H.K. Won g, and B. J iang. 200 5. F i brinolyt ic
enzymes in Asian traditional fermented foods. Food Res.
Int. 38: 243-250.
Adams, A.K. and E.O. Wermuth. 1999. Antioxidant vitamins
and the prevention of coronary heart disease. Am. F am.
Physician 60: 895-905.
Ajith, T.A. a nd K.K. Jana rdha nan. 2002. Anti oxidant and
antihepatotoxic activities of Phellinus rimosus (Berk) Pilat.
J. Ethnopharmacol. 81: 387-391.
Ali, N.A., J. Ludtke, H. Pilgrim, and U. Lindequist. 1996a.
In hibit ion of che mil umi nes cen ce re spons e of hum an
m ononuclear cells and suppres sion of mitogen-induced
proliferation of spleen lymphocytes of mice by hispolon and
hispidin. Pharmazie 51: 667-670.
Ali, N.A.A., H. P ilgrim, K. Liberra, U. Lindequist, and R.
Jansen. 1996b. Hispolon, a yellow pigment from Inonotus
hispidus. Phytochemistry 41: 927-929.
Aruoma, O.I. 1998. Free radicals, oxidative stress, and
antioxidants in human health and disease. J. Am. Oil Chem.
Soc. 75: 199-212.
Awadh, A.N.A., R.A.A. Mothana, A. Lesnau, H. Pilgrim, and U.
Lindequist. 2003. Antiviral activity of Inonotus hispidus.
Fitoterapia 74: 483-485.
Bae, J.S., K.H. Jang, H. Yim, and H.K. Jin. 2005.
Polysa ccharides isolate d from Phellinus gilv us inhibit
melanoma growth in mice. Cancer Lett. 218: 43-52.
Benzie, I.F.F. and J.J. Strain. 1996 The ferric reducing ability of
plasma (FRAP) as a measure of "antioxidant power": the
FRAP assay. Anal. Biochem. 239: 70-76.
Bimla, N. and B. Punita. 2006. Aluminium-induced imbalance
in oxidant and antioxidant determinants in brain regions of
female rats: protection by centrophenoxine. Toxicol. Mech.
Methods 16: 21-25.
Burns, J., P.T. Gardner, D. Matthews, G.G. Duthie, M.E. Lean,
and A. Crozier. 2001. Extraction of phenolics and changes
in antioxidant activity of red wines during vinification. J.
Agric. Food Chem. 49: 5797-5808.
Chen, X., Z. P. Hu, X.X. Yang, M. Huang, Y. Gao, W. Tang,
S.Y. Chan, X. Dai, J. Ye, C.L.P. Ho, W. Duan, H.Y. Yang,
Y.Z. Zhu, and S.F. Zhou. 2006a. Monitoring of immune
responses to a herbal immuno-modulator in patients with
advanced colorecta l cancer. Int. Imm unopha rmac ol. 6:
499-508.
Chen, W., F.Y. He, and Y.Q. Li. 2006b. The apoptosis effect of
hispolon from Phellinus linteus (Berkeley & Curtis) Teng
on human epidermoid KB cells. J. Ethnopharmacol. 105:
280-285.
Chung, K.S., W.C. Lee, and J.M. Sung. 1998. The antioxidant
effect of the basidiocarps of Phellinus spp. J. Agric. Sci. 40:
51-56.
Cheung, L.M. and C.K. Peter. 2005 Mushroom extracts with
antioxidant activity against lipid peroxidation. Food Chem.
89: 403-409.
Cheung, L.M., P.C.K. Cheung, and V.E.C. Ooi. 2003.
Antioxidant activity and total phenolics of edible mushroom
extracts. Food Chem. 81: 249-255.
Diaz, M.N., B. Frei, J.A. Vita, and J.F. Keaney. 1997.
Antioxidants and atherosclerotic heart disease. N. Engl. J.
Med. 337: 408-416.
Diplock, A.T. 1997. Will the ¡¥good fairies¡¦ please proves to us
that vitamin E lessens human degenerative of disease.. Free
Radic. Res. 27: 511-532.
Duh, P.D., Y.Y. Tu, and G.C. Yen. 1999. Antioxidant activity of
water extract of Harng Jyur (Chyrsanthemum morifolium
Ramat). LWT-Food Sci. Technol. 32: 269-277.
Espin, J.C., C. Soler-Rivas, H.J. Wichers, and C. Viguera-Garcia.
2000. Anthocyanin-based natural colorants: a new source of
antiradical activity for foodstuff. J. Agric. Food Chem. 48:
1588-1592.
Hatano, T., R. Edamatsu, M. Hiramatsu, A. Moti, Y. Fujita, T.
Yasuhara, T. Yoshida, and T. Okuda. 1989. Effects of tannins
and related polyphenols on superoxide anion radical, and on
pg_0009
CHANG et al. ¡X Free radical scavenging activity of
Phellinus merrillii
415
1,1-diphenyl-2-picrylhydrazyl radical. Chem. Pharm. Bull.
37: 2016-2021.
Hou W.C., W.C. Wu, C.Y. Yang, H.J. Chen, S .Y. L iu, and
Y.H. Lin. 2004. Antioxidant activities of methanolic and
hot-water extracts from leaves of three cultivars of Mai-
Men-Dong (Liriope spicata L.). Bot. Bull. Acad. Sin. 45:
285-290.
Hu, C. and D.D. Kitts. 2000. Studies on the antioxidant activity
of Echinaceae root extract. J . Agric . F ood Chem. 48:
1466-1472.
Huang, D.J ., C.D. L in, H.J. Chen, W.C. Hou, and Y.H. Lin.
2004. Acti ve recom binant t hioredoxin h prote in with
antioxidant activities from sweet potato (Ipomoea batatas
[L.] Lam ¡¥Tainong 57¡¦) storage roots. J. Agric. Food Chem.
52: 4720-4724.
Huang D.J., C.D. Lin, H.J. Chen, and Y.H. Lin. 2004.
Antioxidant and antiproliferative activities of sweet potato
(Ipomoea batatas [L.] Lam ¡¥Tainong 57¡¦) constituents. Bot.
Bull. Acad. Sin. 45: 179-186.
Huang, D.J., H.J. Chen, C.D. Lin, and Y.H. Lin. 2005.
Antioxidant and antiproliferative activities of water spinach
(Ipomoea aquatica Forsk) constituents. Bot. Bull. Acad.
Sin. 46: 99-106.
Huang, G.J., H.J. Chen, Y.S. Chang, M.J. Sheu, and Y.H. Lin.
2007. Recombinant sporamin and its synthesized peptides
with antioxidant activities in vitro. Bot. Stud. 48: 133-140.
J ong, S.C. and J.M. Birmingham. 1992. Edible mushrooms
i n b io te ch nol og y. P roc e ed in gs of As i a n My co lo gy
Symposium. Seoul, pp. 18-35.
Kim, G.Y., Y.H. Oh, and Y.M. Park. 2003. Acidic polysaccharide
isolated from Phellinus linteus induces nitric oxide-
mediated tumoricidal activity of m acrophages through
protein tyros ine kinas e and protein kinase C. Biochem.
Biophys. Res. Commun. 309: 399-407.
Kim, S.H., H.S. Lee, S. Lee, J. Cho, K. Ze, J. Sung, and Y.C.
Kim. 2004. Mycelial culture of Phellinus linteus protects
primary cultured rat hepatocytes agains t hepatotoxins. J.
Ethnopharmacol. 95: 367-372.
Lai, K.C., Y.C. Liu, M.C. Tseng, and J.H. Lin. 2006. Isolation
and identification of a sildenafil analogue illegally added in
dietary supplements. J. Food Drug Anal. 14: 19-23.
Laranjinha, J., O. Vieira, V. Madeira, and L. Almeida. 1995.
Two related phenolic antioxidants with opposite effects on
vitamin E content in low density lipoproteins oxidized by
ferrylmyoglobin: consumption versus regeneration. Arch.
Biochem. Biophys. 323: 373-381.
Li, G., D.H. Kim, T.D. Kim, B.J. Park, H.D. Park, J.I. Park, M.K.
Na, H.C. Kim, N.D. Hong , K. Lim, B.D. Hwang, and W.H.
Yoon. 2004. Protein-bound polysaccharide from Phellinus
linteus induces G
2
/M phase arrest and apoptosis in SW480
human colon cancer cells. Cancer Lett. 216: 175-181.
Li, L., T.B. Ng, and L. Zhao. 2005. Antioxidant activity with
content of phenolics in extracts from the culinary-medicinal
abalone mushroom Pleurotus abalones, Chen et Chang
(Agaricomycetideae). Int. J. Med. Mushrooms 7: 237-242.
Lin, C.C. and P.C. Huang. 2002. Antioxidant and
hepatoprotective effects of Acathopanax senticosus.
Phytother. Res. 14: 489-494.
Lin, S .B., C.H. Li, S .S. Lee, and L.S. Kan. 2003. Triterpene-
enriched extracts from Ganoderma lucidum inhibit growth
o f hepa tom a c el ls via su ppres s ing p rote in ki nas e C ,
activating mitogen-activated protein kinases and G2-phase
cell cycle arrest. Life Sci. 72: 2381-2390.
Lin, S. Y., H. Y. Liu, Y. L. Lu, and W. C. Hou. 2005. Antioxidant
a ctivitie s of muc ilages from different Taiwanes e yam
cultivars. Bot. Bull. Acad. Sin. 46: 183-188.
Mallavadhani, U., A. Sudhaka r, K.V.S . Sathya narayana, A.
Mahapatra, W. Li, and B. Richard. 2006. Chemical and
a na lyti cal screening of some edible mushroom s. F ood
Chem. 95: 58-64.
Mau, J.L., S.Y. Tsai, Y.H. Tseng, and S.J. Huang. 2005.
A nt io xi da nt p rop er ti e s of ho t wat e r e x tra c ts fro m
Ganoderma tsugae Murrill. LWT-Food Sci. Technol. 38:
589-587.
Meir, S., J. Kanner, B. Akiri, and S.P. Hadas. 1995.
De term inat ion and invol vem ent of aque ous re ducing
c om pounds i n oxi dat ive defe ns e s ys te ms of va rio us
senescing leaves. J. Agric. Food Chem. 43: 1813-1817.
Mo, S ., S. Wang, G. Zhou, Y. Yang, Y. Li, X. Chen, and J.
Shi. 2004. Phelligridins C-F: cytotoxic pyrano[4,3-
c ][2]benzopyran-1,6-dione and furo[3,2-c]pyran-4-one
derivatives from the fungus Phellinus igniarius. J. Nat.
Prod. 67: 823-828.
Nakamura, T., S. Matsugo, Y. Uzuka, S. Matsuo, and H.
Kawagishi. 2004. Fractionation and anti-tumor activity of
the mycelia of liquid-cultured Phellinus linteus. Biosci.
Biotechnol. Biochem. 68: 868-872.
Ragazzi, E. and G. Veronese. 1973. Quantitative analys is of
phenolics compounds after thin-layer chromatographic
separation. J. Chromatogr. A. 77: 369-375.
Re, R., N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, and
C. Rice-Evans . 1999. Antioxidant ac tivity applying an
improved ABTS radical cation decolourization assay. Free
Radic. Biol. Med. 26: 1231-1237.
Repetto, M.G. and S.F. Llesuy. 2002. Antioxidant properties of
natural compounds used in popular medicine for gastric
ulcers. Braz. J. Med. Biol. Res. 35: 523-534.
Rice-Evans, C.A., N.J. Miller, and G. Paganga. 1997.
Antioxidant properties of phenolic compounds. Trends Plant
Sci. 2: 152-159.
Smith, M.A., G. Perry, P.L. Richey, L.M. Sayre, V.E. Anderson,
and M.F. Beal. 1996. Oxidative damage in Alzheimer¡¦s.
Nature 382: 120-121.
Sohn, Y.H. and K.S. Nam. 2001. Antimutagenicity and induction
of anticarcinogenic phase II enzymes by basidiomycetes. J.
Ethnopharmacol. 77: 103-109.
Soler-Rivas, C., J.C. Espin, and H.J. Wichers. 2000. An easy and
fast test to compare total free radical scavenger capacity of
foodstuffs. Phytochem. Anal. 11: 330-338.
pg_0010
416
Botanical Studies, Vol. 48, 2007
Song, Y.S., S.H. Kim, J.H. Sa, C. Jin, C.J. Lim, and E.H. Park.
2003. Anti-angiogenlc, antioxidant and xanthine oxidase
inhibition activities of the mushroom Phellinus linteus. J.
Ethnopharmacol. 88: 113-116.
Tanaka, M., C.W. Kuei, Y. Nagashima, and T. Taguchi. 1998.
Application of antioxidative maillrad reaction products
from histidine and glucose to sardine products. Nippon
Suisan Gakkai Shi 54: 1409-1414.
Tong, L.M., S. Sas aki, D.J. McClements , and E.A. Decker.
2000. Mechanis ms of the antioxidant activity of a high
molecular weight fraction of whey. J. Agric. Food Chem.
48: 1473-1478.
Van-Acker, S.A.B.E., G.P. Van-Balen, D.J. van-den-Berg, A.
Bast, and W.J.F. van-der-Vijgh. 1998. Influence of iron
chelation on the antioxidant activity of flavonoids. Biochem.
Pharmacol. 56: 935-943.
Vinogradov, E. and S.P. Wasser. 2005 The structure of a
pol ysa ccharide isol ated from Inonot us le vis P. Ka rst.
mushroom (Heterobasidiomycetes). Carbohydr. Res. 340:
2821-2825.
Wass er, S.P. and A.L . Weis. 1999. Medicinal properties of
substances occurring in higher basidiomycetes mushrooms.
Int. J. Med. Mushrooms. 1: 31-62.
Yamaguchi, T., H. Takamura, T. Matoba, and J. Terao. 1998.
HPLC method for evaluation of the free radical-scavenging
activity of foods by using 1, 1-diphenyl-2-picrylhydrazyl.
Biosci. Biotechnol. Biochem. 62: 1201-1204.
Yen, G.C. and H.Y. Chen. 1995. Antioxidant activity of various
tea extracts in relation to their antimutagenicity. J. Agric.
Food Chem. 46: 849-854.
Yen, G.C. and D.Y. Chuang. 2000. Antioxidant properties of
water extracts from Cassia tora L. in relation to the degree
of roasting. J. Agric. Food Chem. 48: 2760-2765.
Yen, G.C., P.D. Duh, and C.L. Tsai. 1993. Relationship between
antioxidant activity and maturity of peanut hulls. J. Agric.
Food Chem. 41: 67-70.
Zjawiony, J.K. 2004. Biologically Active Compounds from
Aphyllophorales (Polypore) Fungi. J. Nat. Prod. 67:
300-310.
pg_0011
CHANG et al. ¡X Free radical scavenging activity of
Phellinus merrillii
417
pg_0012