Life Sciences 71 (2002) 469 – 482

www.elsevier.com/locate/lifescie

Protection of oxidative damage by aqueous extract from

Antrodia camphorata mycelia in normal human erythrocytes
You-Cheng Hseu a,1, Weng-Cheng Chang b,1, Yi-Ting Hseu c, Chia-Ying Lee c,
Yi-Jen Yech c, Pei-Chun Chen c, Jing-Yi Chen c, Hsin-Ling Yang c,*
a
Department of Medical Technology, Fooyin Institute of Technology, Kaohssiung, Taiwan
b
Department of Physiology, School of Medicine, China Medical College, Taichung, Taiwan
c
Department of Nutrition, China Medical College, 91 Hsueh Shih Road, Taichung 40421, Taiwan
Received 11 April 2001; accepted 29 January 2002

Abstract

Antrodia camphorata (A. camphorata) is well known in Taiwan as a traditional Chinese medicine. The purpose
of this study was to evaluate the ability of aqueous extract from A. camphorata mycelia to protect normal human
erythrocytes against oxidative damage in vitro. Oxidative hemolysis and lipid/protein peroxidation of erythrocytes
induced by the aqueous peroxyl radical [2,2V-Azobis(2-amidinopropane) dihydrochloride, AAPH] were suppressed
by A. camphorata mycelia in a time-and concentration-dependent manner. A. camphorata mycelia also prevented
the depletion of cytosolic antioxidant glutathione (GSH) and ATP in erythrocytes. Moreover, cultured human
endothelial cell damage induced by AAPH was suppressed by A. camphorata mycelia. Interestingly, A. camphorata
mycelia exhibited significant cytotoxicity against leukemia HL-60 cells but not against cultured human endothelial
cells. These results imply that A. camphorata mycelia may have protective antioxidant and anticancer properties.
D 2002 Elsevier Science Inc. All rights reserved.

Keywords: Erythrocyte; Antrodia Camphorata mycelia; Antioxidants; Hemolysis; Glutathione; ATP; Endothelial cells;
HL-60 cells

Introduction

A new basidiomycete Antrodia camphorata (A. camphorata) in the Polyporaceae (Aphyllophorales),

which causes brown heart rot of Cinnamomun kanehirai (Hay) (Lauraceae) in Taiwan, was identified as

*
Corresponding author. Tel.: +886-4-2205-3366x3366; fax: +886-4-22062891.
E-mail address: hlyang@mail.cmc.edu.tw. (H.-L. Yang).
1
These authors contributed equally to the study.

0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 6 8 6 - 7
470 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482

a new genus of the Antrodia species [1–3]. A. camphorata is rare and expensive since it grows only on
the inner heart wood wall of the endemic evergreen Cinnamonum kanehirai and cannot be cultivated. It
has been used in traditional Chinese medicine for the treatment of food and drug intoxication, diarrhea,
abdominal pain, hypertension, itchy skin, and liver cancer [4], but very few biological activity tests have
been reported.
Increasing evidence suggests that oxidative damage to cell components may have an important
pathophysiological role in several types of human diseases [5,6]. Reactive oxygen species (ROS)
have been implicated in damages of erythrocytes in patients with h-thalassemia, sickle cell anemia,
glucose-6-phosphate dehydrogenase deficiency, and other hemoglobinopathies [7–9]. Erythrocytes are
highly susceptible to oxidative damage as a result of high polyunsaturated fatty acid (PUFA) content
of their membranes and the high cellular concentrations of oxygen and hemoglobin (Hb), a
potentially powerful promoter of oxidative processes [10,11]. Lipid peroxidation is one of the
consequences of oxidative damage and has been suggested as a general mechanism for cell injury
and death (i.e., hemolysis) [12]. Malondialdehyde (MDA), the end product of lipid peroxidation of
erythrocytes, is a highly reactive and bifunctional molecule. It has been shown to cross-link
erythrocyte phospholipids and proteins to impair a variety of the membrane-related functions, which
ultimately lead to diminished erythrocyte survival (hemolysis) [13–16]. Erythrocyte lipid peroxida-
tion may be involved in pathological conditions associated with congenital and acquired defects
which impair the antioxidant protective systems, or with strong oxidative insults which overwhelm
the defense systems [5,6,10]. Oxidants also produce alterations in erythrocyte membranes as
manifested by a decreased cytoskeletal protein content, and production of high-molecular-weight
proteins [17,18], which can lead to abnormalities in erythrocyte shape and disturbances in the micro-
circulation [19].
A. Camphorata is currently popular medicinal mushrooms in Taiwan. Mushroom was reported to
possess antitumour and immunomodulating activities [20,21]. Some chinese herbal are also reported to
exhibit strong antioxidant activity [22,23]. However, little is available about the biological activities of
A. Camphorata. In our research, we used the wild picked air-dried mycelia harvested from submerged
cultures as samples to study the antioxidant properties of aqueous extract from A camphorata due to the
interesting biological activities and the potential clinical applications. In this paper, A camphorata was
used for the inhibition of aqueous peroxyl radicals [2,2V-Azobis (2-amidinopropane) dihydrochloride,
AAPH]-induced oxidative hemolysis and lipid/protein peroxidation of normal human erythrocytes. We
also tested the effects of A. camphorata mycelia on the growth and viability of cultured human
endothelial cells and leukemia HL-60 cells.

Methods

Chemicals

The following reagents were obtained from Sigma Chemical Co. (St Louis, MO): sodium chloride
(NaCl), sodium phosphate dibasic (anhydrous) (Na2HPO4), bovine serum albumin (BSA), 2-thiobar-
bituric acid (TBA), ascorbic acid, 5,5V-dithio-bis 2-nitrobenzoic acid (DTNB), ethylenediaminetetra-
acetic acid (EDTA), glutathione (GSH), ATP kit. 2,2V-Azobis (2-amidinoporpane) dihydrochloride
(AAPH), and phosphoric acid (H3PO4). Trichloroacetic acid was purchased from Wako Pure Che-
 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482 471

mical Ind. (Osaka, Japan). Ammonium persulfate, bisacrylamide (30%), coomassie brilliant blue R-
250, sodium dodecyl sulfate (SDS). N.N.NV.NV-tetramethylethylenediamine (TEMED), dye reagent
concentrate were obtained from the Bio-Rad Co. (Hercules, CA). Fetal bovine serum (FBS), medium
M-199, RPMI-1640, glutamine, penicillin-streptomycin and penicillin-streptomycin-neomycin (PSN)
were obtained from GIBCO Laboratories (Gaithersburg, MD). All other reagents were of reagent
grade quality, and were supplied either by Sigma or Merck Chemical Companies (Darmstadt,
Germany).

Sample preparation

Fresh air-dried A. camphorata mycelia were obtained from the Biotechnology Center, Grape King
Inc., Chungli, Taiwan. Mycelia were filtered through Whatman #1 paper with water three times before
being air-dried. For the preparation of the aqueous extracts, all air-dried mycelia samples were ground
and then shaken with isotonic phosphate saline buffer (PBS) [154 mM NaCl and 10 mM phosphate
buffer at pH 7.4] at the ratio of 1:25 (w/v) at 25 jC for 10 h, and then centrifuged at 3000 g for 10 min,
followed by passing through a 0.2 Am pore size filter. The stock solution was stored at 20 jC before
analysis of the antioxidant properties. The experiments were done using 3f5 different batches of
aqueous extract of A. camphorata mycelia.

Preparation of erythrocyte suspensions

Blood (10–15 ml) was obtained from 10 healthy volunteers (4 female and 6 male college students;
aged 19 to 22 years) via venapuncture after obtaining informed consent. Human erythrocytes from fresh
citrated-blood were isolated by centrifugation at 1500 g for 10 min. Erythrocytes were then washed four
times with PBS, then re-suspended using the same buffer to the desired hematocrit level. Cells were
stored at 4 jC and used within 6 h of sample preparation. In order to induce the free radical chain
oxidation in erythrocytes, the aqueous peroxyl radicals were generated by thermal decomposition of
AAPH (an azo compound) in oxygen. The advantages of this method were that the AAPH decomposes
thermally to generate radicals without biotransformations or enzymes and the rate of radical generation
was easily controlled by adjusting the concentration of initiator [24,25]. Erythrocyte suspension at 5%
hematocrit was incubated with PBS (control), and preincubated with 1:25 (w/v) aqueous extract from A.
camphorata mycelia at the indicated volume for 30 min. Then, it was incubated with and without 25 mM
AAPH (in PBS at pH 7.4). This reaction mixture was shaken gently while being incubated for the
indicated time at 37 jC.

Hemolysis assay

The reaction mixture (200 Al) was removed and centrifuged at 3000 g for 2 min; absorbance of
the supernatant was determined at 540 nm. The absorbance recorded from A. camphorata was
subtracted from those of the supernatants, because the aqueous extract from A. camphorata would
have interfered with the data. Reference values were determined using the same amount of
erythrocytes in a hypotonic buffer (5 mM phosphate buffer at pH 7.4; 100% hemolysis). The per-
centage hemolysis was calculated using the ratio of the readings (absorbance of sample supernatant/
reference value)  100.
472 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482

Quantitative estimation of lipid peroxidation of erythrocytes

In vitro peroxidation was assessed using a thiobarbituric acid (TBA) reaction. A 1 ml reaction mixture,
100 Al of H3PO4 (0.44 M), and 250 Al (0.67%) thiobarbituric acid were added and incubated at 95 jC for 1
h. Following incubation, it was allowed to stand on ice for 10 min before adding 150 Al trichloroacetic
acid (20%). After centrifugation at 12 000 g for 10 min, the peroxide content in the supernatant obtained
was assayed using the TBA reaction with the molar extinction coefficient (O.D532) of malondialdehyde
(MDA). Tetraethoxypropane was used as the standard [26]. MDA values were expressed as pmole/g Hb.
An aliquot of lysate was also used for the determination of the hemoglobin content by colorimetric
method. Briefly, we added 20 Al lysate into a final volume of 5 ml in Drabkins solution. We measure the
absorbance of samples against a reagent blank at 540 nm. Hemoglobin was expressed as g/ml.

Preparation of erythrocyte ghosts and analysis of erythrocyte membrane proteins by SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of erythrocyte ghosts was
prepared from the reaction mixture using hypotonic lysis in 30 volumes of 5 mM NaH2PO4 (pH 7.4) as
previously described [27]. Hemolysate preparations were washed five times in the above buffer, then
centrifuged at 12 000 g for 30 min. Protein concentrations of the erythrocyte ghost pellets were
determined [28] using BSA, fraction V, as the standard. Erythrocyte ghost pellets were dissolved to a
concentration of 2 mg protein/ml in SDS sample buffer, and treated in the same buffer as reported above
for erythrocyte ghosts. The ghosts (2 mg/ml, 12.5Al) were brought to a total volume of 17 Al, and
incubated at 95 jC for 5 min. SDS-PAGE was carried out on a 1.5 mm-thick slab gel with 4% and 10%
gels for condensation and separations, and stained with coomassie brilliant blue [29]. The gel system was
calibrated for molecular weight determination by measuring the migration of the standard proteins. Gel
densitometry was performed by using image analysis software from PDI, Inc. (Huntington Station, NY).
The linear range of image scanner was 0–3 absorbance units.

Determination of glutathione contents in erythrocytes

Intracellular glutathione (GSH) was determined by titration with DTNB as described previously [30].
After centrifugation of the reaction mixtures (2 ml), 0.6 ml water was added to the erythrocyte pellets to
lyse the cells. Then, 0.5 ml of the lysate was precipitated by the addition of 0.5 ml metaphosphoric acid
solution [1.67 g metaphosphoric acid, 0.2 g EDTA (disodium salt) and 30 g NaCl in 100 ml water]. After
5 min, the protein precipitate was separated from the remaining solution by centrifugation at 18 000 g for
10 min. We then combined 0.45 ml of the solution with 0.45 ml of 300 mM Na2HPO4 and the
absorbance at 412 nm was read against a blank consisting of 0.45 ml solution plus 0.45 ml water. Then,
100 Al DTNB solution (20 mg DTNB in 100 ml of 1% solution citrate) was added to the blank and the
sample. The absorbance of the sample was again read against the blank at 412 nm. GSH values were
expressed as Amole/g Hb.

Measurement of ATP contents in erythrocytes

The procedure for measuring the ATP content was based on the reactions described by
Adams [31]. Briefly, we used pipettes to place 1 ml reaction mixture and 1 ml TCA (12%) into
 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482 473

a centrifuge tube, mixed well and let stand for about 5 min in an ice bath. It was centrifuged
at about 800 g for 10 min to obtain a clear supernatant. The supernatant (0.5 ml), 0.3 mg
NADH (reduced form of a-nicotinamide adenine dinucleotide), and 1.0 ml H2O were added into
1.0 ml of phosphoglyceric acid (PGA) buffer. Glyceraldehyde 3-phosphate dehydrogenase/
3-phosphoglyceric phosphokinase (GAPD/PGK) enzyme mixture (0.04 ml) was then added. After
10 min, absorbance was then taken versus water as reference at 340 nm. By determining the
decrease in absorbance at 340 nm that resulted when NADH was oxidized to a-nicotinamide
adenine dinucleotide (NAD), a measure of the amount of ATP originally present was obtained.
We used the calculations to determine blood ATP concentration and ATP was expressed as Amol/g
Hb.

Culture of human umbilical veins endothelial cells

Human umbilical vein endothelial cells (HUVECs) were kindly provided by the Department of
Obstetrics and Gynecology, China Medical College Hospital. The endothelial cells were prepared
from human umbilical veins essentially as previously described [32] and grown in M-199
containing 20% heat-inactivated fetal bovine serum, penicillin (100 IU/ml), streptomycin (100
Ag/ml), and fungizone (0.25 Ag/ml) in a 5% CO2 humidified incubator at 37 jC. After reaching
confluence, the primary cultured cells were detached using trypsin-EDTA and subcultured to in 6-
well tissue culture plates with 20% FBS at a density of 1  105 or 8  105 cells. After assay, the
cells were removed from each well with 0.25% trypsin-EDTA and counted in a hemocytometer.
All experiments were carried out using cells after only one passage and at least 4 days after
passage.

Culture of HL-60 cells

HL-60 leukemic cells, a human acute promyeloblastic leukemic cell line, were obtained from
American Type Culture Collection (Rockville, MD). These cells were grown in RPMI-1640 supple-
mented with 15% heat-inactivated FBS, 2 mM glutamine, 50 U/ml penicillin, and 50 Ag/ml streptomycin
in a 5% CO2 humidified incubator at 37 jC [33]. The cells were resupended in fresh medium at a density
of 2  105/ml cells per 6-well plate.

Cell viability

The cells were grown in growth medium and maintained without refeeding throughout the
experiments. Every 24 h for 3 days, cultures were harvested and monitored for cell numbers by
counting cell suspensions using a hemocytometer. Cell viability was assessed by the exclusion of 0.4%
trypan blue.

Statistical analysis

Data were presented as mean F SEM. All data were analyzed using analysis of variance
(ANOVA), followed by Dunnett’s test for pairwise comparison. Statistical significance was defined
as p < 0.05.
474 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482

Results

Effects of A. camphorata mycelia on AAPH-induced hemolysis in erythrocytes

When human erythrocytes were incubated in air at 37 jC as a 5% suspension in PBS, they were stable
and little hemolysis was observed for 6 h (5.6 F 0.4%) (Fig. 1). When a water-soluble radical initiator,
AAPH (final concentration 25 mM), was added to the erythrocyte suspension, it induced hemolysis in a
time-dependent manner. In this experimental condition, the onset of oxidative hemolysis occurred within
2–3 h. The addition of A. camphorata mycelia and lack of incubation with AAPH did not cause
significant hemolysis in the erythrocyte suspensions after 2 to 6 h of incubation (data not shown).
However, A. Camphorata mycelia inhibited AAPH-induced hemolysis in a concentration-dependent
manner. This inhibition was maintained for 6 h of incubation when 12.5 Al, 25 Al, and 50 Al stock
solution of 1:25 (w/v) aqueous extract from A. camphorata mycelia were used.

Effects of A. camphorata mycelia on AAPH-induced lipid peroxidation in erythrocytes

AAPH (25 mM) induced lipid peroxidation in the erythrocyte suspension reflected by the generation
of MDA (Table 1). The amount of MDA in the erythrocytes in the control group measured
approximately 11.9 F 1.2, 14.8 F 0.4, and 18.3 F 0.2 pmole/g Hb, respectively, at 2, 4, and 6 h.
AAPH caused lipid peroxidation of erythrocytes in a time-dependent manner. The MDA content was
increased to 53.5 F 1.5, 54.9 F 1.1, and 78.3 F 1.1 pmole/g Hb, respectively, at 2, 4, and 6 h after

Fig. 1. Effects of A. camphorata mycelia on the kinetic of AAPH-induced hemolysis in erythrocytes. Erythrocyte suspension at
5% hematocrit was incubated with PBS (control), or preincubated with 1:25 (w/v) aqueous extract from A. camphorata mycelia
at the indicated volume for 30 min. Then it was incubated with and without 25 mM AAPH for 6 h at 37 jC. Values are
expressed as the mean F SEM of 3 – 6 experiments. * indicates a significant difference from control group (p < 0.05). #
indicates a significant difference from AAPH group (p < 0.05).
 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482 475

Table 1
Effect of A. Camphorata Mycelia on AAPH-induced changes in MDA, GSH, and ATP content of erythrocytes
Experimental condition Time (h) MDA (pmole/g Hb) GSH (umole/g Hb) ATP (umole/g Hb)
a a
Control 2 11.9 F 1.2 4.27 F 0.39 3.56 F 0.17a
AAPH 53.5 F 1.5* 3.34 F 0.08* 2.80 F 0.15*
+ 12.5 ul A. Camphorata 47.9 F 0.8* 3.80 F 0.18a 3.34 F 0.08a
+ 25 ul A. Camphorata 37.0 F 0.6*,a 4.13 F 0.31a 4.23 F 0.12*,a
+ 50 ul A. Camphorata 4.5 F 1.5*,a 4.73 F 0.28a 4.81 F 0.20*,a
Control 4 14.8 F 0.4a 4.78 F 0.13a 2.84 F 0.01a
AAPH 54.9 F 1.1* 2.06 F 0.14* 2.03 F 0.14*
+ 12.5 ul A. Camphorata 48.2 F 2.4*,a 2.89 F 0.25*,a 2.85 F 0.25a
+ 25 ul A. Camphorata 35.1 F 1.2*,a 4.49 F 0.12a 4.01 F 0.34*,a
+ 50 ul A. Camphorata 11.7 F 1.2*,a 4.85 F 0.10a 4.76 F 0.12*,a
Control 6 18.3 F 0.2a 4.70 F 0.29a 2.54 F 0.08a
AAPH 78.2 F 1.1* 0.85 F 0.09* 1.44 F 0.21*
+ 12.5 ul A. Camphorata 67.2 F 3.3* 1.13 F 0.07*,a 2.34 F 0.00a
+ 25 ul A. Camphorata 53.3 F 1.1*,a 2.70 F 0.13*,a 2.77 F 0.12*,a
+ 50 ul A. Camphorata 48.6 F 1.5*,a 3.69 F 0.07*,a 4.07 F 0.11*,a
Erythrocyte suspension at 5% hematocrit was incubated with PBS (control), or preincubated with 1:25 (w/v) aqueous extract
from A. Camphorata mycelia at the indicated volume for 30 min. Then it was incubated with 25 mM AAPH for 2, 4, and 6 h at
37 jC. The MDA, GSH, and ATP content were measured as described in Methods. Values are expressed as the mean F SEM of
3 – 6 experiments.
a
Indicates a significant difference from AAAPH group (p < 0.05).
* Indicates a significant difference from control group (p < 0.05).

incubation with 25 mM AAPH. However, A. camphorata mycelia inhibited AAPH-induced MDA

formation in a concentration-dependent manner ( p < 0.05) (Table 1). The addition of A. camphorata
mycelia and lack of incubation with AAPH did not change the MDA value (data not shown).

Effects of A. camphorata mycelia on AAPH-induced changes in erythrocyte membrane proteins

The membrane proteins of erythrocytes are basically composed of band 1, band 2, band 3, band 4.1,
band 4.2 and other accessory proteins. After treatment of human erythrocytes with AAPH for 6 h, the
high-molecular-weight proteins (HMWP) decreased in intensity for all of the major membrane protein
bands (low-molecular-weight proteins, LMWP) (Fig. 2). A. camphorata mycelia inhibited AAPH-
induced changes in erythrocyte membrane proteins in a concentration-dependent manner. The addition
of A. camphorata mycelia and lack of incubation with AAPH did not change the erythrocyte membrane
proteins (data not shown). As shown in Table 2, densitometric analysis of LMW proteins revealed that A.
camphorata mycelia inhibited AAPH-induced changes in the amount of erythrocyte membrane proteins.

Effects of A. camphorata mycelia on AAPH-induced changes in GSH content of erythrocytes

The amount of GSH in the erythrocytes in the control group measured 4.27 F 0.40, 4.78 F 0.13, and
4.70 F 0.29 Amol/g Hb, respectively, at 2, 4, and 6 h (Table 1). In the presence of 25 mM AAPH, AAPH
caused a significant consumption of the cytosolic GSH in a time-dependent manner. The GSH content
decreased to 3.34 F 0.08, 2.06 F 0.14, and 0.85 F 0.09 Amol/g Hb, respectively, at 2, 4, and 6 h after
476 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482

Fig. 2. Effects of A. Camphorata mycelia on AAPH-induced changes in erythrocyte membrane proteins analyzed using SDS-
PAGE. Erythrocyte suspension at 5% hematocrit was incubated with PBS (control), or preincubated with 1:25 (w/v) aqueous
extract from A. camphorata mycelia at the indicated volume for 30 min. Then it was incubated with 25 mM AAPH for 6 h at
37 jC. Lane a: intact erythrocyte membrane proteins. Lane b: erythrocyte oxidized with 25 mM AAPH. Lane c, d, e:
erythrocyte preincubated with aqueous extract from A. camphorata mycelia at 12.5, 25, and 50 Al, then oxidized with 25 mM
AAPH. The amount of protein layered was 25 Ag in each case. HMWP represents high-molecular-weight proteins. This
experiment was repeated three times with similar results. For the relative change in protein bands, see Table 2.

incubation with 25 mM AAPH. The addition of A. camphorata mycelia inhibited AAPH-induced

depletion of the cytosolic GSH in a concentration-dependent manner ( p < 0.05) (Table 1). The addition
of A. camphorata mycelia and lack of incubation with AAPH did not change the GSH content (data
not shown).

Effects of A. camphorata mycelia on AAPH-induced changes in ATP content of erythrocytes

The amount of ATP in the erythrocytes in the control group measured 3.56 F 0.17, 2.84 F 0.01, and
2.54 F 0.08 Amol/g Hb, respectively, at 2, 4, and 6 h (Table 1). In the presence of 25 mM AAPH, AAPH

Table 2
Effect of A. Camphorata Mycelia on AAPH-induced relative change in erythrocyte membrane proteins by densitometric
analysis
Treatment Band 1 and 2 Band 3 Band 4.1 Band 4.2 Band 5 Band 6
+ 25 mM AAPH 77 F 6* 18 F 3* 68 F 5* 46 F 4* 33 F 5* 10 F 3*
+ 12.5 Al A. Camphorata. 85 F 8 60 F 6* 64 F 9* 50 F 8* 39 F 7* 30 F 3*
+ 25 Al A. Camphorata. 93 F 13 51 F 17* 82 F 6 54 F 9* 92 F 7 56 F 4*
+ 50 Al A. Camphorata. 105 F 6 66 F 13* 112 F 6 88 F 6 111 F 8 95 F 6
Erythrocyte suspension at 5% hematocrit was incubated with PBS (control), or preincubated with 1:25 (w/v) aqueous extract
from A. Camphorata mycelia at the indicated volume for 30 min. Then it was incubated with 25 mM AAPH for 6 h at 37 jC.
Expressed as percent of control (0 Ag) taking control as 100%. Values are means F S.E.M. of 3 experiments.
* Indicates a significant difference from control group (p < 0.05).
 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482 477

caused a significant consumption of the cytosolic ATP in a time-dependent manner. The ATP content
decreased to 2.80 F 0.15, 2.03 F 0.14, and 1.44 F 0.21 Amol/g Hb, respectively, at 2, 4, and 6 h
after incubation with 25mM AAPH. The addition of A. camphorata mycelia inhibited AAPH-induced

Fig. 3. Effects of A. camphorata mycelia on the growth of HL-60 cells (A) and HUVECs (B). Cells were incubated with PBS
(control); or preincubated with 1:25 (w/v) aqueous extract from A. camphorata mycelia at the indicated volume for various
periods of time (24 to 72 h) at 37 jC. HL-60 cells were seeded at 2  105/ml cells and HUVECs were seeded at 1  105 cells
per 6-well plate. Cultures were then harvested and cell numbers were obtained by counting cell suspensions with a
hemocytometer. Values are expressed as the mean F SEM of 3 – 6 experiments. * indicates a significant difference from control
group (p < 0.05).
478 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482

depletion of the cytosolic ATP in a concentration-dependent manner ( p < 0.05) (Table 1). In this study,
the ATP level in the presence of A. camphorata mycelia was higher than in the control cells, which may
be due to the carbohydrate content of A. camphorata mycelia, however, the reasons need to be further
characterized.

Effects of A. camphorata mycelia on the growth and viability of HL-60 cells and HUVECs

We also tested the effects of A. camphorata mycelia on the growth and viability of cultured HL-60
cells and HUVECs. The number of HL-60 cells was significantly inhibited by A. camphorata mycelia ( p
< 0.05) (Fig. 3A). After 24–72 h incubation with 50 to 150 Al stock solution of 1:25 (w/v) aqueous
extract from A. camphorata mycelia cells decreased approximately 20 and 80%, respectively. However,
the number of HUVECs was not affected by A. camphorata mycelia at these concentrations after 24–48
h of incubation (Fig. 3B). The fact that there is a decrease in HUVEC cell number at 48 h with highest
dose of A. camphorata.
In this study, exposure of HUVECs to 15 mM AAPH caused significant cell damage and loss of cell
viability (Fig. 4). After 16 h of incubation, cell viability decreased to 16 F 3% compared with the cells
with no exposure to AAPH (control). The addition of A. camphorata mycelia prior to the addition of
AAPH for 24 h protected the cells from oxidative damage and increased cell viability in a concentration-

Fig. 4. Effects of A. camphorata mycelia on cell viability of HUVECs incubated with AAPH. HUVECs were incubated with
PBS (control); or preincubated with 1:25 (w/v) aqueous extract from A. camphorata mycelia at the indicated volume for 24 h,
then incubated with 15 mM AAPH for 16 h at 37 jC. HUVECs were seeded at 8  105 cells per 6-well. Cultures were then
harvested and cell numbers were obtained by counting cell suspensions with a hemocytometer. Values are expressed as the
mean F SEM of 3 experiments. * indicates a significant difference from control group (p < 0.05). # indicates a significant
difference from AAPH group (p < 0.05).
 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482 479

dependent manner. HUVECs preincubated with 25, 50 and 100 Al stock solution of 1:25 (w/v) aqueous
extract from A. camphorata mycelia had increased cell viability of 21 F 3%, 32 F 5% and 44 F 4%,
respectively, compared with the control cells.

Discussion

Oxidative damage of erythrocyte membrane (lipid/protein) may be implicated in hemolysis

associated with some hemoglobinopathies, oxidative drugs, transition metal excess, radiation and
deficiencies in some erythrocyte antioxidant systems [25]. In this study, we first tested the efficacy of
an aqueous extract from A. camphorata mycelia as an inhibitor of AAPH induced erythrocyte
hemolysis and lipid/protein peroxidation. AAPH, a water-soluble free radical generator, was used to
stimulate the in vivo conditions of oxidative stress and the peroxyl radicals were generated by
thermal decomposition of an azo compound in oxygen [34]. Lipid peroxidation is one of the
consequences of oxidative damage and has been suggested as a general mechanism for cell injury
and death (i.e., hemolysis) [12]. The results indicated that lipid peroxidation and oxidative hemolysis
of erythrocytes induced by AAPH were suppressed by A. camphorata mycelia. Oxidants may also
decrease erythrocyte deformations leading to a decreased survival of erythrocytes and to circulatory
impairments [35,36]. Data showed that the formation of HMW proteins and the concomitant decrease
of the LMW proteins of erythrocytes challenged with AAPH were also inhibited by A. camphorata
mycelia. It is well known that AAPH-induced oxidation of erythrocyte membrane protein is
accompanied by an increase in HMW protein formation. Furthermore, the contents of LMW proteins
decreased [36]. The HMW proteins observed may have been formed by direct cross-linking and/or
interaction of the LMW proteins with oxidized lipids [37,38], which could have led to the
abnormalities in erythrocyte shape and disturbances in the microcirculation. The results supported
that A. camphorata mycelia not only suppressed radical-induced erythrocyte lipid peroxidation and
hemolysis but it also prevented protein oxidation in erythrocytes. Thus, A. camphorata mycelia may
act as radical scavengers in erythrocytes.
GSH plays an important role as an erythrocyte antioxidant on different levels. The results indicated
that A. camphorata mycelia prevented an AAPH-induced decrease in intracellular GSH content of
erythrocyte. GSH oxidation can be the result of direct radical attack but can also occur indirectly
through GSH-requiring repair processes such as the reduction of oxidized membrane protein thiol
groups. Most of the time, antiradical properties are linked to the formation of stable radicals that can
react with GSH. GSH oxidation by stable free radicals may also reflect a possible regeneration of
oxidized scavengers in its active reduced form [25]. GSH appeared to provide the primary antioxidant
defense in stored erythrocytes and their decline. This was concurrent with an increase in oxidative
modifications of membrane lipids and proteins may destabilize the membrane skeleton thereby
compromising erythrocyte survival [39]. The results supported that A. camphorata mycelia prevented
AAPH-induced erythrocyte hemolysis and lipid/protein peroxidation while it affected AAPH-caused
depletion of erythrocyte GSH.
ATP was used by the erythrocytes to maintain membrane shape, control deformations, and maintain
osmotic stability [40]. In this study, A. camphorata mycelia prevented AAPH-induced decreases in
intracellular ATP content of erythrocytes. Depletion of ATP showed the existence of a relationship
between the changes in erythrocyte shape and alterations in the submembrane skeletal network
480 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482

components, which was reported to result in decreased filterability and deformations and increased
blood viscosity [41,42]. These changes contributed to microvascular occlusion, local tissue ischemia,
and consequent tissue damage [19]. The results suggested that A. camphorata mycelia prevented
AAPH-induced erythrocyte protein peroxidation while it affected AAPH-caused depletion of eryth-
rocyte ATP.
ROS caused oxidative damage to cellular components and a loss of endothelial cell (EC) function
[43]. Changes in EC membrane function induced by ROS and lipid peroxidation appeared to play
an important role in the pathogenesis of arteriosclerosis [43,44]. The results indicated that the
addition of A. camphorata mycelia prior to the addition of AAPH protected ECs from oxidative
damage and increased cell viability. Therefore, we suggest that the antioxidant functions of A.
camphorata mycelia in the EC membrane of supplemented cells were mainly responsible for blocking
free radical damage to lipids and proteins in the cell membrane when exposed to free radicals produced
by AAPH.
In this study, an aqueous extract from A. camphorata mycelia exhibited significant cytotoxicity
against leukemia HL-60 cells but not against normal mortal ECs. Some researchers reported that a crude
CHCl3/MeOH extract from A. camphorata exhibited significant cytotoxicity against P-388 murine
leukemia cells [45]. Tumors usually exhibit abnormal and immortal cell growth. Tumor cells differ from
normal mortal cells in that they are no longer responsive to normal growth-controlling mechanisms.
Current chemotherapeutic drugs, for the most part, kill cancer cells directly while normal cells are also
seriously damaged. Trials of agents that change the biological properties of cancer cells so that they lose
one of the major characteristics, namely, the ability to divide continuously and indefinitely, have begun.
Work on tumor cell death aims to shift the balance back again, thereby removing the potential for
uncontrolled growth of tumor cells. However, mechanisms of A. camphorata treated-tumor cells need to
be further characterized.
The composition of A. camphorata mycelia contained carbohydrate (53.5%), protein (23.8%), fat
(5.8%), ash (9.7%), and fiber (7.2%) [46]. The reducing sugars were predominantly components in the
mycelia carbohydrate [46]. The antioxidant activity of A. camphorata mycelia may partially be a result
of its reducing power, because the antioxidant activity has been reported to be concomitant with the
development of the reducing power [47]. The aqueous extract of A. camphorata mycelia has strong
chelating activity on ferrous ions, which may be beneficial to its antioxidant properties [46]. Results of
some studies have indicated that compounds isolated from the crude methanol extract of A. camphorata
included ergostan-type tripenoids, a sesuiterpene, and phenyl and biphenyl derivatives etc. [48–50].
However, the possible compound(s) which may explain the antioxidant and anticancer activity of A.
camphorata mycelia need to be further characterized.
In summary, supplementation using A. camphorata mycelia reduced AAPH-induced erythrocyte
hemolysis, lipid/protein peroxidation, and cell damage. These results imply that A. camphorata mycelia
may have protective antioxidant and anticancer properties for application in food and drug products.
However, further investigation of its in vitro or in vivo activity is warranted.

Acknowledgements

This work was supported by grants NSC-89-2314-B-039-041 and CMC-89-NT-01 from the National
Science Council and China Medical College of the Republic of China.
 Y.-C. Hseu et al. / Life Sciences 71 (2002) 469–482 481

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