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 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 b
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:
[email protected]. (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
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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 peroxidation 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 microcirculation [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-thiobarbituric acid (TBA), ascorbic acid, 5,5V-dithio-bis 2-nitrobenzoic acid (DTNB), ethylenediaminetetraacetic 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-
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mical Ind. (Osaka, Japan). Ammonium persulfate, bisacrylamide (30%), coomassie brilliant blue R250, 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 percentage hemolysis was calculated using the ratio of the readings (absorbance of sample supernatant/ reference value) 100.
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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
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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 6well 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 supplemented 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.
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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).
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Table 1 Effect of A. Camphorata Mycelia on AAPH-induced changes in MDA, GSH, and ATP content of erythrocytes Experimental condition Control AAPH + 12.5 ul A. Camphorata + 25 ul A. Camphorata + 50 ul A. Camphorata Control AAPH + 12.5 ul A. Camphorata + 25 ul A. Camphorata + 50 ul A. Camphorata Control AAPH + 12.5 ul A. Camphorata + 25 ul A. Camphorata + 50 ul A. Camphorata
Time (h) 2
4
6
MDA (pmole/g Hb) a
11.9 F 1.2 53.5 F 1.5* 47.9 F 0.8* 37.0 F 0.6*,a 4.5 F 1.5*,a 14.8 F 0.4a 54.9 F 1.1* 48.2 F 2.4*,a 35.1 F 1.2*,a 11.7 F 1.2*,a 18.3 F 0.2a 78.2 F 1.1* 67.2 F 3.3* 53.3 F 1.1*,a 48.6 F 1.5*,a
GSH (umole/g Hb) 4.27 3.34 3.80 4.13 4.73 4.78 2.06 2.89 4.49 4.85 4.70 0.85 1.13 2.70 3.69
F F F F F F F F F F F F F F F
a
0.39 0.08* 0.18a 0.31a 0.28a 0.13a 0.14* 0.25*,a 0.12a 0.10a 0.29a 0.09* 0.07*,a 0.13*,a 0.07*,a
ATP (umole/g Hb) 3.56 2.80 3.34 4.23 4.81 2.84 2.03 2.85 4.01 4.76 2.54 1.44 2.34 2.77 4.07
F F F F F F F F F F F F F F F
0.17a 0.15* 0.08a 0.12*,a 0.20*,a 0.01a 0.14* 0.25a 0.34*,a 0.12*,a 0.08a 0.21* 0.00a 0.12*,a 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 AAPHinduced 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
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Fig. 2. Effects of A. Camphorata mycelia on AAPH-induced changes in erythrocyte membrane proteins analyzed using SDSPAGE. 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
+ + + +
77 F 6* 85 F 8 93 F 13 105 F 6
18 60 51 66
68 64 82 112
46 50 54 88
33 39 92 111
10 30 56 95
25 mM AAPH 12.5 Al A. Camphorata. 25 Al A. Camphorata. 50 Al A. Camphorata.
F F F F
3* 6* 17* 13*
F F F F
5* 9* 6 6
F F F F
4* 8* 9* 6
F F F F
5* 7* 7 8
F F F F
3* 3* 4* 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).
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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).
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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).
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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
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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 erythrocyte 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.
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