) and hydroxy radical (OH․) as well as non free radical species such as hydrogen peroxide (H

329

INTRODUCTION

The univalent reduction of molecular oxygen results in reactive oxygen species (ROS). ROS include free radicals such as superoxide anion (O

2-

) and hydroxy radical (OH․) as well as non free radical species such as hydrogen peroxide (H

2

O

2

).

¹⁾

Although the generation of ROS is an essential defense mechanism in some instances, it can also cause tissue damage and a wide range of common diseases. These include inflam- mation, aging, cancer, arteriosclerosis, hypertension, and diabetes.

^2∼8)

All aerobic organisms have antioxidant defense that protects against these oxidative stress.

¹⁾

As enzymatic defense mechanism, superoxide dismutase (SOD), which catalyses dismutation of superoxide anion to hydrogen peroxide, and catalase (CAT), which converts hydrogen peroxide into

molecular oxygen and water, are important. Non-enzymatic antioxidants include vitamins such as ascorbic acid, α-toco- pherol, β-carotene, glutathione, and flavonoids, as well as micronutrient elements such as zinc and selenium.

⁹⁾

The practice of using macro-fungi especially mushrooms to treat a variety of disease is found in many countries. Extracts obtained from Phellinus mushrooms have received special attention due to their potent pharmacological activities, in- cluding immuno-stimulation,

¹⁰⁾

anti-tumor activity,

¹¹⁾

anti- oxidant, anti-hepatoxicity.

¹²⁾

P. baumii (Hymenochaetaceae) is a wood inhabiting macro-fungus and used as folk medicine for diabetes, cancer, immune-suppression, and detoxification. Re- cently, it is also reported that fruit body of P. baumii inhibited pulmonary inflammation and scavenged free radicals.

^13,14)

In our investigation, we cultured the mycelia from spores of P.

baumii, isolated water extract from the cultured mycelia, and

책임저자：현진원, ꂕ 690-756, 제주시 제주대학로 66 제주대학교 의과대학 생화학교실

Tel: 064-754-3838, Fax: 064-726-4152 E-mail: jinwonh@cheju.ac.kr

접수일：2006년 9월 25일, 게재승인일：2006년 10월 18일

Correspondence to：Jin Won Hyun

Department of Biochemistry, College of Medicine, Cheju National University, 66, Jejudaehak-no, Jeju 690-756, Korea

Tel: +82-64-754-3838, Fax: +82-64-726-4152 E-mail: jinwonh@cheju.ac.kr

Antioxidant Activity of Water Extract from the Cultured Mycelia of Phellinus baumii

Jinny Park¹, Kyoung Ah Kang², Rui Zhang², Mei Jing Piao², Soyoon Park³, Jin Seo Park⁴, Young Kook Kim⁵, Mun Chaul Rho⁵, Hyun Sun Lee⁵ and Jin Won Hyun¹

Departments of ¹Medicine, ²Biochemistry, ³Pathology, College of Medicine, Cheju National University, Jeju 690-756,

4Department of Science & Technology, Youngdong University, Chungbuk 370-701, ⁵Laboratory of Lipid Metabolism, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea

The antioxidant property of cultured mycelia of Phellinus baumii was investigated. The water-soluble extract isolated from these mycelia showed intracellular reactive oxygen species, 1,1-diphenyl-2-pic- rylhydrazyl (DPPH) radical scavenging and lipid peroxidation inhibitory activities. This radical scavenging activity of extract protected cell viability of Chinese hamster lung fibroblast (V79-4) cells exposed to H

2

O

2

. Furthermore, water extract reduced the apoptotic cells induced by H

2

O

2

, as demon- strated by the decreased number of sub G

1

hypo-diploid cells and apoptotic body formation and it increased the activities of cellular antioxidant enzymes, superoxide dismutase and catalase. Taken together, these findings suggest that mycelia of P. baumii exhibit antioxidant property. (Cancer Prev

Res 11, 329-335, 2006)

ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Key Words: Antioxidant activity, Phellinus baumii, Radical scavenging activity, Antioxidant enzymes

investigated the antioxidant activity.

MATERIALS AND METHODS

1. Preparation of water extract

Sporocarps of P. baumii growing on mulberry trees in Chiaksan, Korea were collected. The strain 0713 was identified as Phellinus baumii by Korea Research Institute of Bioscience and Biotechnology (KRIBB). The specimen was deposited in the gene bank of KCTC 10622BP. Taxonomic studies of the strain were carried out by the method of Jukes and Cantor

¹⁵⁾

and Saitou and Nei.

¹⁶⁾

Phellinus baumii 0713 was inoculated into a 1,000 ml baffled flask containing 100 ml of seed medium composed of 2% malt extract and 0.2% yeast extract pH 6.3 before autoclaving (121

^o

C, 15 min). The culture was incubated for 7 days at 26

^o

C on a rotary shaker (200 rpm). 20 ml of the seed culture was transferred into a 5,000 ml baffled flask containing 1,000 ml of production medium consisting of 2% dextrose, 0.9%

soybean floor and 3.5% of liquid sugar, pH 6.5 before autoclaving. Phellinus baumii 0713 was cultivated at 26

^o

C for 9 days on a rotary shaker at 200 rpm. Mycelia were harvested by filtration and were washed several times with distilled water.

The mycelia were extracted with hot water for 3 h. The water-soluble fraction was filtered and the supernatant was concentrated in freeze-drying to give a brown powder. The dried powder dissolved in phosphate buffered saline (PBS) for

in vitro studies.

2. Cell culture

Chinese hamster lung fibroblast, V79-4 cells were main- tained at 37

^o

C in an incubator, with a humidified atmosphere of 5% CO

2

, and cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, streptomycin (100μg/ml) and penicillin (100 unit/ml).

3. Chemicals

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2'7'-di- chlorodihydrofluorescein diacetate (DCF-DA), and Hoechst 33342 were purchased from Sigma, St. Louis, USA, and the thiobarbituric acid (TBA) from BDH laboratories, England.

The other chemicals and reagents used were analytical grade.

4. Intracellular reactive oxygen species (ROS) measurement

To detect intracellular ROS, the DCF-DA method was used.

DCF-DA diffuses into cells, where is hydrolyzed by intracellular esterase to polar 2'7'-dichlorodihydrofluorescein. This non- fluorescent fluorescein analog is trapped in cells and can be oxidized to the highly fluorescent 2'7'-dichlorofluorescein by intracellular oxidants.

¹⁷⁾

The V79-4 cells were seeded in a 96 well plate at a concentration of 1.0×10

⁵

cells/ml, 16 h after plating, cells were treated with various concentrations of water extract from the cultured mycelia of P. baumii and 30 min later 1 mm H

2

O

2

was added to the plate. Cells were incubated for an additional 30 min at 37

^o

C in an incubator. Fluorescence of 2'7'-dichlorofluorescein was detected at 485 nm excitation and at 535 nm emission, using a PerkinElmer LS-5B spectro- fluorometer.

5. DPPH radical scavenging activity

Various concentrations of the water extracts were added to a 1.0×10

^-4

M solution of DPPH in methanol and the reac- tion mixture shaked vigorously. After 5 h, the amount of DPPH remaining was determined at 520 nm.

¹⁸⁾

6. Lipid peroxidation inhibitory activity

Lipid peroxidation was assayed by the measurement of malondialdehyde (MDA).

¹⁹⁾

V79-4 cells were seeded in culture dish at a concentration of 1.0×10

⁵

cells/ml, 16 h after plating, cells were treated with various concentrations of water extract, 1 h later 1 mm H

2

O

2

was added to the plate, which was incubated for a further 1 h. The cells were then washed with cold PBS, scraped and homogenized in ice-cold 1.15% KCl.

100μl of cell lysates was combined with 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid adjusted to pH 3.5 and 1.5 ml of 0.8% thiobarbituric acid (TBA). The mixture was adjusted to a final volume of 4 ml with distilled water and heated to 95

^o

C for 2 h. After cooling to room temperature, 5 ml of a mixture of n-butanol and pyridine (15：1, v/v) was added to each sample and the mixture shaked vigorously. After centrifugation at 1,000×g for 10 min, the supernatant fraction was isolated, and the absorbance measured at 532 nm.

7. Cell viability

The effect of water extract on the viability of V79-4 cells

was determined by MTT assay, which is based on the cleavage of a tetrazolium salt by mitochondrial dehydrogenase in viable cells.

²⁰⁾

V79-4 cells were seeded in a 96 well plate at a concentration of 1.0×10

⁵

cells/ml, 16 h after plating, cells were treated with various concentrations of water extract and 1 h later 1 mm H

2

O

2

was added to the plate. Cells were incubated for an additional 24 h at 37

^o

C. 50μl of MTT stock solution (2 mg/ml) was then added to each well of a total reaction volume of 200μl. After incubating for 4 h, the plate was centrifuged at 800×g for 5 min and supernatants aspirated. The formazan crystals in each well were dissolved in 150μl of DMSO and the A540 read on a scanning multi-well spectrophotometer.

8. Nuclear staining with Hoechst 33342

V79-4 cells were placed in 24 well plate at a concentration of 1.0×10

⁵

cells/ml, and 16 h after plating, cells were treated at various concentrations of water extract and after further incubation for 1 h, 1 mm H

2

O

2

was added to the culture. After 24 h, 1.5μl of Hoechst 33342 (stock 10 mg/ml), a DNA specific fluorescent dye, was added to each well (1.5 ml) and incubated for 10 min at 37

^o

C. The stained cells were then observed under a fluorescent microscope equipped with a CoolSNAP-Pro color digital camera to examine the degree of nuclear condensation.

9. Flow cytometry analysis

Flow cytometry was performed to determine the apoptotic sub G

1

hypo-diploid cells.

²¹⁾

V79-4 cells were placed in 6 well plate at a concentration of 1.0×10

⁵

cells/ml. 16 h after plating, cells were treated at various concentrations of water extract and after a further incubation of 1 h, 1 mm H

2

O

2

was added to the culture. After 24 h, cells were harvested at the indicated times, and fixed in 1 ml of 70% ethanol for 30 min at 4

^o

C. The cells were washed twice with PBS, and then incubated for 30 min in the dark at 37

^o

C in 1 ml of PBS containing 100μg propidium iodide and 100μg RNase A.

Flow cytometric analysis was performed with a FACSCalibur flow cytometer. The proportion of sub G

1

hypo-diploid cells was assessed by histograms generated by the computer program Cell Quest and Mod-Fit.

10. Superoxide dismutase (SOD) activity

V79-4 cells were seeded at a concentration of 1.0×10

⁵

cells/

ml, 16 h after plating, cells were treated with various con- centrations of water extract for 1 h. The harvested cells were suspended in 10 mm phosphate buffer (pH 7.5) and were then lysed on ice by sonicating twice for 15 seconds. 1% Triton X-100 was then added to lysates and incubated for 10 min on ice. The lysates were clarified, by centrifugation at 5,000×g for 10 min at 4

^o

C, to remove cellular debris. The protein content of the supernatant was determined by Bradford me- thod using bovine serum albumin as the standard. Activity of SOD detected level of epinephrine auto-oxidation inhibited by SOD.

²²⁾

50μg of protein was added to 500 mm phosphate buffer (pH 10.2) and 1mm epinephrine. Epinephrine undergoes auto-oxidation rapidly at pH 10.0 to produce adrenochrome, a pink colored product, which was assayed at 480 nm using a UV/VIS spectrophotometer in kinetic mode. SOD inhibits the auto-oxidation of epinephrine. The rate of inhibition was monitored at 480 nm and the amount of enzyme required to produce 50% inhibition was defined as one unit of enzyme activity. The SOD activity was expressed as units/mg protein.

11. Catalase (CAT) activity

50μg of protein was added to 50 mm phosphate buffer (pH 7.0) and 100 mm (v/v) H

2

O

2

and then this mixture was incubated for 2 min at 37

^o

C and the absorbance of the mixture were monitored for 5 min at 240 nm. The change in absor- bance is proportional to the breakdown of H

2

O

2

.

²³⁾

The CAT activity was expressed as units/mg protein.

RESULTS

1. Radical scavenging activity of water extract

The antioxidant activities of water extract from the cultured mycelia of P. baumii were evaluated by the intracellular ROS and DPPH free radical scavenging activities. The intracellular ROS scavenging activity of water extract showed dose dependent manner, 30% in 10μg/ml, 43% in 50μg/ml, and 63% in 100μg/ml (Fig. 1A). This ROS scavenging activity of water extract is consistent with DPPH radical scavenging acti- vity of water extract (Fig. 1B), showing dose dependent man- ner, 14% in 10μg/ml, 18% in 50μg/ml, and 30% in 100μg/

ml. However, DPPH radical scavenging activity of water

extract showed lower activity compared to intracellular ROS

scavenging activity. Taken together, these results suggest that

water extract from the cultured mycelia of P. baumii has

antioxidant effect.

2. Effect of water extract on lipid peroxidation

The ability of water extract to inhibit lipid peroxidation in H

2

O

2

-treated V79-4 cells was also tested. The generation of malondialdehyde (MDA) and related substances that react with thiobarbituric acid (TBA) was inhibited by this extract. This activity of water extract showed 20% in 10μg/ml, 24% in 50 μg/ml, and 30% in 100μg/ml (Fig. 2).

3. Protective effect of water extract on cell survival

The protective effect of water extract on cell survival in

H

2

O

2

-treated V79-4 cells was tested. Cells were treated with extract at various concentrations for 1 h prior to the addition to H

2

O

2

. The cell survival was determined 24 h later by MTT assay. As shown in Fig. 3, treatment with extract induced a dose dependent increase of cell survival; 34% in 10μg/ml, 42% in 50μg/ml, and 57% in 100μg/ml.

4. Reduction of H2O2-induced apoptosis by water extract treatment

In order to analyze the protective effect of water extract on H

2

O

2

-induced apoptosis, nuclei of V79-4 cells were stained with Hoechst 33342 dye for detection of microscopy and with propidium iodide dye for detection of flow cytometry. As

Fig. 1. Effects of water extract from the cultured mycelia of P. baumii on scavenging intracellular ROS (A) and DPPH radical (B). The intracellular ROS was detected by DCF-DA method. The data represent three experiments and expressed as mean±standard error.

Fig. 2. Effect of water extract on inhibition of lipid peroxidation.

Lipid peroxidation was assayed by the measurement of malon- dialdehyde.

Fig. 3. Protective effect of water extract on H2O2 induced oxidative damage of V79-4 cells. H2O2 concentration used was final 1 mm. The viability of V79-4 cells was determined by the MTT assay.

shown in Fig. 4A, control cells revealed intact nuclei, but H

2

O

2

-treated cells showed significant nuclear fragmentation, characteristic of apoptosis, under microscopy. However, when the cells treated with water extract 1 h prior to H

2

O

2

-treat- ment, dramatic decrease in nuclear fragmentation was ob- served. In addition to the morphological evaluation, the pro- tective effect of water extract was confirmed by flow cytometry.

As shown in Fig. 4B, analysis of DNA contents in only H

2

O

2

-treated cells revealed an increase in the proportion of

cells with apoptotic sub-G

1

DNA content to 50%. Treatment at 100μg/ml of water extract decreased the apoptotic sub-G

1

DNA content to 34%. These results suggest that this extract protect cell viability through prevention of H

2

O

2

-induced apoptosis.

5. Effect of water extract on SOD and CAT

In order to investigate whether this antioxidant activity of water extract was mediated by increased activities of anti-

Fig. 4. Apoptotic body formation (A) and apoptotic sub-G1 DNA content (B) of V79-4 cells treated with H2O2 and water extract of mycelia. Concentration of H2O2 and water extract of mycelia used was final 1 mm and 100μg/ml, respectively. Apoptotic bodies are indicated by arrows.

A

0 40 80 120 160 200

0 200 400 600 8001,000

Counts

H O_{2 2}

DNA content

0 40 80 120 160 200

0 200 400 600 8001,000

Counts

Water extract

DNA content

Control H O_{2 2} Water extract

B

Fig. 5. Effects of water extract of mycelia on SOD (A) and CAT (B) activity in V79-4 cells. The data represent three experiments and are expressed as average enzyme unit per mg protein±standard error.

oxidant enzymes, the SOD and CAT activities in water extract treated V79-4 cells were measured. The water extract increased a little SOD activity (Fig. 5A). However, CAT activity in water extract treated cells was significantly increased, showing 22 U/mg protein in 10μg/ml, 26 U/mg protein in 50μg/ml, and 32 U/mg protein in 100μg/ml comparing to 13 U/mg protein of control (Fig. 5B).

DISCUSSION

Mammals constantly form reactive oxygen species (ROS) by oxidative and reductive processes in mitochondria from oxygen (O

2

) derived from respiration or by immune system exposed to foreign antigen, and externally by radiation or various chemicals. However, anti-oxidant enzyme systems of body, which are superoxide dismutase, catalase, glutathione pero- xidase, glutathione reductase, and various anti-oxidant chem- icals such as vitamin C and E, uric acid and bilirubin constantly remove the ROS formed. Therefore, a balance between the generation of ROS and its removal is made, thus maintaining cellular functions. However, under conditions where many ROS are produced or the anti-oxidant system is suppressed, cells are attacked by ROS, a process referred to as “oxidative stress”.

In many clinical disorders, oxidative stress is increased, and a constant stream of reports concern the alleviation of these disorders by the administering of anti-oxidants to those suf- fering from disorders that cause oxidative stress. For example, diabetic patients are subjected to levels of oxidative stress that are much higher than in normal subjects, and some compli- cations are relieved by anti-oxidant treatment.

²⁴⁾

Therefore, researchers have made numerous efforts to find antioxidants.

For this reason, the antioxidant properties of the extract of the cultured mycelia P. baumii have been characterized.

The water extract of the cultured mycelia P. baumii increased intracellular ROS and DPPH radical scavenging activities and then enhanced the viability of V79-4 cells exposed to H

2

O

2

. The protective effect of this water extract on H

2

O

2

-induced apoptosis was observed under microscope and with flow cytometry. The cells exposed to H

2

O

2

exhibited distinct mor- phological features of apoptosis, such as nuclear fragmentation and an increase in percentage of sub G

1

-hypodiploid cells.

However, cells that were pretreated with water extract had significantly reduced numbers of apoptotic cells, as shown by their morphology and sub G

1

DNA contents. This extract

increased the activities of antioxidant enzyme, SOD and CAT.

These enzymes are modulated in various disease caused by free radical attack.

¹⁾

Thus, maintaining the balance between the rate of generation and scavenging of radicals is an essential part of biological homeostasis. SOD catalyses the breakdown of O

2-

to O

2

and H

2

O

2

, prevents formation of OH

^-

and has thereby been implicated as an essential defense against the potential toxicity of oxygen. The ROS scavenging activity of SOD is effective only when it is followed by the actions of CAT, as the SOD generates H

2

O

2

, which needs to be further scavenged by CAT. The water extract activated SOD and CAT, showing more activation in SOD than that in CAT. This indicates that this extract can effectively scavenge H

2

O

2

. In conclusion, the extract form the cultured mycelia of P. baumii had intracellular ROS and DPPH radical scavenging activities, promoted cell viability, inhibited H

2

O

2

induced apoptosis, and enhanced the effects of antioxidant enzymes.

ACKNOWLEDGEMENT

This study was supported by grant from KRIBB-JEJU joint research program by Jeju Provincial Government.

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