Tissue culture and bioavailability of Taiwan's {{Antrodia camphorata

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通識教育年刊第五期第 247 至 255 頁 2003 年 12 月中國醫藥大學通識教育中心

TISSUE CULTURE AND BIOAVAILABILITY OF TAIWAN’S “ANTRODIA CAMPHORATA”

Tay-Yuan Huang

¹

AND Wen–Wen Huang

²

1

: The Department of Chemical Engineering National ChungHsing University

2

: General Education Center

China Medical University, Taichung 404, Taiwan

This study investigated how breeding conditions affected the culturing tissue of mycelia from Antrodia camphorata and evaluated its safety and cytotoxicity against HL-60 leukemia cancer cells. The effect of culturing medium, temperature, intensity of light, and pH value on the growth of mycelia were examined. The use of MEA medium, with no light, at 25℃ and pH 4.5 were found to yield mycelia with the largest diameter. The results indicated that the ethanolic extracts of mycelia from A. camphorata at concentrations of 200 μg/mL inhibited the growth of HL-60 cells while exhibiting under 500 μg/mL no significant cytotoxicity to normal cells (including Chang liver cell, Detroit 551 Human embryonic skin cell and WS-1 Human skin keratinocytes).

Keywords: antrodia camphorata, tissue culture, mycelia, bioavailability, cytotoxicity

Requests for reprints should be sent to Wen-Wen Huang, General Education Center, China Medical University, 91 Hsueh-Shih Road, Taichuang 404, Taiwan.

E-mail: [email protected]

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通識教育年刊第五期 248

Ⅰ. Introduction

Antrodia camphorata of Polyporaceae family (Aphyllophorales), which is parasiticon the rotted trees of Cinnamomum kanehirai. Recent research had reported anticancer, antitumor and immunomodulating effects of the fruiting body of A . camphorata. Unlike Ganoderma lucidum, it was found to exist only on Cinnamomum kanehirai in Taiwan (Wu et al., 1997). Polysaccharides and triterpenoids were found to be two main types of active components of medicinal fungi (Tomoda et al., 1990; Kabir et al., 1988; Liu et al., 1997). Antrodia camphorata and Ganoderma lucidum are medicinal fungi (Lee et al., 2002). This pioneer research studied the tissue culture conditions of A. camphorata and evaluated their safety, cytotoxicity and bioavailability with 50%

ethanolic extracts.

Ⅱ. Materials and Methods

2.1 Culture of mycelia

This research used 70% ethanol to initially sterilize wild fruit bodies and mycelia harvested from a tissue culture of Antrodia camphorata. The influence of various breeding conditions on the tissue culturing of the mycelia from A. camphorata was examined. A. camphorata was cultured using various medium components, temperatures, light intensities (1,500 Lux), and pH values on the growth of mycelia was examined (Fig. 1). Total five parameters such as carbon source, supplements, light, temperature and pH value were investigated. Carbon source and the supplement was first individually screened to obtain the best medium components (2％ glucose and 2％ maltose extracts). Respective examinations followed the lightness in the cultivated environment, temperature distribution of 15℃, 20℃and 25

℃, and the pH value distribution of 4.5, 5.7 and 6.7 were conjugated.

2.2 Chemicals and reagents

Extracts of Antrodia camphorata were prepared by extracting fresh

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Tissue Culture and Bioavailability of Taiwan^，s “Antrodia camphorata” 249

mycelia powder using 50 % ethanol. Stock solutions of extracts were adjusted to 100 mg/ml by dilution in distilled water. For treatment, these extracts were further diluted in distilled water and added to cells in culture medium. Fetal bovine serum (GIBCO BRL, Grand Island, New York, USA), 100 U/mL penicillin, 10 μg/mL streptomycin, 2mM glutamine were purchased from GIBCO. Propidium iodide (PI) and other chemicals were obtained from Sigma Co. (St. Louis, MO). All of the chemicals were reagent grade.

2.3 Cells culture

Human promyelocytic leukemia HL-60 cells were obtained from Culture Collection and Research Center (CCRC) in Taiwan, having come originally from The American Type Culture Collection (ATCC). HL-60 cells, Chang liver cell,

Detroit 551 and WS-1 were cultured in Roswell Park Memorial Institutes medium (RPMI-1640), supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37℃, in a 5% CO

2

humidified atmosphere. Before Antrodia camphorata extract treatments were performed, cells were suspended at a final density of 2.5×10

⁵

cell/mL, and seeded in 24-well plates (1 ml/well).

2.4 Bioavailability

Cell viability was determined by PI exclusion method： The cells were treated with various concentration of mycelia extracts for 24 hours, washed once with phosphate buffered saline (PBS), resuspended in PBS containing 4 μg/mL PI, and then analyzed by flow cytometry (FACS Calibur

^TM

, Becton Dickinson, Mountain View, CA) within thirty minutes. All experiments were performed in triplicate (Jun et al., 1998).

MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide)： Human promyelocytic leukemia HL-60 cells were plated at

density of 2.5×10

⁴

cells / well in 96-well plate. The stock of the extracts of

the mycelia from A. camphorata were diluted with medium, then added to

the wells for the desired final concentrations and the final volume is 100

μl/well. After 24 hours of exposure to the extracts of the mycelia from A ﹒

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camphorata, 10 μl of 5 mg/mL MTT was added to each well and incubated 4 more hours under 37℃, and the liquid in the wells was evaporated. Then, 110 μl of hydrochloric acid – isopropanol (0.02 N) was added. After

dissolving the precipitate, the absorbance was detected in the ELISA reader at 570 nm (Mosmann, 1983).

Ⅲ. Results and Discussion

The influence of the growth medium of mycelia from A. camphorata was examined using several different components in the medium. The best conditions were found to involve MEA medium (including glucose 2%, maltose extract 2%, peptone 0.1%, agar 2% and pH 4.5), as shown in Fig. 1.

Glucose was preferred over fructose in the carbon source, and addition of maltose extracts was preferred over yeast extracts in the supplement (Table 1), darkness was preferred over light for eight hours (1,500 Lux) (Table 1), 25℃ was the best temperature (Table 1), and pH 4.5 was optimal (Table 1).

Figure 2 indicated that at 500μg/mL no significant cytotoxicity against three normal cells (50% ethanolic extracts of A. camphorata) exhibited. Chang liver cell, Detroit 551 and WS-1 came from dissimilar tissues of human body provided with the differential cell characteristics and the cytotoxicity capacity, were also shown in Figure 2. HL-60 cells were treated with 50 μg/mL or 200 μg/mL extracts of mycelia, and the cell-cycle distribution was measured after 24, 48 and 72 hours of incubation.

Experimental results indicated significant inhibition of HL-60 cell proliferation by 200 μg/mL of the 50% ethanolic extracts of A. camphorata after 72 hours of treatment. A dose- dependent decline in A.

camphorata-treated cell proliferation was observed as shown in Figure 3.

The rate inhibition reached 47.5%. Subsequent work will focus on

separation, purification, and bioavailability testing of the active components

of A. camphorata.

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Ⅳ. Conclusions

This was the first successful trial to culture Taiwanese A. camphorata from wild fruit bodies. The best conditions for obtaining mycelia of A.

camphorata with the largest diameter were MEA medium, no light, 25℃, and pH 4.5. Cell proliferation results showed that the 50% ethanolic extracts of A. camphorata at 200 μg/mL inhibited the growth of HL-60 cells after treatment for 72 hours (IC

50

Inhibitory concentration was observed at 250 μg/mL). The extent of inhibition reached 47.5%. No conspicuous cytotoxicity to the normal cells was observed at 500μg/mL (including Chang liver cell, Detroit 551 Human embryonic skin cell and WS-1 human skin keratinocytes). These encouraging results prompted us to further investigate if A. comphorata might be a good anticancer drug.

Table.1 Influence of light, temperature and pH on mycelia formation from Antrodia camphorata

Cultivation conditions *Diameter (cm) Carbon source Glucose (2％)

Fructose (2％)

7.60 ±0.18

^a

6.20 ±0.23

^a

MEA

Supplements Maltose extract(2％) Yeast extract (2％)

7.55 ±0.39

^a

6.15 ±0.28

^a

No light (0 Lux) 7.70 ±0.12

^c

Environment

Light of 8 hr (1,500 Lux) 3.00 ±0.21

^c

15℃ 2.50 ±0.18

^c

20℃ 5.48 ±0.25

^c

Temperature (℃)

25℃ 7.30 ±0.26

^c

4.5 7.55 ±0.29

^a

5.7 5.83 ±0.39

^a

pH

6.3 5.22 ±0.63

^a

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*: Diameter was measured to select an assumption of round of mycelia in the well plate by using A Vernier caliper (MITUTOYO 530-102 series).

^a

p<0.05,

^b

p<0.01

^c

, p<0.001

Fig. 1 Optical photography of growth mycelia of Antrodia camphorata cultured in MEA medium

Antrodia -normal cell viability

0 20 40 60 80 100 120

0 31.2 62.5 125 250 500 1000

Concentration ( ug/ml )

Percent Cell Viability (%) ^WS1

Chang Detroit 551

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Fig. 2 Effect of Antrodia camphorata on normal cell viability (Chang cells, WS-1 and Detroit 551).

Note: Cellular viability was measured by MTT assay.

Cells were exposed to indicated amounts of

Antrodia camphorata

for 24

hours. Viable cells in various treatments were expressed as a percentage of control. Data points represented means ± S.D. for three independent experiments.

HL60-Antrodia camphorata

0 20 40 60 80 100 120

0 12.5 25 50 100 150 200 250

Concentration ( ug/ml )

Percent cell viability (%) 24 hr

48 hr 72 hr

Fig. 3 Effect of ethanol extract of Antrodia camphorata on HL-60 cells viability Note: Cellular viability was measured by PI exclusion. Cells were exposed to

indicated amounts of extracts from Antrodia camphorata for 24, 48 and 72 hours. Viable cells in various treatments were expressed as a percentage of control. Data points represented means ± S.D. for three independent experiments.

V. References

Wu SH, Ryvarden L and Chang TT. Antrodia camphorata (“niu-chang-chih”), new combination of a medical fungus in Taiwan. Bot. Bull. Acad. Sin 1997;38:273-275.

Tomoda M, Shimizu N, Gonda R, Kanari M, Yamada H and Hikino H.

Anti-complementary and hypoglycemic activities of the glycans from the

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seeds of Mlva verticillata. Planta Medica 1990:56:168-170.

Kabir Y, Kimura S and Tamura T. Dietary effect of Ganoderma lucidum mushroom on blood pressure and lipid levels in spontaneously hypertensive rats (SHR). J. Nutr. Sci. Vitaminol 1988;34:433-438.

Liu F, Ooi VEC and Chang ST. Free radical scavenging activities of mushroom

polysaccharide extracts. Life Sciences 1997;60:763-771.

Lee IH, Huang RL, Chen CT, Chen HC, Hsu WC and Lu MK, Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiology Letters 2002;209:63.

Chronic Myeloid Leukemia Trialists' Collaborative Group. Interferon alfa versus chemotherapy for chronic myeloid leukemia: a meta-analysis of seven randomized trials. J. Natl. Cancer Inst 1997;89:1616-1620.

Jun CD, Pae HO, Yoo JC, Kwak HJ, Park RK and Chung HT. Cyclic adenosine monophosphate inhibits nitric-induced apoptosis in human leukemic Hl-60 cells. Cellular Immunology 1998;183:13.

Mosmann T. Rapid colorimetric assay for cellular growth and survival:

application to proliferation and cytotoxicity assays. J. Immunol Method

1983;65:55.

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台灣樟芝之組織培養及生體可用性

黃泰源

¹

、黃雯雯

²

1：中興大學化學工程所

2：中國醫藥大學通識教育中心

Tissue culture and bioavailability of Taiwan&apos;s {{Antrodia camphorata