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Ethanolic Extracts of Antrodia cinnamomea
Mycelia Fermented at Varied Times and Scales
Have Differential Effects on Hepatoma Cells
and Normal Primary Hepatocytes
Y.-S. C
HEN, J.-H. P
AN, B.-H. C
HIANG, F.-J. L
U,
ANDL.-Y. S
HEENABSTRACT: Mycelia of Antrodia cinnamomea (AC), an edible fungus native to Taiwan, were produced by submerged fermentation with various fermentation times in 250 mL, 5 and 500 L fermentors and were evaluated for the effect of fermentation products on the viabilities of Hep3B and HepG2 hepatoma cells and normal primary rat hepatocytes. The results showed that the ethanolic extracts of AC mycelia (from 250 mL fermentation for 8 wk and 5 and 500 L fermentations for 4 wk) possessed high antihepatoma activity. The IC50of ethanolic extract of AC mycelia fermented
for 8 wk in a 250 mL fermentor against Hep3B and HepG2 cells were 82.9 and 54.2μg/mL, respectively. Furthermore, the IC50for Hep3B and HepG2, treated with ethanolic extract of AC mycelia fermented for 4 wk in the 5 L fermentor
were 48.7 and 3.8μg/mL, respectively. Those treated with ethanolic extract of AC mycelia fermented for 4 wk in the 500 L fermentor were 36.9 and 3.1μg/mL, respectively. No adverse effects of all samples on normal primary rat hepatocytes were observed.
Keywords: Antrodia cinnamomea, fermentation times and scales, Hep3B, HepG2, normal primary hepatocytes
Introduction
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ntrodia cinnamomea (AC) is a traditional and prizedTai-wanese medicinal supplement that is well known for its anti-cancer and antidote functions (Wang and Huang 2002). In 1990s, a number of novel triterpenoids and steroid acids were found in the fruit body of this edible fungus (Chen and others 1995; Cherng and others 1995, 1996; Chiang and others 1995; Yang and others 1996; Shen and others 1997; Wu and others 1997). AC only grows on the inner cavity wall of Cinnamomum kanchirai Hay (Lauraceae) in Taiwan. The C. kanchirai Hay, however, has now become scarce in Taiwan’s forests and also the growth rate of AC is very slow (Wu and others 1997). Thus, at present, the edible AC products avail-able to the local population in Taiwan are produced by large-scale fermentation.
Although AC has been used as traditional medicine by the local population in Taiwan for a long time, most of scientific research ar-ticles related to its pharmacological and nutritional aspects were published only recently. AC’s properties include antioxidative ac-tivity, liver protective function, antihypertensive acac-tivity, antiangio-genic activity, anti-inflammatory activity, and anticancer activity (Song and Yen 2002, 2003; Dai and others 2003; Liu and others 2004, 2007; Mau and others 2004; Shen and others 2004; Chen and oth-ers 2005; Hsu and othoth-ers 2005, 2007; Song and othoth-ers 2005b). Ac-cording to the survey, among “main causes of death” for the year 2006 in Taiwan, malignant neoplasm continues to be on the top list of leading causes of death, and chronic liver diseases,
includ-MS 20080388 Submitted 5/23/2008, Accepted 6/10/2008. Authors Chen, Pan, Chiang, and Sheen are with Graduate Inst. of Food Science and Technology, Natl. Taiwan Univ., Taipei 106, Taiwan. Author Lu is with Dept. of Applied Chemistry, Chung Shan Medical Univ., Taichung 402, Taiwan. Direct in-quiries to author Sheen (E-mail: [email protected]).
ing liver cirrhosis, were at the 7th position. Therefore, maintain-ing a healthy liver function is always an important issue in Taiwan. Studies indicated that AC is promising to be an effective health pro-moting edible fungus due to its liver protective function and an-tihepatoma activity. Presently, the material sources of AC that are used by researchers are manufactured from submerged fermenta-tion and solid state fermentafermenta-tion in Taiwan (Chang and others 2001; Song and Yen 2002; Yang and others 2003; Mau and others 2004; Lin and Sung 2006). However, as these material sources of AC are not produced from the same fermentation process, the relations between bioactivity functions and fermentation conditions of AC are not clear. The objective of this research was to evaluate the anti-hepatoma activities of AC products, isolated from AC cultivated for different time intervals at different fermentation scales. Thus, the optimum fermentation conditions for AC were established for sub-merged cultivation, which could produce a product with enhanced antihepatoma activity.
Materials and Methods
Materials
The fermentation products of AC were produced at Gradu-ate Inst. of Food Science and Technology, Natl. Taiwan Univ. (Taipei, Taiwan). Dulbecco’s Modified Eagle Medium (DMEM), antibiotic–antimycotic solution (AA), nonessential amino acid so-lution (NEAA), L-15 medium, insulin, transferrin, fetal bovine serum (FBS), and penicillin–streptomycin solution were pur-chased from Gibco Laboratories (Grand Island, N.Y., U.S.A.). Col-lagenase (type I) was purchased from Worthington Biochem-ical Co. (Lakewood, N.J., U.S.A.). Percoll was purchased from Pharmacia LKB (Piscataway, N.J., U.S.A.). N,N-dimethylfluoramide (DMF) was purchased from the Lab-Scan (Dublin, Ireland). Trypsin-EDTA solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
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tetrazolium bromide (MTT), N-[2-hydroxyethyl]piperazine-N -[2-ethanesulfonic acid] (HEPES), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), reduced β-nicotinamide adenine
din-ucleotide (β-NADH), sodium dodecyl sulfate (SDS), phospho-tungstic acid, 2-thiobarbituric acid, 1,1,3,3-tetraethoxypropane, re-duced glutathione (GSH), oxidized glutathione (GSSG), iodoacetic acid, reduced β-nicotinamide adenine dinucleotide phosphate
(β-NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), sodium azide (NaN3), glutathione reductase (GRd), and collagen were obtained
from Sigma Chemical (St. Louis, Mo., U.S.A.).
Fermentation of AC in 250 mL flask
AC (BCRC35716) was obtained from Bio-resource Collection and Research Center(Hsinchu, Taiwan). The AC hyphae were activated with PDA medium at 25◦C. Then the active AC was inoculated into a 250 mL flask. The fermentation conditions were initial pH 5, malt extract 2%, peptone 0.1%, and carbon sources 2% (glucose, lactose, or galactose) cultured at 22◦C, 100 rpm for 8 wk.
Fermentation of AC in 5 and 500 L fermentors
The AC hyphae were activated with PDA medium at 25◦C. The activated AC was subcultured in a 500 mL flask. The subculture con-ditions were initial pH 5, malt extract 2%, peptone 0.1%, and carbon sources 2% cultured at 22◦C, 100 rpm for 4 wk. The subcultured AC (broth and mycelia) was then inoculated into a 5 or 500 L fermen-tor and fermented for 8 wk. The fermentation conditions were the same as subculture condition.
Preparation of samples (broth filtrates
and mycelia ethanolic extracts)
Each fermentation product of AC was separated into broth and mycelia. The broth was sterilized at 121◦C for 30 min and then fil-trated with 0.22μm filter. The filtrates of broth were lyophilized. In terms of mycelia, 1 g of each sample was extracted with 95% ethanol (20 mL) at 30◦C for 24 h. The filtrates were dried under vacuum.
Hepatoma cell culture
Human hepatoma HepG2 and Hep3B cells were kindly do-nated by Dr. Ming Shi Shiao (Dept. of Medical Research and Ed-ucation, Taipei Veterans General Hospital, Taipei, Taiwan). Cells were cultured in complete DMEM (cDMEM) (pH 7) at 37◦C, 5% CO2, and 90% relative humidity. The cDMEM containing 10% FBS,
100 units/mL penicillin, 100μg/mL streptomycin, and 0.25 μg/mL
amphotericin-B (1% antibiotic–antimycotic solution), and 100μM (1% MEM nonessential amino acid solution) were used. To remove the cells from the culture dish, the cells were trypsinized using 1 mL trypsin-EDTA solution for 3 min at 37◦C.
Antihepatoma activity assay
HepG2 and Hep 3B cells were cultured in 96-well plates at a den-sity of 1× 104cells/100μL cDMEM/well. After 24 h of incubation,
the medium was replaced by 100μL new serum-free DMEM
con-taining 0 or 100μg/mL sample for 48 h. Then the medium was discarded, and 25μL MTT solution (5 mg/mL PBS) and 100 μL
serum-free DMEM were added to every well and reincubated for an additional 4 h. Hundred microliters of MTT lysis buffer (20 g SDS in 50 mL DMF and 50 mL water) were added to dissolve the for-mazan crystals formed. Then the plates were read at 570 nm in a microplate reader (Anthos reader 2001, Salzburg, Austria).
Hepatocytes isolation and culture
Male Narl:SD rats (6- to 8-wk old) were purchased from the Natl. Lab. Animal Center, Taiwan. The rats were housed in plastic cages
with artificial 12-h light/dark cycles, and they were fed with Purina 5001 rodent chow diet (Purina, St. Louis, Mo., U.S.A.), with no wa-ter or food restriction. The method of hepatocyte isolation was as described by Sheen and others (1996). Rats were anesthetized us-ing intraperitoneal injection with sodium pentobarbital (100 mg/kg body weight). To remove the blood, the liver was perfused via the portal vein with 150 mL of 25 mM sodium phosphate buffer (pH 7.6), which contained 3.1 mM KCl, 119 mM NaCl, 5.5 mM glu-cose, 1 g/L BSA, and 5 mg/L phenol red at a flow rate of 25 mL/min. For another 10 min, the liver was perfused with 200 mL of the same buffer supplemented with 80 mg collagenase, 40 mM CaCl2, and
5 mg of trypsin inhibitor at a rate of 18 mL/min. The liver was then removed, sieved, washed, suspended in a Percoll buffer (Kreamer and others 1986), and centrifuged (Hermle Z300K, Germany) at 4◦C to produce a single cell suspension of hepatic parenchymal cells. Hepatocytes were washed twice with washing medium. After the final washing, the isolated hepatocytes were suspended at a den-sity of 5× 105cells/mL in L-15 cell culture medium (pH 7.6),
sup-plemented with 18 mM HEPES, 2.5% FBS, 5 mg/L insulin, 5 mg/L transferrin, 28 mM galactose, 1 μM dexamethasone, 100 units/L penicillin, and 100 mg/L streptomycin. Hepatocytes were plated at a density of 1× 106cells/2 mL in each 35-mm collagen-precoated
culture dish (NUNC, Denmark) and incubated in a humidified in-cubator (NuAire, Plymouth, Minn., U.S.A.) at 37◦C in an air atmo-sphere. The culture medium was replaced with fresh medium at 4 h after plating. The medium was changed using the same cul-ture medium but containing 2 g/L BSA instead of FBS at 24 h after plating.
Treatment of hepatocytes
At 24 h after plating, hepatocytes (1× 106) were treated with 0
and 50, 100, or 200μg/mL AC samples, which were previously
dis-solved in ethanol (the final concentration of ethanol was 0.5%). Af-ter 24 h of treatment, the culture medium was removed, and the hepatocytes were washed with cold PBS. Then the cells were re-moved with a cell scraper and collected for further analysis.
Cell viability of hepatocytes
The effect of samples on the viability of primary hepatocytes was evaluated by lactate dehydrogenase (LDH) leakage (Moldeus and others 1978). After collecting the cell-free medium, the hepa-tocytes were lysed with 20 mM potassium phosphate buffer con-taining 0.5% Triton X-100. The cells were collected and centrifuged at 10000× g for 30 min at 4◦C. A supernatant was used to analyze LDH activity. The samples were mixed in 1 mL of Krebs-Henseleit buffer containing 20 g/L BSA, NADH (0.2 mM final concentration), and pyruvate (1.36 mM final concentration). The rate of change in absorbance at 340 nm due to NADH oxidation was recorded. Intra-and extracellular LDH activities were analyzed, Intra-and the percentages of leakage were counted.
Lipid peroxidation
According to the method of Fraga and others (1988), thio-barbituric acid-reactive substance (TBARS) was used to mea-sure lipid peroxidation. The samples and standards (1,1,3,3-tetramethoxypropane) were mixed in 20 mM potassium phosphate buffer, 3% SDS solution, 0.1 N HCl, 10% phosphotungstic acid, and 0.7% thiobarbituric acid (TBA) and heated in boiling water for 30 min, then cooled to room temperature. The reactive prod-ucts were extracted with n-butanol and analyzed with a fluores-cence spectrophotometer at 515-nm excitation and 555-nm emis-sion. The results were compared to a standard curve expressed in nanomoles of TBARS per milligram of protein.
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Major component analysis by HPLC
We followed the HPLC analytical methodology, which was pub-lished by Chyr and Shiao (1991). The conditions of this HPLC anal-ysis were as follows: Cosmosil C18 column (Nacalai Tesque, Japan), Hitachi L-7420 UV-VIS detector (Hitachi, Tokyo, Japan), UV 243 nm, gradient elution (started with 80% methanol, increased linearly to 84% methanol in 15 min, to 86% in further 15 min, to 88% in 10 min, to 94% in further 10 min, and finally to 100% in 20 min), flow speed (0.7 mL/min). The analytical concentration is 20000 ppm.
Statistical analysis
Statistical analysis was performed using 1-way analysis of vari-ance (ANOVA) and Tukey’s multiple comparison test (SAS Inst. Inc., Cary, N.C., U.S.A.) to determine significant differences among means (P< 0.05).
Results and Discussion
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s the wild fruiting body of AC is now difficult to find in Taiwan’s forests, most of the material sources of AC products are pro-vided from submerged fermentation and solid-state fermentation. Although studies about the traditional edible AC have already been published, these studies did not use the same material sources. Moreover, the relations between bioactivity and fermentation con-ditions of AC are not clear. In this study, AC products were obtained via 250 mL flask, 5 and 500 L fermentation with various cultivation times, and the effects of ethanolic extracts of these AC products on the growth of hepatoma cells and normal primary rat hepatocytes were evaluated.The effect of ethanolic extract of AC
from 250 mL flask fermentation product
on the antihepatoma activity
AC was submerged and fermented in 250 mL flask with 2% glu-cose as carbon sources for 1, 2, 4, 6, and 8 wk. These mycelia and
Table 1 --- The effect of ethanolic extracts of AC mycelia from 250-mL flask fermentation on the growth inhibition of Hep3B and HepG2 cells.
Concentrations of treatment (μg/mL)
0.1 1 10 100 200
Fermentation
Cells time (wk) Cell viability (%) IC50(μg/mL)
Hep3B 1 102.5± 8.1a 103.8± 6.9a 111.4± 6.4a 108.7± 7.2a 109.7± 5.9a – 2 100.1± 5.8a 103.2± 6.9ab 110.7± 6.4b 94.7± 4.8b 96.3± 8.7b – 4 103.8± 10.9a 103.8± 4.9a 114.7± 5.7a 90.5± 5.0b 82.0± 6.1b – 6 99.6± 6.0a 90.6± 6.3ab 81.4± 8.2bc 73.8± 6.1c 48.9± 5.6d 199.5 8 103.2± 5.2a 91.8± 4.1a 75.9± 13.5a 42.3± 1.4c 30.2± 13.5c 82.9 HepG2 1 107.5± 2.9a 102.4± 38a 100.7± 5.2a 111.6± 3.9a 109.1± 4.9a – 2 100.7± 5.1a 96.6± 7.5a 99.3± 3.3a 94.9± 7.1ab 87.3± 6.4b – 4 98.7± 7.2a 99.8± 4.8a 90.3± 5.7b 86.0± 6.6b 73.9± 3.7c – 6 97.5± 7.5a 89.9± 10.5ab 85.1± 4.5b 68.0± 4.1c 41.8± 9.2d 168.2 8 107.5± 2.9a 89.4± 2.5b 47.1± 2.6c 27.6± 1.5d 2.9± 0.4e 54.2
Values bearing different letter in the same row are significantly different (P< 0.05) by 1-way ANOVA and Tukey’s multiple comparison test (n = 6).
Table 2 --- The effects of ethanolic extracts of AC mycelia from 5 L fermentation on the growth inhibition of Hep3B and HepG2 cells.
Concentrations of treatment (μg/mL)
0.1 1 10 100
Fermentation
Cells time (wk) Cell viability (%) IC50(μg/mL)
Hep3B 4 78.8± 1.6a 71.4± 2.2a 53.6± 6.3b 5.2± 4.1c 48.7
8 92.0± 4.8a 74.2± 8.8b 44.1± 1.2c 9.6± 1.3d 52.6
HepG2 4 85.0± 8.6a 56.2± 6.0a 30.2± 3.3b 15.2± 5.8b 3.8
8 59.8± 3.8a 29.8± 1.5b 23.8± 1.5c 17.1± 3.1d 2.9
Values bearing different letter in the same row are significantly different (P< 0.05) by 1-way ANOVA and Tukey’s multiple comparison test (n = 6).
broth were separated by filter paper, extracted with 95% ethanol for 24 h at 30◦C, and lyophilized afterward. The effects of the ethano-lic extracts of AC mycelia and broth (fermentation of 1, 2, 4, 6, and 8 wk) on the cell viability of HepG2 and Hep3B cells were evaluated. The result showed no adverse effect on cell viability of HepG2 and Hep3B cells that were treated with ethanolic extracts from 1, 2, and 4 wk mycelia and broth from all the weeks (data not shown). How-ever, the treatment with ethanolic extracts of AC mycelia from 6 and 8 wk fermentations could significantly decrease the cell viabil-ity of HepG2 and Hep3B cells in a dose-dependent manner (Table 1). The IC50of ethanolic extract of fermentation of 8 wk AC mycelia
to inhibit HepG2 cells growth was 54.2 μg/mL, but to inhibit
Hep3B cells was about 82.9μg/mL. The results showed that the ex-tent of inhibition was more profound in HepG2 cells than Hep3B cells.
The effects of ethanolic extracts of AC
from 5 and 500 L fermentation products
on the antihepatoma activity
The subcultured AC was inoculated into a 5 L fermentor and fer-mented for 4 and 8 wk. The fermentation conditions were initial pH 5, malt extract 2%, peptone 0.1%, and carbon source (glucose) 2% cultured at 22◦C, 100 rpm. The results showed that the treat-ment with ethanolic extract of 4 and 8 wk fertreat-mentation mycelia of AC (5FAC4-Em and 5FAC8-Em) could significantly decrease the cell viability of HepG2 and Hep3B cells in a dose-dependent man-ner (Table 2). However, 5FAC4-Em and 5FAC8-Em possessed sim-ilar ability to inhibit proliferation of HepG2 and Hep3B cells. The IC50of Hep3B and HepG2 treated with 5FAC4-Em were 48.7 and
3.8μg/mL, respectively, while those treated with 5FAC8-Em were 52.6 and 2.9μg/mL, respectively. The inhibition activity of
5FAC4-Em and 5FAC8-5FAC4-Em against HepG2 cells is comparatively higher then Hep3B cells. The results indicate that the ethanolic extracts of 4 weeks’ AC mycelia from 5 L fermentor (5FAC4-Em) could
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Table 3 --- The effect of ethanolic extracts of AC mycelia from 500 L fermentation on the growth inhibition of Hep3B and HepG2 cells.
Concentrations of treatment (μg/mL)
0.1 1 10 100 200
Fermentation
Cells time (wk) Cell viability (%) IC50(μg/mL)
Hep3B 1 94.1± 2.9a 91.1± 4.6a 96.6± 3.6a 69.3± 3.2b 50.3± 8.1c 196.8 2 105.8± 3.2a 84.3± 5.3b 59.0± 1.8c 43.6± 1.6d 40.5± 2.0d 78.9 3 65.9± 4.9a 54.5± 4.0b 43.1± 7.5c 42.4± 2.1c 37.7± 2.1c 48.6 4 76.8± 4.9a 63.3± 4.9b 40.1± 2.0c 29.3± 3.3d 16.6± 5.6e 36.9 6 70.2± 3.1a 58.1± 4.7b 40.3± 3.0c 29.8± 3.9d 3.4± 2.2e 27.0 HepG2 1 89.5± 2.3a 89.7± 8.2a 93.4± 7.6a 64.6± 7.9b 27.8± 8.1c 135.5 2 74.2± 8.4a 69.6± 9.3a 63.9± 5.7a 20.8± 1.7b 7.6± 1.3c 54.0 3 90.3± 5.1a 82.4± 7.5b 39.6± 3.1c 15.4± 0.1d 20.7± 2.5d 17.5 4 77.5± 9.9a 64.8± 5.6b 16.4± 1.3c 19.7± 5.8c 19.9± 2.0c 3.1 6 91.7± 9.6a 61.6± 9.7b 22.0± 4.8c 14.8± 5.7c 2.9± 1.4d 5.1
Values bearing different letter in the same row are significantly different (P< 0.05) by 1-way ANOVA and Tukey’s multiple comparison test (n = 6). significantly decrease the cell viability of HepG2 and Hep3B cells, in
contrast to the ethanolic extract of 4 wk mycelia from the 250 mL flask, which did not show any inhibition. In the 250-mL flask fer-mentor, only ethanolic extract of 8 wk AC mycelia had inhibition activity against HepG2 and Hep3B cells.
Because the lifetime of inducted air filter is no more than 2 mo in the 500 L fermentation, the cultivation time of AC was only for 6 wk. One liter of AC fermentation product was sampled at 1, 2, 3, 4, and 6 weeks. These samples were extracted with 95% ethanol for 24 h at 30◦C and lyophilized. The effect of these ethanolic extracts of AC mycelia (500FAC1-Em, 500FAC2-Em, 500FAC3-Em, 500FAC4-Em, and 500FAC6-Em) on the cell viability of HepG2 and Hep3B cells was also evaluated. The results showed that these samples could significantly decrease the cell viability of HepG2 and Hep3B cells in a dose-dependent manner (Table 3). Again, the inhibition activ-ity of these samples against HepG2 cells was comparatively higher than that of Hep3B cells. The IC50of 500FAC1-Em, 500FAC2-Em,
500FAC3-Em, and 500FAC4-Em decreased as the cultivation time increased. However, the IC50of ethanolic extract of 6 wk AC mycelia
(500FAC6-Em) was not less than 500FAC4-Em.
Figure 1 indicates the variation of morphology of HepG2 and Hep3B cells with 5FAC4-Em treatment. There were marked differ-ences between 10, 100, 200, and 400μg/mL and control treatment on the morphology of HepG2 and Hep3B cells. A similar result was observed on the morphology of HepG2 and Hep3B cells with 500FAC6-Em treatment (data not shown).
The results from cell viability studies showed that Hep3B and HepG2 cells were more inhibited on treatment with the ethano-lic extract of AC mycelia fermented for 8 wk in the 250 mL flask than those fermented for 1, 2, 4, and 6 wk. The IC50of ethanolic
extract of AC mycelia from the 500 L fermentation (500FAC1-Em, 500FAC2-Em, 500FAC3-Em, and 500FAC4-Em) also decreased with cultivation time. These results indicate that the antihepatoma ac-tivity of AC is related to the cultivation time of fermentation. Sim-ilar results have been reported on fermented soy food (doenjang) where increasing the fermentation period when making doenjang, increases its antitumor and antimetastatic effect in vivo (Jung and others 2006). Doenjang fermented for 24 mo exhibited a 2- to 3-fold increase in the antitumor effects on sarcoma-180-injected mice and antimetastatic effects in colon 26-M 3.1 cells in mice compared with the 3- or 6-mo fermented doenjang.
The AC mycelia needed 8 wk of submerged fermentation in a 250 mL flask to exhibit an inhibitory effect against HepG2 and Hep3B cells. However, the ethanolic extracts of AC mycelia fer-mented only for 4 wk in the 5 and 500 L fermentors possessed significant inhibitory effect against Hep3B and HepG2 cells. These
results indicated that the antihepatoma activity of AC products is also related to the scale of fermentation fermentor. When the vol-ume was changed from the 250 mL fermentor into a 5 L fermen-tor, the cultivation time could be decreased from 8 to 4 wk. More-over, the ethanolic extracts of AC mycelia fermented for 4 wk in a 5 L fermentor have slightly lower inhibitory effect against Hep3B
Figure 1 --- The effect of various concentrations of ethano-lic extract of AC mycelia from 5 L fermentors on the mor-phology of (A) HepG2 and (B) Hep3B cells (200×). is 100μm in length
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and HepG2 cells than the samples from a 500 L fermentor. The tank pressure of the 500 L fermentor probably reduced the growth of AC. Therefore, according to these results, we suggest that the cultiva-tion time requires 4 wk in a large fermentor, and the ethanolic ex-tract of AC mycelia possessed antihepatoma activity. Many reports related to anticancer activity of AC have been published in recent years (Hseu and others 2004; Liu and others 2004; Nakamura and others 2004; Hsu and others 2005; Song and others 2005a, 2005b). However, these studies did not use the same material sources of AC. Moreover, the relationship between bioactivity and bioactive com-pounds of AC are not clear.
Effect of ethanolic extract of AC mycelia
from 5 and 500 L fermentation on the growth
of primary rat hepatocytes
In this study, we used liver-perfusion method to isolate nor-mal primary rat hepatocytes from nor-male Narl:SD rats (6 to 8 wk old) and to evaluate the effect of 5FAC4-Em and 500FAC6-Em on the growth of normal primary rat hepatocytes. Figure 2 indicates
Figure 2 --- The effect of various concentrations of ethanolic extract of AC mycelia from 5 and 500 L fermentors on the morphology of primary rat hepatocytes (200×).
--- is 100μm in length.
the variation of morphology of normal primary rat hepatocytes with 5FAC4-Em and 500FAC6-Em treatment. There were no marked differences between 1, 10, and 100μg/mL and control treatment
on the morphology of normal primary rat hepatocytes. However, the growth of normal primary rat hepatocytes was inhibited sig-nificantly at 200 and 400μg/mL treatment with 5FAC4-Em and 500FAC6-Em.
In addition, to evaluate the cell viability of normal primary rat hepatocytes by assessing the percentage of LDH leakage (Figure 3). The result indicated no ill effect of normal primary rat hepatocytes at 1, 10, and 100μg/mL of 5FAC4-Em and 500FAC6-Em treatment. For hepatoma cell lines, however, the IC50of
5FAC4-Em and 500FAC6-5FAC4-Em were 48.7 and 27μg/mL for Hep3B cells and 3.8 and 5.1μg/mL for HepG2 cells, respectively. These dose
con-centrations are well below the 100μg/mL dose concentration of 5FAC4-Em and 500FAC6-Em treatment. Thus, ethanolic extracts of AC mycelia possessing inhibiting activity on HepG2 and Hep3B cells did not show any adverse effect on normal primary rat hepato-cytes, but the LDH leakages of normal primary rat hepatocytes were
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quite significant when treated with 200 and 400μg/mL of 5FAC4-Em and 500FAC6-5FAC4-Em.
The lipid peroxidation of normal primary rat hepatocytes with various concentrations of 5FAC4-Em and 500FAC6-Em treatments was evaluated. The TBARS values of normal primary rat hepato-cytes showed no significant difference from 0 to 200μg/mL of 5FAC4-Em and 500FAC6-Em treatments. This result indicated that the ethanolic extracts of AC mycelia would not cause lipid peroxi-dation in normal primary rat hepatocytes (Figure 4).
Human hepatocytes in primary culture provide the closest
in vitro model to human liver and the only model that can
pro-duce a metabolic profile of a given drug that is similar to that found
in vivo. However, their availability is limited due to the restricted
access to suitable tissue samples (Gomez-Lechon and others 2003). In this study, normal human hepatocytes were replaced by nor-mal primary rat heptatocytes. The results showed that 1, 10, and 100μg/mL of ethanolic extract of AC possessing antihepatoma ac-tivity on HepG2 and Hep3B cells, which did not have any adverse effect on normal primary rat hepatocytes. In addition, HepG2 is a kind of wild type hepatoma cells. Hep3B is a kind of delete-p53 hep-atoma cells. This variation may be the reason for a comparatively higher inhibition activities of FAC4-Em, FAC8-Em and 500FAC4-Em against HepG2 cells than with Hep3B cells.
The HPLC chromatograms of the ethanolic
extracts of AC mycelia from 500 L fermentor
To evaluate the major activity component of AC mycelia from a 500 L fermentor, we followed the HPLC analytical methodology
Figure 3 --- The effect of various concentrations of ethanolic extracts of AC mycelia from 5 and 500 L fermentors on the cell viability of primary rat hepatocytes at 24 h treated. Values bearing different letter are significantly different (P< 0.05) by 1-way ANOVA and Tukey’s multiple comparison test (n= 6).
Figure 4 --- The effect of various concentrations of ethanolic extracts of AC mycelia from 5 and 500 L fermentors on the TBARS of primary rat hepatocytes at 24 h treated.
published by Chyr and Shiao (1991) to analyze ethanolic extracts of AC mycelia from a 500 L fermentor. Their result indicated that the major activity components appeared in the retention time of HPLC profile between 15 and 52 min. In this study, the results showed that the HPLC profile of ethanolic extract of 6 wk fermented AC mycelia possessed more peak areas than those of 3 wk fermented AC mycelia (Figure 5). Besides, the HPLC profile of ethanolic extract of 1 wk fermented AC mycelia possessed fewer peaks in the reten-tion time between 15 and 50 min. This result indicated that there were many possible active components in the ethanolic extract of 6 wk fermented AC mycelia from 500 L fermentor. Moreover, these possible active components could be related to that the ethanolic extract of the 6-wk fermented AC mycelia possessed high antihep-atoma activity.
Conclusions
I
n this study, the antihepatoma activities of AC products fer-mented with different time and scales were evaluated. The result showed that the treatment with ethanolic extracts of AC mycelia from 5 and 500 L fermentation could significantly decrease the cell viability of HepG2 and Hep3B cells, and the 100μg/mL of treatmentwith the same samples did not have any adverse effect on normal primary rat hepatocytes. It indicated that the cultivation time of 5 and 500 L fermentation required only 4 wk to produce AC mycelia which had significant antihepatoma activity. These results could be used as a guide to produce AC products possessing antihep-atoma activity in large-scale fermentation processes. Moreover, the high antihepatoma activity ethanolic extracts of AC mycelia contain
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&
Food
Figure 5 --- The HPLC chromatograms of ethanolic extract of AC mycelia from 500 L fermentor. Analytical concen-tration is 20000 ppm. The captions, (A), (B), and (C), in-dicate that the HPLC profile of ethanolic extract of AC mycelia fermented 1, 3, and 6 wk in 500 L fermentor, re-spectively. The retention time of peak 1 is 15.9 min, peak 2 is 18.8 min, peak 3 is 25.8 min, peak 4 is 32.2 min, peak 5 is 34.1, peak 6 is 40.3 min, peak 7 is 46.3, and peak 8 is 51 min.
many potential active components. The study on the identification of active compounds from AC products with antihepatoma activity is in progress.
Acknowledgments
We thank the Natl. Science Council of Taiwan for financially sup-porting this study and Biotechnology Center, Grape King Inc., Chungli, Taiwan, for providing technical support in the 500 L fer-mentation process.
References
Chang HL, Chao GR, Chen CC, Mau JL. 2001. Non-volatile taste components of
Agar-icus blazei, Antrodia camphorata and Cordyceps militaris mycelia. Food Chem
74(2):203–7.
Chen CH, Yang SW, Shen YC. 1995. New steroid acids from Antrodia
cinnamo-mea, a fungal parasite of Cinnamomum micranthum. J Nat Prod 58(11):1655–
61.
Chen SC, Lu MK, Cheng JJ, Wang DL. 2005. Antiangiogenic activities of polysaccha-rides isolated from medicinal fungi. FEMS Microbiol Lett 249(2):247–54. Cherng IH, Chiang HC, Cheng MC, Wang Y. 1995. 3 New triterpenoids from
Antrodia-cinnamomea. J Nat Prod 58(3):365–71.
Cherng IH, Wu DP, Chiang HC. 1996. Triterpenoids from Antrodia cinnamomea. Phy-tochemistry 41(1):263–7.
Chiang HC, Wu DP, Cherng IW, Ueng CH. 1995. A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia-cinnamomea. Phytochemistry 39(3):613– 6.
Chyr R, Shiao MS. 1991. Liquid chromatographic characterization of the triterpenoid patterns in Ganoderma lucidum and related species. J Chromatogr 542:327–36. Dai YY, Chuang CH, Tsai CC, Sio HM, Huang SC, Chen JC, Hu ML. 2003. The
protec-tion of Antrodia camphorata against acute hepatotoxicity of alcohol in rats. J Food Drug Anal 11(3):177–85.
Fraga CG, Leibovitz BE, Tappel AL. 1988. Lipid-peroxidation measured as thiobarbi-turic acid-reactive substances in tissue-slices—characterization and comparison with homogenates and microsomes. Free Radic Biol Med 4(3):155–61.
Gomez-Lechon MJ, Donato MT, Castell JV, Jover R. 2003. Human hepatocytes as a tool for studying toxicity and drug metabolism. Curr Drug Metab 4(4):292– 312.
Hseu YC, Yang HL, Lai YC, Lin JG, Chen GW, Chang YH. 2004. Induction of apoptosis by Antrodia camphorata in human premyelocytic leukemia HL-60 cells. Nutr Can-cer 48(2):189–97.
Hsu YL, Kuo YC, Kuo PL, Ng LT, Kuo YH, Lin CC. 2005. Apoptotic effects of extract from
Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines.
Cancer Lett 221(1):77–89.
Hsu YL, Kuo PL, Cho CY, Ni WC, Tzeng TF, Ng LT, Kuo YH, Lin CC. 2007. Antrodia
cin-namomea fruiting bodies extract suppresses the invasive potential of human liver
cancer cell line PLC/PRF/5 through inhibition of nuclear factor kappaB pathway. Food Chem Toxicol 45(7):1249–57.
Jung KO, Park SY, Park KY. 2006. Longer aging time increases the anticancer and an-timetastatic properties of doenjang. Nutrition 22(5):539–45.
Kreamer BL, Staecker JL, Sawada N, Sattler GL, Hsia MTS, Pitot HC. 1986. Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell Dev Biol 22(4):201–11.
Lin ES, Sung SC. 2006. Cultivating conditions influence exopolysaccharide produc-tion by the edible Basidiomycete Antrodia cinnamomea in submerged culture. Int J Food Microbiol 108(2):182–7.
Liu DZ, Liang YC, Lin SY, Lin YS, Wu WC, Hou WC, Su CH. 2007. Antihypertensive activities of a solid-state culture of Taiwanofungus camphoratus (Chang-chih) in spontaneously hypertensive rats. Biosci Biotechnol Biochem 71(1):23–30. Liu JJ, Huang TS, Hsu ML, Chen CC, Lin WS, Lu FJ, Chang WH. 2004. Antitumor
ef-fects of the partially purified polysaccharides from Antrodia camphorata and the mechanism of its action. Toxicol Appl Pharmacol 201(2):186–93.
Mau JL, Huang PN, Huang SJ, Chen CC. 2004. Antioxidant properties of methanolic extracts from two kinds of Antrodia camphorata mycelia. Food Chem 86(1):25–31. Moldeus P, Hogberg J, Orrenius S. 1978. Isolation and use of liver cells. Methods
En-zymol 52:60–71.
Nakamura N, Hirakawa A, Gao JJ, Kakuda H, Shiro M, Komatsu Y, Sheu CC, Hattori M. 2004. Five new maleic and succinic acid derivatives from the mycelium of Antrodia
camphorata and their cytotoxic effects on LLC tumor cell line. J Nat Prod 67(1):46–
8.
Sheen LY, Lii CK, Sheu SF, Meng RHC, Tsai SJ. 1996. Effect of the active principle of garlic—diallyl sulfide—on cell viability, detoxification capability and the antioxida-tion system of primary rat hepatocytes. Food Chem Toxicol 34(10):971–8. Shen YC, Yang SW, Lin CS, Chen CH, Kuo YH, Chen CF. 1997. Zhankuic acid F: a
new metabolite from a Formosan fungus Antrodia cinnamomea. Planta Med 63(1): 86–8.
Shen YC, Chou CJ, Wang YH, Chen CF, Chou YC, Lu MK. 2004. Anti-inflammatory activity of the extracts from mycelia of Antrodia camphorata cultured with water-soluble fractions from five different Cinnamomum species. FEMS Microbiol Lett 231(1):137–43.
Song TY, Yen GC. 2002. Antioxidant properties of Antrodia camphorata in submerged culture. J Agric Food Chem 50(11):3322–7.
Song TY, Yen GC. 2003. Protective effects of fermented filtrate from Antrodia
cam-phorata in submerged culture against CCl4-induced hepatic toxicity in rats. J Agric
Food Chem 51(6):1571–7.
Song TY, Hsu SL, Yeh CT, Yen GC. 2005a. Mycelia from Antrodia camphorata in submerged culture induce apoptosis of human hepatoma HepG2 cells possibly through regulation of Fas pathway. J Agric Food Chem 53(14):5559–64.
Song TY, Hsu SL, Yen GC. 2005b. Induction of apoptosis in human hepatoma cells by mycelia of Antrodia camphorata in submerged culture. J Ethnopharmacol 100(1– 2):158–67.
Wang BC, Huang RC. 2002. The development and marketing of Ganoderma lucidum and Antrodia camphorata. Food Ind 34(5):3–14.
Wu SH, Ryvarden L, Chang TT. 1997. Antrodia camphorata (“niu-chang-chih”), new combination of a medicinal fungus in Taiwan. Bot Bull Acad Sinica 38(4):273–5. Yang FC, Huang HC, Yang MJ. 2003. The influence of environmental conditions on the
mycelial growth of Antrodia cinnamomea in submerged cultures. Enzyme Microb Technol 33(4):395–402.
Yang SW, Shen YC, Chen CH. 1996. Steroids and triterpenoids of Antrodia
cin-namomea—a fungus parasitic on Cinnamomum micranthum. Phytochemistry
