Research Papers of Antrodia camphorata Papers from PubMed, key word: Antrodia cinnamomea as of 4-6-2012 http://www.ncbi.nlm.nih.

gov/sites/entrez Table of Contents 1: Antrodia cinnamomea fruiting bodies extract suppresses the invasive potential of human liver cancer cell line PLC/PRF/5 through inhibition of nuclear factor kappaB pathway. ............................................................................................................... 3 2: Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media. ................................................................................................................................................................ 4 3: Apoptotic effects of Antrodia cinnamomea fruiting bodies extract are mediated through calcium and calpain-dependent pathways in Hep 3B cells. ......................................................................................................................................................... 4 4: Protective effects of a neutral polysaccharide isolated from the mycelium of Antrodia cinnamomea on Propionibacterium acnes and lipopolysaccharide induced hepatic injury in mice. .................................................................................................. 4 5: Statistical optimization of medium components for the production of Antrodia cinnamomea AC0623 in submerged cultures. ..................................................................................................................................................................................... 5 6: Adenosine as an active component of Antrodia cinnamomea that prevents rat PC12 cells from serum deprivation-induced apoptosis through the activation of adenosine A2A receptors. .................................................................................................. 5 7: Cultivating conditions influence exopolysaccharide production by the edible Basidiomycete Antrodia cinnamomea in submerged culture. ..................................................................................................................................................................... 5 8: Antiangiogenic activities of polysaccharides isolated from medicinal fungi. ........................................................................ 6 9: Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells. .............. 6 10: Zhankuic Acid F: A New Metabolite from a Formosan Fungus Antrodia cinnamomea. ..................................................... 7 11: New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum. ...................................... 7 12: Fermented Antrodia cinnamomea Extract Protects Rat PC12 Cells from Serum Deprivation-Induced Apoptosis: The Role of the MAPK Family. ........................................................................................................................................................ 7 13: Identification and characterization of a lipase gene from Antrodia cinnamomea. ............................................................... 7 14: Maleimide and maleic anhydride derivatives from the mycelia of Antrodia cinnamomea and their nitric oxide inhibitory activities in macrophages. .......................................................................................................................................................... 8 15: Isolation and analysis of genes specifically expressed during basidiomatal development in Antrodia cinnamomea by subtractive PCR and cDNA microarray. .................................................................................................................................... 8 16. Development of an activation tagging system for the basidiomycetous medicinal fungus Antrodia cinnamomea. ............ 8 17. Inhibitory effects of antrodins A-E from Antrodia cinnamomea and their metabolites on hepatitis C virus protease. ........ 9 18. Ethanolic extracts of Antrodia cinnamomea mycelia fermented at varied times and scales have differential effects on hepatoma cells and normal primary hepatocytes. ...................................................................................................................... 9 19. Cloning and heterologous expression of a novel ligninolytic peroxidase gene from poroid brown-rot fungus Antrodia cinnamomea. .............................................................................................................................................................................. 9 1

20. Compound MMH01 possesses toxicity against human leukemia and pancreatic cancer cells. ........................................... 9 21: Anti-angiogenic effects and mechanisms of polysaccharides from Antrodia cinnamomea with different molecular weights. .................................................................................................................................................................................... 10 22. A triterpenoid methyl antcinate K isolated from Antrodia cinnamomea promotes dendritic cell activation and Th2 differentiation. ......................................................................................................................................................................... 10 23. Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea. ........................... 10 24. Characterization and heterologous expression of a novel lysophospholipase gene from Antrodia cinnamomea. ............. 11 25: Fructification of Antrodia cinnamomea Was Strain Dependent in Malt Extract Media and Involved Specific Gene Expression. .............................................................................................................................................................................. 11 26. Cloning and characterization of the lanosterol 14alpha-demethylase gene from Antrodia cinnamomea. ......................... 11 27. The 4-acetylantroquinonol B isolated from mycelium of Antrodia cinnamomea inhibits proliferation of hepatoma cells. ................................................................................................................................................................................................. 12 28. A 90-day subchronic toxicological assessment of Antrodia cinnamomea in Sprague-Dawley rats. .................................. 12 29. Antioxidant activities of extracts and metabolites isolated from the fungus Antrodia cinnamomea.................................. 12 30. Antroquinonol from ethanolic extract of mycelium of Antrodia cinnamomea protects hepatic cells from ethanol-induced oxidative stress through Nrf-2 activation. ................................................................................................................................ 13 31. Evaluation of Genotoxicity of Antrodia cinnamomea in the Ames Test and the In Vitro Chromosomal Aberration Test. 13 32. Metabolite Profiles for Antrodia cinnamomea Fruiting Bodies Harvested at Different Culture Ages and from Different Wood Substrates. ..................................................................................................................................................................... 14 33. Anti-inflammatory effects of methanol extract of Antrodia cinnamomea mycelia both in vitro and in vivo..................... 14 34. 4-Acetylantroquinonol B Isolated from Antrodia cinnamomea Arrests Proliferation of Human Hepatocellular Carcinoma HepG2 Cell by Affecting p53, p21 and p27 Levels. ................................................................................................................ 15 35. Developmental Toxicity Assessment of Medicinal Mushroom Antrodia cinnamomea T.T. Chang et W.N. Chou (Higher Basidiomycetes) Submerged Culture Mycelium in Rats. ........................................................................................................ 15 36. Cytochrome P450 Genes in Medicinal Mushroom Antrodia cinnamomea T.T. Chang et W.N. Chou (Higher Basidiomycetes) are Strongly Expressed During Fruiting Body Formation. ........................................................................... 16 37. Methanol extract of Antrodia cinnamomea mycelia induces phenotypic and functional differentiation of HL60 into monocyte-like cells via an ERK/CEBP- signaling pathway. ................................................................................................. 16 38. Proteomic analysis of differently cultured endemic medicinal mushroom antrodia cinnamomea T.T. Chang et W.N. Chou from Taiwan. ............................................................................................................................................................................ 16 39. Medium modification to enhance the formation of bioactive metabolites in shake flask cultures of Antrodia cinnamomea by adding citrus peel extract. ................................................................................................................................................... 17 40. Ethanol Extracts of Fruiting Bodies of Antrodia cinnamomea Suppress CL1-5 Human Lung Adenocarcinoma Cells Migration by Inhibiting Matrix Metalloproteinase-2/9 through ERK, JNK, p38, and PI3K/Akt Signaling Pathways. ........... 17 2

41. Ethanol extracts of fruiting bodies of Antrodia cinnamomea exhibit anti-migration action in human adenocarcinoma CL1-0 cells through the MAPK and PI3K/AKT signaling pathways. ..................................................................................... 18 Below are from ScienceDirect ................................................................................................................................................. 19 1: Antioxidant properties of water-soluble polysaccharides from Antrodia cinnamomea in submerged culture ..................... 19 2: The hepatoprotective activity against ethanol-induced cytotoxicity by aqueous extract of Antrodia cinnamomea............. 19 3: Influence of nutritional components and oxygen supply on the mycelial growth and bioactive metabolites production in submerged culture of Antrodia cinnamomea ........................................................................................................................... 19 4: Characterization and biological functions of sulfated polysaccharides from sulfated-salt treatment of Antrodia cinnamomea ............................................................................................................................................................................. 20 5: The influence of environmental conditions on the mycelial growth of Antrodia cinnamomea in submerged cultures ....... 20 6: Triterpenoids from Antrodia cinnamomea ........................................................................................................................... 21 7: Purification and partial characterization of a lipase from Antrodia cinnamomea ................................................................ 21 8: A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea ..................................................... 21 9: Glucose stimulates production of the alkaline-thermostable lipase of the edible Basidiomycete Antrodia cinnamomea ... 21 10: Antrodia cinnamomea sp. nov. on Cinnamomum kanehirai in Taiwan .............................................................................. 22 11. Steroids and triterpenoids of Antodia cinnamomeaA fungus parasitic on Cinnamomum micranthum .......................... 22 12. Characterization and functional elucidation of a fucosylated 1,6--D-mannogalactan polysaccharide from Antrodiacinnamomea ...................................................................................................................................................... 22 13. Chemical profiling of the cytotoxic triterpenoid-concentrating fraction and characterization of ergostane stereo-isomer ingredients from Antrodia camphorata ................................................................................................................................... 22 14. Analysis of volatile compounds of Antrodia camphorata in submerged culture using headspace solid-phase microextraction ........................................................................................................................................................................ 23 15. Development of a LCMS/MS method for the determination of antrodin B and antrodin C from Antrodia camphorata extract in rat plasma for pharmacokinetic study ................................................................................... 23

1: Antrodia cinnamomea 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. 2007 Jul;45(7):1249-57. Epub 2007 Jan 17. Hsu YL, Kuo PL, Cho CY, Ni WC, Tzeng TF, Ng LT, Kuo YH, Lin CC. Department of Pharmacy, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan. In this study, we first report the anti-invasive effect of ethylacetate extract from Antrodia cinnamomea (EAC) fruiting bodies in the human liver cancer cell line PLC/PRF/5. Treatment with EAC decreased the cancer invasion of PLC/PRF/5 cells in a dose-dependent manner. This effect was strongly associated with a concomitant decrease in either the level or activity of VEGF, MMP-2, MMP-9 and MT1-MMP, and an increase in the expression of TIMP-1 and TIMP-2. EAC inhibited constitutively activated and inducible NF-kappaB in both its DNA-binding activity and transcriptional activity. Furthermore, EAC also inhibited the TNF-alpha-activated NF-kappaB-dependent reporter gene expression of MMP-9 and VEGF, and the invasion of cancer cells. EAC also exhibited an inhibitory effect on angiogenesis in a Matrigel Plug Angiogenesis Assay. Further investigation revealed that EAC's inhibition of cancer cell growth and invasion was also 3

evident in a nude mice model. Our results indicate that EAC inhibits the activation of NF-kappaB, and may provide a molecular basis for drug development using EAC as an anti-invasive agent in the prevention and treatment of cancer. PMID: 17316946 [PubMed - in process] 2: Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media. Bioresour Technol. 2007 Sep;98(13):2511-7. Epub 2006 Oct 27. Lin ES, Chen YH. Department of Cosmetic Science, Vanung University, No. 1 Van-Nung Road, Chung-Li, Taoyuan 320, Taiwan, ROC. Submerged cultures were used to identify growth-limiting nutrients by Antrodia cinnamomea strains. The mycelial biomass and EPS production by A. cinnamomea BCRC 35396 were markedly higher than other A. cinnamomea strains. A relatively high C/N ratio was favorable for both the mycelial growth (5.41g/l) and EPS production (0.55g/l); the optimum ratio was 40. The glucose was available utilized preferentially for mycelial growth, rather than for EPS production. Flushing the culture medium with nitrogen had a stimulating effect on both mycelial growth and EPS production. In addition, peptone, yeast extract and malt extract appeared to be important and significant component for EPS production. Phosphate ion, magnesium ion and thiamine were probably not essential for mycelial growth. By optimizing the effects of additional nutrition, the results showed that 5% (w/v) glucose, 0.8% (w/v) peptone, 0.8% (w/v) yeast extract, 0.8% (w/v) malt extract, 0.03% (w/v) KH(2)PO(4), 0.1% (w/v) MgSO(4).7H(2)O and 0.1% (w/v) thiamine could lead to the maximum production of EPS (1.36g/l). PMID: 17071080 [PubMed - in process] 3: Apoptotic effects of Antrodia cinnamomea fruiting bodies extract are mediated through calcium and calpain-dependent pathways in Hep 3B cells. Food Chem Toxicol. 2006 Aug;44(8):1316-26. Epub 2006 Feb 28. Kuo PL, Hsu YL, Cho CY, Ng LT, Kuo YH, Lin CC. Department of Biotechnology, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan. Antrodia cinnamomea is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. cinnamomea (EAC) fruiting bodies in Hep 3B, a liver cancer cell line. EAC decreased cell proliferation of Hep 3B cells by inducing apoptotic cell death. EAC treatment increased the level of calcium (Ca2+) in the cytoplasm and triggered the subsequent activation of calpain and caspase-12. EAC also initiated the mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 in Hep 3B cells. Furthermore, the mitochondrial apoptotic pathway amplified the calpain pathway by Bid and Bax interaction and Ca2+ translocation. We have therefore concluded that the molecular mechanisms during EAC-mediated proliferation inhibition in Hep 3B cells were due to: (1) apoptosis induction, (2) triggering of Ca2+/calpain pathway, (3) disruption of mitochondrial function, and (4) apoptotic signaling being amplified by cross-talk between the calpain/Bid/Bax and Ca2+/mitochondrial apoptotic pathways. Publication Types: Research Support, Non-U.S. Gov't PMID: 16600460 [PubMed - indexed for MEDLINE] 4: Protective effects of a neutral polysaccharide isolated from the mycelium of Antrodia cinnamomea on Propionibacterium acnes and lipopolysaccharide induced hepatic injury in mice. Chem Pharm Bull (Tokyo). 2006 Apr;54(4):496-500. Han HF, Nakamura N, Zuo F, Hirakawa A, Yokozawa T, Hattori M. Institute of Natural Medicine, University of Toyama, Japan. Mycelia of Antrodia cinnamomea were extracted with chloroform and hot water. A neutral polysaccharide named ACN2a separated from the water extract was purified using 10% CCl3COOH, and repeated column chromatography on HW-65 and DE-52 cellulose. Its structure was determined by chemical and spectroscopic analyses. ACN2a was composed of Gal, Glc, Fuc, Man and GalN (in the ratio 1:0.24:0.07:0.026:faint), in which an alpha-D-(1-->6)-Gal linkage accounted for 73% of all linkages. The ratio of branch points was about 16% of the total residual numbers, and branches were attached to C-2 of galactosyl residues of the main chain. ACN2a had an average molecular weight of 12.9x10(5) Daltons, [alpha]D25=+115 degrees (c=0.44, H2O); [eta]=0.0417dl.g-1, Cp=0.2663 cal/(g. degrees C). The hepatoprotective effect of ACN2a was 4

evaluated using a mouse model of hepatic injury that was induced by Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS). The administration of ACN2a (0.4, 0.8 g/kg/d, p.o.), significantly prevented increases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzyme activities in mice treated with P. acnes-LPS, indicating hepatoprotective activity in vivo. Publication Types: Research Support, Non-U.S. Gov't PMID: 16595952 [PubMed - indexed for MEDLINE] 5: Statistical optimization of medium components for the production of Antrodia cinnamomea AC0623 in submerged cultures. Appl Microbiol Biotechnol. 2006 Oct;72(4):654-61. Epub 2006 Feb 23. Chang CY, Lee CL, Pan TM. Institute of Microbiology and Biochemistry, National Taiwan University, 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan. The nutritional medium requirement for biomass and triterpenoid production by Antrodia cinnamomea AC0623 strain was optimized. Box-Behnken was applied to optimize biomass and triterpenoid production. According to response surface methodology (RSM), the optimum concentrations of N-source were determined. The results indicate that when a submerged culture in shake flasks was operated at 28 degrees C, initial pH 5.5, and rotation speed 105 rpm, the biomass and triterpenoid content in dry basis could be increased to 3.20% (w/w) and 31.8 mg/g, respectively. The experiments were further scaled up to 100- and 700-l fermentors. Higher content of triterpenoids (63.0 mg/g) was obtained in 700-l fermentations by means of the control of cultural conditions and the modification of medium composition based on the RSM. PMID: 16496140 [PubMed - indexed for MEDLINE] 6: Adenosine as an active component of Antrodia cinnamomea that prevents rat PC12 cells from serum deprivation-induced apoptosis through the activation of adenosine A2A receptors. Life Sci. 2006 Jun 13;79(3):252-8. Epub 2006 Jan 27. Lu MK, Cheng JJ, Lai WL, Lin YR, Huang NK. National Research Institute of Chinese Medicine, Taipei, Taiwan, No 155-1, Section 2, Li-Nung Street., Shipai, Pei-tou District (112), Taipei, Taiwan, ROC. Antrodia cinnamomea (formerly named Antrodia camphorata) is a rare medicinal fungus. We previously reported that it exhibits antioxidative, vasorelaxative, anti-inflammatory, and anti-angiogenic effects. When serum deprivation-induced apoptosis in neuronal-like PC12 cells was used as a stress model, the extract of A. cinnamomea displayed effectiveness in preventing serum-deprived apoptosis. Since our previous data show that the extract of A. cinnamomea contains adenosine (ADO), we attempt to investigate if the active component is ADO and to identify its targeting site in this study. After pre-incubation with ADO deaminase, neither ADO nor the extract of A. cinnamomea exerted any protection, demonstrating that the active component of A. cinnamomea is ADO. Furthermore, an ADO A(2A) receptor (A(2A)-R) antagonist was used and was able to block the protective effects of ADO and the extract of A. cinnamomea, demonstrating that the ADO targeting site in this model is A(2A)-R. Taken together, the protective effect of A. cinnamomea is owed to its active component, ADO, which acts through activation of A(2A)-R to prevent serum deprivation-induced PC12 cell apoptosis. Publication Types: Research Support, Non-U.S. Gov't PMID: 16443241 [PubMed - indexed for MEDLINE] 7: Cultivating conditions influence exopolysaccharide production by the edible Basidiomycete Antrodia cinnamomea in submerged culture. Int J Food Microbiol. 2006 Apr 25;108(2):182-7. Epub 2006 Jan 23. Lin ES, Sung SC. Department of Cosmetic Science, Vanung University, No. 1 Van-Nung Rd, Chung-Li, Taoyuan (32045), Taiwan, ROC. eslin@msa.vnu.edu.tw Antrodia cinnamomea is a medicinal fungus that has been used in Taiwan as a traditional medicine for the treatment of tumorigenic diseases. We prove that controlling the culturing conditions (i.e., temperature and pH) and modifying the composition of the medium (i.e., carbon, nitrogen, mineral sources and vitamins) can dramatically enhance the production of the exopolysaccharide of A. cinnamomea. We have found that the temperature, initial pH, and agitation time are all 5

critical for exopolysaccharide production during the cultivation of A. cinnamomea in submerged cultures; our optimized conditions were 28 degrees C, pH 5.5, and 14 days, respectively. In addition, when optimizing the effects of additional nutrition, we found that 5% (v/v) glucose, 0.5% (v/v) calcium nitrate, 0.1% (v/v) ferrous sulfate, and 0.1% (v/v) nicotinic acid led to the greatest production of exopolysaccharides; the exopolysaccharide production, mycelial biomass and specific product yield reached 0.49 g/l, 2.60 g/l and 0.19 g/g, respectively. The results indicate that nutrients can be utilized to improve the production of exopolysaccharide and that good mycelial growth does not seem to be a determining factor for a high production yield of exopolysaccharide in A. cinnamomea. Publication Types: Research Support, Non-U.S. Gov't PMID: 16434117 [PubMed - indexed for MEDLINE] 8: Antiangiogenic activities of polysaccharides isolated from medicinal fungi. FEMS Microbiol Lett. 2005 Aug 15;249(2):247-54. Chen SC, Lu MK, Cheng JJ, Wang DL. Department of Medicine, Taipei Medical University-Wan-Fang Hospital, Taiwan; Institute of Clinical Medicine, National Yang-Ming University Medical College, Taipei 116, Taiwan. Extracted polysaccharides from medicinal fungi, including Antrodia cinnamomea, Antrodia malicola, Antrodia xantha, Antrodiella liebmannii, Agaricus murrill, and Rigidoporus ulmarius, were investigated for their effects on vascular endothelial growth factor (VEGF)-induced tube formation in endothelial cells (ECs). Chemical analysis revealed that myo-inositol, sorbitol, fucose, galactosamine, glucosamine, galactose, glucose, and mannose were the neutral sugars in these polysaccharides. These fungal polysaccharides showed no toxicity to ECs. For the inhibition of endothelial tube formation, extracted polysaccharides from A. xantha and R. ulmarius were shown to produce greater inhibition compared to those from other fungi. Fucose, glucose and mannose were the predominant monosaccharides from these two fungi. These results suggest that monosaccharides may play a role in the inhibitory effect of these fungi on endothelial tube formation. In contrast to the inhibition on tube formation from polysaccharides of A. cinnamomea and A. malicola, polysaccharides from A. xantha and R. ulmarius, with molecular weight between 2693-2876 and 304-325 kDa, were critical for this inhibitory activity. Our results show that polysaccharides isolated from A. xantha and R. ulmarius provide greater antiangiogenesis than those from commercialized A. murrill (Brazilian mushroom) and A. cinnamomea. These studies provide a basis for the potential development of these polysaccharides for antiangiogenesis usage. Publication Types: Research Support, Non-U.S. Gov't PMID: 16046081 [PubMed - indexed for MEDLINE] 9: Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells. Life Sci. 2005 May 13;76(26):3029-42. Cheng JJ, Huang NK, Chang TT, Wang DL, Lu MK. National Research Institute of Chinese Medicine, Room 739, No155-1, Section 2, Li-Nung Street, Pei-tou District (112), Taipei, Taiwan. The main purposes of this study were to investigate the regulation of polysaccharides isolated from A. cinnamomea on vascular endothelial growth factor (VEGF)-induced cyclin D1 expression and down stream signaling pathway that may correlate with their anti-angiogenc effects in endothelial cells (ECs). Crude and fractionated polysaccharides (Fra-1 to Fra-4) of A. cinnamomea showed slightly toxicity to ECs as compared with their inhibition concentration on angiogenic-related gene expression. The crude extract and fractionated fractions, except for Fra-2, of A. cinnamomea polysaccharides significantly decreased VEGFR2 phosphorylation on tyrosine 1054/1059, cyclin D1 promotor activity, and protein expression induced by VEGF. Crude extract of A. cinnamomea polysaccharides inhibited the binding of VEGF to KDR/flk-1 in a dose-dependent manner. These results indicated that inhibition of VEGF interaction with VEGF receptor 2 is the mechanism serves A. cinnamomea as a protective mechanism composing the anti-angiogenesis function. Furthermore, A. cinnamomea polysaccharides also blocked VEGF-induced migration and capillary-like tube formation of ECs on Matrigel. Taken together, these results indicate that A. cinnamomea polysaccharides inhibit cyclin D1 expression through inhibition of VEGF receptor signaling, leading to the suppression of angiogenesis. Publication Types: Research Support, Non-U.S. Gov't PMID: 15850596 [PubMed - indexed for MEDLINE] 6

10: Zhankuic Acid F: A New Metabolite from a Formosan Fungus Antrodia cinnamomea. Planta Med. 1997 Feb;63(1):86-8. Shen YC, Yang SW, Lin CS, Chen CH, Kuo YH, Chen CF. Institute of Marine Resources, National Sun Yat-sen University, 70 Lien-Hai Rd., Kaohsiung, Taiwan, Republic of China. Zhankuic acid F ( 1), a new steroid acid, was isolated from the fruit bodies of ANTRODIA CINNAMOMEA Chang & Chou, SP. NOV. (Polyporaceae). The structure of 1 was elucidated by detailed analysis of 1D- and 2D-NMR spectra. Compound 1 appeared as a major product from the microbial transformation ( MUCOR RACEMOSUS) of zhankuic acid A ( 2). PMID: 17252333 [PubMed - in process] 11: New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum. J Nat Prod. 1995 Nov;58(11):1655-61. Chen CH, Yang SW, Shen YC. School of Pharmacy, National Taiwan University, Taipei, Republic of China. Three new steroids, zhankuic acids A [1], B [2], and C [3], were isolated from the fruiting bodies of Antrodia cinnamomea by bioassay-guided fractionation. The structures of these compounds were elucidated by chemical reactions and detailed analysis of their 1H- and 13C-nmr spectra. Biological studies revealed that 1 exhibited cytotoxic activity against P-388 murine leukemia cells and 2 showed weak anticholinergic and antiserotonergic activities. Publication Types: Research Support, Non-U.S. Gov't PMID: 8594142 [PubMed - indexed for MEDLINE] 12: Fermented Antrodia cinnamomea Extract Protects Rat PC12 Cells from Serum Deprivation-Induced Apoptosis: The Role of the MAPK Family. J Agric Food Chem. 2008 Jan 11 [Epub ahead of print] Lu MK, Cheng JJ, Lai WL, Lin YJ, Huang NK. andrew@mail.nricm.edu.tw. Antrodia cinnamomea (formerly A. camphorata) has recently and commercially been used in the formulation of nutraceuticals and functional foods in Taiwan. Because of its diverse properties, the neuroprotective effect was investigated using a fermented A. cinnamomea extract in this study. Serum deprivation-induced apoptosis in neuronal-like pheochromocytoma (PC12) cells was used as a cell stress model, and it was found that A. cinnamomea was effective in preventing serum-deprived apoptosis according to results of an MTT assay and Hoechst staining. Serum deprivation resulted in decreased phosphorylation of extracellular signal-regulated kinase (ERK) and increased phosphorylations of c-Jun NH 2-terminal kinase (JNK) and p38, of the family of mitogen-activated protein kinases (MAPKs); however, A. cinnamomea reversed these phenomena, supporting the antagonistic effects between ERK and JNK-p38 in regulating cell survival. The previously identified active component of A. cinnamomea, adenosine (ADO), also exerted the same effects as A. cinnamomea in preventing apoptosis and regulating phosphorylations of MAPKs. Although an inhibitor of the ERK upstream activator blocked A. cinnamomea-induced ERK phosphorylations, it failed to block the protection of A. cinnamomea and ADO. A protein kinase A (PKA) inhibitor blocked the protection by both A. cinnamomea and ADO. Both JNK and p38 inhibitors were effective in preventing the phosphorylations of JNK and p38 and serum deprivation-induced apoptosis. Collectively, A. cinnamomea prevented serum deprivation-induced PC12 cell apoptosis through a PKA-dependent pathway and by suppression of JNK and p38 activities. PMID: 18186605 [PubMed - as supplied by publisher] 13: Identification and characterization of a lipase gene from Antrodia cinnamomea. Mycol Res. 2008 Jul 4. [Epub ahead of print] Chu FH, Wang SY, Lee LC, Shaw JF. School of Forestry and Resource Conservation, National Taiwan University, Taipei 106, Taiwan. A partial (634bp) cDNA clone, AF1229, obtained from ESTs of solid-cultured basidiomes of Antrodia cinnamomea is homologous to the lipase gene in Rhizomucor miehei. 5'-RACE and 3'-RACE amplification showed that the full-length lipase gene, Ac-LIP, has a 912bp open reading frame (ORF), a 183bp 5' non-coding region, and a 144bp 3' non-coding region. Ac-LIP contains the lipase consensus sequence, VTVVGHSLGA, and encodes a 303-amino acid polypeptide that 7

appears to be an extracellular protein with a calculated molecular mass of 31.8kDa. RT-PCR analysis suggested that Ac-LIP was strongly expressed during the basidiomatal formation stage of A. cinnamomea. When over-expressed in Escherichia coli, Ac-LIP yielded a protein that was capable of performing hydrolysis of trilinolein by gas chromatography/mass spectrometry (GC/MS) analysis. A. cinnamomea lipase represents the first enzyme of the lipase family from a basidiomycetous fungus, which has been characterized at the molecular level. PMID: 18652894 [PubMed - as supplied by publisher] 14: Maleimide and maleic anhydride derivatives from the mycelia of Antrodia cinnamomea and their nitric oxide inhibitory activities in macrophages. J Nat Prod. 2008 Jul;71(7):1258-61. Epub 2008 Jun 4. Wu MD, Cheng MJ, Wang BC, Yech YJ, Lai JT, Kuo YH, Yuan GF, Chen IS. Food Industry Research and Development Institute, No 331, Shih-Pin Road, Hsinchu 300, Taiwan, Republic of China. On cultivation of the fungus Antrodia cinnamomea (BCRC 36799) on a medium, the mycelium was extracted and evaluated for nitric oxide (NO) inhibitory activity. Bioactivity-directed fractionation led to the isolation of two new maleimide derivatives, antrocinnamomins A (1) and B (2), and two new maleic anhydride derivatives, antrocinnamomins C (3) and D (4), along with three known compounds, 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione (5), 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5-dione (6), and 3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione (7). Structural elucidation of compounds 1-4 was carried out by spectroscopic data. Compound 1 displayed significant inhibitory effect on nitric oxide (NO) production. PMID: 18522430 [PubMed - in process] 15: Isolation and analysis of genes specifically expressed during basidiomatal development in Antrodia cinnamomea by subtractive PCR and cDNA microarray. FEMS Microbiol Lett. 2008 Mar;280(2):150-9. Epub 2008 Jan 22. Chu FH, Lee YR, Chou SJ, Chang TT, Shaw JF. School of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan. cDNAs specifically expressed at the basidiome stage were isolated by using PCR-selected cDNA subtraction in order to study gene regulation during porous-hymenium basidiomatal formation in Antrodia cinnamomea. blastx results suggested that most of the expressed sequence tags (52.4-69.5%) had no significant protein homology to genes from other published living things. cDNAs particularly expressed at different growing conditions were identified using cDNA microarray analysis. Reverse transcriptase PCR analyses confirmed that the clone putative to P-type ATPase, various cytochrome P450s and some unknown genes were abundant at natural basidiomes while endoglucanase was abundant at the tissue from artificial medium. PMID: 18218021 [PubMed - indexed for MEDLINE] 16. Development of an activation tagging system for the basidiomycetous medicinal fungus Antrodia cinnamomea. Mycol Res. 2008 Nov 27. [Epub ahead of print] Chen EC, Su YH, Kanagarajan S, Agrawal DC, Tsay HS. Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, 168 Gifeng E Road, Wufeng, Taichung County 41349, Taiwan, ROC. This study describes the development of an efficient and reliable activation tagging system for the medicinal fungus Antrodia cinnamomea. For successful Agrobacterium tumefaciens-mediated transformation, different parameters were considered. The Agrobacterium concentration of 5x10(8)cfuml(-1), 1mm acetosyringone, 25-d-old mycelia at 0.2gml(-1), and co-culture period of 6d were found to be the most optimal conditions for enhancing the transformation efficiency. The mitotic stability of transferred DNA (T-DNA) was demonstrated by growing eight randomly selected putative transformants in malt extract agar medium for five subcultures. Insertion of T-DNA into the genome of transformants was confirmed by PCR and Southern hybridization. Results showed that 88% of the mutants contained a single T-DNA insertion. Two of the mutants were observed with different triterpenoid profiles compared with the untransformed cultures. Our results suggest a new functional genomics approach to tag the triterpenoid biosynthesis genes in A. cinnamomea. PMID: 19059480 [PubMed - as supplied by publisher] 8

17. Inhibitory effects of antrodins A-E from Antrodia cinnamomea and their metabolites on hepatitis C virus protease. Phytother Res. 2008 Nov 11. [Epub ahead of print] Phuong DT, Ma CM, Hattori M, Jin JS. Institute of Natural Medicine, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan. Antrodia cinnamomea is a highly valued folk medicine used for liver cancer, a disease often caused by the long term infection of hepatitis C virus (HCV). In the present study, the maleic and succinic acid constituents (antrodins A-E) of this medicinal fungus, the in vivo metabolites of antrodin C and the analogue of one of the metabolites were tested for their inhibitory activity on HCV protease. Most of the compounds showed potent inhibitory activity, with antrodin A being the most potent (IC(50) = 0.9 microg/mL). Antrodin A was isolated as one of the constituents of A. cinnamomea and was also detected as an in vivo metabolite of the major constituent antrodin C. The mode of inhibition for antrodin A on HCV protease was revealed by a Lineweaver-Burk plot as competitive inhibition. These results strongly support the use of this folk medicine for liver cancer and HCV infection which is a global problem. Copyright (c) 2008 John Wiley & Sons, Ltd. PMID: 19003946 [PubMed - as supplied by publisher 18. Ethanolic extracts of Antrodia cinnamomea mycelia fermented at varied times and scales have differential effects on hepatoma cells and normal primary hepatocytes. J Food Sci. 2008 Sep;73(7):H179-85. Epub 2008 Aug 18 Chen YS, Pan JH, Chiang BH, Lu FJ, Sheen LY. Graduate Inst of Food Science and Technology, Natl Taiwan Univ, Taipei, Taiwan. 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 IC(50) of ethanolic extract of AC mycelia fermented for 8 wk in a 250 mL fermentor against Hep3B and HepG2 cells were 82.9 and 54.2 microg/mL, respectively. Furthermore, the IC(50) for Hep3B and HepG2, treated with ethanolic extract of AC mycelia fermented for 4 wk in the 5 L fermentor were 48.7 and 3.8 microg/mL, respectively. Those treated with ethanolic extract of AC mycelia fermented for 4 wk in the 500 L fermentor were 36.9 and 3.1 microg/mL, respectively. No adverse effects of all samples on normal primary rat hepatocytes were observed. PMID: 18803715 [PubMed - in process 19. Cloning and heterologous expression of a novel ligninolytic peroxidase gene from poroid brown-rot fungus Antrodia cinnamomea. Microbiology. 2009 Feb;155(Pt 2):424-33. Huang ST, Tzean SS, Tsai BY, Hsieh HJ. Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 10617, Taiwan, ROC. A novel ligninolytic peroxidase gene (ACLnP) was cloned and characterized from a poroid brown-rot fungus, Antrodia cinnamomea. The genomic DNA of the fungus harboured two copies of ACLnP, with a length of 2111 bp, interlaced with 12 introns, while the full-length cDNA was 1183 bp, with a 66 bp signal peptide and an ORF of 990 bp. The three-dimensional molecular structure model was comparable to that of the versatile peroxidase of Pleurotus eryngii. ACLnP was cloned into vector pQE31, successfully expressed in Escherichia coli strain M15 under the control of the T5 promoter and produced a non-glycosylated protein of about 38 kDa, pI 5.42. The native and recombinant ACLnP was capable of oxidizing the redox mediator veratryl alcohol, and also decolorized bromophenol blue and 2,6-dimethoxyphenol dyes, implicating a functional extracellular peroxidase activity. The significance of discovering a functional ACLnP gene in A. cinnamomea in terms of wood degradation and colonization capacity in its unique niche is discussed. PMID: 19202090 [PubMed - indexed for MEDLINE] 20. Compound MMH01 possesses toxicity against human leukemia and pancreatic cancer cells. Toxicol In Vitro. 2009 Apr;23(3):418-24. Epub 2009 Jan 22. Chen YJ, Chou CJ, Chang TT. 9

Department of Radiation Oncology, Mackay Memorial Hospital, 92 Chung San North Road, Section 2, Taipei 104, Taiwan. chenmdphd@yahoo.com MMH01 is a compound isolated from Antrodia cinnamomea. MMH01 markedly inhibited growth of human leukemia U937 and pancreatic cancer BxPC3 cells. It resulted in distinct patterns of cell cycle distribution in U937 (G2/M, sub-G1 and polyploidy) and BxPC3 cells (G0/G1 and sub-G1). The modes of cell death in U937 cells include apoptosis and mitotic catastrophe, whereas apoptosis-associated events or necrosis in BxPC3 cells. Neither mitochondrial membrane permeabilization nor caspase dependence was noted. Proteins involving mitotic catastrophe-associated cell death such as cyclin B1 and checkpoint kinase 2 were activated in U937 cells. Only slight to moderate viability inhibition was noted to human monocytes, the normal counterpart of these myeloid leukemic cells. In conclusion, MMH01 possesses cytotoxicity against human leukemia and pancreatic cancer cells. PMID: 19344682 [PubMed - indexed for MEDLINE 21: Anti-angiogenic effects and mechanisms of polysaccharides from Antrodia cinnamomea with different molecular weights. J Ethnopharmacol. 2009 Jun 25;123(3):407-12. Epub 2009 Mar 31. Yang CM, Zhou YJ, Wang RJ, Hu ML. Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan, ROC. ETHNOPHARMACOLOGICAL RELEVANCE: Antrodia cinnamomea is a popular medicinal mushroom in Taiwan that has been widely used for treatment of various cancers and liver diseases. AIM OF THE STUDY: This study aimed to investigate the immunomodulatory effect on angiogenesis of polysaccharides from mycelia of Antrodia cinnamomea (PMAC). MATERIALS AND METHODS: PMAC were extracted in boiling water, precipitated with 95% ethanol, and separated into four different molecular weights (<5, 5-30, 30-100, > 100 kDa). Tube formation and chorioallantoic membrane (CAM) assay were used to determine the in vitro and ex vivo anti-angiogenic effects. RESULTS: Only the PMAC-mononuclear cells (MNCs)-conditioned medium (CM) with MW > 100 kDa significantly and concentration-dependently decreased the secretion of vascular endothelial growth factor in human leukemia cells and inhibited the matrigel tube formation in human umbilical vein endothelial cells. Similarly only the PMAC-MNC-CM with MW > 100 kDa significantly and concentration-dependently increased the levels of interleukin (IL)-12 and interferon-gamma (IFN-gamma). In addition, the ex vivo CAM assay revealed that only the PMAC with MW>100 kDa significantly and dose-dependently inhibited neovascularization. CONCLUSIONS: PMAC with MW > 100 kDa are anti-angiogenic in vitro and ex vivo, and the effects are likely through immunomodulation. PMID: 19501273 [PubMed - in process] 22. A triterpenoid methyl antcinate K isolated from Antrodia cinnamomea promotes dendritic cell activation and Th2 differentiation. Eur J Immunol. 2009 Sep;39(9):2482-91. Yu YL, Chen IH, Shen KY, Huang RY, Wang WR, Chou CJ, Chang TT, Chu CL. Vaccine Research and Development Center, National Health Research Institutes, Miaoli, Taiwan. Dendritic cells (DC) play a central role in the initiation and regulation of immune responses. Increasing evidence has indicated that manipulation of DC can serve as a therapeutic mechanism for immunomodulation. In this study we tested some unique compounds isolated from Antrodia cinnamomea, a medicinal fungus in Taiwan, on mouse bone marrow-derived DC activation. A triterpenoid methyl antcinate K (me-AntK) promoted DC maturation by enhancing the expression of MHC class II, CD86, and reducing the endocytosis. TNF-alpha, MCP-1, and MIP-1beta were secreted by DC after me-AntK treatment, indicating augmentation of innate immunity by me-AntK. Interestingly, the me-AntK-activated DC induced Ag-specific T-cell proliferation and facilitated Th2 differentiation. Examining signaling responses, we found that me-AntK treatment uniquely activated JNK and ERK in DC. Our results demonstrate that me-AntK is the first natural triterpenoid to promote the ability of DC to prime Th2 responses. This suggests that me-AntK can potentially be applied to enhance immune responses and modulate DC function in immunotherapy. PMID: 19701888 [PubMed - in process] 23. Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea. 10

Mycol Res. 2009 Feb 9. [Epub ahead of print] Chu FH, Chen YR, Lee CH, Chang TT. School of Forestry and Resource Conservation, National Taiwan University, Taipei 106, Taiwan. Mago nashi (Mago) and Y14 proteins, highly conserved among eukaryotes, participate in mRNA localization and splicing, and as such play important roles in oogenesis, embryogenesis and germ-line sex determination during animal development. Here we identified mago (Acmago) and Y14 (AcY14) homologues derived from Antrodia cinnamomea. Acmago encodes 149 amino acids and AcY14 encodes 168 amino acids. Multiple amino acid sequence alignment as well as secondary and tertiary structure prediction showed that AcMago and AcY14 have similar protein structure to the reported crystal structures of other Mago and Y14 proteins. During fungal development both Acmago and AcY14 genes were abundantly expressed in natural basidiomes. This is the first report of the molecular characterization and expression analysis of the mago and Y14 genes from fungi. PMID: 19640396 [PubMed - as supplied by publisher] 24. Characterization and heterologous expression of a novel lysophospholipase gene from Antrodia cinnamomea. J Appl Microbiol. 2009 Sep 29. [Epub ahead of print] Hsu KH, Wang SY, Chu FH, Shaw JF. School of Forestry and Resource Conservation, Nation Taiwan University, Taipei, Taiwan. Abstract Aims: A novel lysophospholipase (LysoPL) from the basidiomycetous fungi Antrodia cinnamomea named ACLysoPL was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: The gene encoding ACLysoPL was obtained from expressed sequence tags from A. cinnamomea. The full length of this gene has a 945 -bp open reading frame encoding 314 amino acids with a molecular weight of 35.5 kDa. ACLysoPL contains a lipase consensus sequence (GXSXG) motif and a Ser-His-Asp catalytic triad. A putative peroxisomal targeting signal type 1 was found in the C-terminal. Heterologous expression of ACLysoPL in E. coli showed that the enzyme preferentially hydrolyses long-chain acyl esterases at pH 7 and 30 degrees C. ACLysoPL is a psychrophilic enzyme about 40% of whose maximum activity remained at 4 degrees C. The LysoPL activities with lysophospholipids as substrate were analysed by gas chromatography/mass spectrometry. Conclusion: We have identified and characterized a gene named ACLysoPL encoding a protein performing LysoPL and esterase activities. Significance and Impact of the Study: This is the first LysoPL of A. cinnamomea identified and characterized at the molecular level. PMID: 19849771 [PubMed - as supplied by publisher] 25: Fructification of Antrodia cinnamomea Was Strain Dependent in Malt Extract Media and Involved Specific Gene Expression. J Agric Food Chem. 2009 Nov 13. [Epub ahead of print] Chu YC, Yang RM, Chang TT, Chou JC. Department of Natural Resources and Environmental Studies, National Dong Hwa University, Shou-feng, Hualien 97401, Taiwan. Antrodia cinnamomea is an expensive medicinal fungus that grows only inside the rotten trunk of Cinnamomum kanehirae . In vitro culture of A. cinnamomea fruiting body is difficult and, therefore, of value for further investigation. To study whether the fructification of A. cinnamomea is strain dependent in artificial media, we grew four different A. cinnamomea strains on malt extract agar (MEA) media. The standard MEA and a series of dilution of the MEA nutrient components were made to culture A. cinnamomea. The formation of fruiting body was determined by visual and microscopic observation on A. cinnamomea's porous morphogenesis and HPLC analysis. All A. cinnamomea strains cultured grew best in 50% MEA, but carried different capabilities of fructification. In addition, we studied four antioxidation- or senescence-related genes, including a cytochrome P450, a glutathione-S-transferase, a peroxiredoxin, and a manganese superoxide dismutase. We found both cytochrome P450 and glutathione-S-transferase were expressed 3.66- and 2.75-fold in fruiting body compared with mycelium, respectively, and perxoiredoxin and manganese superoxide dismutase were found with similar expressions in both fruiting body and mycelium. PMID: 19911844 [PubMed - as supplied by publisher] 26. Cloning and characterization of the lanosterol 14alpha-demethylase gene from Antrodia cinnamomea. J Agric Food Chem. 2010 Apr 28;58(8):4800-7. 11

Lee CH, Hsu KH, Wang SY, Chang TT, Chu FH, Shaw JF. School of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan. Abstract: Sterol 14alpha-demethylase (CYP51) is one of the key enzymes for sterol biosynthesis in fungi; it is widely distributed in all members of the cytochrome P450 superfamily. In this study, AcCyp51, encoding a cytochrome P450 sterol 14alpha-demethylase, was obtained from the sequences of EST libraries of Antrodia cinnamomea by using 5' RACE and genome walking methods. The open reading frame of AcCyp51 is 1635 bp and encodes 544 amino acids. The recombinant protein of AcCYP51 fused with glutathione-S-transferase from Escherichia coli revealed the demethylating activity by using lanosterol as substrate and GC-MS analysis. Gene expression levels of AcCYP51 were higher in natural basidiomes than in other cell types. Transcription of AcCYP51 increased in various culture conditions including adding squalene, lanosterol, itroconazole, and oleic acid as inducers. These reveal the important functions of AcCYP51 in basidiomatal formation and suggest that it might participate in other biological processes. PMID: 20334412 [PubMed - in process] 27. The 4-acetylantroquinonol B isolated from mycelium of Antrodia cinnamomea inhibits proliferation of hepatoma cells. J Sci Food Agric. 2010 May 19;90(10):1739-1744. [Epub ahead of print] Lin YW, Pan JH, Liu RH, Kuo YH, Sheen LY, Chiang BH. Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan, ROC. Abstract: BACKGROUND: Antrodia cinnamomea is known for its antihepatoma activity, yet the identity of its active compound was unclear. In this study, a 5-ton fermenter was used to prepare sufficient mycelium of A. cinnamomea for active compound isolation and identification. RESULTS: Using antiproliferative activity toward HepG2 cells as guidance in the isolation process, 4-acetylantroquinonol B was purified and identified to be the major bioactive compound of A. cinnamomea cultivated by submerged fermentation. The median effective doses (EC(50)) of 4-acetylantroquinonol B for HepG2 cells were 0.10 +/- 0.00 and 0.08 +/- 0.00 microg mL(-1) for 72 and 96 h treatments, respectively. The selective indices of 4-acetylantroquinonol B were 100 and 125 for 72 and 96 h treatments, respectively, indicating that this compound had high selective activity for hepatoma cells. CONCLUSION: 4-Acetylantroquinonol B is the major antihepatoma constituent of Antrodia cinnamomea mycelium produced by submerged fermentation. Copyright (c) 2010 Society of Chemical Industry. PMID: 20564437 [PubMed - as supplied by publisher] 28. A 90-day subchronic toxicological assessment of Antrodia cinnamomea in Sprague-Dawley rats. Food Chem Toxicol. 2010 Nov 18. [Epub ahead of print] Chen TI, Chen CC, Lin TW, Tsai YT, Nam MK. Grape King Biotechnology Inc., Chungli, Taiwan. Abstract: Antrodia cinnamomea (Ac) is a medicinal mushroom widely used for the treatment of abdominal pain, hypertension and hepatocellular carcinoma, but subchronic toxicity of this material has not yet been investigated. This present study was conducted to assess the 90-day oral toxicity of A. cinnamomea from submerged culture in male and female Sprague-Dawley (SD) rats. Eighty rats were divided into four groups, each consisting of ten male and ten female rats. Test articles were administered by oral gavage to rats at 3000, 2200 and 1500mg/kg BW/day for 90 consecutive days and reverse osmosis water was used as control. All animals survived to the end of the study. During the experiment period, no abnormal changes were observed in clinical signs, body weight and ophthalmological examinations. No significant differences were found in urinalysis, hematology and serum biochemistry parameters between the treatment and control groups. Necropsy and histopathological examination indicated no treatment-related changes. According to the above results, the no-observed-adverse-effect level (NOAEL) of Antrodia cinnamomea is identified to be greater than 3000mg/kg BW/day in Sprague-Dawley rats. Copyright 2010. Published by Elsevier Ltd. PMID: 21093523 [PubMed - as supplied by publisher] 29. Antioxidant activities of extracts and metabolites isolated from the fungus Antrodia cinnamomea. 12

Nat Prod Res. 2011 Feb 15:1-9. [Epub ahead of print] Wu MD, Cheng MJ, Wang WY, Huang HC, Yuan GF, Chen JJ, Chen IS, Wang BC. Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), Hsinchu 300, Taiwan. Abstract: Three different solvent partitions (n-hexane, ethyl acetate [EtOAc] and n-BuOH) of the culture broth from Antrodia cinnamomea were assayed with two different radical scavenging methods: 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and superoxide radical scavenging (SOD) assay. The EtOAc layer exhibited the best antioxidant activity. Two major antioxidant metabolites were isolated from the active EtOAc layer. The antioxidant activities of compounds 1-6 were further evaluated by DPPH, SOD and trolox equivalent antioxidant capacity (TEAC) assays. Compounds 3 and 5 showed stronger free radical scavenging than the reference BHA, ED(50) = 1.36 and 34.24 M. Compound 5 displayed moderate SOD activity (ED(50) = 310.0 M), and its antioxidant capacity of TEAC value was 2.2 mM trolox equivalency. PMID: 21337253 [PubMed - as supplied by publisher] 30. Antroquinonol from ethanolic extract of mycelium of Antrodia cinnamomea protects hepatic cells from ethanol-induced oxidative stress through Nrf-2 activation. J Ethnopharmacol. 2011 Jun 14;136(1):168-77. Epub 2011 Apr 20. Kumar KJ, Chu FH, Hsieh HW, Liao JW, Li WH, Lin JC, Shaw JF, Wang SY. Source: Department of Forestry, National Chung Hsing University, Kou Kung Road, Taichung 402, Taiwan. Abstract: AIM OF THE STUDY: In recent years, the medicinal mushroom Antrodia cinnamomea, known as "niu-chang chih" has received much attention with regard to its possible health benefits; especially its hepatoprotective effects against various drugs, toxins, and alcohol induced liver diseases. However, the molecular mechanism underlying this protective effect of Antrodia cinnamomea and its active compound antroquinonol was poorly understood. In the present study we evaluated to understand the hepatoprotective efficacy of antroquinonol and ethanolic extracts of mycelia of Antrodia cinnamomea (EMAC) in vitro and in vivo. MATERIALS AND METHODS: The protective mechanism of antroquinonol and EMAC against ethanol-induced oxidative stress was investigated in cultured human hepatoma HepG2 cells and ICR mice model, respectively. HepG2 cells were pretreated with antroquinonol (1-20M) and oxidative stress was induced by ethanol (100mM). Meanwhile, male ICR mice were pretreated with EMAC for 10 days and hepatotoxicity was generated by the addition of ethanol (5g/kg). Hepatic enzymes, cytokines and chemokines were determined using commercially available assay kits. Western blotting and real-time PCR were subjected to analyze HO-1 and Nr-2 expression. EMSA was performed to monitor Nrf-2 ARE binding activity. Possible changes in hepatic lesion were observed using histopathological analysis. RESULTS: Antroquinonol pretreatment significantly inhibited ethanol-induced AST, ALT, ROS, NO, MDA production and GSH depletion in HepG2 cells. Western blot and RT-PCR analysis showed that antroquinonol enhanced Nrf-2 activation and its downstream antioxidant gene HO-1 via MAPK pathway. This mechanism was then confirmed in vivo in an acute ethanol intoxicated mouse model: serum ALT and AST production, hepatocellular lipid peroxidation and GSH depletion was prevented by EMAC in a dose-dependent manner. EMAC significantly enhanced HO-1 and Nrf-2 activation via MAPKs consistent with in vitro studies. Ethanol-induced hepatic swelling and hydropic degeneration of hepatocytes was significantly inhibited by EMAC in a dose-dependent manner. CONCLUSIONS: These results provide a scientific basis for the hepatoprotective effects of Antrodia cinnamomea. Data also imply that antroquinonol, a potent bioactive compound may be responsible for the hepatoprotective activity of Antrodia cinnamomea. Moreover, the present study highly supported our traditional knowledge that Antrodia cinnamomea as a potential candidate for the treatment of alcoholic liver diseases. Copyright 2011 Elsevier Ireland Ltd. All rights reserved. PMID: 21540101, [PubMed - in process] 31. Evaluation of Genotoxicity of Antrodia cinnamomea in the Ames Test and the In Vitro Chromosomal Aberration Test. In Vivo. 2011 May-Jun;25(3):419-23. Wu MF, Peng FC, Chen YL, Lee CS, Yang YY, Yeh MY, Liu CM, Chang JB, Wu RS, Yu CC, Lu HF, Chung JG. 13

Source: China Medical University, No 91, Hsueh-Shih Road, Taichung City 404, Taiwan, R.O.C. Tel: +886 4220533662500, jgchung@mail.cmu.edu.tw. Abstract: Antrodia cinnamomea is an expensive and highly valued folk medicinal fungus that grows only inside the rotten trunk of Cinnamomum kanehirae, an evergreen broad-leaved tree. This fungus has recently been used commercially in the formulation of nutraceuticals and functional foods in Taiwan. It has been used for centuries as a detoxificant in cases of food poisoning, diarrhea, vomiting, hepatic disease and various kinds of cancers. The present study investigated the effects of Antrodia cinnamomea on mutagenicity using a bacterial reverse mutation assay employing the Salmonella typhimurium strains TA97, TA98, TA100, TA102, and TA1535. The effects of Antrodia cinnamomea on chromosome structure were tested in Chinese hamster ovary (CHO) cells. Antrodia cinnamomea was not mutagenic in all bacterial strains and it was not genotoxic in CHO cells. PMID: 21576417, [PubMed - in process] 32. Metabolite Profiles for Antrodia cinnamomea Fruiting Bodies Harvested at Different Culture Ages and from Different Wood Substrates. J Agric Food Chem. 2011 Jun 21. [Epub ahead of print] Lin TY, Chen CY, Chien SC, Hsiao WW, Chu FH, Li WH, Lin CC, Shaw JF, Wang SY. Source: Department of Forestry, National Chung-Hsing University, Taichung, Taiwan. Abstract: Antrodia cinnamomea is a precious edible fungus endemic to Taiwan that has long been used as a folk remedy for health promotion and for treating various diseases. In this study, an index of 13 representative metabolites from the ethanol extract of A. cinnamomea fruiting body was established for use in quality evaluation. Most of the index compounds selected, particularly the ergostane-type triterpenoids and polyacetylenes, possess good anti-inflammation activity. A comparison of the metabolite profiles of different ethanol extracts from A. cinnamomea strains showed silmilar metabolites when the strains were grown on the original host wood (Cinnamomum kanehirai) and harvested after the same culture time period (9 months). Furthermore, the amounts of typical ergostane-type triterpenoids in A. cinnamomea increased with culture age. Culture substrates also influenced metabolite synthesis; with the same culture age, A. cinnamomea grown on the original host wood produced a richer array of metabolites than A. cinnamomea cultured on other wood species. We conclude that analysis of a fixed group of compounds including triterpenoids, benzolics, and polyacetylenes constitutes a suitable, reliable system to evaluate the quality of ethanol extract from A. cinnamomea fruiting bodies. The evaluation system established in this study may provide a platform for analysis of the products of A. cinnamomea. PMID: 21668009 [PubMed - as supplied by publisher] 33. Anti-inflammatory effects of methanol extract of Antrodia cinnamomea mycelia both in vitro and in vivo. J Ethnopharmacol. 2011 Jun 16. [Epub ahead of print] Wen CL, Chang CC, Huang SS, Kuo CL, Hsu SL, Deng JS, Huang GJ. Source: Taiwan Seed Improvement and propagation Station, Council of Agriculture, Propagation Technology Section, Taichung, Taiwan; School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan; Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan. Abstract: AIMS OF THE STUDY: Antrodia cinnamomea is a folk medicinal mushroom commonly used in Taiwan for the treatment of several types of cancers and inflammatory disorders. This study aimed to explore the folk use of Antrodia cinnamomea on pharmacological grounds to characterize the scientific basis of anti-inflammatory activity. MATERIALS AND METHODS: The in vitro anti-inflammatory activity of methanol extract of liquid cultured mycelia of Antrodia cinnamomea (MEMAC) was judged by the measurement of the produced levels of pro-inflammatory cytokines and mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and human peripheral blood mononuclear cells (PBMCs). The in vivo anti-inflammatory activity of MEMAC was evaluated using carrageenan-induced hind paw edema in mice, the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver and the levels of malondialdehyde (MDA) and nitrite oxide (NO) in the edema paw. The levels of serum NO and TNF- were measured. The MEMAC was administered at 14

the concentrations of 100, 200, and 400mg/kg body weight of mouse. RESULTS: MEMAC inhibited the production of LPS-induced pro-inflammatory cytokines (TNF- and IL-6) and mediators (NO and PGE2) in RAW264.7 cells and human PBMCs. Data from Western blotting showed that MEMAC decreased the levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in LPS-stimulated RAW264.7 macrophages. In vivo, MEMAC showed significant (p<0.05) anti-inflammatory activity by reducing the edema volume in carrageenan-induced paw edema in mice. MEMAC (400mg/kg) also reduced the carrageenan-induced leukocyte migration (50.925.71%). Further, MEMAC increased the activities of CAT, SOD, and GPx in the liver tissue and decreased the levels of serum NO and TNF- after carrageenan administration. CONCLUSIONS: Our results showed that MEMAC has the anti-inflammatory property both in vitro and in vivo, suggesting that it may be a potential preventive or therapeutic candidate for the treatment of inflammatory disorders. Copyright 2011. Published by Elsevier Ireland Ltd. PMID: 21704694 [PubMed - as supplied by publisher] 34. 4-Acetylantroquinonol B Isolated from Antrodia cinnamomea Arrests Proliferation of Human Hepatocellular Carcinoma HepG2 Cell by Affecting p53, p21 and p27 Levels. J Agric Food Chem. 2011 Jul 26. [Epub ahead of print] Lin YW, Chiang BH. Institute of Food Science and Technology, National Taiwan University , Taipei, Taiwan. Abstract: The 4-acetylantroquinonol B isolated from the mycelium of Antrodia cinnamomea could inhibit proliferation of hepatocellular carcinoma cells HepG2 with IC (50) 0.1 g/mL. When the HepG2 cells were treated with 4-acetylantroquinonol B for 72 h, the proportion of cells in the G1 phase of the cell cycle increased and that in the S phase decreased significantly, and the proportion of G2/M phase cells were not obviously changed. In addition, the 4-acetylantroquinonol B treatment resulted in the decreases of CDK2 and CDK4, and an increase of p27 in a dose-dependent manner. The protein levels of p53 and p21 proteins were also increased when the cells were treated with low dosage (0.1 g/mL) of 4-acetylantroquinonol B. Higher dosages, however, decreased the expression of p53 and p21 proteins. Assay of RT-PCR indicated that, corresponding to the increases of p53 and p21 proteins at the dosage of 0.1 g/mL, the mRNAs of p53 and p21 showed 1.66- and 1.61-fold upregulations, respectively. Corresponding to the decreases of CDK2 and CDK4 proteins, the mRNAs of CDK2 and CDK4 showed -1.02- and -1.13-fold downregulations, respectively. However, level of p27 mRNA showed -1.2-fold downregulation in spite of the increase in p27 protein. This observation, again, confirms the fact that the p27 gene rarely undergoes homozygous inactivation in cancer cells. Our finding suggested that the 4-acetylantroquinonol B inhibits proliferation of HepG2 cells via affecting p53, p21 and p27 proteins, and can be considered as a potential cancer drug. PMID: 21739974, [PubMed - as supplied by publisher] 35. Developmental Toxicity Assessment of Medicinal Mushroom Antrodia cinnamomea T.T. Chang et W.N. Chou (Higher Basidiomycetes) Submerged Culture Mycelium in Rats. Int J Med Mushrooms. 2011;13(6):505-11. Chen TI, Chen CW, Lin TW, Wang DS, Chen CC. Source: Grape King Inc., 60 Sec. 3, Lung-Kang Rd, Chung-Li City 320, Taiwan. Abstract: Antrodia cinnamomea is a Taiwanese medicinal mushroom with high antioxidant and polysaccharide content. The objective of this study is to investigate developmental toxicity of A. cinnamomea in pregnant Sprague-Dawley rats. Animals were daily gavaged with A. cinnamomea mycelium at dosage levels of 0 (reverse osmosis water), 50, 150, and 500 mg/kg from gestation day (GD) 6 to 15. All dams were sacrificed on GD 20 and were subjected to cesarean section. Fetuses were examined for external, visceral, and skeletal abnormalities. All copulated females survived until the end of the study. No significant differences were recorded in body weight change, food consumption, and maternal gestational parameters. Only two fetal malformations were noted in 970 fetuses from the treatment groups. Some variations, such as enlarged fontanel, split sternebrae, absent sacral, absent caudal vertebral centra, absent thoracic centra, absent 13th-14th ribs, and fused ribs, were found during the skeletal examination, but no treatment-induced abnormalities occurred. No dose dependency was observed in any of the developmental variations. Overall observation of foetal malformations from rats given A. cinnamomea mycelium during pregnancy demonstrates that this material is not teratogenic at doses up to 500 mg/kg. It is concluded that A. cinnamomea BCRC 35398 mycelium has no teratogenic effects in female rats and is safe to 15

be used as a functional food ingredient. PMID: 22181838, [PubMed - in process] 36. Cytochrome P450 Genes in Medicinal Mushroom Antrodia cinnamomea T.T. Chang et W.N. Chou (Higher Basidiomycetes) are Strongly Expressed During Fruiting Body Formation. Int J Med Mushrooms. 2011;13(6):513-23. Hsu KH, Lee YR, Lin YL, Chu FH. Source: School of Forestry and Resource Conservation, National Taiwan University, Taipei 106, Taiwan. Abstract: Medicinal mushroom Antrodia cinnamomea is a higher Basidiomycetes endemic to Taiwan, where it is commonly used as a traditional folk medicine. It is well known for its multiple biologic activities and its potential for commercial development. Here, ten full lengths of cytochrome P450 (CYP) genes (ac-1 to ac-10) from A. cinnamomea were cloned and identified. With the exception of ac-3 and ac-8, which will probably be assigned as new CYP families, these genes had more than 40% amino acid identity and close evolutionary relationships to known CYPs. Among the ten genes, only Ac-7 did not possess a transmembrane domain but had an N-terminal signal peptide, so it was considered a novel extracellular CYP. The ten A. cinnamomea CYPs had different expression profiles in different growth conditions. In general, they were strongly expressed during the formation of fruiting bodies, especially in natural basidiomycetes. The expression of six CYPs of A. cinnamomea (ac-1 to ac-3 and ac-5 to ac-7) were strictly inhibited in the mycelia cell type. It was therefore concluded that these CYPs are most active in the fruiting bodies of A. cinnamomea. PMID: 22181839, [PubMed - in process] 37. Methanol extract of Antrodia cinnamomea mycelia induces phenotypic and functional differentiation of HL60 into monocyte-like cells via an ERK/CEBP- signaling pathway. Phytomedicine. 2012 Mar 15;19(5):424-35. Epub 2012 Jan 30. Wen CL, Teng CL, Chiang CH, Chang CC, Hwang WL, Kuo CL, Hsu SL. Source: Taiwan Seed Improvement and Propagation Station, Council of Agriculture, Propagation Technology Section, Taichung, Taiwan; School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung, Taiwan. Abstract: Antrodia cinnamomea (named as Niu-chang-chih), a well-known Taiwanese folk medicinal mushroom, has a spectrum of biological activities, especially with anti-tumor property. This study was carried out for the first time to examine the potential role and the underlying mechanisms of A. cinnamomea in the differentiation of human leukemia HL60 cells. We found that the methanol extract of liquid cultured mycelia of A. cinnamomea (MEMAC) inhibited proliferation and induced G1-phase cell cycle arrest in HL60 cells. MEMAC could induce differentiation of HL60 cells into the monocytic lineage, as evaluated by the morphological change, nitroblue tetrazolium reduction assay, non-specific esterase assay, and expression of CD14 and CD11b surface antigens. In addition, MEMAC activated the extracellular signal-regulated kinase (ERK) pathway and increased CCAAT/enhancer-binding protein (C/EBP) expression. Reverse transcriptase polymerase chain reaction analysis showed that MEMAC upregulated the expression of C/EBP and CD14 mRNA in HL60 cells. DNA affinity precipitation assay and chromatin immunoprecipitation analyses indicated that MEMAC enhanced the direct binding of C/EBP to its response element located at upstream of the CD14 promoter. Furthermore, inhibiting ERK pathway activation with PD98059 markedly blocked MEMAC-induced HL60 monocytic differentiation. Consistently, the MEMAC-mediated upregulation of C/EBP and CD14 was also suppressed by PD98059. These findings demonstrate that MEMAC-induced HL60 cell monocytic differentiation is via the activating ERK signaling pathway, and downstream upregulating the transcription factor C/EBP and differentiation marker CD14 gene, suggesting that MEMAC might be a potential differentiation-inducing agent for treatment of leukemia. Copyright 2011 Elsevier GmbH. All rights reserved. PMID: 22293124 [PubMed - in process] 38. Proteomic analysis of differently cultured endemic medicinal mushroom antrodia cinnamomea T.T. Chang et W.N. Chou from Taiwan. Int J Med Mushrooms. 2011;13(5):473-81. Lin YL, Wen TN, Chang ST, Chu FH. Source: School of Forestry and Resource Conservation, National Taiwan University, Taipei 106, Taiwan. 16

Abstract: Antrodia cinnamomea is peculiar to Taiwan. It only grows on one host and is highly valued as an important component of several traditional Chinese medicines. In this study, the different protein expression profiles of artificially cultivated vegetative mycelium and wild-type basidiomatal fruiting bodies were compared and unique protein spots from wild-type basidiomatal fruiting body were investigated using 2D polyacrylamide gel electrophoresis and LC-MS/MS protein identification. Most of the wild-type proteins not seen in the artificially cultivated mycelium were associated to function in metabolism, cell stress, ROS scavenging, and cell growth. Several proteins from wild-type basidiomes, such as catalase, aryl-alcohol dehydrogenase, S-adenosyl-L-homocysteine hydrolase, intradiol dioxygenase, haloacid dyhydrogenase, alpha- and beta-form tubulin, prohibitin, septin, chaperone, and HSP90 ATPase, showed higher expression than those from artificially cultured mycelium at the mRNA level. PMID: 22324413 [PubMed - in process] 39. Medium modification to enhance the formation of bioactive metabolites in shake flask cultures of Antrodia cinnamomea by adding citrus peel extract. Bioprocess Biosyst Eng. 2012 Feb 26. [Epub ahead of print] Yang FC, Ma TW, Chuang YT. Source: Department of Chemical and Materials Engineering, Tunghai University, Taichung, 40704, Taiwan, fcyang@thu.edu.tw. Abstract: Antrodia cinnamomea has recently become a well-known medicinal mushroom in Taiwan. Bioactive compounds found in A. cinnamomea include: polysaccharide, sesquiterpene lactone, steroids and triterpenoids. The aim of this study was to evaluate the feasibility of adding citrus peel extract to enhance the formation of bioactive metabolites in the submerged culture of A. cinnamomea. With the exception of grapefruit, citrus peel extracts tested were proved to be beneficial to mycelial growth and to the production of intracellular polysaccharide. Lemon was the most effective for enhancing bioactive metabolite production. With an addition of 2% (v/v), the mycelium biomass concentration and intracellular polysaccharide content rose from 11.96 g DW/L of the control and 123.6 mg/g DW to 21.96 g DW/L and 230.8 mg/g DW, respectively, on day 8. The production of triterpenoids also increased from 86.7 to 282.9 mg/L. Moreover, this study also demonstrates that although the addition of peel extract could cause the lengthening of the exponential phase and reduce the specific growth rate, the production rate of biomass, intracellular polysaccharide and triterpenoids was still enhanced significantly. PMID: 22367480 [PubMed - as supplied by publisher] 40. Ethanol Extracts of Fruiting Bodies of Antrodia cinnamomea Suppress CL1-5 Human Lung Adenocarcinoma Cells Migration by Inhibiting Matrix Metalloproteinase-2/9 through ERK, JNK, p38, and PI3K/Akt Signaling Pathways. Evid Based Complement Alternat Med. 2012;2012:378415. Epub 2012 Feb 21. Chen YY, Liu FC, Chou PY, Chien YC, Chang WS, Huang GJ, Wu CH, Sheu MJ. Source: School of Pharmacy, China Medical University, 91 Hsueh-Shih Road, Taichung 404, Taiwan. Abstract: Cancer metastasis is a primary cause of cancer death. Antrodia cinnamomea (A. cinnamomea), a medicinal mushroom in Taiwan, has shown antioxidant and anticancer activities. In this study, we first observed that ethanol extract of fruiting bodies of A. cinnamomea (EEAC) exerted a concentration-dependent inhibitory effect on migration and motility of the highly metastatic CL1-5 cells in the absence of cytotoxicity. The results of a gelatin zymography assay showed that A.cinnamomea suppressed the activities of matrix metalloproteinase-(MMP-) 2 and MMP-9 in a concentration-dependent manner. Western blot results demonstrated that treatment with A. cinnamomea decreased the expression of MMP-9 and MMP-2; while the expression of the endogenous inhibitors of these proteins, that is, tissue inhibitors of MMP (TIMP-1 and TIMP-2) increased. Further investigation revealed that A. cinnamomea suppressed the phosphorylation of ERK1/2, p38, and JNK1/2. A. cinnamomea also suppressed the expressions of PI3K and phosphorylation of Akt. Furthermore, treatment of CL1-5 cells with inhibitors specific for PI3K (LY 294002), ERK1/2 (PD98059), JNK (SP600125), and p38 MAPK (SB203580) decreased the expression of MMP-2 and MMP-9. This is the first paper confirming the antimigration activity of this potentially beneficial mushroom against human lung adenocarcinoma CL1-5 cancer cells. PMID: 22454661, [PubMed - in process] PMCID: PMC3291113

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41. Ethanol extracts of fruiting bodies of Antrodia cinnamomea exhibit anti-migration action in human adenocarcinoma CL1-0 cells through the MAPK and PI3K/AKT signaling pathways. Phytomedicine. 2012 Mar 29. [Epub ahead of print] Chen YY, Chou PY, Chien YC, Wu CH, Wu TS, Sheu MJ. Source: School of Pharmacy, China Medical University, 91 Hsueh-Shih Road, Taichung 404, Taiwan. Abstract: Cancer metastasis is a primary cause of cancer death. Antrodia cinnamomea (A. cinnamomea), a medicinal mushroom in Taiwan, has been shown antioxidant and anticancer activities. In this study, we first observed that ethanol extract of fruiting bodies of A. cinnamomea (EEAC) exerted a concentration-dependent inhibitory effect on migration and motility of CL1-0 cells in the absence of cytotoxicity. The results of a gelatin zymography assay showed that A. cinnamomea suppressed the activity of matrix metalloproteinase (MMP)-2 and MMP-9 in a concentration-dependent manner. Western blot results demonstrated that treatment with A. cinnamomea decreased the expression of MMP-9 and MMP-2; while the expression of the endogenous inhibitors of these proteins, i.e., tissue inhibitors of MMP (TIMP-1 and TIMP-2) increased. Two major compounds from EEAC codycepin and zhankuic acid A alone and together inhibited MMP-9 and MMP-2 expressions. Further investigation revealed that A. cinnamomea suppressed the phosphorylation of p38, and JNK1/2. A. cinnamomeaalso suppressed the expressions of PI3K and phosphorylation of AKT. This is the first report confirming the anti-migration activity of this potentially beneficial mushroom against human lung adenocarcinoma CL1-0. Crown Copyright 2012. Published by Elsevier GmbH. All rights reserved. PMID: 22464013, [PubMed - as supplied by publisher]

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Below are from ScienceDirect 1: Antioxidant properties of water-soluble polysaccharides from Antrodia cinnamomea in submerged culture Food Chemistry Volume 104, Issue 3, 2007, Pages 1115-1122 Ming-Chi Tsai, Tuzz-Ying Song, Ping-Hsiao Shih and Gow-Chin Yen a Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan b Department of Nutrition and Health Science, Chungchou Institute of Technology, 6, Lane 2, Sec 3, Shan-chiao Road, Yuanlin, Changhwa 51003, Taiwan Received 27 September 2006; revised 10 January 2007; accepted 10 January 2007. Available online 25 January 2007. Abstract: Antrodia cinnamomea, a well-known tradition Chinese medicine, possesses anti-tumor, anti-oxidation activities and stimulates the immune system. The aim of this study was to investigate the protective effect of water-soluble polysaccharides from the fermented filtrate and mycelia of Antrodia cinnamomea in submerged culture (ACSC) on hydrogen peroxide-induced cytotoxicity and DNA damage in Chang liver cells. Oxidative DNA damage was evaluated by single cell gel electrophoresis (Comet assay) or by the formation of 8-hydroxy-deoxyguanosine (8-OHdG) adducts. The polysaccharides isolated by ion-exchange chromatography contained glucose, xylose, galactose, arabinose, and mannose. The results showed that incubation of Chang liver cells with isolated polysaccharides at 200 g/mL for 5 h prior to H2O2 treatment (50 M, 30 min) significantly reduced oxidative DNA damage as detected by the formation of comet tail DNA and 8-OhdG adducts by 89% and 69%, respectively. Pre-treatment Chang liver cells with polysaccharides also reduced the levels of thiobarbituric acid reactive substances (TBARS) (p < 0.01) and intracellular reactive species (ROS) (p < 0.01) induced by H2O2. Moreover, glutathione S-transferase (GST) and the GSH/GSSG ratio were significantly increased in Chang liver cells pre-incubated with the polysaccharides (p < 0.01). These results demonstrate that polysaccharides in ASCS have antioxidant properties which may involve up-regulation of GST activity, maintenance of normal GSH/GSSG ratio, and scavenging of ROS. Corresponding author. Tel.: +886 4 22879755; fax: +886 4 22854378. 2: The hepatoprotective activity against ethanol-induced cytotoxicity by aqueous extract of Antrodia cinnamomea doi:10.1016/j.jcice.2008.03.008, Journal of the Chinese Institute of Chemical Engineers, Volume 39, Issue 5, September 2008, Pages 441-447 Yi-Chien Hoa, Ming-Tse Lina, , , Kow-Jen Duana and Yen-Shang Chenb a Department of Bioengineering, Tatung University, Taipei 104, Taiwan b Glory Biotech Co., Ltd., Chia-Yi Hsien 621, Taiwan Abstract: The objective of this study was to evaluate the hepatoprotective activity against the ethanol-induced cytotoxicity by Antrodia cinnamomea which were produced from a large-scale fermentation. The production of biomass, extracellular polysaccharide (EPS), intracellular polysaccharide (IPS), and triterpenoids was 27.1, 1.2, 0.7, and 2.0 g/L, respectively, using a 5000-L agitated bioreactor with 3500 L of A. cinnamomea medium (ACM). The hepatoprotective effects of the water extract from the mycelia of A. cinnamomea (WAC) were evaluated in vitro using ethanol-induced cytotoxicity on AML12 hepatocytes. The cytotoxicity and the apoptosis-associated phosphatidyl serine redistribution of plasma membrane to AML12 cells induced by 300 mM ethanol were effectively reduced by adding 500 mg/L of WAC. From the compositional analysis, the major component in WAC was polysaccharides that showed a high galactose content (70.9 mg/g crude polysaccharide). Hence, the large-scale fermentation was efficient in producing cell mass and its metabolites which were able to protect hepatocytes from the damage by ethanol. Corresponding author. Tel.: +886 2 25925252x3315x29; fax: +886 2 25854735. 3: Influence of nutritional components and oxygen supply on the mycelial growth and bioactive metabolites production in submerged culture of Antrodia cinnamomea doi:10.1016/j.procbio.2005.12.005, Process Biochemistry, Volume 41, Issue 5, May 2006, Pages 1129-1135 Ing-Lung Shiha, Kelly Panb and Chienyan Hsiehc, , a Department of Environmental Engineering, Da-Yeh University, Chang-Hwa, Taiwan b Department of Bioindustry Technology, Da-Yeh University, Chang-Hwa, Taiwan c Department of Biotechnology, National Formosa University, Yun-Lin, Taiwan 19

Abstract: Effects of carbon sources, nitrogen sources, plants oils and oxygen supply on the cell growth and production of bioactive metabolites such as exopolysaccharide (EPS), intracellular polysaccharide (IPS) and triterpenoid in the submerged culture of Antrodia cinnamomea CCRC36716 were studied in detail. Malt extract (ME), yeast extract (YE) and corn steep powder (CSP) were favorable nitrogen sources to the mycelial growth. The highest cell growth (12.52 0.03 g/l cell dry weight), EPS production (1861 62 mg/l) and IPS content (41 12 mg/g DW) can be obtained on day 10 of cultivation in the medium containing 3% CSP, 3% YE, and 3% ME, respectively. The highest overall triterpenoid production (30 mg/g DW) was obtained in 3% CSP medium after 14 d of cultivation. Amongst five carbon sources examined, maltose and glucose yielded relatively high mycelial biomass; high cell densities of 8.29 0.05 and 8.69 0.05 g DW cells/l were obtained after 10 d of cultivation when 4% of maltose and glucose was used, respectively. Pronounced production of EPS, 1482 63 and 1318 48 mg/l, was obtained after 10 d of cultivation when 4% of lactose and sucrose was used, respectively. The maximum IPS content (49 9 mg/g DW) was achieved in 4% glucose medium after 10 d of cultivation; the highest overall triterpenoid production (31 mg/g DW) was obtained in 2% glucose medium after 14 d of cultivation. All plant oils tested stimulated cell growth of A. cinnamomea, enhanced the production of IPS, but inhibited the triterpenoid production. EPS production was slightly inhibited with soy oil but enhanced by the other oils tested, and the maximal EPS production (1147 47 mg/l) was obtained when 0.5% of peanut oil was supplemented. The high O2 supply in the A. cinnamomea culture was favorable for cell growth and polysaccharide production, but was inhibitory on the triterpenoid production. The results obtained are useful in regulation and optimization of A. cinnamomea culture for efficient production of cell mass and bioactive metabolites such as EPS, IPS and tripterpenoids in the submerged culture. Corresponding author at: 64, Wenhua Rd., Huwei, Yunlin, Taiwan 63208. Fax: +886 5 6315502. 4: Characterization and biological functions of sulfated polysaccharides from sulfated-salt treatment of Antrodia cinnamomea doi:10.1016/j.procbio.2008.12.012, Process Biochemistry, Volume 44, Issue 4, April 2009, Pages 453-459 Jing-Jy Chenga, Nai-Kuei Huanga, Huu-Sheng Lurb, Chung-Io Kuob and Mei-Kuang Lua, , a National Research Institute of Chinese Medicine, Taipei, Taiwan b Department of Agronomy, National Taiwan University, Taipei, Taiwan Abstract: Sulfated polysaccharides (SPSs) of Antrodia cinnamomea were extracted by an exhaustive papain digestion and characterized, and their biological functions were evaluated. In this study, we demonstrated the existence of SPSs in the medicinal fungus, A. cinnamomea. Since no SPSs had previously been identified in any fungal organism, we attempted to characterize those from A. cinnamomea. SPSs from A. cinnamomea inhibited in vitro Matrigel tube formation, in an angiogenesis model, in a dose-dependent manner. Furthermore, using serum deprivation-induced apoptosis in neuronal-like PC12 cells as a stress model, the SPSs of A. cinnamomea were effective in preventing serum-deprived apoptosis. Compositional analysis revealed that myo-inositol, fucose, galactose, and glucose were the neutral sugars in SPSs of A. cinnamomea, and these SPSs had a high sulfate content. The sulfation degree paralleled their antiangiogenic and neuroprotective activities. In this work, we report novel data on the structure, antiangiogenic, and neuroprotective effects of these fungal SPSs. Corresponding author at: National Research Institute of Chinese Medicine, 155 1 Li-Nung St., Sec. 2, Shipai, Peitou, Taipei 112, Taiwan, ROC. Tel.: +886 2 28201999x7391; fax: +886 2 28264276. 5: The influence of environmental conditions on the mycelial growth of Antrodia cinnamomea in submerged cultures doi:10.1016/S0141-0229(03)00136-4, Enzyme and Microbial Technology, Volume 33, Issue 4, 10 September 2003, Pages 395-402 The 8th Symposium of Young Asian Biochemical Engineers' Community (YABEC 2002) Fan-Chiang Yang, Hui-Chin Huang and Ming-Je Yang Department of Chemical Engineering and Life Science Research Center, Tunghai University, Taichung 40704, Taiwan, ROC Abstract: Antrodia cinnamomea Chang & Chou, sp. nov. growing rarely on the inner wall of Cinnamomum kanehirai Hay was identified as a new species of Antrodia, which is an expensive folk medicine and used as an antidote, anticancer and anticnesmatic drug. In this study the influence of different physical and chemical factors on the growth of mycelium by A. cinnamomea CCRC35396 was investigated in submerged cultures of using shake flasks. In addition, response surface methodology (RSM) was also used to optimize medium composition for the enhancement of biomass productivity. The results indicate that when a submerged culture in shake flasks was operated at 25 C, initial pH 5 and rotation speed 20

100 rpm, the mycelium concentration reached 486.8 mg/100 ml at 14th day. High rotation speed was unfavorable to the growth of mycelium and the optimum was around 100 rpm. According to the RSM, optimal initial pH and the concentrations of C-source and N-source were determined and the mycelium concentration of A. cinnamomea rose to 2164.2 mg/100 ml at the 7th day and the biomass productivity had a eightfold increase, compared to the growth in basal medium. The results proved that the growth rate of mycelium of A. cinnamomea could be highly enhanced by means of the control of cultural conditions and the modification of medium compositions based on the RSM. Corresponding author. Tel.: +886-4-235-90262x214; fax: +886-4-235-90009. 6: Triterpenoids from Antrodia cinnamomea doi:10.1016/0031-9422(95)00541-2, Phytochemistry, Volume 41, Issue 1, January 1996, Pages 263-267 I-Hwa Cherng, De-Peng Wu and Hung-Cheh Chiang Institute of Chemistry, National Taiwan Normal University, Taipei 117, Taiwan Abstract: Four novel ergostane-type triterpenoids (antcins E and F and methyl antcinates G and H) were isolated from the fruiting body of the fungus Antrodia cinnamomea, a newly identified species of Antrodia, Polyporaceae in Taiwan. Their structures were elucidated by spectroscopic methods. Corresponding author. Author to whom correspondence should be addressed 7: Purification and partial characterization of a lipase from Antrodia cinnamomea Chin-Hang Shu, Chun-Jun Xu and Gee-Chen Lin Process Biochemistry, Volume 41, Issue 3, March 2006, Pages 734-738, doi:10.1016/j.procbio.2005.09.007 Department of Chemical and Materials Engineering, National Central University, Taoyuan, Taiwan, ROC Abstract: Extracellular lipase from Antrodia cinnamomea BCRC 35396 was first purified by ammonium sulphate precipitation and DEAE-Sepharose chromatography. The yield and purification factor were 33.7% and 17.2 folds, respectively. Lipase production from A. cinnamomea was enhanced by 0.01% olive oil supplementation as additional carbon source in an aerated bioreactor with final titer 26 U/ml after 18 days of fermentation. The molecular weight of the purified enzyme was estimated to be 60 kDa by SDS-PAGE. The enzyme was found to be alkaline tolerant (pH 710) with optimum activity at pH 8.0, but both activity and stability decreased significantly as pH value greater than 10. The enzyme activity was clarified to be stable within the temperature range of 2560 C, with maximal activity at 45 C. This observation was similar to those of mesophiles as expected. Corresponding author at: # 300 Jung-Da Road, Chung-Li, Taoyuan, Taiwan, ROC. Tel.: +886 3 4263749; fax: +886 3 4263749. 8: A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea Phytochemistry, Volume 39, Issue 3, June 1995, Pages 613-616, doi:10.1016/0031-9422(95)00025-3 Hung-Chen Chiang**, De-Peng Wu, I-Wha Cherng and Chuen-Her Ueng Institute of Chemistry, National Taiwan Normal University, Taipei 117, Taiwan, Republic of China Abstract: Three novel compounds, a sesquiterpene, phenyl and biphenyl derivatives, have been isolated from the crude methanol extract of the fungus Antrodia cinnamomea, a new genus of Antrodia species. Their structures were all determined by spectroscopic data and confirmed by X-ray analysis. 9: Glucose stimulates production of the alkaline-thermostable lipase of the edible Basidiomycete Antrodia cinnamomea Enzyme and Microbial Technology, Volume 37, Issue 2, 1 July 2005, Pages 261-265, doi:10.1016/j.enzmictec.2005.03.012 En-Shyh Lin and Hui-Ching Ko Department of Cosmetic Science, Vanung University, No. 1 Van-Nung Road, Chung-Li, Taoyuan 320, Taiwan, ROC Abstract: This paper describes a study of the extracellular lipase synthesised by a submerged culture of Antrodia cinnamomea in two different media (modified ME and modified YM). Lipase production increased substantially upon the addition of glucose; when it comprised 8% of the modified ME and YM media, the lipase production increased remarkably from 0 to 3.35 mU/ml and from 0.59 to 6.17 mU/ml, respectively. The lipase activity decreased, however, beyond certain concentrations of glucose. The lipase synthesis in the modified YM medium was superior to that which occurred in the 21

modified ME medium. In addition, the optimum values of pH and temperature for the crude lipase were 11.0 and 60 C, respectively. The enzyme was stable at values of pH between 7.0 and 12.0 and at temperatures up to 80 C. Thus, this novel lipase from A. cinnamomea is particularly alkaline-resistant and thermostable. Corresponding author. Tel.: +886 3 451 5811x894; fax: +886 3 434 5846. 10: Antrodia cinnamomea sp. nov. on Cinnamomum kanehirai in Taiwan Mycological Research, Volume 99, Issue 6, June 1995, Pages 756-758, doi:10.1016/S0953-7562(09)80541-8 T.T. Chang1 and W.N. Chou2 1 Division of Forest Protection, Taiwan Forestry Research Institute, 53 Nan-Hai Road, Taipei, Taiwan 2 National Museum of Natural Science, Taichung, Taiwan A new basidiomycete Antrodia cinnamomea sp. nov., causing brown heart rot of Cinnamomum kanehirai in Taiwan, is described and illustrated. 11. Steroids and triterpenoids of Antodia cinnamomeaA fungus parasitic on Cinnamomum micranthum Phytochemistry, Volume 41, Issue 5, March 1996, Pages 1389-1392, doi:10.1016/0031-9422(95)00767-9 Shu-Wei Yang*, Ya-Ching Shen, and Chung-Hsiung Chen*, * School of Pharmacy, National Taiwan University, Taipei, Taiwan, Republic of China Institute of Marine Resources, National Sun Yat-sen University, 70 Lien-Hai Rd, Kaohsiung, Taiwan, Republic of China Available online 10 September 1998. Abstract: Two ergostane related steroids, zhankuic acids D and E together with three lanosta related triterpenes, 15-acetyl-dehydrosulphurenic acid, dehydroeburicoic acid, dehydrosulphurenic acid were isolated from the fruit body of the fungus Antrodia cinnamomea. Their structures were determined by spectral analyses and comparison with known compounds. 12. Characterization and functional elucidation of a fucosylated 1,6--D-mannogalactan polysaccharide from Antrodiacinnamomea Jing-Jy Cheng1, Mei-Kuang Lu1, Cha-Yui Lin, Chia-Chuan Chang National Research Institute of Chinese Medicine, 155-1 Li-Nung St., Sec. 2, Shipai, Peitou, Taipei 112, Taiwan Received 14 July 2010; revised 4 August 2010; Accepted 9 August 2010. Available online 17 August 2010. Abstract: Antrodiacinnamomea is a valuable polyporaceous edible fungus native to Taiwan. It was reported to provide a number of pharmaceutical benefits. A. cinnamomea was cultured, the polysaccharides (PSs) were extracted and chromatographically fractionated, and their biological functions were evaluated. The PS subfractions (B85PS-IV) showed differential inhibition of in vitroMatrigel tube formation (an indicator of angiogenesis inhibition) with IC50 values of 7.44, 16.41, 7.07, 7.98, and 16.33 g/ml, respectively. Furthermore, A. cinnamomea PSs also blocked vascular endothelial growth factor (VEGF)-induced endothelial cell migration. B85PS-III and -V dose-dependently decreased angiogenic-related protein expressions, including inhibition of VEGF receptor (VEGFR) phosphorylation and angiopoietin-2 protein expression. To further purify and determine the structure of the bioactive PS, B85PS-III was chromatographically purified to give a water-soluble partial fucosylated 1,6--D-mannogalactan (B85PS-III-1) composed of a nonadecasaccharide repeating unit with a molecular mass of 4.17 102 kDa as shown below. The chemical structure of B85PS-III-1 was characterized by a monosaccharide analysis along with 1H, 13C, and 2D nuclear magnetic resonance spectroscopy. 13. Chemical profiling of the cytotoxic triterpenoid-concentrating fraction and characterization of ergostane stereo-isomer ingredients from Antrodia camphorata Ying-Chi Dua, 1, Tung-Ying Wua, 1, Fang-Rong Changa, b, c, d, Wan-yu Lina, Yu-Ming Hsua, Fu-Ting Chenga, Chi-Yu Lue, Ming-Hong Yena, Ya-Ting Tsuia, Hsuan-Lun Chena, Ming-Feng Houc, Mei-Chin Lua, f, g, 2, , Yang-Chang Wua, h, i, , a Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan b Research and Development Center of Chinese Herbal Medicines and New Drugs, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan c Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan d Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan e Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan 22

Graduate Institute of Marine Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan National Museum of Marine Biology & Aquarium, Pingtung 944, Taiwan h Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan i Natural Medicinal Products Research Center, and Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan Received 28 June 2011; revised 9 September 2011; Accepted 12 September 2011. Available online 16 September 2011. Abstract: Antrodia camphorata (AC), also known as A. cinnamomea, an endemic species in Taiwan, is one of the treasured medicinal mushrooms. AC is traditionally used for its chemopreventive biofunctions. In this investigation, we report a convenient method for concentrating the antiproliferative active triterpenoid-rich fraction (FEA), from ethanolic extract of AC (EEAC). A series of stereo-isomers of zhankuic acids (1-8) from the FEA was purified by HPLC using an efficient acidic solvent system. The structures of compounds 1-8 were elucidated based on spectroscopic data analysis, and the absolute configuration of -chiral carboxylic acid at C-25 in the structures was assigned based on reaction with (R)- and (S)-1-(9-anthryl)-2,2,2-trifluoroethanol. Major ingredients of FEA (eight ergostanes 1-8 and two lanostanes 9-10) were further characterised by high-performance liquid chromatography-photodiode array detection/mass spectrometry (HPLC-PDA/MS). Compounds 1-8 and their pair mixture forms (antcin K, antcin C, zhankuic acid C, and zhankuic acid A) were subjected to anti-proliferative assay against three human leukemia cell lines. Among them, the derivatives with carbonyl group at C-3 showed cytotoxicity with IC50 values ranging from 16.44 to 77.04 g/ml.
g

14. Analysis of volatile compounds of Antrodia camphorata in submerged culture using headspace solid-phase microextraction Zhen-Ming Lua, b, Wen-Yi Taob Hong-Yu Xua, Joanne Limb, Xiao-Mei Zhanga, Li-Ping Wangc, Jing-Hua Chena, Zheng-Hong Xua, b a Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, PR China b The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China c State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China Received 22 June 2009; revised 29 October 2010; Accepted 29 December 2010. Available online 8 January 2011. Abstract: In this work a headspace solid-phase microextraction (HS-SPME) coupled with gas chromatographymass spectrometry (GCMS) and GColfactometry (GCO) was developed to evaluate the profile of the volatile compounds that contribute to the aroma of Antrodia camphorata in submerged culture. For this purpose, the HS-SPME sampling method for the volatile compounds of A. camphorata in submerged culture was optimised by a D-optimal design. A HS extraction of the culture broth of A. camphorata followed by incubation on a carboxen/polydimethylsiloxane (CAR/PDMS) fibre during 31.8 min at 54.6 C gave the most effective and accurate extraction of the volatile compounds. By the optimised method, a total of 49 volatile compounds were identified in culture broth of A. camphorata, while a total of 55 volatile compounds were identified in the mycelia. A series of C8aliphatic compounds (mushroom-like odour), several lactones (fruity odour) and L-linalool (citrus-like odour) were the most potent key odourant in both the mycelia and culture broth. This combined technique is fast, simple, sensitive, inexpensive and useful to monitor volatile compounds associated to A. camphorata. Research highlights Volatiles of fermented Antrodia camphorata were analysed by HS-SPMEGCMS. Aroma of fermented A. camphorata was evaluated by HS-SPMEGCO. HS-SPME condition for the volatiles was extensively optimised by a D-optimal design. 15. Development of a LCMS/MS method for the determination of antrodin B and antrodin C from Antrodia camphorata extract in rat plasma for pharmacokinetic study Yongli Liua, b, Xin Dia, Xingchao Liua, Wenjin Shena, Kelvin Sze-Yin Leungb a School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China b Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong, PR China Received 18 March 2010; revised 14 May 2010; Accepted 15 May 2010. Available online 12 June 2010. Abstract: A selective and sensitive liquid chromatographytandem mass spectrometry (LCMS/MS) method was developed and validated for the determination of antrodin B and antrodin C in rat plasma. Both target compounds, together with the internal standard (diazepam), were extracted from rat plasma samples by liquidliquid extraction with ethyl acetate. 23

Chromatographic separation was carried out on an Agilent XDB-C8 column with an isocratic mobile phase consisting of acetonitrile and water (70:30,V/V) at a flow rate of 0.5 mL/min. The mass spectrometric detection was performed by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source operating in positive ionization mode. The assay exhibited a linear dynamic range of 47.64760 ng/mL for antrodin B and 56.65660 ng/mL for antrodin C. The intra- and inter-day precision was less than 5.3% and the accuracy was less than 2.7% for both analytes. The validated method has been applied to the pharmacokinetic study of antrodin B and antrodin C in rats following oral administration of Antrodia camphorata extract.

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