Antrodia camphorata is a Taiwan-unique fungus which grows on the walls inside the core of decayed Cinnamomum kanehirae. Antrodia camphorata provides many bioactive substance and it is cultivated mainly via mycelium nowadays. First section of this research is focused on Antrodia camphorata (CCRC-35716) in liquid cultivation medium and the changes within extracellular polysaccharides, trierpenoids, pH and growth. It also uses HPLC to analyze the differences of mycelium triterpenoids. This experiment showed when Antrodia camphorata was cultivated in PDB liquid medium for 14 days; it had the highest extracellular polysaccharides (0.23

6 pp. / We injected AGRA PHOS (Potassium Phosphite) 0-2.4-2, Propiconizole – 0.05%, Zn, Mn and Fe 0.105, 0.112, and 0.10% respectively, Zn, Mn and Fe 0.210, 0.220, and 0.200% respectively and Propiconizole – 0.05% + Zn, Mn and Fe 0.105, 0.112, and 0.10% respectively using a low pressure injection system for the control of Antrodia sinuosa in lemon trees. No treatment led to a significant reduction in fungal growth.

8 pp. / We injected AGRA PHOS (Potassium Phosphite) 0-2.4-2, Propaconizole – 0.05%, Propaconizole plus Azoxystrobin – 0.117 and 0.135% respectively, Zn, Mn and Fe 0.105, 0.112, and 0.10% respectively, and Azoxystrobin – 0.137% using a low pressure injection system for the control of Antrodia sinuosa in lemon trees. The Propaconizole + Azoxystrobin treatment, the Azoxystrobin treatment, and the Zn + Mn + Fe treatment led to significantly less fungal lesion growth when applied prior to the introduction of the fungus, as compared to their application after fungal introduction.

Breast cancer (BC) is one of the most common cancers among women worldwide that ~25% of new cancer cases diagnosed every year would be BC; moreover, ~15% of cancer deaths per year caused by BC makes it the leading cause of cancer death among women worldwide. To date, though the cause of a large proportion of BC are still unclear, recent studies have revealed that a supportive breast tissue microenvironment is critical for the development and progression of BC, especially the communication with immune cells within breast tissue. Therefore, breast inflammatory microenvironment is currently received a substantial attention in the prevention and treatment of BC. Research on breast cancer immunology suggests that inflammatory mediators, estrogen and several inflammation-related tumorigenic pathways are potentially contributors for inflammatory breast tumorigenesis. It is evidenced that elevated levels of inflammatory mediators, such as cytokines, chemokines, prostaglandins, and enhanced estrogen production while suffering from chronic inflammation is responsible for not only activating oncogenic pathways, for example NF-κB, STAT3 and Wnt signaling pathways, but also reducing the efficacy of cancer-specific immunity against tumor cells. Accordingly, targeting the chronic inflammatory status in breast tissue has become a promising strategy for breast cancer therapy. Recently, due to the annoying side effects accompanying by traditionally anticancer drugs, there is an increased interest in finding out natural sources to treat BC. Herein, we report that antroquinonol (AQ) and/or 4-acetylantroquinonol B (4-AAQB) isolated from Antrodia Camphorata were able to modulate the expression of several inflammatory mediators, IL-6 and IFN-γ in particular, and downregulate the aromatase expression and Wnt signaling responses induced by inflammatory status. Taken together, the present findings provide new insights into the role of AQ and 4-AAQB in inflammatory breast tumors and also suggest them as promising candidates for breast cancer immunotherapy.

牛樟芝 (Antrodia camphorata; AC) 為台灣特有的藥用真菌,是常見的傳統 中藥,富含三萜類化合物及多醣體。本研究使用牛樟芝、葛花、枳椇子與山楂共 四種萃取物製成之牛樟芝複方飲品 (A. camphorate drink, ACD)為原料,評估此一 複方飲品之抗疲勞功效。本研究從運動表現、疲勞生化指標、肌肉損傷生化指數 以及能量儲存等做為抗疲勞評估標準。將 5 週齡雄性 ICR (Institute of Cancer Research) 小鼠,分為三組,每組各 10 隻,分組如下:(1) 對照組 (vehicle, V); (2) 餵食 15.625 mL/kg 牛樟芝複方飲品 (ACD-1X);(3) 餵食 31.25 mL/kg 牛樟芝 複方飲品 (ACD-2X),連續經口餵食 28 天後進行以下試驗: 1. 測試小鼠前肢抓 力、2. 測試 5%負重游泳力竭時間、3. 進行游泳運動 15 分鐘後立即進行採血觀 察乳酸、血氨、血糖及肌酸激酶、4. 小鼠犧牲後,採集肝臟及肌肉組織進行肝醣 分析與切片。結果顯示,ACD-1X 組 (1124 g) 與 ACD-2X 組 (125 6 g) 在前肢 抓力與對照組 (95 6 g) 相比,分別顯著提升 1.19 與 1.32 倍 (p < 0.05)。ACD-1X 組 (56.714.3 min) 與 ACD-2X 組 (60.717.7 min) 在游泳力竭時間與對照組 (18.23.7 min) 比較分別延長 3.12 及 3.34 倍,均有顯著差異 (p < 0.05)。游泳運 動結束後,ACD-1X 組與 ACD-2X 組之乳酸、血氨及肌酸肌酶均顯著低於對照組 (p < 0.05),而血糖方面則顯著高於對照組(p < 0.05)。肌肉肝醣部分,ACD-1X 組 (1.79 ± 0.12 mg/g) 與 ACD-2X 組 (2.36 ± 0.21 mg/g) 相較於對照組 (1.54 ± 0.06 mg/g),2倍牛樟芝複方飲品組顯著提升1.53倍。肝臟肝醣部分,ACD-1X組 (48.9± 4.1 mg/g) 與 ACD-2X 組 (54.1 ± 5.8 mg/g) 與對照組 (27.0 ± 2.4 mg/g) 比較分別 增加 1.18 及 2.00 倍。研究結果顯示,牛樟芝複方飲品具有增加肝臟及肌肉組織 中的肝醣儲存量,且在游泳運動過程中降低血乳酸、血氨及肌酸激酶的產生。進 一步延長負重游泳力竭的時間及前肢抓力表現。因此,本研究證實牛樟芝複方飲 品具有抗疲勞及提升運動表現之功效。 / Antrodia camphorata is an endemic medical mushroom in Taiwan and has been reported to have multi-biological activities such as anti-fatigue, liver protection, anti-cancer, and anti-inflammation. This study was designed to ascertain the ergogenic and anti-fatigue properties of an A. camphorata drink (ACD) (comprising A. camphorata, Pueraria flos flowers, Hovenia dulcis Thunb, and Crataegus pinnatifida) by forelimb grip strength, load-weighted swimming test, and biochemical examinations in mouse model. Thirty male ICR mice (5-week-old) were randomly divided into three groups (n=10 per group): (1) vehicle; (2) a recommended dosage of 15 mg per 75 mL once per day for adult human (ACD-1X); (3) a 2-fold of recommended usage for adult human (ACD-2X). Mice were given orally either vehicle or A. camphorate drink by gavage for 28 days. The groups ACD-1X and ACD-2X showed significant increases in forelimb grip strength, swimming time to exhaustion skeletal, and muscle glycogen as compared to the vehicle group. Blood lactate and ammonia levels in the ACD-1X and ACD-2X groups were significantly lower than in the vehicle group post a swimming test. Moreover, the activity of plasma creatine kinase (CK), a marker of muscular damage, was significantly decreased in the ACD-1X and ACD-2X groups than vehicle group after swimming exercise. These results suggest that A. camphoratahas great potential for application in relevant fields for its ergogenic and anti-fatigue activities. / 第一章 緒論 .........................................................................................................................1 第一節 研究目的..............................................................................................................2 第二章 文獻探討 .................................................................................................................3 第一節 牛樟芝成份及相關研究......................................................................................3 第二節 葛花成份及相關研究..........................................................................................6 第三節 枳椇子成份及相關研究......................................................................................7 第四節 山楂成份及相關研究..........................................................................................8 第五節 運動與疲勞..........................................................................................................9 第三章 材料與方法 ...........................................................................................................12 第一節 實驗測試樣品....................................................................................................12 第二節 實驗動物之飼養與實驗流程............................................................................14 第三節 血液與組織樣本之收集與前處理....................................................................16 第四節 統計分析............................................................................................................17 第四章 結果與討論 ...........................................................................................................18 第一節 補充牛樟芝複方飲品對於小鼠游泳運動能力表現之測試............................18 第二節 補充牛樟芝複方飲品對於小鼠游泳運動後血液中乳酸、血氨、血糖濃 度以及 CK 活性之分析..................................................................................................19 第三節 補充牛樟芝複方飲品對於小鼠前肢抓力表現之測試....................................20 第四節 補充牛樟芝複方飲品對於小鼠肌肉與肝臟部位肝醣含量之分析................21 第五章 結論 .......................................................................................................................22 參考文獻.............................................................................................................31

Leaky buildings are those that show elevated moisture contents of the framing timber, which can subsequently lead to the establishment of fungal and bacterial decay. Prior to this study, the causative agents of the decay in these leaky buildings were unknown, though it was suspected to be one or more species of decay fungi. Therefore, the overall goal of this multi-disciplinary PhD thesis research was to determine the causative agents of decay in leaky buildings of New Zealand in an effort to develop solutions for both their remediation and future prevention. Use of molecular biology methodology and classical mycological techniques based on morphology enabled identification of decay fungi from framing timber and air samples of leaky New Zealand buildings and provided insight into relative importance based on isolation frequency. In most cases, fungi colonising Pinus radiata D. Don were isolated to produce pure cultures. Mycelia from these cultures on agar media were collected to extract DNA. To identify the fungi to the species level, polymerase chain reaction (PCR) with fungal specific DNA primer pairs were performed followed by DNA sequencing of the internal transcribed spacer (ITS) region. Identification was by BLAST (Basic Local Alignment Search Tool) search on sequences in known GenBanks. In total, 421 samples from leaky buildings were processed, predominately untreated P. radiata decayed framing timber and also fibre cement boards and building paper. From these, sixty-eight fungal identifications were made. The only taxa that were isolated with significant frequency were identified as 4 basidiomycete species, as follows, along with the number of times they were isolated from the 421 samples: • Gloeophyllum sepiarium (Wulf.: Fr.) Karst. 13x • Oligoporus placenta (Fries 1865) Gilb. In Ryv.1985 11x • Antrodia sinuosa (Fr.) Karst. 8x • Gloeophyllum trabeum (Fr.) Murr. 4x Although these species were identified repeatedly, in total they represent less than 10% of the total samples and, therefore, it is concluded that the leaky building decay samples represent high fungal biodiversity. An aerial spore study of internal air, wall cavity air and exterior air of leaky buildings was carried out using a Merck MAS-100 instrument which collects spores directly onto selective media plates. Viable fungal aerial spores were detected at every sampling location tested at the leaky buildings, by the criteria of culturing, with a highest mean of 3714 colony-forming units (CFU) per cubic metre found in the cavities of water-damaged walls. This aerial spore study in conjunction with isolation from decayed wood samples from the same leaky buildings enabled identification of G. sepiarium and A. sinuosa at the same test site. The use of carboxymethylcellulose medium further demonstrated the presence of potential cellulose-degrading fungi within and around the location. Overall, the combination of direct sampling of timber and air sampling proved useful for detection of fungal species variability at a multi-unit building. Four decay fungi isolated from New Zealand leaky buildings and two standard control decay fungi (Coniophora puteana and Serpula lacrymans) were submitted to laboratory wood block testing to determine the effectiveness of currently used wood framing preservatives under laboratory conditions before and after a standard leaching regime. P. radiata blocks were treated with water based boron copper azole and solvent based IPBC propiconazole plus tebuconazole (1:1) preservatives and exposed to the basidiomycetes for 12 weeks. Mass loss for the fungal decay-infected samples was recorded of up to 55% for preservative-treated samples, up to 62% mass loss for leached samples and up to 58% mass loss for un-preservative treated samples. Additionally, well defined dosage responses and approximate toxic thresholds were obtained for all preservatives tested. Results suggested that the minimum IPBC retention specified by Hazard Class 1.2 of NZS3640:2003 (0.025% m/m) is on the low side, and demonstrated after the 2 week leaching regime complete loss of efficacy of boron at 0.4% m/m boric acid equivalent (BAE). This PhD research gave a first overview of fungi occurring in New Zealand leaky buildings, and it demonstrated the following key aspects of wood preservation: 1. The isolated test fungus Antrodia sinuosa was more difficult to control with propiconazole plus tebuconazole at retention 0.007% m/m than the known tolerant fungus Oligoporus placenta; 2. Boron at Hazard Class 1.2 retention of 0.4% m/m BAE was not toxic to Oligoporus placenta; 3. Serpula lacrymans exhibited tolerance to the highest retention of 0.06 %m/m tebuconazole plus propiconazole; and 4. Gloeophyllum species appeared susceptible to all wood preservatives. In order to correlate fungal colonisation and wood decay, colonised wood blocks were studied using light microscopy (LM) and field- emission scanning electron microscopy (FE-SEM). Microscopic observations of P. radiata wood blocks following a standard wood decay test of twelve weeks of fungal colonisation by Serpula lacrymans, Antrodia sinuosa, Oligoporus placenta and Gloeophyllum sepiarium revealed that the two microscopic techniques employed were complementary by allowing features such as pit membranes, chlamydospores or S3/S2 compound middle lamella interface to be photographed in greater detail, allowing for more precise analyses and interpretation of key findings, as follows: 1. Brown rot fungi directly target their apical growth towards degraded pit apetures; 2. Reliance on light microscopy and observed birefringence as a tool to record changes in cell wall crystallinity associated with brown rot decay alone could be misleading; 3. Presence of fine (≤ 1 m) to wide (≥ 3.5 m) bore-hole and hyphal size ranges, and nearly unchanged cell wall thickness of all wood/test fungal combinations, confirmed active decay at moderate to late stages; 4. Some ray parenchyma cells for Antrodia sinuosa, Oligoporus placenta and Gloeophyllum sepiarium colonised blocks were intact throughout late stages of decay, outlining that they were not preferentially degraded early in the brown rot decay process, and 5. Presence of bore-holes, clamp and medallion clamp formation and resting spores (chlamydospores and arthrospores) are fungal specific, can aid in their differentiation and identification, and should be recorded during wood decay studies, as especially resting spores are an important factor when planning remediation strategies. In summary, this PhD thesis research provided the first comprehensive investigation into the biodiversity of fungi from leaky New Zealand buildings, identified the dominant species and presented details about their micromorphology and their decay patterns. It also demonstrated substantial differences in efficacy of preservative formulations currently (December 2008) approved for framing treatments in New Zealand and possible deficiencies where framing may be subjected to severe leaching. This study also provided the first comparative analyses of viable fungal aerial spores between leaky wall cavities and the surrounding air environment. Subsequently, this research added to the knowledge of the decay fungal species diversity in and around New Zealand leaky buildings, outlined their capabilities to degrade treated and un-treated P. radiata framing timber and illustrated the efficacy of New Zealand approved wood preservatives for their potential as remedial treatment and future prevention.

83 Abstract The work focuses on the physiology of wood-rotting fungi and the possibilities of their inhibition by physical methods (microwave and gamma irradiations, dry heat) or newly developed chemical preparatives contaning metallic nanoparticles. The effect of abovementioned treatments on mycelial growth, activities of extracellular enzymes responsible for wood degradation and on spore germination or viability was studied in selected strains of wood-rotting fungi obtained from culture collection (Coniophora puteana, Fibroporia vaillantii, Gloeophyllum sepiarium) or isolated from infected buildings (Serpula lacrymans).