靈芝在中草藥中屬上品藥，赤芝(Ganoderma lucidum)及松杉靈芝(Ganoderma tsugae)為靈芝屬(Ganoderma)裡目前常被大量栽種的品種，其多方面之生物活性包含抗腫瘤、免疫調節及抗氧化;但是在抗微生物之活性上，只有赤芝有相關文獻報導◦細菌抗藥性在近數十年來逐漸成為臨床上的一大難題，尤其是針對免疫力低下的病患細菌抗藥性增加臨床治療上之難度，本研究目的在於探討松杉靈芝萃取物之抗菌活性及其機轉◦本研究發現松杉靈芝乙醇萃取物對於革蘭氏陽性菌的最小抑制生長濃度約為 2 mg/ml，並證實能減少枯草桿菌及金黃色葡萄球菌之細胞數，進一步針對松杉靈芝乙醇萃取物對金黃色葡萄球菌在不同生長期之機制探討，利用蛋白質體學之技術發現松杉靈芝乙醇萃取物對金黃色葡萄球菌在對數期及靜止期之作用機制不同，對於金黃色葡萄球菌之分泌體學的研究中也發現松杉靈芝乙醇萃取物能改變一般分泌途徑之分泌活性◦本研究發現松杉靈芝乙醇萃取物對金黃色葡萄球菌之多方位機轉顯示松杉靈芝乙醇萃取物具發展成為抗微生物藥物的潛力◦

Ganoderma (Lingzhi) is one of the top grade drugs in traditional Chinese medicine (TCM). The biological activities have been well reported including antitumor, immunomodulatory and antioxidation in two well cultivated species: G. lucidum and G. tsugae. The antimicrobial activity has been studied in different extracts from G. lucidum but no related study focused on G. tsugae. The various resistances in bacteria isolates have raised the difficulty of the emergent treatment in clinical medicine, especially in immunocompromised patients. This study is to investigate whether the extracts of G. tsugae have the potential to be antimicrobial agents. The ethanolic extract (GTEE) has minimum inhibitory concentration (MIC) around 2 mg/ml to Gram-positive bacteria. We also found GTEE could decrease the cell viability and inhibit the cell growth of Bacillus subtilis and Staphylococcus aureus. Due to the impact of GTEE on growth curve of S. aureus, cells at both exponential phase (4-h) and stationary phase (10-h) were picked for mechanism study. Our data indicates that GTEE affects the intracellular protein profiles in S. aureus and the profile changes are different between 4-h and 10-h samples. The effect of GTEE on secretory protein is also observed that GTEE may affect the secretion in the general secretory pathway (Sec translocation). The antimicrobial activity of GTEE and its multi-targeted phenomenon imply that GTEE can be potentially developed as an antibacterial agent.

Content
Acknowledgement i
Content iii
Abbreviations v
Content of Figures vi
Content of Tables vi
摘要 vii
Abstract viii
Chapter 1. Introduction 1
Traditional Chinese medicine & Ganoderma tsugae research 1
Bacterial resistance 2
Antibiotics 5
TCM and antimicrobial agents 6
Chapter 2. Experimental design 7
Chapter 3. Results and Discussion 8
Results 8
Bacterial culture 8
Different extracts of Ganoderma tsugae in inhibiting growth of bacteria 8
Effect of GTEE on Gram-positive bacteria 9
The impact of GTEE on S. aureus growth 9
The intracellular protein profiles of S. aureus by proteomics approach 10
The effect of GTEE on S. aureus secretory proteins 10
The gene expression of general secretory pathway 11
Discussion 11
GTEE has antimicrobial activity to Gram-positive bacteria 11
GTEE inhibits the growth of S. aureus 12
The protein profiles changed after GTEE treatment in S. aureus 13
GTEE affects the secretion activity in S. aureus 15
The multi-targeted effect of GTEE on S. aureus 16
Figures 17
Tables 30
Chapter 4. Conclusion 35
Summary 35
Contribution 35
Chapter 5. Experimental methods and materials 36
Preparation of Ganoderma tsugae extracts 36
Bacterial Culture 36
Minimum Inhibitory Concentration 37
Colony Forming Unit 38
Sample preparation and Two-Dimensional Gel Electrophoresis 38
Gel to Gel Comparison and Analysis 40
Collection of secretory proteins 42
RNA isolation and qPCR 43
Statistical analysis 46
Reagents 47
Instruments 49
Kits 50
Solutions 51
Appendix 57
Preparation of Ganoderma tsugae extract 57
Supplementary data 57
References 62

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