臺灣博碩士論文加值系統

抗生素的濫用，使抗生素抗藥性的問題變的非常嚴重，其中金黃色葡萄球菌對penicillin和methicillin的抗藥性更是深受重視。自從1980年代，PRSE (penicillin-resistant Staphylococcus epidermidis)和 MRSA (methicillin-resistant S. aureus)更變成院內重要的致病菌，PRSE和MRSA易造成嚴重的感染包括心內膜炎和致死性的休克。研究中，我們測試四十種中藥對抗PRSA (penicillin-resistant S. aureus), MRSA和PRSE， 測試抗菌的方法有瓊脂紙錠擴散法、連續稀釋法和殺菌力評估試驗。我們發現訶子的最小抑菌濃度雖然大於1mg，訶子256 ug/ml粗萃取物併用4 ug/ml oxacillin，對於MRSA(ATCC33591)具有殺菌作用。將訶子粗萃取物經有機溶媒分配之後，分別得到正己烷層, 氯仿層, 乙酸乙酯層, 正丁醇層和水層，其中正丁醇層256 ug/ml併用2 ug/ml oxacillin，對於MRSA(ATCC33591)具有殺菌作用，而1024 ug/ml氯仿層或乙酸乙酯層併用2 ug/ml penicillin G，對於PRSA(ATCC11632)也可達到殺菌效果。將正丁醇層利用管柱層析的方法，得到兩個抗菌成分chebulagic acid (CA)和1,2,3,4,6-penta-O-B-D-glucose。其中CA對MRSA(ATCC33591)的抑菌濃度為 512 ug/ml，當oxacillin併用CA256 ug/ml，可使oxacillin的最小抑菌濃度，由8降至0.5 ug/ml。我們測試CA對兩株MRSA臨床菌株和三株ORSA (oxacillin-resistant S. aureus)臨床菌珠的抗菌活性，其中對MRSA臨床菌株在 512 ug/ml併用0.5 ug/ml oxacillin時可達到抑菌作用;對ORSA臨床菌株則在 512 ug/ml分別併用0.5 ug/ml和2 ug/ml oxacillin時，亦達到抑菌的作用。總之，CA併用抗生素對於MRSA、PRSA、PRSE，具有良好的協同性抑菌效果。

Because people abuse antibiotics, bacterial resistance to antibiotics becomes very serious. Staphylococcus aureus also produce resistance to penicillin and methicillin. Since 1980s, PRSE (penicillin- resistant staphylococcus epidermidis) and MRSA (methicillin-resistant S. aureus) have become important nosocomial pathogens. PRSE and MRSA can cause serious infections, including endocarditis and toxic shock syndrome. In this study, we tested in vitro antibacterial activities of forty traditional Chinese medicines against PRSA (penicillin-resistant S. aureus), MRSA and PRSE. We tested antibacterial activities by using disc agar diffusion method, broth dilution test, and time kill assay test. Although we have found that the minimum inhibitory concentration(MIC) of Chebulae Fructus>1mg, we combined the crude extract 256 ug/ml with 4 ug/ml oxacillin, it could inhibit MRSA(ATCC33591). We isolated Chebulae Fructus by partition, and we got hexane fraction, chloroform fraction, ethyl acetate fraction, n-butanol fraction, and H2O fraction. When we combined the n-butanol fraction 256 ug/ml with 2 ug/ml oxacillin, it could inhibit MRSA(ATCC33591). We combined the chloroform fraction or the ethyl acetate fraction 1024 ug/ml with 2 ug/ml penicillin G, it could inhibit PRSA(ATCC11632). We isolated the n-butanol fraction by column chromatography, and we got two active compounds, chebulaic(CA) acid and 1,2,3,4,6-penta-O-B-D- glucose. The MIC of CA against MRSA(ATCC33591) was 512 ug/ml. .When oxacillin was combined with 256 ug/ml CA against MRSA (ATCC33591), the MIC of oxacillin was falling from 8 to 0.5 ug/ml. We tested the antibacterial activitiy of CA against two clinical isolates of MRSA and three clinical isolates of ORSA (oxacillin-resistant S. aureus). We combined CA 512 ug/ml with 0.5 ug/ml oxacillin, it could inhibit two clinical isolates of MRSA. We combined CA 512 ug/ml with 0.5 ug/ml and 2 ug/ml oxacillin, it could inhibit clinical isolates of ORSA. Abouve all, CA combined with antibiotics against MRSA、PRSA and PRSE has good synergical effect.

目錄
目錄--------------------------------------------------------------------------------I
縮寫表----------------------------------------------------------------------------IV
表目錄-----------------------------------------------------------------------------V
圖目錄----------------------------------------------------------------------------VI
中文摘要-----------------------------------------------------------------------VIII
英文摘要--------------------------------------------------------------------------X
總論------------------------------------------------------------------------------XII
第一章 中藥材抗菌活性篩選
I.前言-------------------------------------------------------------------------------1
II.實驗材料
II-1.抗菌中藥材----------------------------------------------------------------4
II-2.菌種-------------------------------------------------------------------------5
II-3.儀器-------------------------------------------------------------------------6
II-4.試藥-------------------------------------------------------------------------6
III.實驗方法
III-1.藥材的萃取---------------------------------------------------------------7
III-2.檢測藥物紙錠之製備---------------------------------------------------7
III-3.菌株之解凍與活化------------------------------------------------------7
III-4.培養液及培養基之配製------------------------------------------------8
III-5.瓊脂紙錠擴散法---------------------------------------------------------8
III-6.連續稀釋法---------------------------------------------------------------9
III-7.中藥材合併臨床抗生素之最低有效抑制濃度試驗-------------10
III-8.殺菌效力評估----------------------------------------------------------10
IV.實驗結果
IV-1.瓊脂紙錠擴散法之結果----------------------------------------------12
IV-2.連續稀釋法之結果----------------------------------------------------15
IV-3.中藥材合併抗生素之結果-------------------------------------------17
V.討論--------------------------------------------------------------------------19
第二章 訶子抗菌成分分離和抗菌成分對抗藥性金黃葡萄球菌抑制活性
I.前言------------------------------------------------------------------------------21
II.實驗材料
II-1.材料-----------------------------------------------------------------------25
II-2.菌種-----------------------------------------------------------------------25
II-3.儀器-----------------------------------------------------------------------26
II-4.溶媒及試藥--------------------------------------------------------------27
II-5.管柱層析-----------------------------------------------------------------28
II-6.薄層層析-----------------------------------------------------------------28
II-7.薄層層析之展開溶媒--------------------------------------------------28
II-8.TLC檢測法--------------------------------------------------------------28
III.實驗方法
III-1.訶子的萃取-------------------------------------------------------------29
III-2.各分劃層的製備-------------------------------------------------------29
III-3.n-Butanol層的分離---------------------------------------------------30
III-4.成分結構測定----------------------------------------------------------30
III-5.有效成分對抗藥性菌株之生長曲線測定法----------------------30
III-6.有效成分合併臨床抗生素對抗藥性菌株之生長曲線測定法-31
IV.實驗結果
IV-1.各分劃層之連續稀釋法試驗----------------------------------------33
IV-2.各分劃層合併抗生素之結果----------------------------------------33
IV-3.成分結構鑑定----------------------------------------------------------36
IV-4.抽出成分的結構-------------------------------------------------------43
IV-5.chebulagic acid之抗菌活性------------------------------------------44
V.討論-----------------------------------------------------------------------------48
第三章chebulagic acid對臨床抗oxacillin與methicillin抗藥性金黃葡萄球菌抑制活性
I.前言------------------------------------------------------------------------------51
II.實驗材料
II-1.抑菌活性成分-----------------------------------------------------------53
II-2.菌種-----------------------------------------------------------------------53
II-3.儀器-----------------------------------------------------------------------54
II-4.試藥-----------------------------------------------------------------------54
III.實驗方法
III-1.chebulagic acid對抗藥性菌株之生長曲線測定法---------------55
III-2.chebulagic acid合併臨床抗生素對抗藥性菌株之生長曲線測定法------------------------------------------------------------------------55
IV.結果---------------------------------------------------------------------------57
V.討論----------------------------------------------------------------------------61
結論-------------------------------------------------------------------------------63
參考文獻-------------------------------------------------------------------------64

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