臺灣博碩士論文加值系統

近年來由於抗生素的濫用，導致抗藥性菌株逐年增加，造成病人出現無抗生素可用的嚴重現象，促使醫學界不斷尋求新的抗生素替代性產品。大蒜為天然抗菌植物，其有效抗菌成份來自於大蒜受到擠壓後釋出的含硫化合物，即為蒜素(Allicin)。本研究目的為檢測大蒜水萃物--蒜素對抗藥性菌株之抑菌功效與生物活性，評估其作為抗生素替代物之可能性。
本研究檢測之抗菌產品為由廠商提供之蒜素水溶液，廠商之製備方式是先從大蒜粉提煉純化出蒜素(Allicin)，再使用專利的超臨界流體萃取系統來濃縮蒜素水溶液，使蒜素水溶液濃度達到1000 μg/ml時分流並收集。測試菌株使用十種共一百株，經脈衝式電泳(Pulsed Field Gel Electrophoresis ; PFGE)基因分型之抗藥性臨床菌株，以測試對不同基因型之同種菌株的抗菌結果。本研究以瓊脂紙錠擴散法(agar disc diffusion method)作蒜素水溶液初步抗菌活性分析，再進一步偵測最低抑菌濃度(minimum inhibitory concentration; MIC )與最低殺菌濃度(minimum bacteriocidal concentration; MBC )及MBC與MIC之比值(MBC/MIC ratio)，接著以細菌之0.5、1、2、4倍MIC值進行殺菌效率與動態變化試驗(time-killing test)。
抗菌活性分析結果顯示，以紙錠擴散法偵測蒜素水溶液對所有測試之菌株皆產生明顯抑制圈，MIC介於15.6至125 μg/ml之間，MBC則介於31.25至500 μg/ml之間。且MBC與MIC之比值皆介於1到4 之間，顯示其作用模式可能為殺菌作用。殺菌效率動態變化試驗結果顯示，1倍MIC之蒜素水溶液對革蘭氏陽性菌中之MRSA、MSSA、Staphylococcus epidermidis在24小時可完全抑制，但對VRE、VSE則須2倍MIC以上濃度方可完全抑制; 1倍MIC之蒜素水溶液對革蘭氏陰性菌Pandrug resistant Acinetobacter baumannii、K. pneumonia (ESBL) 、 E. coli (ESBL) 、 Salmonella、 P. aeruginosa在2至8小時即可完全抑制，對全部十種測試菌株皆可持續抑制達48小時。
接著，我們以儲存於四種溫度之蒜素水溶液，在儲存後連續每天測定蒜素抗菌活性以觀察蒜素水溶液儲存後剩餘之生物活性，依抗菌活性衰減時間來計算其生物半衰期。結果顯示，儲存於室溫(22~23℃)之蒜素水溶液對S. aureus ATCC25923抗菌活性檢測換算之生物半衰期為6.6天，對E. coli ATCC25922之生物半衰期為4.3天。儲存於4℃之蒜素水溶液對S. aureus ATCC25923抗菌活性檢測換算之生物半衰期延長為19.6天，對E. coli ATCC25922之生物半衰期延長為18.3天。蒜素水溶液儲存在-20℃與-80℃之抗菌活性檢測換算之生物半衰期可維持1年半以上。為避免蒜素水溶液受氧化而失活故以數種方式隔絕氧氣，結果顯示添加抗氧化劑維生素C與維生素E、真空包裝、加礦物油隔絕空氣均無法延長生物半衰期，故低溫儲存仍為我們所測得之最有效延長蒜素水溶液生物半衰期的方式。

In the recent years, abuse of antibiotics has caused drug-resistant bacteria strains increasing every year. Due to a lack of effective antibiotics, the research of new antibiotic alternatives is in demand. Garlic is a natural anti-bacterial plant. The effective antibacterial ingredient Allicin is a sulfur compound from extrusion of garlic. This study is to assess garlic water extraction of Allicin as an antibiotic alternative product by detecting its antibacterial effects against drug-resistant strains and biological stability.
The Allicin aqueous solution was extracted from garlic powder, and then concentrated garlic aqueous solution was collected at an Allicin concentration of 1000 ppm by patented supercritical fluid extraction system. We used 100 drug-resistant clinical strains of ten species, prepared by PFGE genotyping, to test the antibacterial results against different genotypes of the same strain species. The experiment applied agar disc diffusion method for preliminary antibacterial activity analysis, and further detected the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and the ratio of MBC and MIC. Then, all of the strains were tested to find the bactericidal efficiency and dynamic change (time-killing test) according to 0.5, 1, 2, and 4 times of their MIC values.
Preliminary antibacterial activity analysis showed that all strains had apparent inhibition zones with agar disc diffusion method. The MICs were between 15.6 and 125 μg /ml, and the MBCs were between 31.3 and 500 μg /ml. The ratios of MBC to MIC were between 1 and 4, and implied their mode of action was bactericidal effect. Dynamic change of bactericidal efficiency showed that the MIC concentration of Allicin aqueous solution with MIC concentrations can completely inhabit MRSA, MSSA, and Staphylococcus epidermidis of the Gram-positive bacteria in the first 24 hours, and five species of Gram-negative bacteria in 2 to 8 hours. The above inhibition effects remained 48 hours. Complete inhibition of VRE and VSE needed more than double MIC concentrations of Allicin aqueous solution.
The biological half-life was determined by the antibacterial activity decay time after some storage duration with stored in specific environment. With room temperature storage (22~23℃), the biological half-life of Allicin aqueous solution against standard S. aureus ATCC25923 strain was 6.6 days, and against standard E. coli ATCC25922 strain was 4.3 days. With 4℃ storage, its biological half-life against standard S. aureus ATCC25923 strain extended to 19.6 days, and against standard E. coli ATCC25922 was 18.3 days. The biological half-life of Allicin aqueous solution stored in -20 ℃ to -80 ℃ can extend to 1.5 years or more. Isolating the Allicin aqueous solution from oxygen by adding vitamin C and vitamin E, vacuum package, or immersion in mineral oil did not extend its biological half-life. Therefore, low temperature storage was the best way to prolong the biological half-life of Allicin aqueous solution.

中文摘要............................................................................................................................i
英文摘要..........................................................................................................................iii
誌謝...................................................................................................................................v
目次................................................................................................................................. vi
表目錄............................................................................................................viii
圖目錄..........................................................................................................................x
附圖目錄....................................................................................................................xi
縮寫對照表...................................................................................................................xii
第一章 文獻回顧.............................................................................................................1
1-1 細菌的構造與分類............................................................................................1
1-2 抗生素的作用機轉與發展........................................................................…....3
1-2-1 抗生素的發展史.....................................................................................3
1-2-2 抗生素的作用機制與分類.....................................................................4
1-3 細菌對抗生素產生抗藥性的方式.......................................................….........7
1-4 抗藥性菌株流行現況........................................................................................8
1-5 大蒜Garlic (Allium Sativum)介紹 .................................................................10
1-6 大蒜有效抗菌成份蒜素(Allicin)介紹............................................................11
1-7 蒜素的醫學應用..............................................................................................12
1-7-1 蒜素的抗細菌功效...............................................................................13
1-7-2 蒜素抗黴菌功效...................................................................................13
1-7-3 蒜素抗原蟲功效...................................................................................13
1-8 蒜素水溶液的穩定性探討............................................................................13
第二章 研究目的...........................................................................................................15
第三章 材料與方法.......................................................................................................16
3-1 研究架構..........................................................................................................16
3-2 研究材料..........................................................................................................17
3-2-1 菌株………….......................................................................................17
3-2-2 培養用培養基及試劑與耗材..............................................................19
3-2-3 儀器裝置...............................................................................................20
3-3 實驗方法.....................................................................….................................21
3-3-1大蒜水溶液製造流程............................................................................21
3-3-2 細菌培養.....................................................................................21
3-3-3 瓊脂紙錠擴散試驗(Kirby-Bauer Disk-diffusion test).....................22
3-3-4最低抑菌濃度測定(Minimum inhibitory concentration;MIC).....24
3-3-5 最低殺菌濃度測定(Minimum bactericidal concentration; MBC)….25
3-3-6 殺菌效率、動態變化試驗(Time-kill curves).................................25
3-3-7 蒜素水溶液檢測生物活性試驗……………………………………26
第四章 實驗結果...........................................................................................................29
4-1蒜素水溶液之瓊脂紙錠擴散試驗..................................................................29
4-1-1蒜素水溶液對革蘭氏陽性菌之瓊脂紙錠擴散試驗……..…………..29
4-1-2蒜素水溶液對革蘭氏陰性菌之瓊脂紙錠擴散試驗………………....30
4-2蒜素水溶液之培養液稀釋試驗.......................................................................32
4-2-1蒜素水溶液對革蘭氏陽性菌之最低抑菌濃度與最低殺菌濃度........32
4-2-2蒜素水溶液對革蘭氏陰性菌之最低抑菌濃度與最低殺菌濃度........32
4-3蒜素水溶液之殺菌效率、動態變化試驗……………………….....................33
4-3-1蒜素水溶液對革蘭氏陽性菌之殺菌效率、動態變化試驗................. 33
4-3-2蒜素水溶液對革蘭氏陰性菌之殺菌效率、動態變化試驗.................34
4-4蒜素水溶液的生物活性試驗...........................................................................34
第五章 討論.....................................................………………..………………………37
第六章 結論.......................................................………………………………………44
參考文獻..........................................................………………………………………...45

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