- 學位論文
五倍子對Methicillin與Penicillin抗藥性金黃葡萄球菌與表皮葡萄球菌之抗菌活性研究
The antibacterial activity of Galla Rhois against methicillin resistant Staphylococcus aureus and penicillin resistant Staphylococcus epidermidis
摘要
金黃葡萄球菌(Staphylococcus aureus)與表皮葡萄球菌(Staphylococcus epidermidis)為造成嚴重感染與院內感染的重要病源菌，其中methicillin resistant staphylococcus aureus (MRSA)與pencillin resistant Staphylococcus epidermidis (PRSE)更是造成今日台灣地區院內感染的禍源菌。因此為尋找新抗生物質以對抗威脅病人生命的抗藥性菌株，本研究以紙錠擴散法(disc agar diffusion test)與連續稀釋法(broth dilution test)檢測五十種中藥材後，發現五倍子粗萃取物具有良好之抑菌活性其對MRSA與PRSE最低抑菌濃度(minimum inhibitory concentration；MIC)分別為512 microgram/ml與256 microgram/ml；在與臨床抗生素Prostaphlin ®合併使用於MRSA時，五倍子粗萃物劑量為256 microgram/ml併用Prostaphlin ® 2 microgram/ml即可抑制細菌生長，而在PRSE方面，五倍子粗萃物劑量為128 mg/ml併用Penicilllin G ® 3.36 unit/ml即可抑制細菌生長。而在殺菌效力評估試驗（time kill assay），其劑量為1024 mg/ml時，在24小時內，即具有99 %的殺菌效果，因此進行管柱層析法（column chromatography）並配合活性追蹤分離出五個有效成分，再經結構鑑定與抑菌活性評估，發現GR5（1, 2, 3, 4, 6 - penta - O - galloyl - beta - D — glucose）抑菌效果最佳，其對MRSA與PRSE的MIC分別為15.625 microgram/ml與62.5 microgram /ml。而在抗生素與活性成分合併方面，GR5在3.91 microgram/ml 合併penicillin G 1.25 unit/ml即對PRSE有抑制生長的效果；而在由醫院所收集到十株對methicillin具有抗藥性的金黃葡萄球菌（clinical methicillin resistant Staphylococcus aureus； CMRSA）的抑菌活性評估中，GR5對其中四株菌株MICs為125 microgram/ml，另外六株為62.5 microgram/ml，而在十株對oxacillin具有抗藥性的金黃葡萄球菌（clinical oxacillin resistant Staphylococcus aureus； CORSA）的抑菌活性評估中，GR5對其中四株菌株MICs為62.5 microgram/ml，另外六株為125 microgram/ml，而在GR5使用劑量為原先的1/2至1/4時，合併抗生素oxacillin 2 microgram/ml或2 microgram/ml以下的劑量，均可具有抑菌生長的效果；另一方面在對MRSA中所含的beta -lactamase抑制率表現上，GR5隨著劑量的增加其抑制率更是可高達80 %以上，因此GR5在抗生物質的開發上具有相當的潛力。
並列摘要
Abstract Staphylococcus aureus and Staphylococcus epidermidis are becoming major pathogens and causing infections within the hospital. Especially, the methicillin resistant staphylococcus aureus (MRSA) and the penicillin resistant Staphylococcus epidermidis (PRSE) are causing serious trend of infections, and is also threatening human lives in Taiwan today. In order to find new and more effective antibiotics to against MRSA and PRSE, disc agar diffusion, and broth dilution are used for the experiment and research to test the fifty antibacterial Chinese herbs. During the experiment, we found that Galla Rhois crude extract has the best antibacterial activity, according to our experimentation we found that the crude extract can effectively resist MRSA and PRSE with minimum inhibitory concentrations（MICs）of 512 microgram/ml and 256 microgram/ml. In combination clinical antibiotics with the crude extract, when combination Prostaphlin ® 2 microgram/ml with the crude extract 256 microgram/ml will be able to resist MRSA and otherwise when combination Penicilllin G ® 3.36 unit/ml with the crude extract 128 microgram/ml will be able to resist PRSE. In time kill assay, the crude extract 1024 microgram/ml approaches 99 % bactericidal activity, so we used column chromatography and followed its antibacterial activity to isolation the five compound from crude extract and the structures of these compound were determined based on the analysis of NMR spectroscropic data. We found that GR5（1, 2, 3, 4, 6 - penta - O - galloyl - beta - D - glucose）can effectively resist MRSA and PRSE with MICs (minimum inhibitory concentration) of 15.625 microgram/ml and 62.5 microgram/ml. In combination antibiotics with compound, when combination penicillin 1.25 unit/ml with GR5 3.91 microgram/ml will be able to resist PRSE. In antibacterial activity assay the GR5 showed good effect against 10 clinical isolates of MRSA from hospital. The GR5 can effectively resist four strains with MICs of 125 microgram/ml, and six strains MICs of 125 microgram/ml. Otherwise the GR5 can effectively resist four strains with MICs of 62.5 microgram/ml, and six strains MICs of 125 microgram/ml that 10 clinical isolates of ORSA from hospital. In combination antibiotics aspect, the GR5 inhibitory concentration indices were between 1/2 ~ 1/4 MICs in combination with 2 microgram/ml and under 2 microgram/ml. Besides, accompanied dose to raise, GR5 has the highest percentage of up 80 % on resisting beta - lactamase. Conclude the above advantages, the GR5 has the pontential ability for the development of future antibacterial agent.