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研究生:林佩琳
研究生(外文):Pei-Lin Lin
論文名稱:到手香精油之抑菌性研究及其在生物支架上之應用
論文名稱(外文):Study on the antibacterial activity of Plectranthus amboinicus (Lour.) Spreng essential oil and its application on biological scaffold
指導教授:賴麗旭賴麗旭引用關係
口試委員:林麗雲金安兒
口試日期:2013-01-17
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:184
中文關鍵詞:到手香抑菌生物支架
外文關鍵詞:Plectranthus amboinicus (Lour.) Sprengantibacterialbiological scaffold
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到手香 (Plectranthus amboinicus (Lour.) Spreng.),為唇形科(Labiatae)多年生草本植物,具濃郁香氣。目前有關到手香之文獻資料多著重於精油之成分分析,以及抗發炎、抑制黴菌等功效上,對細菌之抑菌效果的研究則較少看到。因此,本研究將五月、六月和八月份採收之到手香以40℃烘箱乾燥成七成乾、室溫陰乾成五成乾及日光曬乾三種方式乾燥並磨成粉後,以蒸氣蒸餾法(Steam distillation)萃取精油,再以氣相層析質譜儀(GC/MS)分析精油組成及其對皮膚上之四種病原菌金黃色葡萄球菌 (Staphylococcus aureus)、表皮葡萄球菌(Staphylococcus epidermidis)、痤瘡丙酸桿菌(Propionibacterium acnes)、綠膿桿菌(Pseudomonas aeruginosa) 及二種食品中毒菌沙門氏桿菌 (Salmonella enterica subsp. enteric)、大腸桿菌 (Escherichia coli )之抑菌效果。結果顯示以八月份日光曬乾之到手香葉精油的萃取率最佳,約為1.0%。以GC/MS分析出精油之主成分為芹香酚 (carvacrol),佔36.69%,另以GC測定其含量為0.61 mg/mL。在抑菌性結果方面,20%到手香精油對Propionibacterium acnes具顯著抑菌性,抑菌圈達32.4±0.4 mm,對Pseudomonas aeruginosa之抑菌圈最小為12.2±0.6 mm。抑菌率則隨著精油濃度增加而上升,0.05%到手香精油對Staphylococcus aureus之抑菌率最佳達83.45%, Salmonella enterica subsp. enterica 次之為77.12%,Escherichia coli第三為64.62%。之後將不同濃度的到手香精油應用於以0.9%刺槐豆膠和0.9%幾丁聚醣為主成分之生醫材料的製備中,發現製作完成的生物支架樣品對六種細菌之抑菌效果並不理想,推斷可能是支架的結構限制了精油的移動,或是精油在支架製作過程中揮發喪失所致。以掃描式電子顯微鏡(SEM) 及影像分析軟體觀察生物支架樣品之微細結構發現,以未添加到手香精油之生物支架樣品其結構較緊密,平均孔洞直徑最小,添加1% 精油及0.1% Tween 80之樣品平均孔洞直徑最大。之後進行性質的測定,發現所有生物支架樣品的厚度約介於2.2~3.3 mm之間。壓縮試驗的結果顯示,以未添加到手香精油之樣品壓縮應力值最大,添加0.5%精油、0.05% Tween 80之樣品壓縮應力值最小。綜合以上結果可知,未添加到手香精油之樣品結構較緊密、機械性質較硬,而添加0.5%精油、0.05% Tween 80之生物支架樣品則較柔軟。到手香精油對皮膚上的三種病原菌及食品中常見的二種細菌具有良好之抑菌效果,但添加到手香精油之生物支架對六種細菌之抑菌效果並不理想,未來值得進一步探討如何在生醫材料之製備中將到手香精油做更好的應用。
Plectranthus amboinicus (Lour.) Spreng is one of the Labiatae perennial herbs with a rich aroma. The research works in the literatures about P. amboinicus (Lour.) Spreng mostly focus on the composition analysis of the essential oil and its anti-inflammation and mold inhibition activity. In contrast, researches on its antibacterial activity are quite limited. Therefore, in this study, the P. amboinicus (Lour.) Spreng harvested in May, June and August were oven dried at 40℃ to 30% moisture, room temperature dried to 50% moisture, or sun dried followed by pulverization. The essential oils in different samples were then extracted by using steam distillation method. The compositions of the essential oils were analyzed by gas chromatography-mass spectrometry (GC/MS), and its antibacterial activity to six strains of bacteria commonly found on the infected skin or food-borne pathogen, including Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, Salmonella enterica subsp. enteric, Pseudomonas aeruginosa, and Propionibacterium acnes were studied. It was found that the sun-dried P. amboinicus (Lour.) Spreng leaves harvested in August showed the highest extraction yield (about 1.0%) of essential oils among the samples tested. As analyzed by GC/MS, the main compound in essential oil was found to be carvacrol (about 36.69%), and its concentration was found to be 0.61 mg/mL as detected by GC. Antibacterial activity as determined by agar-diffusion method revealed that 20% P. amboinicus (Lour.) Spreng essential oil was most effective to inhibit the growth of Pro. acnes, as evidenced by a large inhibition zone of 32.4±0.4 mm, and least effective to inhibit the growth of P. aeruginosa, as evidenced by a small inhibition zone of 12.2±0.6 mm. Furthermore, the inhibition rate increased with increasing the concentration of essential oil, and 0.05% P. amboinicus (Lour.) Spreng essential oil was most effective to inhibit the growth of Staphylococcus aureus, as evidenced by reaching an inhibition rate of 83.45%, followed by Salmonella enterica subsp. Enteric (77.12%) and Escherichia coli (64.62%). The P. amboinicus (Lour.) Spreng essential oil was then applied to fabricate a bio-scaffold with 0.9% locust bean gum and 0.9% chitosan. It was found that the antibacterial activity of the bio-scaffold with essential oils to 6 strains of bacteria was not significant, implying either the movement of essential oils were restricted to the matrix of bio-scaffold or the essential oils were loss during the manufacture process of bio-scaffold. SEM and image analysis results revealed that the pore size of the bio-scaffold made of 0.9% locust bean gum and 0.9% chitosan was the smallest, and the pore size of the sample made of 1% essential oil, 0.1% Tween 80, 0.9% chitosan and 0.9% locust bean gum was the largest. The thickness of all bio-scaffold samples was in the range of 2.2~3.3 mm. Mechanical analysis revealed that the sample made of 0.9% locust bean gum and 0.9% chitosan had the largest compressive stress, and the compressive stress of the sample made of 0.5% essential oil , 0.05% Tween 80, 0.9% chitosan and 0.9% locust bean gum was the smallest. Based on the above results, we concluded that the bio-scaffold made of 0.5% essential oil, 0.05% Tween 80, 0.9% chitosan and 0.9% locust bean gum showed flexible texture with less tight pores. In contrast, bio-scaffold made of 0.9% locust bean gum and 0.9% chitosan had tight construction and hard texture. P. amboinicus (Lour.) Spreng essential oil had pronounced antibacterial activity to six strains of pathogenic bacteria, and is worthy of further investigating for a better way to incorporate the essential oils during the fabrication of bio-scaffold.
目錄
摘要I
AbstractIII
縮寫表XV
壹、前言1
貳、文獻回顧3
一、唇形科植物3
(一) 唇形科植物特徵 3
(二) 分布及藥用4
二、簡介本研究所使用之藥用植物—『到手香』5
(一) 形態5
(二) 藥用8
(三) 方例8
三、簡介植物精油9
(一) 認識精油9
(二) 精油的化學成分11
(三) 精油的種類15
四、精油之萃取方式16
(一) 水蒸餾法(Water distillation)17
(二) 蒸氣蒸餾法(Steam distillation)17
(三) 溶劑萃取法(Organic solvent extraction)18
(四) 脂吸法(Enfleurage)18
(五) 壓榨法(Expression)19
(六) 超臨界流體萃取法(Supercritical fluid extraction,簡稱SFE)19
五、簡介精油之分析方法20
(一) 氣相層析法 (Gas chromatography, GC) 及氣相層析/質譜儀 (GC/MS)20
(二) 液相層析法 (Liquid chromatography, LC)22
(三) 濾紙層析法 (Paper chromatography, PC)、薄層層析法 (Thin-layer chromatography, TLC)23
(四) 高效液相層析法 (High performance liquid chromatography, HPLC)24
(五) 超臨界流體層析法 (Supercritical fluid chromatography, SFC)24
(六) 核磁共振光譜分析 (Nuclear magnetic resonance spectroscopy, NMR)24
六、簡介純露25
七、簡介本研究所使用之人體中常發現之微生物27
(一) 金黃色葡萄球菌 (Staphylococcus aureus)27
(二) 綠膿桿菌 (Pseudomonas aeruginosa )31
(三) 痤瘡丙酸桿菌 (Propionibacterium acnes )33
(四) 大腸桿菌 (Escherichia coli )36
(五) 表皮葡萄球菌 (Staphylococcus epidermidis)39
(六) 沙門氏桿菌 (Salmonella enterica subsp. enterica )41
七、多孔性生物支架 (Porous bio-scaffold)45
(一) 簡介生物支架45
(二) 簡介幾丁聚醣 (Chitosan) 46
參、研究目的53
肆、材料與方法54
一、實驗架構54
二、實驗材料與方法55
(一) 到手香55
(二) 樣品前處理55
(三) 到手香乾燥粉末之基本成分分析62
(四) 『到手香』精油和純露的製備69
(五) 精油之組成性質分析73
(六) 精油表面張力 (Surface tension)之測定77
(七) 精油和純露之微生物抑菌試驗78
(八) 精油對細菌之抑菌率試驗89
(九) 精油對細菌之最小抑菌濃度 (Minimal Inhibitory Concentration, MIC) 試驗91
(十) 精油對細菌之最小殺菌濃度 (Minimal Bactericidal Concentration, MBC) 試驗94
(十一) 精油在生物支架上之應用95
(十二) 生物支架性質測定101
(十三) 統計分析104
伍、結果與討論106
一、基本成分分析106
二、『到手香』水萃取精油之萃取率109
三、精油之組成性質分析113
(一) 八月份經日光曬乾之到手香萃取精油成分分析113
(二) 主成分 Carvacrol 之 GC 定量分析123
四、精油表面張力 (Surface tension) 之測定125
五、到手香精油和純露之微生物抑菌試驗127
(一) 六種細菌之抑菌圈分析 (定性)127
(二) 六種細菌之抑菌率分析 (定量)133
(三) 六種細菌之最小抑菌濃度 (MIC)135
六、到手香精油在生物支架上之應用139
(一) 生物支架樣品製備時不同乳化劑對精油之影響139
(二) 生物支架對六種微生物之抑菌試驗142
(三) 微結構分析151
(四)生物支架性質測定157
陸、結論167
柒、參考文獻170
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