臺灣博碩士論文加值系統

本研究目的係以台灣傳統藥用植物－巴豆 (Croton tiglium L.) 之根、莖、葉及種子等各部位為試材，測其含水率，然經冷浸法或熱迴流萃取法取得其乙醇萃取液及測定其 pH 值、顏色變化及收率，且利用氣相層析質譜分析儀 (Gas Chromatography-Mass Spectrophotometer, GC/MS) 分析巴豆各部位之主成分，藉以探討其對常見於文物之黴菌，諸如：黑麴黴 (Aspergillus niger)、黃麴黴 (Aspergillus flavus)、綠色木黴 (Trichoderma viride) 與自然落菌時之抗黴性，且以其不同濃度萃取物測定不同黴菌之最低抑制濃度 (Minimum Inhibitory Concentration, MIC)，並將萃取液添加於糊劑進行抗黴應用試驗；然又以其探討病原菌，諸如：金黃色葡萄球菌 (Staphylococcus aureaus)、枯草桿菌 (Bacillus subtilis)、大腸桿菌 (Escherichia col)、大腸桿菌 (Escherichia col) 及綠膿桿菌 (Pseudomonas aeruginosa) 等之抑菌性。
基本性質方面，以葉部含水率最高，為 71.94 %。各部位萃取液 pH 值大致呈偏弱鹼性，其值介於 6.38 - 8.49，而最高者為巴豆莖部乙醇之熱迴流萃取液，其 pH 值為 8.49。顏色變化由巴豆各部位於不同萃取法所得萃取液之 △E＊ 值得知，介於 1.18 – 44.04，且各部位萃取液隨濃度增加而有偏黃綠色之現象。經冷浸法或熱迴流萃取法取得之乙醇萃取液收率介於 1.51 – 10.51 %，其中以熱迴流萃取之葉部最高，為 10.51 %。
經 GC/MS 分析巴豆各部位乙醇萃取物之主成分可分為：酯類、酸類、胺類、醇類及酚類等，其相對含量分別為 10.13 - 78.04 %、4.95 - 20.40 %、0.00 - 83.04 %、0.00 - 3.15 % 及 1.58 - 1.86 % 等。酯類化合物為亞油酸乙酯 (Linoleic acid ethyl ester) 及油酸乙酯 (Ethyl oleate) 等，其相對含量最高者為巴豆種子經乙醇冷浸法之萃取物，值為 26.84 % 及 13.21 %，其次為巴豆根部經乙醇冷浸法萃取物，其值為 3.46 % 及 10.84 %。以酸類化合物而言，各部位均分析出棕櫚酸 (Palmitic acid) 之成分，最高者為葉部經熱迴流萃取法者，其值為 29.91 %，而油酸 (Oleic acid) 部分則以根部經乙醇冷浸法者之相對含量最高，其次為根部之熱迴流萃取物及種子經冷浸法者，其值分別為 4.88 %、4.54 % 及 4.22 %。以胺類化合物而言，除經冷浸法之種子者無此化合物外，其餘者皆具油酸酰胺 (Oleamide) 之成分，最高者為莖部經乙醇熱迴流法者，其值為 70.56 %。醇類部分則以根部熱迴流萃取物之柏木醇 (Cedrol) 的相對含量最高，為 3.15 %。酚類者則以莖部熱迴流萃取物之 2,6- 二叔丁基對甲酚 (2,6-Di-tert-butyl-4-methylphenol) 的相對含量最高，為 1.86 %。
抗黴性結果得知，巴豆各部位無論經冷浸法或熱迴流萃取法所得之乙醇萃取物皆具有抗黴性，且對自然落菌及綠色木黴之抗黴性較佳，又知各乙醇萃取物於濃度 10 mg/mL 即具抗黴性。MIC 結果顯示，巴豆根部以熱迴流萃取法所得之乙醇萃取物具有較低的 MIC 值，且對黑麴黴而言，其值為最低 (0.78 mg/mL)，最高者則為巴豆莖部以冷浸法所得者，其對綠色木黴之 MIC 值為 25 mg/mL。添加於糊劑之抗黴試驗結果顯示，無論莖或葉部之萃取液加入糊劑後進行抗黴試驗均具有抗黴效果，並於接種黑麴黴 56 天後觀察可知，葉部萃取液之黑麴黴生長率已達 100 %，而莖部者則為 50 %，其莖部者抑制黑麴黴生長程度較葉部者有 2 倍之多。以菌種而言，冷浸法之莖部萃取物以濃度 50 mg/mL，其接菌 56 天後，以自然落菌最佳 (10 %)；黑麴黴次之 (20 %)；黃麴黴則達 (35 %)。依成分分析及抗黴性試驗結果顯示，冷浸法之莖部萃取物其亞油酸乙酯及油酸乙酯含量為 11.2 %，優於葉部者之 4.54 %，因而推論添加膠料之抗黴應用試驗結果可知，莖部者較具抗黴性能。
抑菌試驗方面，各試材巴豆乙醇萃取液皆具有抑菌性，以莖部萃取部位者之抑制效果較佳，其中熱迴流萃取法對五種病原菌皆為完全抑制，並於巴豆莖部經熱迴流萃取法所得之乙醇萃取物於濃度 50 mg/mL 時，對枯草桿菌具有最佳抑制能力，其抑菌圈為 18.71 mm；而根部次之，其抑菌圈為 16.95 mm，又由其冷浸法所得者除濃度 10 mg/mL 外，其他濃度者皆為完全抑菌。另依成分分析及抑菌性試驗結果顯示，莖部乙醇萃取物之亞油酸乙酯及油酸乙酯等所佔相對含量達 11.2 %，而根部熱迴流法者則為 9.99 %，但葉部者之冷浸法及熱迴流萃取法為最低，分別為 4.54 % 及 0.00 %，由此推斷無論冷浸法或熱迴流萃取法所得莖部者之抑菌性對五種病原菌的效果較優於葉部者。
由上述結果得知，本研究所用巴豆於不同萃取方法所得各部萃取液對文物黴菌及對病原菌之抑菌性與其酯類成分的亞油酸乙酯、油酸乙酯及油酸等相對含量有關。

The objectives of this study were to use traditional medical plants, Croton tiglium L., as the experimental materials, in Taiwan. The specimens of Croton tiglium are included root, stem, leaves and seed. The basic properties of moisture content for each portion of Croton tiglium were investigated. The yield, pH value and color difference (△E＊) of each portion of extracts obtained from the cold soaking processes or the hot backset refined methods with ethanol was measured. The main compounds of each portion of Croton tiglium were analyzed by using Gas Chromatography-Mass Spectrophotometer (GC/MS). The extracts were to investigate the antifungal (Aspergillus niger, Aspergillus flavus, trichoderma viride and air fungi particles) activities, to determine of Minimum Inhibitory Concentration (MIC) of the extracts with different concentrations for various fungi, and were used to combined with adhesives to carry out the antifungal tests; as well as to examine the antibacterial (Staphylococcus aureaus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa) activities.
As the results of the basic properties, leaves of Croton tiglium had the highest moisture content, 71.94 %. The pH value of each portion of Croton tiglium was from 6.38 to 8.49, and the highest one (8.49) was the stem of ethanolic extracts by using the hot backset refined methods. The extracts of color difference (△E＊) with different extracted method obtained that the value was between 1.18 and 44.04, and it became yellowish green with the increase of the concentration. The yield, 1.51 – 10.51 %, of the ethanolic extracts by using the cold soaking processes or the hot backset refined methods was obtained and the leaves portion, 10.51 %, was the highest one.
By GC/MS analysis, the main compounds from each ethanolic extracts of Croton tiglium were included esters, acids, amines, alcohols and phenols, and their relatived contents were 10.13 - 78.04 %, 4.95 - 20.40 %, 0.00 - 83.04 %, 0.00 - 3.15 % and 1.58 - 1.86 %, respectively. The main compounds of the esters were linoleic acid ethyl ester and ethyl oleate. The highest one was the extracts of the seed portion, 26.84 % and 13.21 % and followed by the root, 3.46 % and 10.84 %, with the ethanolic cold soaking methods. For the acid compounds, the extracts of each portion were analyzed to the compounds of palmitic acid, and the highest one, 29.91 %, was the leaves extracts with the hot backset refined methods. The compounds of oleic acid was 4.88 %, 4.54 % and 4.22 % for the root extracts with the cold soaking processes, the root extracts with the hot backset refined methods, and the seed extracts with the cold soaking processes in that order. From the results of amine compounds, expect for the seed extracts with the cold soaking processes, the others of each portion obtained the compounds of oleamide, and the highest one, 70.56 %, was the stem extracts with the hot backset refined methods. For the compounds of alcohols, cedrol was obtained and its relative content was 3.15 %. From the results of phenol compounds, the highest one was 2,6-Di-tert-butyl-4-methylphenol from the stem extracts with the hot backset refined methods and its value was 1.86 %.
The results of the antifungal activities obtained that regardless of by using the cold soaking or the hot backset refined methods with ethanol, the antifungal activities of the Croton tiglium extracts of root, stem, leaves and seed was all effectiveness. These extracts were with a better antifungal activity to air fungi particles and Trichoderma viride, and each ethanolic extracts with 10 mg/mL concentration were effective to the antifungal activity. The results of the MIC showed that the root extracts of Croton tiglium by using the hot backset refined methods with ethanol had a lower MIC value, and for Aspergillus niger, it was the lowest value, 0.78 mg/mL. The highest one was the stem, using the cold soaking method, and the MIC value against Trichoderma viride was 25 mg/mL. The results of the antifungal tests with the adhesives showed that both the stems and leaves extracts after adding the extracts with the adhesives were antifungal effect, and the 56th days after inoculated with Aspergillus niger showed that the antifungal activities of the leaf extracts for Aspergillus niger growth rate were 100 %, but it was 50 % for the stem ones, indicating that the antifungal activities of the stem extracts to the growth rate of Aspergillus niger were two times of the leaves ones. For the type of fungi, the antifungal activities of the stem extracts, using the hot backset refined methods, with 50 mg/mL of the concentration for the 56th days after the inoculation, the best one was air fungi particles (10 %); followed by Aspergillus niger (20 %) and Aspergillus flavus amounted to (35 %). According to the results of the compounds and the antifungal activities, the stem extracts, using the ethanolic cold soaking methods, with 11.2 % of ethyl linoleate and ethyl oleate that was higher than the leaves ones with 4.45 %. Therefore, it is inferred that the antifungal activities of the stem extracts are better effectiveness.
The results of the antibacterial tests obtained that the extracts were all effective to antibacterial activity. The stem extracts, using the hot backset refined methods, were the best one with inhibitory completely to the antibacterial activity for five sorts of bacteria used, and at 50 mg/mL was the best effective to Bacillus subtilis, as well as the diameter of growth inhibition was 18.71 mm, following by the root ones, 16.95 mm, but not for the extracts by using the cold soaking method at 10 mg/mL. According to the results of the compounds and the antibacterial activities, the stem extracts, using the ethanolic cold soaking methods, with 11.2 % of ethyl linoleate and ethyl oleate that was higher than the leaves ones, regardless of by using the cold soaking or the hot backset refined methods with ethanol, with 4.45 % and 0.00 %. It is suggested the antibacterial activities of the stem extracts are effective to five sorts of bacteria used than these of the leaves ones.
The above results concluded that the antifungal and antibacterial activities of the extracts of each portion of Croton tiglium with different extractive method were concerned with the relative contents of ethyl linoleate, ethyl oleate and oleic acid.

摘要……………………………………………………………………...I
Abstract…………………………………………………………………V
目錄………………………………………………………………....IX
表目錄………………………………………………………...………XIII
圖目錄…………………………………………………………….....XVII
第一章 前言……………………………………………………………..1
第二章 文獻回顧………………………………………………………..6
一、藥用植物之發展現況……………………………………………...6
二、萃取技術………………………………………………………….14
三、巴豆之應用……………………………………………………….16
四、巴豆氣相層析質譜儀 (GC-MS) 分析.……………………………20
五、黴菌……………………………………………………………….24
(一) 黑麴黴 (Aspergillus niger)………………………………...24
(二) 黃麴黴 (Aspergillus flavus)………………………………..24
(三) 綠色木黴 (Trichoderma viride)……………………………24
(四) 自然落菌 (Air fungi particles)…………………………….25
六、紙質文物生物性劣化及防治方法………………………………..26
七、病原菌……………………………………………………………..30
(一) 金黃色葡萄球菌 (Staphylococcus aureaus)…………….30
(二) 枯草桿菌 (Bacillus subtilis)………………………………31
(三) 大腸桿菌 (Escherichia coli)………………………………31
(四) 綠膿桿菌 (Pseudomonas aeruginosa)……………………32
第三章 巴豆之基本性質與成分分析………………………………..34
一、材料與方法………………………………………………………..37
(一) 試驗材料…………………………………………………..37
(二) 試驗方法………………………………………………….37
1. 巴豆之乙醇萃取………………………………………37
2. 各試樣之代號…………………………………………..38
3. 巴豆各部位含水率之測定…………………………….40
4. 巴豆各部位乙醇萃取液pH值之測定…………………40
5. 濾紙浸漬巴豆各萃取液顏色變化之測定……….……..40
6. 巴豆乙醇萃取物收率之測定…………………………...41
7. 濾紙浸漬巴豆各萃取液重量增加率之測定…………...41
8. 巴豆各部位組成分之分析…………………………...42
(三) 統計分析…………………………………………………...43
二、結果與討論………………………………………………………..44
(一) 巴豆各部位之含水率……………………………………...44
(二) 巴豆各部位乙醇萃取液之 pH值………………………...44
(三) 濾紙浸漬巴豆各萃取液之顏色變化……………………...45
(四) 巴豆各部位乙醇萃取物之收率…………………………...46
(五) 濾紙浸漬巴豆各萃取液之重量增加率…………………...47
(六) 巴豆各部位之組成分…………………………………...49
三、結語………………………………………………………………..62
第四章 巴豆乙醇萃取物抗黴性之評估…………………………….65
一、材料與方法………………………………………………………..67
(一) 試驗材料…………………………………………………...67
(二) 試驗方法…………………………………………………...68
1. 抗黴性…………………………………………………...68
2. 最低抑制濃度 (MIC)…………………………………...70
3. 添加於糊劑之抗黴性應用……………………………...70
二、結果與討論………………………………………………………..72
(一) 抗黴性……………………………………………………...72
1. 萃取部位………………………………………………...72
2. 萃取液濃度……………………………………………...75
3. 萃取方法………………………………………………...77
4. 接菌菌種………………………………………………...79
(二) 最低抑制濃度……………………………………………...81
(三) 添加於糊劑之抗黴性應用………………………………...88
1. 萃取部位………………………………………………...88
2. 萃取液濃度……………………………………………...89
3. 萃取方法………………………………………………...91
4. 接菌菌種………………………………………………...92
5. 不同濕度環境…………………………………………...93
三、結語………………………………………………………………..95
第五章 巴豆乙醇萃取物抑菌性之評估…………………………….97
一、材料與方法………………………………………………………..100
(一) 試驗材料………………………………………………….100
(二) 試驗方法………………………………………………….101
(三) 統計分析………………………………………………….102
二、結果與討論………………………………………………………..103
(一) 抑菌性…………………………………………………….103
三、結語………………………………………………………………..107
第六章 結論………………………………………………………......109
參考文獻……………………………………………………………...113

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