臺灣博碩士論文加值系統

1909年Tanret於黑麥角菌(Claviceps purpurea)中分離純化出一含硫結晶物，並鑑定其為2-硫醇-L-組胺酸-甜菜鹼(2-Mercapto-L-histidine-betaine, ERGO)，後命名為麥角硫因(L-Ergothioneine)。麥角硫因可從菇類、微生物發現，動物則無法自行合成，需藉由飲食攝取。近幾十年來的研究發現，麥角硫因具有保護細胞免受紫外線輻射破壞、防止DNA斷裂和突變，及維持生物體系氧化還原平衡等功能。本論文的研究目的是將新鮮菇類：秀珍菇、杏鮑菇、香菇及金針菇子實體以乙醇進行超音波震盪萃取並減壓濃縮，將所得之萃取物進行麥角硫因含量分析及抗氧化能力試驗。在麥角硫因含量測試中，本研究利用毛細管電泳暨雷射誘導螢光偵測器(CE-LIF)及毛細管電泳暨紫外光吸收偵測器(CE-UV)兩種方法進行分析。結果發現，秀珍菇其麥角硫因含量為本實驗探討菇類之首，於CE-LIF及CE-UV測得麥角硫因含量分別為1353±41 mg/kg DW及1085±23 mg/kg DW，而金針菇萃取物其麥角硫因含量為最低，CE-LIF及CE-UV分析求得麥角硫因含量分別為824.2±6.4 mg/kg DW及549±1 mg/kg DW。麥角硫因在CE-LIF其偵測極限(Limit of detection, LOD)及定量極限(Limit of quantification, LOQ)分別為0.217 nM及0.723 nM；CE-UV其LOD及LOQ分別為41.36 nM及53.86 nM。最後本研究使用兩種不同自由基系統來對菇類乙醇萃取物進行抗氧化能力分析，分別為氧自由基吸收能力法測定(ORAC)及氫氧自由基轉移能力法測定(HORAC)。結果發現，秀珍菇萃取液其ORAC及HORAC抗氧化能力最高，ORAC值及HORAC值分別為9.48±0.20 eq. TE mM/100 g DW及2.77±0.05 eq. GA mM/100 g DW。

In 1909, Tanret was first scientist who discovered a crystalline sulfur-containing compound from Claviceps purpurea. Later, it was identified as a 2-Mercapto-L-histidine-betaine, then this compound was named L-ergohioneine (ERGO). ERGO, is known to be synthesized by fungi and bacteria, though ERGO cannot be synthesized in animals, but it can be accumulated through diet. Throughout the past few decades, there have been several reports on ERGO regarding special physiological function, protecting DNA against oxidation damage and inhibiting cell death caused by reactive oxygen species (ROS) and UV radiation. In this study, we bought various mushrooms: Pleurotus ostreatus, Pleurotus eryngii, Lentinula edodes and Flammulina velutipes. The ERGO was first using ultrasonication-assisted solvent (95% ethanol) extraction, then concentrated by vacuum evaporation. To analyze the ERGO content in these mushrooms, the capillary electrophoresis -laser induced fluorescence detection (CE-LIF) and ultraviolet detection (CE-UV) were employed. The results revealed that the highest ERGO content: 1353±41 mg/kg DW and 1085±23 mg/kg DW were found in P. ostreatus extract for CE-LIF and CE-UV, respectively. The lowest ERGO content was found in F. velutipes with value of 824.26.4 mg/kg DW and 549±1 mg/kg DW for CE-LIF and CE-UV, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) for CE-LIF was found to be 0.217 nM and 0.723 nM, respectively. The LOD and LOQ for CE-UV was 41.36 nM and 53.86 nM, respectively. The antioxidant activity was conducted by oxygen radical absorbance capacity (ORAC) and hydroxyl radical averting capacity (HORAC) methods. The P. ostreatus extract exhibited an especially high antioxidant capacity among these mushrooms with ORAC and HORAC values of 9.48±0.20 eq. TE mM/100 g DW and 3.68±0.03 eq. mM GA/100 g DW, respectively.

摘要………………………………………………………………………Ⅰ
Abstract………………………………………………………………………Ⅲ
致謝………………………………………………………………………Ⅴ
目錄………………………………………………………………………Ⅶ
表目錄………………………………………………………………………Ⅹ
圖目錄………………………………………………………………………ⅩⅡ
第一章 緒論………………………………………………………………………1
1-1 前言………………………………………………………………………1
1-2 研究目的………………………………………………………………………4
第二章 文獻回顧………………………………………………………………………5
2-1 金針菇………………………………………………………………………5
2-1.1 金針菇簡介………………………………………………………………………5
2-1.2 金針菇生物活性………………………………………………………………………6
2-2 杏鮑菇………………………………………………………………………7
2-2.1 杏鮑菇簡介………………………………………………………………………7
2-2.2 杏鮑菇生物活性………………………………………………………………………7
2-3 香菇………………………………………………………………………8
2-3.1 香菇簡介………………………………………………………………………8
2-3.2 香菇生物活性………………………………………………………………………9
2-4 秀珍菇………………………………………………………………………10
2-2.1 秀珍菇簡介………………………………………………………………………10
2-2.2 秀珍菇生物活性………………………………………………………………………10
2-5 麥角硫因………………………………………………………………………11
2-5.1 麥角硫因簡介………………………………………………………………………11
2-5.2 結構與性質………………………………………………………………………11
2-5.3 抗氧化性質與應用………………………………………………………………………14
2-6 氧自由基吸收能力法………………………………………………………………………15
2-7 氫氧自由基轉移能力法………………………………………………………………………16
第三章 菇類麥角硫因含量分析及其抗氧化能力探討………………………………………………………………………19
3-1 前言………………………………………………………………………19
3-2 實驗方法………………………………………………………………………22
3-2.1 材料與方法………………………………………………………………………22
3-2.2 四種菇類萃取方法………………………………………………………………………24
3-2.3 CE-LIF偵測麥角硫因含量之分析………………………………………………………………………25
3-2.4 CE-UV偵測麥角硫因分離條件探討及含量之分析………………………………………………………………………27
3-2.5 氧自由基吸收能力法測定………………………………………………………………………27
3-2.6 氫氧自由基吸收能力法測定………………………………………………………………………30
3-3 實驗結果與討論………………………………………………………………………32
3-3.1 CE-LIF偵測菇類麥角硫因含量之分析………………………………………………………………………32
3-3.2 CE-UV偵測菇類麥角硫因分離條件探討………………………………………………………………………39
3-3.3 CE-UV偵測菇類麥角硫因含量分析………………………………………………………………………45
3-3.4 氧自由基吸收能力法測定………………………………………………………………………50
3-3.5 氫氧自由基吸收能力法測定………………………………………………………………………50
第四章 結論與未來展望………………………………………………………………………55
參考文獻………………………………………………………………………57
作者經歷………………………………………………………………………66

表目錄
表3-1 可與硫醇衍生之螢光其成色衍生物其性質表………………………………………………………………………35
表3-2 菇類其CE-LIF所測得之麥角硫因含量表………………………………………………………………………38
表3-3 菇類其CE-UV所測得之麥角硫因含量表………………………………………………………………………47
表3-4 菇類於ORAC及HORAC其相對抗氧化劑之指數………………………………………………………………………52

圖目錄
圖1-1 麥角硫因於體內可能扮演的角色與交互作用………………………………………………………………………3
圖2-1 麥角硫因結構圖………………………………………………………………………12
圖2-2 麥角硫因互變異構體………………………………………………………………………13
圖2-3 Fenton反應………………………………………………………………………17
圖3-1 HORAC與ORAC其自由基關係圖………………………………………………………………………21
圖3-2 菇類萃取之流程圖………………………………………………………………………24
圖3-3 樣品衍生物及CE-LIF分析步驟流程圖………………………………………………………………………26
圖3-4 ORAC分析步驟流程圖………………………………………………………………………29
圖3-5 HORAC分析步驟流程圖………………………………………………………………………31
圖3-6 麥角硫因與5-IAF反應機制………………………………………………………………………34
圖3-7 麥角硫因標準品(50 nM)與5-IAF反應時間圖與波峰面積之探討………………………………………………………………………36
圖3-8 菇類萃取物與5-IAF衍生反應之CE-LIF分析圖………………………………………………………………………37
圖3-9 不同分離緩衝溶液所對秀珍菇萃取物CE-UV分析圖之影響………………………………………………………………………41
圖3-10 不同濃度之分離緩衝溶液其秀珍菇萃取物CE-UV分析圖………………………………………………………………………42
圖3-11 不同pH之分離緩衝溶液其秀珍菇萃取菇CE-UV分析圖………………………………………………………………………43
圖3-12 不同分離電壓其秀珍菇萃取物CE-UV分析圖………………………………………………………………………44
圖3-13 菇類萃取物其CE-UV分析圖………………………………………………………………………46
圖3-14 CE-LIF與CE-UV其菇類麥角硫因含量比較圖………………………………………………………………………49
圖3-15 ORAC與HOARC其菇類抗氧化比較圖………………………………………………………………………54

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