臺灣博碩士論文加值系統

綠藻為現今熱門的天然藻類保健素材之ㄧ，因部分保健功效與糖苷酵素(α-glucosidase、β-glucosidase及β-glucuronidase)活性之抑制有潛在相關，故本研究探討綠藻(Chlorella vulgaris)各種區分物之分離、分子性質鑑定及對上述三種酵素活性之影響。樣品包括: 綠藻熱水萃取物 (WS)、綠藻熱水不溶物經Cellulase+Pectinase降解之水可溶物(E1)、綠藻粉經cellulase降解之凍乾物的熱水萃物 (E2) ，上述樣品再經超濾膜處理次劃分成大(F1)、小(F3)分子區分物。再自綠藻熱水萃不溶物製備70 %酒精萃取(WIS-E70S) 與耐酵素降解之不溶物(E1-WIS、E2-WIS)等。分子篩層析結果顯示，上述產物分子呈雙元分佈者有: WS-F1 (重量平均分子量為3395 kDa及170 kDa)、WS-F3 (5 kDa及1 kDa)、E1-F1 (14 kDa及5 kDa); 呈單分佈者為: E1-F3 (6 kDa)、E2-F1 (158 kDa)、E2-F3 (6 kDa)。WS-F1的主要單糖組成為核糖(49 %); E1-F1為半乳糖(38 %); E1-WIS為阿拉伯糖(40 %); WS-F3、E1-F3、E2-F1、E2-F3及E2-WIS等皆為葡萄糖(約30~85 %不等)。由酵素抑制與酵素動力學實驗結果得知，在萃取物濃度10 mg/mL下，對α-glucosidase之抑制以WIS-E70S (61 %)效果較佳，經Lineweaver-Burk作圖顯示其抑制作用為混合型非競爭性抑制; 對β-glucosidase以WS-F1(56 %)與E2-F3(48 %)效果較佳，分別為競爭性抑制與混合型非競爭性抑制; 而對β-glucuronidase以WIS-E70S抑制效果較佳(在 10 mg/mL下達89 %)，為一個強的混合型非競爭性抑制劑。

Chlorella vulgaris is one of the popular healthy materials in natural algae, with healthy efficiency potentially relating to glycosidase (α-glucosidase, β-glucosidase and β-glucuronidase) inhibition. Therefore, this study was to investigate the isolation, preparation, characterization, and glycosidase inhibition of various fractions from Chlorella vulgaris. The samples included: hot water extract of Chlorella (WS), enzymatic hydrolyte of Chlorella hot water insoluble (WIS) degraded by both cellulase and pectinase (E1), and hot water extract of cellulase hydrolyte from freeze-dried Chlorella powder (E2), and the large (F1) and small (F3) molecular fractions of the above samples separated by ultrafiltration. The 70 % ethanol soluble of WIS (WIS-E70S) and resistant enzyme hydrolysis insoluble substance (E1-WIS, E2-WIS) were further prepared. The results of high-performance molecular size exclusion chromatography indicated that the samples of two molecular distributions were: WS-F1 (3395 kDa and 170 kDa), WS-F3 (5 kDa and 1 kDa), E1-F1 (14 kDa and 5 kDa); those of mono-distribution included: E1-F3 (6 kDa), E2-F1 (158 kDa), E2-F3 (6 kDa). The major monosaccharides was ribose for WS-F1 (49%), galactose for E1-F1 (38 %), arabinose for E1-WIS (40 %), and glucose for the others (WS-F3, E1-F3, E2-F1, E2-F3 and E2-WIS) (approximately 30~85%). The results of enzyme inhibition and enzymatic kinetics suggested that, at a concentration of 10 mg/mL, WIS-E70S exhibited the best inhibitive effect against α-glucosidase (61%), in a mixed-type noncompetitive way implied by Lineweaver-Burk plot. WS-F1 (56%) and E2-F3 (48%) showed the greatest inhibition on β-glucosidase, in competitive and mixed-noncompetitive types, respectively. And, WIS-E70S was the most effective to reduce β-glucuronidase activity (89% at 10 mg/mL) with a strongly mixed-type noncompetitive manner.

目錄

頁次
中文摘要...................................................I
英文摘要..................................................II
謝誌......................................................IV
目錄.......................................................V
圖目錄....................................................VI
表目錄...................................................VII
一、 前言..................................................1
二、 文獻回顧...............................................2
1.綠藻之分類............................................2
2.綠藻的營養特質與組成...................................3
3.綠藻萃取方法..........................................6
4.綠藻之生理功效........................................7
4.1.具有抗氧化能力....................................7
4.2.促進免疫活性......................................7
4.3.提高脂質代謝......................................8
4.4.預防細菌感染......................................9
5.綠藻在工業上之應用....................................10
6.植物細胞壁組成.......................................11
6.1.初生細胞壁.......................................11
6.2.次生細胞壁.......................................12
6.3.中膠層..........................................12
7.水解細胞壁之酵素種類與功用.............................15
7.1.纖維素分解酵素...................................15
7.2.果膠分解酵素.....................................16
8.消化道中糖苷酵素之抑制................................18
8.1. α-Glucosidase.................................18
8.2. β-glucosidase及β-glucuronidase.................20
三、 研究目的..............................................22
四、 材料與方法............................................23
1.1.樣品制備流程.......................................23
1.2.實驗流程..........................................24
2.化學藥品............................................25
3.樣品制備............................................25
3.1.未破璧綠藻......................................25
3.2.酵素破璧綠藻....................................26
3.3.製備綠藻乙醇萃取物................................26
4.綠藻藻體細胞之顯微鏡觀察...............................26
5.化學分析.............................................26
5.1.總醣測定.........................................26
5.2.醣醛酸測定.......................................27
5.3.蛋白質測定.......................................27
5.4.UV/Visible 光譜及染劑錯合鑑定.....................29
(1)高濃度碘溶液試驗.................................29
(2)Congo red......................................29
(3)Aniline blue...................................29
5.5.α-amylase降解對碘呈色的影響........................29
6.分子性質之測定........................................30
6.1.分子量分布........................................30
6.2.單糖組成分析......................................30
7.酵素抑制活性測定......................................33
7.1. α-Glucosidase之活性測定..........................33
7.2. β-glucosidase之活性測定..........................34
7.3. β-glucuronidase之活性測定........................34
8.酵素抑制動力學........................................37
五、 結果與討論............................................39
1.顯微鏡觀察藻體外觀.....................................39
2.E1-酵素降解時pH值之變化................................39
3.基本組成分析..........................................39
4.分子量分布............................................43
5.單糖組成分析..........................................43
6.綠藻萃取物之染劑錯合鑑定...............................48
7.α-amylase酵素降解....................................48
8.酵素活性抑制..........................................52
8.1. α-Glucosidase..................................52
8.2. β-glucosidase..................................52
8.3. β-glucuronidase................................52
9.酵素抑制動力學........................................58
9.1. α-Glucosidase..................................58
9.2. β-glucosidase..................................67
9.3. β-glucuronidase................................68
六、 結論.................................................70
七、 參考文獻..............................................71

圖目錄
頁次
圖一 顯微鏡下之Chlorella Vulgaris......................3
圖二 植物細胞壁之構造..................................13
圖三 1-deoxynojirimycin之結構式.......................19
圖四 總醣檢量線.......................................28
圖五 醣醛酸檢量線.....................................28
圖六 單糖標準品之HPAEC檢量線...........................32
圖七 4-nitrophenyl α-D-glucopyranoside檢量線.........35
圖八 4-nitrophenyl β-D-glucopyranoside檢量線.........35
圖九 4-nitrophenyl β-D-glucuronide檢量線.............36
圖十 綠藻藻體外觀.....................................40
圖十一 綠藻在水中進行E1酵素降解時pH之變化.................41
圖十二 綠藻萃取物之分子量分布圖...........................44
圖十三 綠藻萃取物之染劑錯合鑑定...........................50
圖十四 綠藻萃取物之α-amylase酵素降解物的碘染呈色試驗.......51
圖十五 各種綠藻萃取物和劃分物對α-glucosidase之抑制活性.....54
圖十六 各種綠藻萃取物和劃分物對β-glucosidase之抑制活性.....55
圖十七 各種綠藻萃取物和劃分物對β-glucuronidase之抑制活性...56
圖十八 WIS-E70S對α-glucosidase之抑制型態(A)，斜率(B) 及
1/Vm’(C)對抑制濃度做圖...........................59
圖十九 Acarbose對α-glucosidase之抑制型態(A) 及斜率(B)對抑制
濃度做圖.........................................60
圖二十 Green tea對α-glucosidase之抑制型態(A)，斜率(B) 及
1/Vm’(C)對抑制濃度做圖...........................61
圖二十一 WS-F1對β-glucosidase之抑制型態(A) 及斜率(B)對抑制濃度
做圖............................................62
圖二十二 E2-F3對β-glucosidase之抑制型態(A) ，斜率(B) 及
1/Vm’(C) 對抑制濃度做圖..........................63
圖二十三 WIS-E70S對β-glucuronidase之抑制型態(A) ，斜率(B) 及
1/Vm’(C) 對抑制濃度做圖..........................64
圖二十四 Green tea對β-glucuronidase之抑制型態(A) 及斜率(B)
對抑制濃度做圖...................................65

表目錄
頁次
表一 綠藻乾燥藻體中含有之營養素...........................4
表二 綠藻萃取方法之文獻整理..............................6
表三 近年來綠藻生理機能性之研究..........................10
表四 已知之綠藻多醣及細胞壁組成..........................14
表五 酵素降解綠藻細胞壁之研究............................17
表六 腸道酵素抑制之研究.................................21
表七 單糖組成之HPAEC 分析條件...........................31
表八 單糖分析之回歸方程式...............................32
表九 綠藻成分分析......................................42
表十 綠藻萃取物之分子量分布.............................45
表十一 綠藻萃取物之單糖組成...............................46
表十二 綠藻萃取物及劃分物在10 mg/mL下對酵素之抑制活性.......57
表十三 抑制物對酵素之抑制型態及動力學參數(Ki、αKi及α值).....66

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