臺灣博碩士論文加值系統

摘要
金門大豆屬（Glycine tomentella Hayata）一條根為台灣民眾廣泛
使用之本土特有中草藥，業者宣稱金門一條根的效用類似古籍記載
之千觔拔屬（Flemingia philipinensis Merr ＆ Rolfe ），亦具有鎮痛解
熱、舒筋活血等功效，但其藥效及有效成分並無研究報告，故本研
究首先比較金門大豆屬及千觔拔屬一條根的生理活性，並純化大豆
屬一條根95 %乙醇萃取物，鑑定其功能特性及有效成分。
大豆屬一條根之水、50 ％乙醇及95 ％乙醇粗萃取物，多酚類
含量分別約為千觔拔屬各相關萃取物的4.5、2 及1.5 倍。以產率換
算時，大豆屬之95 ％乙醇粗萃取物多酚類含量高於千觔拔屬，其餘
兩種萃取物無顯著差異。掃除自由基DPPH (α,
α-diphenyl-β-picrylhydrazyl)、抑制脂質自氧化及抑制脂氧合酶之能
力，皆以大豆屬活性較高。抑制環氧合酶的能力，以千觔拔屬95 ％
乙醇粗萃取物高於大豆屬，且為環氧合酶抑制劑indomethacin 之
30,000 倍。
大豆屬一條根95 ％乙醇粗萃取物經polyamide、silica gel 管柱
可得十一區分（fractions），對於人血液流變性質的影響，添加大豆
屬純化之十一區分化合物之血液（fluid behavior index, n=0.72∼
0.78），皆較未添加一條根分離物之血液（n=0.68）更接近牛頓型流
體（n=1），其黏稠指標（fluid consistency, K）為57∼79 cP，較控制
組（98 cP）低，一條根分離物可降低血液黏稠度，而增加血液流動
性。
在抗氧化力方面，由silica gel 管柱所得五區分抑制不飽和脂肪
酸自氧化力高於95％乙醇粗萃取物，IC50 為粗萃取液之15∼70 ％；
所有區分掃除自由基之IC50 為95 %乙醇粗萃取物之2∼21 倍。
大豆屬一條根之十一區分對脂氧合酶之抑制力為NDGA
（nordihydroguaiaretic acid）的0.36 ~ 1.67 ％、95 ％乙醇粗萃取物
之2.6 ~ 17.8 倍，其中以第VIII 區分抑制力最高，其再經製備型HPLC
分離、TLC、呈色反應及UV 吸收光譜，可推測為不含glycoside 之
異黃酮，以核磁共振光譜及HPLC 鑑定比對為異黃酮類之daidzein
化合物，其抑制脂氧合酶的能力為第VIII 區分及95 %乙醇粗萃取物
之1.4 及23 倍，故daidzein 為大豆屬一條根有效抑制脂氧合酶的化
合物。

Abstract
The I-Tiao-Gung, root of Glycine tomentella Hayata has been used
extensively as a herbal medicine in Taiwan. It was claimed to have similar
functions in analgesic, anti-pyretic, muscle relaxative and blood-circulation
enhancing effects to those of the Flemingia philipinensis Merr ＆ Rolfe.
However, no extensive research regarding its bioactivities nor its active
compounds was found in literature. The objectives of this study were to
compare the bioactivities of the roots of the 2 species, and to separate and
identify the effective compounds in the Glycine root.
The polyphenols content extracted from Glycine, using water, 50 %
ethanol and 95 % ethanol as solvents were 4.5, 2, and 1.5 times of the
corresponding extract of the Flemingia, respectively. The yield in dry
weight of polyphenols of 95 % ethanolic extract of Glycine was higher than
that of Flemingia. The two other solvent extracts showed no significant
difference between the 2 types of I-Tiao-Gung. Glycine was higher in free
radical-scavenging activity determined with DPPH, reduction in
hemoglobin-catalyzed lipid autoxidation and inhibition of the lipoxygenase
(LOX)-catalyzed arachidonate oxidation. But the inhibition on the
cyclooxygenase-catalyzed arachidonate oxidation was higher for the 95 %
ethanolic extract of Flemingia than that of Glycine, and was 30,000 times
of the positive control, indomethacin.
The 95 % ethanolic extract of Glycine was further fractionated using
polyamide and silica gel column into 11 fractions. Blood thinning effects
were observed in vitro by mixing the Glycine root extracts with human red
blood cells. The addition of the 11 fractions of 95 % ethanolic extract of
Glycine root increased the flow behavior index (n) from 0.68 to 0.72~0.78,
and reduced fluid consistency (K) from 98 to 57~79 cP indicative of a
change in flow behavior becoming closer to Newtonian type and a thinning
consistency.
The antioxidant activities of the 5 fractions eluted from silica gel
column were higher than the 95 % ethanolic crude extract. The IC50 values
were 15~70 % of the latter. The IC50 values of all fractions on DPPH free
radical-scavenging activity were 2~21 times than the 95 % ethanolic crude
extracts.
The 11 fractions of the 95 % ethanolic extract showed inhibitory ability
on LOX being 0.36~1.67 % of the NDGA (nordihydroguaiaretic acid), and
2.6~17.8 times of the 95 % ethanolic extract. The inhibitory activity of the
fraction VIII was the highest among the 11 fractions. Fraction VIII was
further separated using HPLC, and preliminarily identified using TLC
sprayed with coloring agents, and UV-Vis spectrum. Isoflavone without
glycoside was considered the bioactive component, which was further
identified with NMR to be daidzein and its inhibitory ability on LOX was
1.4 and 23 times of the fraction VIII and the 95 % ethanolic extract.

目錄
中文摘要........................................................................................................ I
英文摘要...................................................................................................... III
壹、前言........................................................................................................ 1
貳、文獻整理................................................................................................ 5
一、黃酮類之基本結構及分類.................................................................... 5
二、黃酮類之生理功能................................................................................ 7
1. 預防紅血球溶血............................................................................. 7
2. 預防心血管疾病............................................................................. 8
三、異黃酮類之生理功能............................................................................ 9
四、黃酮類之抗氧化機制.......................................................................... 10
1. 螯合金屬離子............................................................................... 10
2. 清除活性氧或自由基...................................................................12
3. 保護內因性抗氧化劑...................................................................12
五、15-脂氧合酶與動脈粥狀硬化的關係.................................................13
六、15-脂氧合酶的抑制劑.........................................................................15
1. vitamin E.......................................................................................15
2. 黃酮類(flavonoids) .......................................................................16
3. PD 146 176 ...................................................................................16
七、產生發炎及過敏反應的化學媒介......................................................17
八、發炎及過敏反應的抑制......................................................................18
1. 環氧合酶作用：生成物前列腺素的生理功能...........................19
2. 5-脂氧合酶作用：生成物白三烯素的生理功能.......................20
3. 以天然物抑制環氧合酶及5-脂氧合酶的作用..........................21
(1)大蒜.........................................................................................21
(2)綠茶.........................................................................................23
(3)橄欖油.....................................................................................23
九、黃酮類之分析方法..............................................................................24
參、材料與方法..........................................................................................26
一、材料......................................................................................................26
二、方法......................................................................................................26
實驗流程......................................................................................................27
1. 一條根粗萃取物之製備...............................................................28
2. 一條根粗萃取物之分離...............................................................28
(1) polyamide 管柱層析.............................................................28
(2) silica gel 管柱層析.................................................................30
(3)製備型HPLC 層析.................................................................31
(4) TLC 分析................................................................................32
(5)呈色反應.................................................................................32
(6)分離物之HPLC 分析.............................................................34
3. 分析方法.......................................................................................35
(1)總多酚類之定量.....................................................................35
(2)脂氧合酶總活性測定.............................................................36
(3)環氧合酶總活性測定.............................................................36
(4)對亞麻油酸自氧化之抑制作用.............................................37
(5)掃除自由基試驗.....................................................................38
(6)血液滲透壓之測定.................................................................39
(7)血液黏度測定.........................................................................40
4. 核磁共振光譜（Nuclear Magnetic Resonance）分析方法........41
5. 統計分析.......................................................................................41
肆、結果與討論..........................................................................................42
一、大豆屬與千觔拔屬一條根粗萃取物功能特性之比較......................42
1. 萃取產率及總多酚類含量...........................................................42
2. 掃除DPPH 自由基之能力..........................................................45
3. 對亞麻油酸自氧化之影響...........................................................47
4. 對脂氧合酶活性之抑制...............................................................49
二、大豆屬一條根活性成分之分離鑑定..................................................51
1.分離步驟.........................................................................................51
(1) polyamide gel 管柱層析.......................................................51
(2) silica gel 管柱層析.................................................................52
2. 活性及純度篩選...........................................................................54
(1)對脂氧合酶之抑制.................................................................54
(2)環氧合酶測定方法之修飾.....................................................57
(3)對環氧合酶之抑制.................................................................58
(4)對血色素催化脂質自氧化之影響.........................................61
(5)掃除DPPH 自由基之能力.....................................................63
(6)對人血液黏度之影響.............................................................65
(7)分離物之純度.........................................................................68
(8)以TLC 及呈色反應判別化合物種類....................................70
3.分離鑑定.........................................................................................72
(1)以製備型HPLC 分離化合物.................................................72
(2)以NMR 鑑定化合物結構......................................................75
(3)以HPLC 鑑定化合物.............................................................80
4. 95EE-70E-f-Hex/EA-VIII-2 對脂氧合酶之抑制.........................80
伍、結論......................................................................................................83
陸、參考文獻..............................................................................................85
圖次
附圖一、黃酮類之基本結構........................................................................5
附圖二、黃酮類之結構分類....................................................................... 6
附圖三、綠茶萃取物之主要成分................................................................8
附圖四、quercetin, kaempferol, morin 之化學結構..................................11
附圖五、PD146176 之結構........................................................................17
附圖六、大蒜中主要活性成份..................................................................21
附圖七、橄欖油之水溶性多酚類..............................................................24
圖一、實驗流程..........................................................................................27
圖二、金門產一條根95 % 乙醇粗萃取物，及經silica gel 管柱之第
VIII 區分HPLC 圖譜......................................................................67
圖三、金門產一條根95EE-f-70E-Hex/EA-VIII 經製備型HPLC 分離
三區分之HPLC 圖譜......................................................................73
圖四、金門產一條根95 ％乙醇粗萃取物經管柱之區分物
（95EE-70E-Hex/EA-VIII-2）全波長掃描圖譜.............................74
圖五、金門產一條根95 ％乙醇粗萃取物經管柱區分物
(95EE-70E-f- Hex/EA-VIII-2)之NMR 1H 圖譜...............................77
圖六、金門產一條根95 ％乙醇粗萃取物經管柱區分物
(95EE-70E-f-Hex/EA-VIII-2)之NMR 13C 圖譜............................78
圖七、金門產一條根95EE-70E-f-Hex/EA-VIII-2 及daidzein
之HPLC 圖譜..................................................................................81
表次
表一、金門產一條根95 %乙醇粗萃取物經polyamide 管柱分離，
沖提溶劑之比例與體積....................................................................29
表二、金門產一條根95 %乙醇粗萃取物經silica 管柱分離，
沖提溶劑之比例與體積................................................................31
表三、多酚類化合物之呈色反應..............................................................34
表四、大豆屬與千觔拔屬一條根萃取液產率及總多酚類含量之比較..44
表五、大豆屬與千觔拔屬一條根萃取液掃除DPPH 自由基之能力.....46
表六、大豆屬與千觔拔屬一條根萃取液抑制血紅素催化亞麻油酸
自氧化之比較..................................................................................48
表七、以冷光法測定大豆屬與千觔拔屬一條根萃取液對於脂氧合
酶作用亞麻油酸之抑制作用..........................................................50
表八、一條根70 ％乙醇分離物經silica gel 管柱區分之分離物乾重..53
表九、大豆屬一條根70 %乙醇分離物，經silica gel 管柱層析後各
層分離物，對脂氧合酶之抑制作用..............................................56
表十、大豆屬、千觔拔屬一條根95 %乙醇粗萃取物，及大豆屬經
silica gel 管柱層析後第VIII 區分對環氧合酶之抑制作用.........60
表十一、大豆屬一條根70 %乙醇分離物，經silica gel 管柱層析後各
層分離物抑制血紅素催化亞麻油酸自氧化之比較..................62
表十二、大豆屬一條根70 %乙醇分離物，經silica gel 管柱層析後各
層分離物清除DPPH 自由基之能力..........................................64
表十三、大豆屬一條根70 %乙醇分離物，經silica gel 管柱層析後各
層分離物對人血液流體特性依冪次模式σ＝K γn 之影響........67
表十四、大豆屬一條根70 %乙醇分離物，經silica gel 管柱層析後
第VIII 層分離物之呈色反應.....................................................71
表十五、金門產一條根95 ％乙醇粗萃取物經管柱區分物
(95EE-70E-f-Hex/EA-VIII-2)與標準品daidzein 之
1H-NMR and 13C-NMR 圖譜數據...............................................79
表十六、大豆屬一條根分離物95EE-70E-Hex/EA-VIII-2 及
標準品daidzein、genistein 對脂氧合酶之抑制作用................82

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