臺灣博碩士論文加值系統

膽固醇經過度加熱或長期貯存時會發生自氧化反應，而衍生出一系列的膽固醇氧化物（cholesterol oxidation products, COPs）。COPs經食用後，可引起許多不良病變，如毒害細胞、抑制膽固醇合成、動脈粥瘤狀損傷、致癌或突變等作用。因此有必要減少熱加工或冷藏時膽固醇自氧化之程度。本研究採用20％(w/v)膽固醇標準品，添加0.002％檞皮酮(quercetin)作為氧化抑制劑，利用乙醇為助溶劑，將兩者共溶於paraffin/lauryl alcohol(LA)(4/1,v/v)之溶劑系統中，在氧氣及150℃下，以TLC定性及高效率液相層析儀(HPLC)定量分析膽固醇及COPs，觀察槲皮酮抑制各COPs之生成情形並藉此推導各抑制反應動力學模式及生成速率。
膽固醇於150℃醇溶劑中加熱會有裂解及自氧化反應，其中自氧化包含兩大途徑：碳七氧化及環氧化反應。COPs濃度會隨膽固醇加熱時間增加而增加。以非線性回歸之統計分析碳七氧化反應，發現COPs生成速率常數依序為：7-hydroperoxycholesterol (7-OOH, k1=453.5 h-1) ＞7-hydroxycholesterol (7-OH, k2=270.3 h-1)＞7-ketocholesterol (7-Keto, k3=175.5 h-1)。環氧化反應除了生成5,6-expoxycholesterol (5,6-EP, k4=683.1 h-1)外，溶劑系統中含有LA，會使5,6-EP產生醇化反應(Alcoholysis)反應，而生成具毒性的Triol(5α-cholestan-3β,5,6β-triol, k6'=0.3967 h-1)，Triol會進一步脫氫反應而形成cholestan-3β, 5α-diol-6-one (diol-6-one) (k7=5.353 h-1)。膽固醇添加槲皮酮後，加熱時間30分鐘之前，槲皮酮非常有效抑制COPs之生成，之後由於槲皮酮因本身氧化降解或是熱裂解反應而被耗盡。在150℃，膽固醇存在下，槲皮酮濃度下降速率常數(ki)為3.302 h-1。這導致60分鐘以後各COPs的生成有明顯的上升，故膽固醇受熱之動力學研究分為二個時期來研究：(1)初期槲皮酮存在時期(0-60分鐘)及(2)後期為槲皮酮存在量消耗至抗氧化力明顯下降時期(30-120分鐘)。在初期，膽固醇形成7-OOH之生成速率常數(k1)下降至1.2×10-5 h-1，槲皮酮下降5,6-EP的生成速率常數(k4)亦由683.1 h-1降至0.0157 h-1，初期時槲皮酮藉由提供氫原子捕捉自由基，以中止膽固醇自由基連鎖反應(速率常數(kp)為3.279 h-1)，避免自由基攻擊膽固醇及後續之自氧化反應。在後期，各膽固醇自氧化反應幾乎與對照組相似，k1、k2及k3值分別為111.70 h-1、76.77 h-1及30.99 h-1，與對照組比較仍明顯降低。環氧化反應部份，k4值下降為551.4 h-1，k6'值為1.6313 h-1。膽固醇熱裂解的反應速率常數(k5)也因槲皮酮存在，不論在初期 (k5 = 0.193 h-1)或是後期(k5 = 0.281 h-1)，皆較對照組(k5 = 0.656 h-1)有明顯下降。以上各方程式迴歸分析之R2，除了添加對照組中由Triol生成cholestan-3β, 5α, 6-one的脫氫反應的R2 (0.64)較低外，其餘皆在0.82-1.00，顯示本實驗所提動力學方程式符合膽固醇之自氧化及槲皮酮抑制模式，可作為將來槲皮酮抑制膽固醇自氧化反應的參考依據。

A series of cholesterol oxidation products (COPs) are formed through oxidative degradation during intensive heating at high temperature or long-term storage. COPs are cytotoxic, and can inhibit cholesterol biosynthesis, induce carcinogenesis, mutagenicity and antherosclerosis. Intake of COPs may cause adverse effects to our health. Therefore, it is important to reduce the degree of cholesterol oxidation in food products during thermal processing or cold storage. 0.002％ of quercetin was added into cholesterol standard as an antioxidant, using paraffin/laury alcohol (LA) as solvent and ethanol as co-solvent. Cholesterol was heat at 150℃ for up to 120 min with continuous oxygen purging. In order to study the antioxidative mechanism of quercetin, the various COPs were analyzed by TLC and HPLC and were kinetically studied using non-linear regression models.
Two major reaction pathways－degradation and autoxidation involved during heating of cholesterol at 150℃ in the alcohol solvent system. Among these, the autoxidation pathway includes C-7 and epoxidation routes. Results showed that the COPs concentration increased with increasing heating time. After nonlinear regression analyses on reactions in the C-7 route, the rate constants of COPs formation were observed as follows: 7-hydroperoxycholesterol (7-OOH, k1=453.5 h-1) ＞7-hydroxycholesterol (7-OH, k2=270.3 h-1)＞7-ketocholesterol (7-Keto, k3=175.5 h-1). In the solvent system containing LA, the formation of 5, 6-expoxycholesterol (5, 6-EP, k4 = 709.1 h-1) in the epoxidation route may lead to alcoholysis and the formation of Triol (5α-cholestan-3β,5,6β-triol, k6'=0.3967 h-1). Triol could further proceed dehydrogenation and formed cholestan-3β, 5α-diol-6-one (diol-6-one) (k7=5.353 h-1).
For the degradation pathway, quercetin could effectively reduce the formation of COPs during the initial heating time (<30min). Thereafter, due to oxidative and thermal degradations decrease of antioxidative activity was observed when depletion of quercetin was occurred. The degradation rate constant for quercetin in presence of cholesterol at 50℃ was 3.302 h-1. The depletion of quercetin led to increase of COPs formation. Thus, kinetic studies for heating of cholesterol at 150℃ can be divided into two stages：(1) initial stage in presence of quercetin (0-60min) and (2) final stage at almost depletion of quercetin of which antioxidative activity is highly reduced (30-120min). In the initial stage, the rate constants for 7-OOH formation decreased to 1.2×10-5 h-1 from 453.5 h-1and 5, 6-EP to 0.0157 h-1 from 683.1 h-1. In the initial stage, quercetin acts as free radical scavenger, which terminates the propagation of cholesterol free radical. The scavenging rate constant for quercetin was 3.279 h-1. In the final stage, the rate constants of COPs formation were similar to those of the control, with 3.8 to 5.0 times of reduction. k1、k2 and k3 for the C-7 route were respectively 111.70 h-1, 76.77 h-1and 30.99 h-1,whereas k4 and k6' for the epoxidation route were 551.4 and 1.6313 h-1, respectively. The rate constants for thermal degradation of cholesterol (k5) was 0.656 h-1 for the control and in presence of quercetin they reduced to 0.193 h-1 in the initial stage and to 0.281 h-1 in the final stage. Except for the dehydrogenation reaction of Triol→cholestan-3β,5α,6-one, the correlation coefficients (r2) for the reactions ranged 0.82-1.00. The kinetic model developed in this study can be used to predict the concentration changes of COPs inhibited by quercetin during heating of cholesterol.

目 錄
第一章 緒言…………………………….……………………….....1
第二章 文獻回顧………………………………………………….3
一、膽固醇及其氧化物………………………………….………...3
(一) 膽固醇簡介………………………………….….…….....3
(二) 膽固醇氧化產物簡介……………………….………..…5
(三) 膽固醇自氧化機制………………………….….…….....9
(1)A、B環自氧化反應…………..……….…………….9
(2)側鏈自氧化反應………………..………………..…..12
(四) 膽固醇自氧化動力學……………………….………….14
(1)油脂氧化動力學……………………………………..14
(2)抗氧化劑抑制油脂氧化動力學……………………..22
(3)膽固醇自氧化動力學方程式……………………..…24
(4)膽固醇氧化動力學研究…………………………..…29
(五) 影響膽固醇氧化因子……………………….….……....32
(1)加工方法及溫度……………………………………..32
(2)儲藏時間……………………………….……..…..….34
(3)酸鹼值..……………………………….……………...35
(4)照射處理..……………………………..………….….35
(5)食品的成分………..……………………..……….….37
(6)氧源……..………………………………..………..…39
(7)抗氧化劑..…………………………..……………..…41
(六) 膽固醇氧化物對生理的影響..…………….……….…45
(1)抑制酵素活性…………………………….………….45
(2)細胞毒性……………………………….……..…..….45
(3)心血管疾病..………………………….……………...47
(4)致突變性與致癌性..…………………..………….….47
二、類黃酮物質…………………………………………………...48
(一) 類黃酮物質簡介……………………….….…….……...48
(1)類黃酮物質……………………………….………….48
(2)類黃酮物質之生合成……………….……..…..….....50
(3)類黃酮物質的分類………………….…………….....50
(4)類黃酮物質的分布與飲食來源……..……..…….….56
(二) 類黃酮物質之生理活性..………………….………….61
(1)具防治光氧化之作用…………………….………….61
(2)抗腫瘤與抗癌作用…………………….……..…..….61
(3)對血液循環系統的作用…………………………......63
a.類黃酮與冠狀動脈心臟病…….……………….......63
b.類黃酮的抗過敏、抗血栓與保護血管效果……...63
(4)抗骨質疏鬆效果…...………………..………….…....64
(5)其他作用…..……………………………..……….….64
三、類黃酮物質抗氧化性…………………….….…….…...…….65
(一) 類黃酮化學結構與抗氧化性………….….…….……...65
(二) 槲皮酮的抗氧化性…………………….….…….……...72
(三) 槲皮酮的裂解反應…………………….….…….……...79
第三章 材料與方法……………………………………………...83
一、實驗材料………………………………………..….………...83
(一) 標準品………………………………….……...…….....83
(二) 溶劑及其他試藥……………………….…………....…83
(三) 薄層層析板………………………….………...…….....84
(四) 固相萃取管柱……………………………...….…….....84
二、實驗方法………………………………………..….………...84
(一) 膽固醇氧化物之製備及加熱………….……...…….....84
(二) COPs萃取及純化……..……………….…………....….85
(三) 膽固醇過氧化物及COPs之定性及定量分析……......85
(1)顯色劑Wurster dye之製備…………..….………….85
(2)薄層層析法(TLC)……….…………….……..…..….85
(3)膽固醇及COPs之定量分析……………………......86
(四) 膽固醇自氧化動力學之研究……………...….……....87
(五) 槲皮酮殘存量之定量分析………….…...….……...…87
第四章 結果與討論..………………………………………..…...89
一、定性及定量分析………...……………………..….……..…...89
(一) 膽固醇之熱裂解……...……………….……...…….......89
(二) 膽固醇過氧化物……………………………………..…92
(三) 膽固醇氧化物……………...……….………...…….......93
(四) 槲皮酮殘存量……………………………...….………..104
二、膽固醇受熱之動力學研究…………………..….…………....108
(一) 槲皮酮存在下膽固醇自氧化及熱裂解動力學方程式..108
(二) 膽固醇熱裂解及自氧化動力學方程式…….……....….114
(三) 速率常數……………………………………….…….....120
第五章 結論…………………………………………………...…..128
第六章 參考文獻……………………………..……………....…..129
附錄一…………………………………………...……….…………….144

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