臺灣博碩士論文加值系統

本論文之目的在於探討40ºC水、100ºC熱水、50%酒精及正己烷等四種溶劑之蓮子心(蓮Nelumbo nucifera Gaertn)萃取物對免疫細胞及streptozotocin (STZ)誘發之糖尿病BALB/c小鼠免疫調節功能的影響，利用體外(in vitro)及體內(in vivo)等實驗模式，進行抗發炎功效的評估，並對蓮子心的四種溶劑萃取物進行成分分析。
成分分析的結果顯示，蓮子心的四種不同溶劑萃取物成分組成略有差異，總酚及總醣含量是在50%酒精萃取物中較高，而總蛋白及總類黃酮含量則在40ºC水萃取物中較高。
體外免疫細胞實驗的結果顯示，蓮子心四種溶劑萃取物的免疫調節功效略有不同，40ºC水萃取物傾向促發炎趨勢的免疫調節，100ºC熱水及50％酒精萃取物則傾向抗發炎趨勢的免疫調節，正己烷萃取物的免疫調節趨勢則不確定。
以蓮子心及其酒精萃取物連續28天餵食STZ誘發之BALB/c糖尿病小鼠的結果顯示，實驗組及控制組的體重皆無顯著改變，大多呈緩慢增加的趨勢。一般生化指標顯示，餵食低(1mg/mouse/day)及高劑量(20 mg/mouse/day)蓮子心使小鼠胸腺重量下降，並降低血清中葡萄糖及IgG的濃度；餵食中劑量(5 mg/mouse/day)蓮子心，降低IgG、IgA及IgM的濃度。而餵食低劑量(0.2 mg/mouse/day)蓮子心50%酒精萃取物，則使副睪脂肪組織重量下降，並降低HDL、IgG與IgA的濃度；餵食中劑量(1 mg/mouse/day)蓮子心50%酒精萃取物，使胸腺重量下降，並降低TG、HDL、IgG及IgA的濃度；餵食高劑量(4 mg/mouse/day)蓮子心50%酒精萃取物，降低葡萄糖、HDL及IgG的濃度。免疫指標顯示，餵食蓮子心對腹腔細胞具有傾向抗發炎調節趨勢的功效，對脾臟細胞為傾向Th2免疫反應的調節趨勢；餵食蓮子心50%酒精萃取物對腹腔細胞具有傾向抗發炎調節趨勢的功效，但對脾臟細胞則傾向促發炎調節趨勢及傾向Th2免疫反應的調節趨勢。
綜合體外及體內實驗的結果顯示，蓮子心酒精萃取物對腹腔細胞具有抗發炎功效，但對脾臟細胞則呈現不同的功效。

The aim of this study was to investigate the immunomodulatory effects of lotus (Nelumbo nucifera Gaertn) plumule and its extracts using 40°C water, 100°C hot water, 50% ethanol and n-hexane on primary immune cells and streptozotocin (STZ) -induced diabetic mice. The chemical compositions including total phenol, sugar, protein and flavonoids of different extracts from lotus plumule were determined.
The results showed that differential constitutes existed in the four lotus plumule extracts. Ethanol extracts of lotus plumule consisted of abundant phenolic and sugar contents, however higher protein and flavonoid contents were existed in 40°C water extracts of lotus plumule.
In an in vitro study using primary immune cells, the immunomodulatory effects of different extracts from lotus plumule were differential. The 40°C water extracts demonstrated a pro-inflammatory potential, however the 100°C hot water and 50% ethanol extracts exhibited an anti-inflammatory potential in vitro. The immunomodulatory potential of n-hexane extracts was still uncertain.
In 28 days consecutive tube feeding study using STZ-induced female BALB/c diabetic mice, lotus plumule and its ethanol extracts were conducted to test the immunomodulatory potential under a diabetic disease stress. The results showed that supplementation with lotus plumule and its ethanol extracts did not significantly affect the mouse body weight compared to the control group. Supplementation with low (1 mg/mouse/day) and high dose (20 mg/mouse/day) crude sample significantly decreased thymus weight, serum glucose and IgG levels. Supplementation with medium dose (5 mg/mouse/day) crude sample significantly decreased serum IgG, IgA and IgM levels. Supplementation with low dose (0.2 mg/mouse/day) ethanol extracts from lotus plumule significantly decreased adipose tissue weight, serum HDL-cholesterol, IgG and IgA levels. Supplementation with medium dose (1 mg/mouse/day) ethanol extracts from lotus plumule significantly decreased thymus weight, triglycerides, HDL-cholesterol, IgG and IgA levels. Supplementation with high dose (4 mg/mouse/day) ethanol extracts significantly decreased serum glucose, HDL-cholesterol and IgG levels. According to the secretion and ratio of Th1 and Th2 cytokines by splenocyte and peritoneal cell cultures from STZ-induced diabetic mice, crude lotus plumule supplementation demonstrated anti-inflammatory effects on peritoneal cells and exhibited Th2-skewed immunodulatory effects on splenocytes. Supplementation with ethanol extracts of lotus plumule also demonstrated anti-inflammatory effects on peritoneal cells and exhibited Th2-skewed immunomodulatory effects on splenocytes, however supplementation with ethanol extracts of lotus plumule seemed to have pro-inflammatory effects on splenocytes.
In conclusion, the ethanol extracts of lotus plumule demonstrated an anti-inflammatory effect on peritoneal cells in both in vitro and in vivo studies. However, ethanol extracts of lotus plumule exhibited differential effects on splenocytes in vivo.

總 目 次
表目次……………………………………………………….…………….......…vi
圖目次…………………………………………………...……………….……..viii
縮寫對照表………………………………………...…………………………….ix
中文摘要……………………………………………….………………………....x
英文摘要……………………………………………….…………………………xi


第一章 緒言……………………………………….…….….……….…………1
第一節 前言…………………………………..………………….…………1
第二節 文獻整理……………………………..………………….…………2
壹、 免疫系統簡介….…………………….…………………….….……2
一、 先天性免疫反應………………….…………………....….….…2
二、 適應性免疫反應…………………..………………...…..………3
貳、 細胞激素之功能………………….….……………………..………3
參、 Th1與Th2細胞亞群之平衡………...…….…….………….…...…4
肆、 糖尿病與發炎反應……………...…..…………….………..………8
伍、 傳統中草藥與免疫反應…………….……...……………..……….9
陸、 蓮的背景介紹……………………….….……………..…..………10
柒、 蓮各部位之成份與效用……………..…….……………….…..…11
第三節 研究動機及目的………..………….……………………….……15

第二章 蓮子心不同溶劑萃取物之製備與其體外免疫調節功能之研究..17
第一節 前言………………………..………...………..…….……………17
第二節 材料與方法………………………….………………...…………17
壹、 材料製備……………………………..………………….…………17
貳、 成分分析……………….………………...…………….….………17
參、 蓮子心不同溶劑萃取物體外免疫調節功能評估…...……….....20
一、 實驗動物…………….………………….………………………20
1. 初代免疫細胞之來源…………..…………………………...……20
2. 腹腔細胞之取得與培養………….………………………...……20
3. 脾臟細胞之取得與培養………….………………………...……21
二、 蓮子心不同溶劑萃取物對免疫細胞之調節功能評估………22
1. 實驗設計……………………………..…..……………….………22
2. 脾臟細胞增生之測定………………...………….……….………23
三、 發炎媒介物之測定…………………..………………...………23
1. 一氧化氮(nitric oxide, NO)分析………..………………....……23
2. IL-1β、IL-6、TNF-α及IL-10之測定…..…………….….……25
四、 統計分析…………………………………...………..............…27
第三節 結果…………………………………………....…………….……28
壹、 蓮子心不同溶劑萃取物的基本化學成分分析…………….……28
貳、 蓮子心不同溶劑萃取物體外免疫調節功能評估…...…….……28
一、 四種蓮子心萃取物對脾臟細胞存活之影響………...…...……28
二、 四種蓮子心萃取物對脾臟細胞分泌細胞激素之影響.….……29
1. 蓮子心40ºC水萃取物……………………………….……….……29
2. 蓮子心100ºC水萃取物……………………………..................…29
3. 蓮子心50%酒精萃取物…………………………………………...29
4. 蓮子心正己烷萃取物…………………………………………...…30
三、 四種蓮子心萃取物對腹腔細胞分泌細胞激素之影響….…….30
1. 蓮子心40ºC水萃取物…………………….………………….……30
2. 蓮子心100ºC水萃取物……………………….…...………...……30
3. 蓮子心50%酒精萃取物………………………….……………..…31
4. 蓮子心正己烷萃取物……………………………….……………..31
第四節 討論………………………………………………………….……42
ㄧ、萃取率及成分組成……………………………………………..……42
二、脾臟之抗發炎功效……………………………………………..……42
三、腹腔之抗發炎功效……………………………………………..……42
第五節 結論………………………………………………….…....………44

第三章 蓮子心及其萃取物對Streptozotocin誘發之糖尿病BALB/c雌鼠免疫調節功能的研究………………………………………….……45
第一節 前言…………………………………………………………….…45
第二節 材料與方法…………………………………………………….…45
壹、 STZ糖尿病誘發模式之建立…………………….…………….…45
一、 實驗動物…………………………………………….……………45
二、 實驗設計……………………………………………….…………46
三、 不同濃度STZ的配製及糖尿病誘導之方式……...……..…….46
四、 葡萄糖耐受性試驗…………………...…………………........…49
五、 初代免疫細胞之取得與培養…….………………..……………49
1. 腹腔細胞………………………………..………………..…………49
2. 脾臟細胞…………………………………..…………………..……50
六、 一般生化指標之分析…………………….…………………..…51
1. 臟器組織重量之測定…………………………..………………….51
2. 血糖之測定…………………………………………..………….…51
七、 免疫指標之分析……………………………………..…………..52
1. 一氧化氮(nitric oxide, NO)分析………………………….....……52
2. IL-1β、IL-2、IL-4、IL-5、IL-6、TNF-α及IL-10之測定…..…52
八、 統計分析………………………...…………………..…………..…53
貳、 糖尿病誘發型BALB/c小鼠之蓮子心及其酒精萃取物28天餵食試驗……………………………….……………………..…………53
一、 實驗動物……………………………………………...........……53
二、 實驗設計…………………………………………...………….....54
三、 葡萄糖耐受性試驗……..……….……………….………...……54
四、 初代免疫細胞之取得與培養………....………….…..…………54
1. 腹腔細胞……………………………………….…….……..………54
2. 脾臟細胞……………………………………………..………..……54
五、 一般生化指標之分析…………………...…..………………..…54
1. 體重變化……………………………………………………..…..…54
2. 臟器絕對組織及相對組織重量變化……………………………..54
3. 血糖之測定……………………………..…………...……….….…57
4. 血脂之測定……………………………………..………...……..…57
a. Total Cholesterol……………………………………...….…....…57
b. Triglycerides…………….…………..……………………………58
c. HDL-cholesterol……………….….……..…………………….…59
d. LDL-cholesterol…………………….………………………….…60
5. 血清抗體（IgG、IgA、IgM、IgE）之測定………........………61
六、 免疫指標之分析………….………………………………...……62
1. 一氧化氮(nitric oxide, NO)分析……….………………..........…62
2. IL-1β、IL-2、IL-4、IL-5、IL-6、TNF-α及IL-10之測定...…62
七、 統計分析………………………………………………..……..…62
第三節 結果………………………………………………………...……..63
壹、 STZ糖尿病誘導模式建立…………………..……………………63
一、 單次腹腔注射STZ試驗模式…………....…..…………….……63
1. 臟器重量變化………………………………...…...………….……63
2. 一週血糖變化…………………………………….....….…….……63
3. 葡萄糖耐受試驗之血糖變化…………………..……...…….……63
二、 二次腹腔注射STZ試驗模式……………....…………….……64
1. 臟器重量變化………………..………………………...…….……64
2. 一週血糖變化……………………..……………………...….……64
3. 葡萄糖耐受試驗之血糖變化………..……………………………65
4. 腹腔細胞激素之分泌…………...………..………………….……65
5. 脾臟細胞激素之分泌………………...………..…………….……65
貳、 蓮子心及其酒精萃取物28天餵食以STZ誘發糖尿病誘發的BALB/c小鼠……………….………………………..……….……73
一、 餵食期間體重變化………………...…………………..………..73
二、 臟器重量變化……………………………..………………..……73
三、 STZ誘發一週後之血糖變化……..………..……………..……73
四、 葡萄糖耐受試驗之血糖變化…………………..………….……73
五、 血脂變化………………..……………………..…………………74
六、 血清抗體變化……………..……………………..………………74
七、 腹腔及脾臟細胞NO分泌變化……...……………….…………74
八、 腹腔細胞激素分泌變化…………………..………………….…74
九、 脾臟細胞激素分泌變化……………….……..………...…….…74
第四節 討論………………………………………………….….......……88
壹、 STZ糖尿病誘導模式建立……………………………..……....…88
貳、 糖尿病誘發之BALB/c小鼠的蓮子心及其酒精萃取物28天餵食試驗…………………………….…………………………..….…...88
第五節 結論……………………………………………………............…89


第四章 總結論………………………………...………….………………….92


第五章 參考文獻………………………………...…….……….……………93














表 目 次
表一 蓮子心不同溶劑萃取之萃取率……………………………..…….……18
表二 蓮子心不同溶劑萃取物化學成分分析……………………..…….…...32
表三 蓮子心40ºC水萃取物對BALB/c雌鼠脾臟細胞發炎媒介物NO及細胞激素分泌的影響………………….….…………………………………..…..34
表四 蓮子心100ºC熱水萃取物對BALB/c雌鼠脾臟細胞發炎媒介物NO及細胞激素分泌的影響.………………….……..………………………………..35
表五 蓮子心50%乙醇萃取物對BALB/c雌鼠脾臟細胞發炎媒介物NO及細胞激素分泌的影響…………………………………………….………………..36
表六 蓮子心正己烷萃取物對BALB/c雌鼠脾臟細胞發炎媒介物NO及細胞激素分泌的影響…………..……………………………………………..……..37
表七 蓮子心40ºC水萃取物對BALB/c雌鼠腹腔細胞發炎媒介物NO及細胞激素分泌的影響……………………………..….….………………………..38
表八 蓮子心100ºC熱水萃取物對BALB/c雌鼠腹腔細胞發炎媒介物NO及細胞激素分泌的影響……….…………………………………………………..39
表九 蓮子心50%乙醇萃取物對BALB/c雌鼠腹腔細胞發炎媒介物NO及細胞激素分泌的影響………….…………………………………………………..40
表十 蓮子心正己烷萃取物對BALB/c雌鼠腹腔細胞發炎媒介物NO及細胞激素分泌的影響…………………………………....…………………………..41
表十一 蓮子心及其酒精萃取物餵食STZ誘發之糖尿病小鼠28天試驗之分組………. …………..…………………………………………………………...55
表十二 不同濃度STZ對BALB/c雌鼠單次腹腔注射一週後對其臟器重量之影響….…………………………………………….………………………….67
表十三 不同濃度STZ對BALB/c雌鼠單次腹腔注射後一週內血糖的變化………….…………………….………………………………………………..68
表十四 不同濃度STZ對BALB/c雌鼠單次腹腔注射一週後對其葡萄糖耐受試驗的影響……………………………………….…………………………..68
表十五 不同濃度STZ對BALB/c雌鼠二次腹腔注射一週後對其臟器重量之影響….…………………….……...…………………………………………..69
表十六 不同濃度STZ對BALB/c雌鼠二次腹腔注射後一週內血糖的變化………….………………………………………….…………………………..70
表十七 不同濃度STZ對BALB/c雌鼠二次腹腔注射一週後對其葡萄糖耐受試驗的影響………………………………………….………………………..70
表十八 不同濃度STZ二次腹腔注射BALB/c雌鼠一週後對其腹腔細胞細胞激素分泌的影響…………………………………….………………………..71
表十九 不同濃度STZ二次腹腔注射BALB/c雌鼠一週後對其脾臟細胞細胞激素分泌的影響…………….………………………………………………..72
表二十 餵食不同劑量蓮子心28天對STZ誘發之糖尿病BALB/c雌鼠臟器重量的影響……………………………………………….……………………..78
表二十一 餵食不同劑量蓮子心萃取物28天對STZ誘發之糖尿病BALB/c雌鼠臟器重量的影響…….………………………………………...…………..79
表二十二 餵食不同劑量蓮子心及其萃取物28天期間對STZ誘發之糖尿病BALB/c雌鼠餵食期間禁食血糖的影響……………………….……………..80
表二十三 餵食不同劑量蓮子心及其萃取物28天對STZ誘發之糖尿病BALB/c雌鼠之葡萄糖耐受試驗的影響……………….……………………..81
表二十四 餵食不同劑量蓮子心及其萃取物28天對STZ誘發之糖尿病BALB/c雌鼠血清血脂的影響.………………………………………...……...82
表二十五 餵食不同劑量蓮子心及其萃取物28天對STZ誘發之糖尿病BALB/c雌鼠血清抗體含量的影響…….……………………………………...83
表二十六 餵食不同劑量蓮子心及其萃取物28天對STZ誘發之糖尿病BALB/c雌鼠腹腔及脾臟細胞分泌NO的影響………………………..……..84
表二十七 餵食不同劑量蓮子心及其萃取物28天對STZ誘發之糖尿病BALB/c雌鼠腹腔細胞分泌細胞激素的影響…………………………….…..85
表二十八 餵食不同劑量蓮子心及其萃取物28天對STZ誘發之糖尿病BALB/c雌鼠脾臟細胞分泌細胞激素的影響……………………….………..86
表二十九 餵食不同劑量蓮子心及其萃取物28天對STZ誘發之糖尿病BALB/c雌鼠脾臟細胞分泌細胞激素的影響...………………………..……..87
表三十 不同濃度STZ二次腹腔注射BALB/c雌鼠一週後對其脾臟細胞細胞激素IL-2/IL-4的影響………………………….……………….……………90
表三十一 餵食不同劑量蓮子心及其萃取物28天對STZ誘發之糖尿病BALB/c雌鼠脾臟細胞細胞激素IL-2/IL-4的影響………………………..…91



圖 目 次
圖一 Th 2反應的免疫過程可能促成自體免疫疾病腦脊髓炎的病變..……..5
圖二 Th 1/Th 2免疫細胞的分化機制…………….……………………………6
圖三 從處女T細胞生成Th 1與Th 2細胞….…………...…………………….7
圖四 蓮子心樣品收集過程...……………...….…………………….…………12
圖五 本論文實驗架構…………….…………………..…………….…………16
圖六 蓮子心不同溶劑之萃取流程………………………………....…………18
圖七 蓮子心免疫調節功能體外細胞模式之實驗設計圖……………..……24
圖八 蓮子心不同溶劑萃取物對BALB/c雌鼠脾臟細胞存活的影響………33
圖九 單次腹腔注射STZ以誘發小鼠形成糖尿病之流程…………………..47
圖十 二次腹腔注射STZ以誘發小鼠形成糖尿病之流程…………………..48
圖十一 蓮子心及其酒精萃取物對STZ誘發之糖尿病小鼠28天餵食試驗……………………………...………………………………………….………56
圖十二 連續餵食不同劑量蓮子心28天對以STZ誘發糖尿病之雌鼠體重……………………...……………………………………………….…………76
圖十三 連續餵食不同劑量蓮子心萃取物28天對以STZ誘發糖尿病之雌鼠體重………………………………………...…...…………………….…………77

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