臺灣博碩士論文加值系統

諾麗，被視為一種具有多種醫療效果的熱帶果樹，長久以來被應用在民俗療法上。而近幾年研究也顯示，諾麗的果實、葉子、根等部位可分離出各種不同的營養成分，且具良好的醫療保健功效。本計畫研究目的在於探討諾麗葉多醣體萃取物對水晶蝦之免疫能力的影響。於實驗中利用煮萃方式萃取出諾麗葉多醣體，利用酚-硫酸呈色定量法(phenol-sulfuric acid method)進行總醣定量，方便配製後續實驗所要用的水晶蝦免疫調節劑。實驗採用100 μg / L 及250 μg / L兩種濃度的諾麗葉多醣體作為實驗組的免疫調節濃度，對水晶蝦進行免疫調節，與未添加任何調節劑之陰性對照組及添加市售商品AZOO水晶蝦維他命劑 (100 μl / L)之陽性對照組進行比較。免疫調節時間為期四週，後續進行免疫指標分析、水溫變動適應性分析及病菌清除率實驗。依其結果，來評估諾麗葉多醣體萃取物做為免疫調節劑是否對水晶蝦之非特異性免疫有增強的效果。免疫指標分析結果顯示，諾麗葉多醣體可有效提升水晶蝦血球之酚氧化酶(phenoloxidase, PO)活性、超氧陰離子(superoxide anion, O2-)產生率及超氧歧化酶 (superoxide dismutase, SOD)活性。而在水溫變動適應性分析及病菌清除率分析結果顯示，諾麗葉多醣體萃取物可提升水晶蝦之溫度變化耐受性及增強清除病菌能力。綜合本研究結果顯示，以浸泡諾麗葉多醣體萃取物方式，可有效提升水晶蝦非特異性免疫能力。

Morinda citrifolia, commonly called as “noni”, is considered to be a tropical fruit with variety of health effects, has been used in folk remedies for over 2000 years. In recent years, some research indicated different components separate from noni fruit, leaf and root have lot of medical benefits. The purpose of this research was to investigate the immunoregulatory effects of polysaccharide extracts from noni leaf on Caridina serrata. In this study, we used hot water to extract noni leaf polysaccharide, and quantified the total sugar by using phenol sulfuric acid method, which allowed us to prepare the amount of immuoregulatory buffer accordingly. In the test of immunity, we used 100 μg/L and 250 μg /L doses of noni leaf polysaccharide respectively as the experimental groups to stimulate Caridina serrata’s immunity, and compared to the groups of one without any immuoregulatory buffer, and one with commercial product (AZOO vitamin). It was taken four weeks to regulate Caridina serrata’s immunity, and then analyzed the immune parameter, adaptability of temperature change and clearance efficiency of D. hansenii to make sure if noni leaf polysaccharide could enhance Caridina serrata’s non-specific immunity or not. The result of immune parameter shows that noni leaf polysaccharide can enhance Caridina serrata’s haemocytes phenoloxidase activity, superoxide anion yield and superoxide dismutase activity. Also, in the aspect of adaptability of temperature change and clearance efficiency indicate that noni leaf polysaccharide can improve the ability of temperature adaptation and clearance efficiency. In conclusion, immersing Caridina serrata in noni leaf polysaccharide can enhance Caridina serrata’s non-specific immunity.

目錄------------------------------------------------------------------------------------Ⅰ
表目錄---------------------------------------------------------------------------------Ⅱ
圖目錄---------------------------------------------------------------------------------Ⅲ
附錄目錄------------------------------------------------------------------------------Ⅳ
中文摘要-------------------------------------------------------------------------------1
英文摘要-------------------------------------------------------------------------------2
第一章 前言---------------------------------------------------------------------------3
1.1 觀賞水族產業--------------------------------------------------------------------3
1.1.2水晶蝦---------------------------------------------------------------------------4
1.2 甲殼類免疫防禦機制----------------------------------------------------------4
1.2.1 物體屏障-----------------------------------------------------------------------5
1.2.2 非特異性免疫-----------------------------------------------------------------5
1.2.2.1 血球的種類與功能---------------------------------------------------------7
1.2.3 黑色素形成--------------------------------------------------------------------8
1.2.4 吞噬作用-----------------------------------------------------------------------8
1.2.5 血淋巴凝集--------------------------------------------------------------------9
1.3 水產生物疾病防治-------------------------------------------------------------9
1.3.1 免疫刺激物-------------------------------------------------------------------10
1.3.1.1 諾麗--------------------------------------------------------------------------12
1.3.1.2 諾麗萃取物-----------------------------------------------------------------13
1.4 研究目的------------------------------------------------------------------------14
第二章 材料與方法--------------------------------------------------------------15
2.1 實驗材料------------------------------------------------------------------------15
2.1.1 藥品與試劑-------------------------------------------------------------------15
2.1.2 儀器裝置----------------------------------------------------------------------16
2.1.3 植物來源----------------------------------------------------------------------17
2.1.4 菌株來源----------------------------------------------------------------------17
2.1.5 實驗動物----------------------------------------------------------------------17
2.2 實驗方法------------------------------------------------------------------------17
2.2.1 樣本前處理-------------------------------------------------------------------17
2.2.2 多醣體的製備與定量-------------------------------------------------------17
2.2.2.1 粗萃萃取物製備-----------------------------------------------------------18
2.2.2.2 多醣製備--------------------------------------------------------------------18
2.2.2.3 總醣定量--------------------------------------------------------------------18
2,2,2,4 多醣體水溶液的醣含量分析--------------------------------------------19
2.2.3 免疫調節劑的開發----------------------------------------------------------19
2.2.4 免疫動物試驗----------------------------------------------------------------19
2.2.5 免疫調節期間累積死亡率統計-------------------------------------------20
2,2,6 水晶蝦免疫能力分析-------------------------------------------------------20
2.2.6.1 水晶蝦度水溫變動的適應性試驗--------------------------------------20
2.2.6.2 血淋巴採集-----------------------------------------------------------------21
2.2.6.3 總血球數分析--------------------------------------------------------------21
2.2.6.4 酚氧化酵素活性分析----------------------------------------------------22
2.2.6.5 超氧離子產生率分析----------------------------------------------------22
2.2.6.6 超氧歧化酶活性分析----------------------------------------------------23
2.2.6.7 病菌清除率分析----------------------------------------------------------24
2.2.6.8 統計分析-------------------------------------------------------------------25
第三章 結果-----------------------------------------------------------------------26
3.1 諾麗多醣體萃取---------------------------------------------------------------26
3.2萃取物總醣定量----------------------------------------------------------------26
3.3 多醣體水溶液的醣含量分析------------------------------------------------27
3.4 免疫調節期間累積死亡率統計---------------------------------------------27
3.5 水晶蝦對水溫變動適應性試驗---------------------------------------------27
3.6不同水族添加劑對於水晶蝦總血球數之影響分析----------------------28
3.7 不同水族添加劑對於水晶蝦酚氧化酵素活性之影響分析------------28
3.8 不同水族添加劑對於水晶蝦超氧離子產生率之影響分析------------29
3.9 不同水族添加劑對於水晶蝦超氧歧化酶活性之影響分析------------29
3.10 不同水族添加劑對於水晶蝦病菌清除率之影響分析----------------30
第四章 討論-----------------------------------------------------------------------31
第五章 結論-----------------------------------------------------------------------35
表--------------------------------------------------------------------------------------36
圖--------------------------------------------------------------------------------------41
參考文獻-----------------------------------------------------------------------------51
附錄-----------------------------------------------------------------------------------59

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