黑豆 (Glycine max L.)具有許多抗氧化活性成分，根據文獻指出黑豆在經過適當的預熱處理可以顯著提升抗氧化物質如多酚與花青素的萃取率與抗氧化能力。本實驗室先前研究發現黑豆水萃取物具有減緩四氯化碳所誘導的肝臟損傷。黑豆的護肝效能推測可能與抗氧化物質有關，因此本研究探討烘烤預熱處理對於黑豆水萃取物的護肝效能之影響。本研究選用台南三號的青仁黑豆，分為未烘烤黑豆、烘烤黑豆(130℃，5 分鐘)、未烘烤黑豆種皮，以熱水浸泡萃取 (80℃，30分鐘)，經減壓濃縮後凍乾製成粉末。
護肝效能評估的動物實驗選用6週齡sprague-dawely (SD)大鼠，以隨機方式分為六組，組別分別為未誘導的控制組、四氯化碳誘導組 (carbon tetrachloride, CCl4)、Silymarin (CCl4+Silymarin 200 mg/kg bw)、未烘烤黑豆組 (CCl4+ 200 mg/kg bw)、烘烤黑豆組 (CCl4+ 200 mg/kg bw)、未烘烤黑豆種皮組 (CCl4+ 200 mg/kg bw)，實驗進行八週餵食試驗。血清分析結果顯示，在第一、三、六及八週，四氯化碳誘導肝指數alanine aminotransferase (ALT)及aspartate aminotransferase (AST)值明顯提高，在給予Silymarin、烘烤黑豆及未烘烤黑豆種皮水萃取物能顯著降低血清中ALT與AST。在八週犧牲大鼠後，取其血清分析發現四氯化碳提高serum triglyceride (TG)、total cholesterol (TC)、blood urea nitrogen (BUN)等生化數值，但降低 total antioxidant capacity (TAC)。四氯化碳也導致肝臟中抗氧化酵素活性的下降，superoxide dismutase (SOD)、glutathione peroxidase (GPx)、catalase (CAT)、glutathione reductase (GR)、glutathione S-transferases (GST)的活性皆有明顯下降SD大鼠的肝臟組織中glutathione (GSH)與total SH groups (TSH)含量的降低及oxidized glutathione (GSSG)、malondialdehyde (MDA)的顯著增加。顯示四氯化碳誘發肝臟的氧化壓力在給予Silymarin、烘烤黑豆組及未烘烤黑豆種皮組處理能顯著降低血清中TC及BUN並提升肝臟抗氧化酵素SOD、CAT、GR、GST活性與GSH含量，及降低GSSG與MDA含量。整體來說以未烘烤黑豆種皮的護肝效果較優異，能額外提高TAC、GPx、TSH (P<0.05)，降低TG、GSSG。肝臟組織切片染色顯示四氯化碳誘導組有顯著細胞空泡化 (vacuoles)、細胞壞死 (necrosis)及纖維化 (fibrosis)的情形，然而未烘烤黑豆種皮組能顯著降低細胞壞死的情況。分析三種黑豆萃取物中總多酚、總類黃酮及總花青素的含量亦顯示，黑豆種皮水萃取物含有最多的總多酚 (0.11±0.01 g/g extract)、總類黃酮 (0.39±0.04 g/g extract)、總花青素 (2.82±0.01 mg/g extract)。推測黑豆種皮組有較佳的護肝效果可能與種皮上的花青素有關。
花青素的萃取通常以含微酸的乙醇進行萃取，因此本研究再探討黑豆種皮乙醇萃取物的護肝效果，黑豆種皮以80％乙醇(含0.1％乙酸)進行4℃ 48小時萃取，經過減壓濃縮後凍乾成粉末。動物實驗同樣選用6週齡SD大鼠進行為期八週的實驗，實驗組分為五組，分別是控制組、四氯化碳誘導組、Silymarin組、低劑量組 (CCl4+ 200 mg/kg bw)、高劑量組 (CCl4+ 400 mg/kg bw)。血清分析結果顯示，Silymarin組與黑豆種皮乙醇萃取物組於第一、三、六、八週血清中ALT、AST能顯著降低，同時能降低TC、TG、BUN數值並提升TAC，並且呈現劑量的效應。Silymarin組與樣品組皆能提升肝臟中抗氧化酵素SOD、CAT、GST、GR、GPx活性，又以高劑量組表現最佳。乙醇萃取物顯著提升肝臟中的GSH、TSH，並降低GSSG、MDA含量。高劑量黑豆乙醇萃取物能顯著降低vacuoles、necrosis、fibrosis數值表示對於四氯化碳肝損具有保護的作用。
由上述實驗結果得知，黑豆種皮的乙醇萃取物亦具有護肝的效能。並且肝臟病理切片結果顯示黑豆種皮乙醇萃取物之護肝效果較水萃取物為佳，因此本研究在尋找以乙醇與丙酮有機溶劑萃取黑豆抗氧化物質的最適條件。依照部分複因子設計，每個因子有兩個不同的萃取條件，分別為烘烤溫度 (130℃、180℃)、烘烤時間 (5 min、30 min)、萃取溶劑 (乙醇、丙酮)、萃取濃度 (60%、80%)、固液態比 (1:4、1:10)、萃取溫度 (4℃、40℃)、萃取時間 (30 min、48 hr)、pH值 (4、6)以上條件分為16組別。分析總多酚、總類黃酮、總花青素，DPPH自由基清除能力與ABTS･+自由基清除能力，並且利用HPLC分析花青素C3G的含量。實驗結果顯示黑豆最佳的萃取條件為，預熱130℃烘烤5分鐘，以60%丙酮 (pH=4) 1：4在4℃萃取48小時，該條件能獲得較多總多酚與總類黃酮並且有較高的抗氧化能力。
黑豆種皮的水萃取物與乙醇萃取物都有對抗四氯化碳誘導的氧化壓力之效果，可能是因為種皮含有大量的抗氧化物質，特別是花青素。因此黑豆具有開發維護肝產品及作為天然抗氧化產品的潛力。

Black soybean (Glycine max L.) contains high amount of antioxidants, and it has been reported that preheating treatment can significantly increase the antioxidants, such as polyphenolic compounds and anthocyanin, and antioxidative activity. Our previous study indicated that black soybean had liver protective function which might be due to the antioxidant in black soybean. Therefore, in this study we investigated the effect of pre-roasting on the liver protective function of the water extract from black soybean. We selected Tainan No. 3 black soybean, the samples were roasted black soybean, unroasted black soybean, and unroasted seed coat of black soybean. After immerged in 80oC hot water for 30 min, the samples were vaccum concentrated, freeze-dried and stored until use.
Six weeks-ago Sprague-Dawely (SD) rats were used and randomized into six groups: they were the control group, carbon tetrachloride group, and carbon tetrachloride with silymarin group (200 mg/kg bw), as well as the water extracts from unroasted black soybean, roasted black soybean and the seed coat of black soybean groups (200 mg/kg bw). At 1, 3, 6 and 8 weeks, the serum samples were took from the rats and analyzed. It was found that carbon tetrachloride increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, however, the supplement of silymarin and all the water extracts significantly reduced both ALT and AST levels. The rats were scarified at 8 week, the serum and liver tissue samples were collected and analyzed. The results showed that carbon tetrachloride increased the levels of triglyceride (TG), total cholesterol (TC) and blood urea nitrogen (BUN) in the serum but decreased total antioxidant capacity (TAC). Carbon tetrachloride also resulted in the declines of antioxidant enzymes activities in the liver tissues, including the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, glutathione reductase (GR), glutathione S-transferase (GST). Glutathione (GSH) and total SH groups (TSH) contents in the liver tissue were decreased by carbon tetrachloride, but the oxidized glutathione (GSSG) and malondialdehyde (MDA) levels were increased. However, the antioxidant enzyme activities of SOD, GR, GST and caltase were significantly increase with the supplement of silymarin, the water extracts from roasted black soybean and the seed coat. Water extract from the seed coat had the highest liver protective function against carbon tetrachloride induced liver damage in SD rats. The seed coat extract also significantly reduce cell necrosis in the liver tissues induced by carbon tetrachloride. It was suggested that high amount of anthocyanin in the water extract from the seed coat might be the important factor contributed to the liver protective function, even though the water extract also had high total phenolic and total flavanoid contents.
The common practice to extract anthocyanin from natural plants is using ethanol in slightly acid condition. Therefore, we also extract the anthocyanin from the seed coat of black soybean using 80% ethanol (containing 0.1% acetate) at 4oC for 48 hr. SD rats were randomly assigned to five groups: the control group, carbon tetrachloride group, carbon tetrachloride with low (200 mg/kg bw) and high (400 mg/kg bw) amount of the ethanol extract. It was noted that the ethanol extract could significantly reduce ALT and AST levels in the serum, enhance all the antioxidant enzymes activities and GSH and TSH levels in the liver. At 400 mg/kg, the ethanol extract could also reduce the cell vacuoles, necrosis and fibrosis of the liver tissues.
Our data indicated that ethanol extract from the seed coat of black soybean also showed liver protective function against carbon tetrachloride induced liver damage, and such protective function was better than the water extracts. Thus, we proposed to search the optimal conditions for extracting antioxidants from black soybean using organic solutions, like ethanol and acetone. Based on a fractional factorial design with 8 factors having two experimental conditions, we selected a total of 16 extraction runs. The experimental combinations were: roasting temperature (130, 180oC), roasting time (5, 30 min), organic solution (ethanol, acetone), solvent concentration (60, 80%), solid and liquid ratio (1:4, 1:10 v/v), extraction temperature (4, 40oC), extraction time (0.5, 48 hr) and pH (4, 6). The best extraction condition was pre-roasting black soybean at 130oC for 5 min and using 60% acetone at 1:4 v/v, 4oC for 48 hr, pH 4. Such extraction condition obtained high antioxidant from black soybean and showed high antioxidative activities.
Both the water and ethanol extracts from the seed coat of black soybean showed liver protective function against carbon tetrachloride induced liver damage in SD rats. Anthocyanins in the seed coat were suggested to be the maim active compounds contributed to such protective function. Thus, black soybean can be considered as a natural source for both antioxidant and liver protective agents.

摘要………………………………………………………………….……………………I

Abstract…………………………………………………………………….……………IV

目次…………………………………………………………………..…………………IX

表次…………………………………………………………………………..…………XI

圖次……………………………………………………………………….……………XII

壹、 前言……………………………………………………………………..………1

貳、 文獻回顧………………………………………………………………..………3
一、 黑豆………………………………………………………………………..……3
二、 多酚化合物………………………………………………………………..……4
三、 類黃酮……………………………………………………………………..……8
四、 花青素…………………………………………………………………………10
五、 熱加工對抗氧化之影響………………………………………………………12
六、 自由基與氧化壓力……………………………………………………………12
七、 肝臟與肝損機制………………………………………………………………16
八、 肝臟保護機制…………………………………………………………………20

參、 實驗藥品與儀器…………………………………………………………………24

實驗架構Ⅰ、烘烤黑豆熱水萃取物對四氯化碳誘導肝損傷之保護效益……..……27
實驗Ⅰ方法…………………………………………………………………………..…28

實驗架構Ⅱ、黑豆種皮乙醇萃取物對四氯化碳誘導肝損傷之保護效益………..…36
實驗Ⅱ方法………………………………………………………………………..……37

實驗架構Ⅲ、烘烤黑豆萃取最適條件之探討……………………………………..…39
實驗Ⅲ方法………………………………………………………………………..……40

實驗Ⅰ結果討論…………………………………………………………………..……46
實驗Ⅱ結果討論…………………………………………………………………..……53
實驗Ⅲ結果討論…………………………………………………………………..……60

肆、 結論………………………………………………………………………………66

伍、 參考文獻…………………………………………………………………..…114

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