臺灣博碩士論文加值系統

中草藥在許多文獻中證實具有保健的功效。本研究參考衛生署所公佈之食品用中草藥，並配合藥典中記載具有保肝功能的配方，搭配出兩種以熱水萃取所得之複方飲品，其一包含有靈芝、菊花、桑葉、枸杞、薄荷及白茅根(以下簡稱為靈芝茶)，其二則是採用黨參、苡仁、黃精、黃耆、茯苓、橘皮及枸杞(以下簡稱為元氣飲)。分別探討兩種複方飲品的嗜好性、安全性以及功能性。透過嗜好性的官能品評分析後，發現在添加了7%的果糖於二種複方中草藥茶當中，能夠提高其整體的接受度。並且在與市售產品的比較上，兩種複方飲品的接受度均高於對照組。
在安全性試驗部分，兩種複方中草藥茶水萃物在1.0~5.0 mg/plate的濃度範圍內，對鼠傷寒桿菌TA98及TA100的試驗系統均未呈現毒性及致突變性，而在低於5.0 mg/mL的濃度下，對大鼠肝細胞株Clone 9亦無明顯的毒性效應。由此初步結果，顯示兩種複方中草藥茶不論在真核或原核細胞的系統中，並無安全性的疑慮。
在抗氧化試驗方面，元氣飲與靈芝茶清除DPPH自由基的活性在3.0 mg/mL時分別達到91.0及91.4%，螯合亞鐵離子能力在3.0 mg/mL時分別達到10.4及48.7%。在還原力的部分，兩者在3.0 mg/mL的濃度時，在700 nm下吸光值分別為1.3及2.2。總抗氧化能力則是在3.0 mg/mL時分別達到84.8及99.7%。清除超氧陰離子的能力則是在5.0 mg/mL時具有73.3及75.7%的效果。而在總酚及類黃酮的含量測定中，元氣飲的部份為18.38及6.43 mg/g extract，靈芝茶的含量分別是27.33及10.76 mg/g extract。透過抗氧化性質EC50分析後，得知靈芝茶的抗氧化能力較優於元氣飲。在測定細胞內抗氧化酵素的活性中，結果呈現二種複方中草藥茶皆能提升胞內GR與GPx的酵素活性。
在抑制肝癌細胞存活率的結果顯示，二種複方中草藥茶皆能夠造成肝癌細胞的存活率下降，其中又以靈芝茶的功效較佳，在5.0 mg/mL的濃度下作用24小時，使肝癌細胞存活率下降至33.2%。在細胞週期的分析上，發現二種複方中草藥茶能夠使細胞在G0/G1期呈現大量的累積，說明了其抑制肝癌細胞的增生，在於調控了細胞週期的進行。
综合以上結果，顯示出兩種複方飲品並無安全上的疑慮，在體外試驗中呈現出抗氧化的能力以及抑制肝癌細胞的增生，並且在嗜好性的評估中獲得較佳的接受度，說明了兩種中草藥複方飲品具有成為保健食品的潛力。

Health protection efficiency of Chinese herbs had been proven in many references. In the present study, food grade Chinese herbs which were announced by the Department of Health, coupled with hepatoprotective formulas according to the pharmacopoeias, were used to make up two compound beverages. One formula contained Ling Chih (Ganoderma Lucidum), Ju Hua (Chrysanthemum morifolium Ramat.), Sang Ye (Morus alba), Gou Qi (Lycium chinensis), Bo He (Mentha spicata), and Bai Mao Gen (root of Imperata cylindrica); therefore, abbreviated as Ling Chih Tea. The other formula contained Dang Shen (Codonopsis pilosula), Yi Ren (seed of Coix lacryma-jobi), Huang Jing (Polygonatum sibiricum), Huang Qi (Astragalus membranaceu), Fu Ling (Poria cocos), Ju Pi (Pericarpium Citri Reticulatae), , Bo He (Mentha spicata) and Gou Qi (Lycium chinensis); therefore, abbreviated as Genki Drink. The preference, safety, and functionality of the two compound Chinese herb teas were evaluated, respectively.
The results of sensory evaluation analysis showed that the total acceptance of the two Chinese herb teas was increased after adding 7% of fructose. In comparison with commercial product, both teas had higher scores in acceptance. With respect to the safety assay, hot water extracts of the two Chinese herb teas didn’t exhibited toxicity or mutagenicity between concentrations of 1.0~5.0 mg/plate in Salmonella typhimurium TA98 and TA100 models. In addition, they didn’t show toxic effect to rat liver cell Clone 9 below 5.0 mg/mL. Hence the two herb teas had no safety anxiety in neither eukaryotic nor prokaryotic systems.
In the antioxidant aspect, the DPPH radical scavenging activity of Genki Drink and Ling Chih Tea reached 91.0 and 91.4%; whereas the ferrous ion chelating activity reached 10.4 and 48.7% respectively in the concentration of 3.0 mg/mL. The reducing power was 1.3 and 2.2 at 3.0 mg/mL. The total antioxidant activity reached 84.8 and 99.7% at 3.0 mg/mL. The superoxide anion scavenging ability was 73.3 and 75.7% at 5.0 mg/mL. The total phenolics and flavonoids content of Genki Drink reached 18.38 and 6.43 mg/g extract; whereas Ling Chih Tea reached 27.33 and 10.76 mg/g extract, respectively. As a result of the EC50 analysis in antioxidant properties, the antioxidant activity of Lin Chih Tea was better than Genki drink. Furthermore, the result of the intracellular antioxidant enzyme activities showed both Chinese herb teas could raise the activity of GR and GPx.
The inhibition of hepatoma cell (HepG2) viability result showed both teas reduced the viability of HepG2 cells, and Ling Chih Tea exhibited stronger efficiency (the viability was 33.2% at 5.0 mg/mL after treated for 24 hours). The analysis of cell cycle showed a great quantity of accumulation at G0/G1 phase after treated by the two Chinese herb teas, which illustrated the inhibition of hepatoma cell proliferation was subject to the progress modification of cell cycle.
In summary, the two compound Chinese herb teas had no safety anxiety, showed antioxidant activity and inhibition of hepatoma cell proliferation in vitro assays, and had better acceptance in sensory evaluation. As a result, the two compound Chinese herb teas exhibited great potential as new nutraceuticals.

中文摘要………………………………………………………………..I
英文摘要……………………………………………………………….III
目錄……………………………………………………………………..V
表次……………………………………………………………………..X
圖次……………………………………………………………………XII
附表次………………………………………………………………...XIV
附圖次…………………………………………………………………XV
第一章、前言…………………………………………………………...1
第二章、文獻回顧……………………………………………………...3
一、藥材之簡介...............................................................................3
1.靈芝……………………………………………………………3
2.菊花……………………………………………………………3
3.枸杞子…………………………………………………………4
4.黨蔘……………………………………………………………5
5.黃耆……………………………………………………………5
6.黃精……………………………………………………………6
7.薄荷……………………………………………………………7
8.桑葉……………………………………………………………7
9.茯苓……………………………………………………………8
10.薏苡仁........................................................................................8
11.橘皮……………………………………………………………9
12.白茅根…………………………………………………………9
二、自由基簡介..………………………………………………...10
1. 自由基的種類………………………………………………10
2. 自由基的來源………………………………………………13
3. 自由基所引起的疾病………………………………………13
4. 體內抗氧化防禦系統………………………………………14
三、食品的安全性評估..………………………………………...17
Ames test……………………………………………………….19
1. 檢驗原理……………………………………………………19
2. 試驗菌株……………………………………………………19
3. 藥物轉化酵素S9…………………………………………...20
四、肝臟的生理與病理………………………………………......22
1. 肝臟的簡介………………………………………………….22
2. 肝臟相關疾病……………………………………………….22
3. 肝癌………………………………………………………….23
4. 中醫藥治療肝癌的原則…………………………………….23
五、細胞週期簡介………………………………………………..26
1. 細胞週期…………………………………………………….26
2. 細胞週期的調控…………………………………………….26
3. 細胞死亡…………………………………………………….28
第三章、材料與方法…………………………………………………..29
一、實驗材料與試劑.…………………………………………….29
二、樣品製備流程.……………………………………………….31
三、嗜好性試驗(官能品評).……………………………………..32
四、儲藏試驗(微生物的檢測)..………………………………….32
五、理化性質的分析.…………………………………………….32
六、Ames test..................................................................................33
1. 毒性試驗…………………………………………………….33
2. 致突變性試驗……………………………………………….33
3. 抗致突變性試驗…………………………………………….34
七、抗氧化能力測定……………………………………………..35
1. 捕捉DPPH自由基的能力…..……………………………...35
2. 螯合亞鐵離子能力…………………..……………………...35
3. 總抗氧化能力的測定…………………...…………………..35
4. 還原力的測定……………...………………………………..36
5. 清除超氧陰離子的能力測定………...……………………..36
八、抗氧化成分含量測定.…………………………………….....36
1. 總酚類化合物……..………………………………………...36
2. 類黃酮含量測定……..……………………………………...37
九、細胞的培養.……………………………………………….....37
十、細胞毒性試驗.…………………………………………….....38
1. 對大鼠肝細胞Clone 9的毒性試驗…………………………38
2. 對人類肝癌細胞株( HepG2 )存活能力的影響…………….39
十一、胞內抗氧化酵素的測定..………………………………….39
1. 細胞質液的製備…………………………………………….39
2. GSH peroxidase ( GPx )活性測定…………………………...40
3. GSH reductase ( GR )活性測定..…………………………….40
十二、細胞週期分析……………………………………………..41
十三、統計分析…………………………………………………..41
第四章、結果與討論…………………………………………………..42
一、二種複方的中草藥比例配置……….……………………….42
二、二種複方中草藥水萃物之萃取率….……………………….42
三、嗜好性官能品評結果……………….……………………….42
1. 嗜口性的調整………………………..……………………...42
2. 與市售產品的比較…………………..……………………...42
3. 殺菌前後之差異性三角試驗.................................................43
四、儲藏試驗(微生物的檢測結果)……………………………...43
五、理化性質的分析……………………….…………………….43
六、複方中草藥萃取物之毒性、致突變性及抗致突變性……..43
1. 毒性試驗………………………………………………….....43
2. 致突變性分析…………………………………………….....44
3. 抗致突變能力…………………………………………….....44
七、二種複方中草藥萃取物之抗氧化特性……………………..45
1. 捕捉DPPH自由基的能力……………………………….....45
2. 螯合亞鐵離子能力……………………………………….....45
3. 總抗氧化能力…………………………………………….....46
4. 還原力………………………………………….....................46
5. 清除超氧陰離子的能力………………………………….....46
6. 抗氧化性質之EC50…………………………………………46
八、二種複方中草藥萃取物之抗氧化成分含量………………..47
九、細胞毒性試驗結果…………………………………………..47
1. 對大鼠肝細胞株Clone 9的影響…………………………..47
2. 抑制人類肝癌細胞株HepG2的能力………………….......48
十、對胞內抗氧化酵素活性的影響……………………………..48
1. GSH peroxidase (GPx)活性變化…………………………….48
2. GSH reductase (GR)活性變化…………………………….....48
十一、對人類肝癌細胞週期的影響.……………………………..49
第五章、結論…………………………………………………………..50
參考文獻………………………………………………………………..51


















表次
表一、二種複方中草藥的比例配置…………………………………..57
表二、二種複方中草藥飲品殺菌前後之三角試驗…………………..58
表三、經殺菌後的二種複方中草藥飲品於37℃儲藏10天後
微生物的檢測…………………………………………………..59
表四、元氣飲於殺菌前後之理化性質………………………………..60
表五、靈芝茶於殺菌前後之理化性質………………………………..61
表六、複方中草藥水萃物元氣飲對鼠傷寒沙門桿菌TA98及
TA100菌株之毒性試驗………………………………………..62
表七、複方中草藥水萃物靈芝茶對鼠傷寒沙門桿菌TA98及
TA100菌株之毒性試驗………………………………………..63
表八、複方中草藥水萃物元氣飲對鼠傷寒沙門桿菌TA98及
TA100菌株之致突變性試驗…………………………………..64
表九、複方中草藥水萃物靈芝茶對鼠傷寒沙門桿菌TA98及
TA100菌株之致突變性試驗…………………………………..65
表十、二種複方中草藥水萃物對NQNO在鼠傷寒沙門桿菌TA98
及TA100菌株之抗致突變性之影響………………………….66
表十一、二種複方中草藥水萃物對B[a]p在鼠傷寒沙門桿菌
TA98及TA100菌株之抗致突變性之影響…………………...67
表十二、二種複方中草藥水萃物之抗氧化性質EC50……………….68
表十三、二種複方中草藥水萃物之總酚及類黃酮含量……………..69
表十四、二種複方中草藥水萃物對大鼠肝細胞 Clone 9
之毒性試驗…………………………………………………..70






























圖次
圖一、以嗜好性官能品評法分析添加不同果糖濃度對
元氣飲嗜口性的影響………………………………………….71
圖二、以嗜好性官能品評法分析添加不同果糖濃度對
靈芝茶嗜口性的影響………………………………………….72
圖三、以嗜好性官能品評法分析二種複方中草藥飲品與
市售產品的整體接受程度…………………………………….73
圖四、二種複方中草藥水萃物之捕捉DPPH自由基能力………….74
圖五、二種複方中草藥水萃物之亞鐵離子螯合能力……………….75
圖六、二種複方中草藥水萃物之總抗氧化能力…………………….76
圖七、二種複方中草藥水萃物之還原力測定……………………….77
圖八、二種複方中草藥水萃物清除超氧陰離子之能力測定……….78
圖九、二種複方中草藥水萃物抑制人類肝癌細胞 HepG2
存活的能力…………………………………………………….79
圖十、二種複方中草藥水萃物對大鼠肝細胞 Clone 9
之抗氧化酵素麩胱甘肽過氧化酶的影響…………………….80
圖十一、二種複方中草藥水萃物對大鼠肝細胞 Clone 9
之抗氧化酵素麩胱甘肽還原酶的影響…………………….81

圖十二、以流式細胞儀分析複方中草藥水萃物元氣飲對
人類肝癌細胞 HepG2 細胞週期的影響………………….82
圖十三、以流式細胞儀分析複方中草藥水萃物靈芝茶對
人類肝癌細胞 HepG2 細胞週期的影響………………….83






























附表次
附表一、自由基的種類…………………………………………………11
附表二、台灣地區民國九十五年十大死亡原因統計資料……………24
































附圖次
附圖一、ROS誘導癌症發生的可能機制……………………………..15
附圖二、細胞內的抗氧化系統………………………………………16

參考文獻
王淑苑，(2000)。Ames test 之原理及其於產品安全性評估之應用。食品工業。32 : 8-17。
行政院衛生署，(1999)。健康食品管理法。
行政院衛生署，(2007)。95年主要死因統計結果。
呂曄，吳春節，陸庭譯，(1998)。結合中西醫治療型肝炎及其病變。聯華書報社。
李少君，陳同偉，楊永寧，蕭碧瑩，張淑芬 編譯，(2005)。分子細胞生物學。藝軒圖書出版社。
李岩，王艷紅，(1999)。肝癌防治錦囊。大作文化。台北。
徐蔚泓，(2004)。肝好就漂亮。尚書文化。
彭秋妹，王家仁，(1991)。食品官能檢查手冊。食品工業發展研究所。
顏正華，(2003)。中藥學。知音出版社。台北市。

Ames, B. N., McCann J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella /mammali an microsome mutagenicity test. Mutat. Res. 31 : 347-364.
Anderson, D. and Phillips, B.J. (1999) Comparative in vitro and in vivo effects of antioxidants. Food Chem. Toxicol. 37 : 1015-1025.
Bellomo, G., Mirabelli, F., DiMonte, D., Richelmi, P., Thor, H., Orrenius, C. and Orrenius, S. (1987) Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone). Biochem Pharmacol. 36 : 1313–1320.
Casciano, D.A. (1982) Mutagenesis assay methods. Food Technol. 36(3) : 48-52.
Chiu, J.H., Kao, H.L., Wu, L.H., Chang, H.M. and Lui, W.Y. (1992) Prediction of relapse or survival after resection in human hepatomas by DNA flow cytometry. J.Clin. Invest. 89 : 539–545.
Cho, C.S. William and Leung, N. Kwok. (2007) In vitro and in vivo anti-tumor effects of Astragalus membranaceus. Cancer Letters. 252 : 43–54.
Chor, S.Y., Hui, A.Y., To, K.F., Chan, K.K., Go, Y.Y., Chan, H.L.Y., Leung, W.K. and Sung, J.J.Y. (2005) Anti-proliferative and pro-apoptotic effects of herbal medicine on hepatic stellate cell. Journal of Ethnopharmacology. 100 : 180–186.
Dinis, T.C.P., Madeira, V.M.C. and Almeida, L.M. (1994) Action of phenolic derivatives (acetaminophen, salicylate, and 5-amino salicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys. 315 : 161-169.
Fang, Y.Z., Yang, S. and Wu, G.Y. (2002) Free radicals, antioxidants and nutrition. Nutrition. 18 : 872–879.
Fridovich, I. and Picker, S.D. (1984) On the mechanism of production of superoxide radical by reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium. Arch. Biochem. Biophys. 228 : 155-158.
Halliwell, B. (1994) Free radicals, antioxidants and human disease: curiosity, cause or consequence? Lancet. 344 : 721-723.
Halliwell, B. and Gutteridge, J.M.C. (1998) Free radicals in biology and medicine. Oxford University Press, New York, pp 27-30.
Huang, Q., Zhang, L., Cheung, C.K. Peter and Tan, X. (2006) Evaluation of sulfated a-glucans from Poria cocos mycelia as potential antitumor agent. Carbohydrate Polymers. 64 : 337–344.
Hutchison, C. and Glover, D.M. (1995) Cell cycle control. New York ：Oxford University Press.
Jacob, R.A. (1995) The integrated antioxidant system. Nutr. Res 15 : 755–766.
Jia, Z., Tang, M. and Wu, J. (1999) The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64 : 555-559.
Jin, Y., Zhang, L., Zhang, M., Chen, L., Cheung, C.K. Peter., Oi, V.E.C. and Lin, Y. (2003) Antitumor activities of heteropolysaccharides of Poria cocos mycelia from different strains and culture media. Carbohydrate Research. 338 : 1517-/1521.
Kuan, N.K. and Passaro, E. Jr. (1998) Apoptosis: programmed cell death. Arch Surg. 133 : 733-735.
Lakshmi, B., Ajith, T.A., Jose, N. and Janardhanan, K.K. (2006) Antimutagenic activity of methanolic extract of Ganoderma lucidum and its effect on hepatic damage caused by benzo[a]pyrene. Journal of Ethnopharmacology. 107 : 297-303.
Lakshmi, B., Ajith, T.A., Sheena, N., Gunapalan, N. and Janardhanann K. K. (2003) Antiperoxidative, Anti-Inflammatory, and Antimutagenic Activities of Ethanol Extract of the Mycelium of Ganoderma lucidum Occurring in South India. Teratogenesis, Carcinogenesis, and Mutagenesis Supplement. 1 : 85–97.
Lander, H.M. (1997) An essential role for free radicals and derived species in signal transduction. FASEB J. 11 : 118-124.
Lawerence, R.A. and Burk, R.F. (1976) Glutathione peroxidase activity in selenium-deficient rat liver. Biochem. Biophys. Res. Commun. 71 : 952-958.
Li, D. and Friedman, S.L. (1999) Liver fibrogenesis and the role of hepatic stellate cells : new insights and prospects for therapy. J. Gastroenterol Hepatol. 4 : 618-633.
Lin, J.M., Lin, C.C., Chen, M.F., Ujiie, T. and Takad, A. (1995) Radical scavenger and antihepatotoxic activity of Ganoderma formosanum, Ganoderma lucidum and Ganoderma neo-japonicum. Journal of Ethnopharmacology. 47 : 33-41.
Lundberg, A.S. and Weinberg, R.A. (1999) Control of the cell cycle and apoptosis. Eur. J. Cancer. 35 : 531-539.
MATE´ S, J.M., MEZ, C.P.R.G. and CASTRO, I.N.N. (1999) Antioxidant Enzymes and Human Diseases. Clinical Biochemistry. 32 : 595–603.
Matés, J.M. and Sánchez-Jiménez, F.M. (2000) Role of reactive oxygen species in apoptosis: implications for cancer therapy. The International Journal of Biochemistry & Cell Biology. 32 : 157-170.
Miller, N.J. and Rice-Evans C.A. (1997) The relative contributions of ascorbic acid and phenolic antioxidants to the total antioxidant activity of orange and apple fruit juices and blackcurrant drink. Food Chem. 60 : 331-337.
Morrissey, P.A. and O’Brien, N.M. (1998) Dietary antioxidants in health and disease. Int. Dairy J. 8 : 463-472.
Oyaizu, M. (1986) Studies on products of browning reaction: Antioxidative activities of products of browning reaction prepared from glucosamine. Jpn. J. Nutr. 44 : 307-315.
Pietenpol, J.A. and Stewart, Z.A. (2002) Cell cycle checkpoint signaling: cell cycle arrest versus apoptosis. Toxicology. 181-2 : 475-481.
Pines, J. (1993) Cyclins and cyclin-dependent kinases: take your partners. Trends Biochem. Sci. 18 : 19-27.
Pines, J. (1999) Four-dimensional control of the cell cycle. Nat cell Biol. 1 : E73-79.
Shao, B.M., Xu, W., Dai, H., Tu, P., Li, Z. and Gao, X.M. (2004) A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb. Biochemical and Biophysical Research Communications. 320 : 1103–1111.
Shapiro, G.I. and Harper, J.W. (1999) Anticancer drug targets: cell cycle and checkpoint control. J. Clin. Invest 104 : 1645-1653.
Shiao, M.S. (2003) Natural Products of the Medicinal Fungus Ganoderma lucidum: Occurrence, Biological Activities, and Pharmacological Functions. The Chemical Record. Vol. 3, 172–180.
Shimada, K., Fujikawa, K., Yahara, K. and Nakamura, T. (1992) Autioxidative properties of xanthan on the autoxidation of soybean oil in cycodextrin emulsion. J. Agic. Food Chem. 40 : 945-948.
Skaper, S.D., Fabris, M., Ferrari, V., Carbonare, M.D. and Leon, A. (1997) Quercetin protects cutaneous tissue-associated cell types including sensory neurous from oxidative stress induced by glutathione deleption cooperative effect of ascorbic acid. Free Radic. Biol. Med. 22：669-678.
Taga, M.S., Miller, E.E. and Pratt, D.E. (1984) Chia seeds as a source of natural lipid antioxidants. J. Am. Oil Chem. 61 : 928-931.
Waleh, N. S., Rapport, S. J. and Mortelmans, K. E. (1982) Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of construction of the required strauns. Mutat. Res. 97: 247-256.
Yu, M.C., Lee, C.H., Lai, C.W., Chiang, I.P., Wang, Y.Y. and Hsu, C.H. ( 2003) The hepatoprotective effect of tranditional Chinese medicine in galatosamine-induced acute hepatotoxicity. Journal of Chinese Medicine. 14 : 99-108.
Yu, T.W., Xu, M. and Dashwood, R.H. (2004) Antimutagenic activity of Spearmint. Environmental and Molecular Mutagenesis. 44 : 387–393.