臺灣博碩士論文加值系統

珍珠是傳統的中藥之一。含有多種胺基酸、礦物質。許多動物實驗結果指出，珍珠粉具有抗氧化、抗衰老、清除自由基等功效，而這些功效可能來自於珍珠粉中的蛋白質。
本研究，採用珍珠粉、珍珠粉蛋白質萃取物及去除蛋白質之珍珠粉進行抗氧化性、清除自由基之活性探討，結果顯示，珍珠粉與珍珠粉蛋白質萃取物皆具有抗氧化性、清除自由基的活性，並隨著劑量呈正相關：珍珠粉中的蛋白質可能是珍珠粉抗氧化之主要貢獻成分。
且以線蟲為模式探討珍珠粉之抗衰老作用，發現，珍珠粉之蛋白質萃取物可延長線蟲的壽命，具有抗衰老作用。
本研究目的在於了解服用珍珠粉後，人體之抗氧化性，本實驗採隨機、安慰劑對照之設計，共計19位受試者參與，實驗組之受試者每天服用珍珠粉3g，而對照組之受試者則每天服用安慰劑3g。本實驗共進行10週，試驗期為8週，試驗後2週為觀察期，試驗期間的第零、二、四、六、八、十週採集血液檢體。結果顯示，食用珍珠粉後，對體重、體脂肪及身體質量指數無顯著影響，在抗氧化性方面，可顯著增加受試者體內總抗氧化力、血漿含硫官能基及麩胱甘肽含量，顯著降低脂質之過氧化，顯著提高紅血球中麩胱甘肽過氧化酶及麩胱甘肽還原酶。此外，連續服用8週後，LDL氧化的時間亦顯著地由43分鐘延長至58分鐘。由本研究可知，每天服用珍珠粉確實可有效增加人體之抗氧化能力，並增加LDL之氧化耐受性，且可延長線蟲的壽命，應該具有抗衰老的作用，而蛋白質可能是主要的貢獻成分。

Pearl is one traditional Chinese medicine, which contains a lot of amino acids and minerals. Many animal studies indicate that pearl has antioxidation and anti-aging effect.
The study was focused on antioxidant effect and anti-aging ability of pearl powder. Results showed that both pearl powder and protein extract of pearl powder had antioxidation and scavenging efficiency on free radicals.
For anti-aging function, wild-type Caenorhabditis elegans was used. Protein extract of pearl powder effectively extended the life span. Protein was probably the main contribution of antioxidation for pearl. To further evaluate the effect of pearl powder on antioxidation in human body. The study employed a randomized, placebo-controlled design. Nineteen subjects were enrolled respectively in a ten-week trial. Each subject ate 3g of pearl powder capsule or placebo every day for continuous eight weeks. The total experimental period was ten weeks, including eight weeks of administration and two weeks of follow-up. Anthropometric measurements, blood collecting were performed at the initial, 2nd, 4th, 6th, 8th, 10th weeks of the testing period. Pearl powder greatly increased total antioxidant capacity, total thiol group levels, contents of glutathione, and reduced thiobarbituric acid reactive substance (TBARS) in plasma. The antioxidant enzyme activity in erythrocytes was also increased, including glutathione peroxidase and glutathione reductase. In additional, oxidative lag time of LDL was significantly increased from 43.50 to 58.00 mins.
In conclusion, pearl powder showed great antioxidation. The main contribution was probably from its protein.

頁次
表次........................................................I
圖次.......................................................II
附錄.......................................................III
中文摘要...................................................IV
英文摘要...................................................VI
壹、 前言.................................................1
貳、 文獻整理.............................................2
一、 珍珠簡介............................................2
(一) 珍珠的歷史........................................2
(二) 珍珠的形成........................................2
(三) 珍珠的物理性質....................................3
(四) 珍珠的分類........................................3
(五) 珍珠的養殖........................................5
二、 珍珠的成份..........................................6
三、 珍珠的生理活性......................................7
(一) 抗衰老作用........................................7
(二) 免疫調節作用......................................9
(三) 其他功能.........................................12
四、 胺基酸作用...........................................13
(一) 胺基酸的抗氧化性..................................13
(二) 胜肽的作用........................................13
(三) 胜肽的抗氧化性....................................14
五、線蟲..................................................15
六、抗衰老................................................17
(一) 衰老的定義與機制..................................17
(二) 自由基與衰老......................................18
(三) 酵素與衰老........................................18
參、 研究目的............................................20
肆、 材料與方法..........................................21
一、 試驗材料............................................21
(一) 珍珠粉膠囊........................................21
(二) 人體試驗..........................................21
1. 基本資料........................................22
2. 血液樣品收集....................................22
二、 試驗用藥............................................24
三、 試驗儀器........................................29
四、 試驗設計............................................31
五、 試驗方法............................................32
(一) 珍珠粉中之蛋白質分析.............................32
1. 珍珠粉蛋白質萃取................................32
2. 珍珠粉蛋白質含量測定............................32
3. SDS-PAGE.......................................33
4. 珍珠粉之蛋白質去除..............................34
(二) 抗氧化性測定.....................................34
1. 抑制亞麻油酸自氧化之能力測定....................34
2. 總抗氧化能力測定................................35
3. 還原力測定......................................36
4. 螯合亞鐵離子之能力測定..........................36
5. 清除活性氧能力之測定............................37
a. DPPH自由基之清除............................37
b. 超氧陰離子之清除作用.........................37
(三) 野生線蟲培養模式之建立與壽命期之計算.............38
1. 線蟲固態培養基..................................38
2. E. coli OP50的培養...............................39
3. 蟲卵分離........................................39
4. 線蟲培養與壽命期計算............................40
5. 珍珠蛋白質對野生型線蟲老化之影響................40
(四) 血漿抗氧化活性之測定.............................41
1. 血漿中總抗氧化能力測定..........................41
2. 血漿中脂質過氧化物含量測定......................41
3. 血漿中含硫官能基之含量測定......................42
4. 血漿中麩胱甘肽(GSH)之含量測定...................43
5. 血漿中LDL氧化延滯期之測定......................43
6. 血漿中ascorbic acid之含量測定.....................45
（五） 抗氧化酵素活性分析...............................46
1. 紅血球中超氧歧化酶(superoxide dismutase；SOD)活性測定..............................................46
2. 紅血球中麩胱甘肽過氧化酶(glutathione peroxidase；GPx)活性測定........................................48
3. 紅血球中麩胱甘肽還原酶(glutathione reductase；GSH Rd)活性測定........................................49
（六） 血清生化分析.....................................50
1. 三酸甘油脂測定..................................50
2. 總膽固醇測定....................................50
3. HDL-C測定......................................50
4. LDL-C測定......................................51
六、 統計分析............................................52
伍、 結果與討論..........................................53
一、 珍珠粉中之蛋白質分析...............................53
二、 抗氧化性...........................................53
(一) 抑制亞麻油酸自氧化能力測定...................53
(二) 總抗氧化能力測定.............................56
(三) 還原力測定...................................59
(四) 螯合亞鐵離子之能力測定.......................59
（五） 清除活性氧能力之測定.........................61
1. DPPH自由基之清除能力.......................61
2. 超氧陰離子之清除能力........................64
三、 抗衰老.............................................66
(一) 延長線蟲生命期...............................66
四、 人體試驗...........................................69
(一) 受試者特性...................................69
(二) 對受試者血漿抗氧化性之影響...................69
(三) 對受試者紅血球中抗氧化酵素活性之影響.........81
(四) 受試者血清生化分析...........................85
陸、 結論................................................91
柒、 參考文獻............................................92
捌、 附錄...............................................98

王永革(2005)，羊胎素-珍珠膠囊抗衰老及免疫增強作用的實驗性研究，山西醫科大學，藥理學研究所

田清淶、殷瑩(1991)，羥脯氨酸和動物衰老相關性的研究，老年學
雜誌11 (3)：169

江琰、劉克武、李靜、劉鑫、石安靜(2003)，三角帆蚌黃白黑珍珠
元素分析及藥用價值評價，化學研究與應用15 (3)：433~434

李桂萍(1994)，不同品種珍珠母中胺基酸的含量測定，北京中醫藥大學學報17 (1)：60~62

李丹、李煜馨、李曉磊(2006)，高蛋白質含量大豆胜肽的理化特性和抗氧化活性，食品與發酵工業32 (11)：11~13

李端、徐翔、李丹、孫建國、吳佩君、盧靜(1996)，水解珍珠粉對D-半乳糖或臭氧所致衰老模型小鼠的抗氧化作用，中藥藥理與臨床
5：32~34

李端、徐翔、吳佩君、李丹、孫建國、盧靜(1996)，水解珍珠粉在
小鼠體內的抗氧化作用，中成藥18 (12)：30~31

李震、李祝、房秋寒(1997)，中藥抗皮膚衰老劑對小鼠皮膚羥脯氨
酸含量的影響，山東中醫藥大學學報21 (2)：142~143

呂明方、王福大(1996)，常見中藥材圖鑑，中國醫藥雜誌社主編，
渡假出版社

余麟(2005)，珍珠及其貝殼的成分與結構研究，華東理工大學，碩
士學位論文

林志彬(2000)，靈芝的現代研究（第二版），北京醫科大學出版社

林富楨、許士凱(2001)，抗衰老醫藥學，合計圖書出版社

周佩玲(1999)，珍珠，北京，地質出版社

周佩玲(1995)，有機寶石與投資指南，武漢，中國地質大學出版社

徐鴻華(2004)，中草藥彩色圖鑑，廣東科技出版社

孫福祥、孫成元、楊明霞、王志民(2004)，珍珠丸清除氧自由基的
實驗研究，中國實驗方劑學雜誌10 (2)：65

梁金龍、王倞濤、彭明生(1999)，貝殼珍珠層的研究與應用，礦物
岩石地球化學通報18 (4)：403~406

張文華、劉玉娥、魏欣冰、劉慧青、李愛國、陳曉陽、李朝武、張釉美(2003)，復方珍珠散調節免疫功能作用的實驗研究，山東中醫藥大學學報27 (6)：459~461

張剛生、童銀洪(2000)，軟體動物貝殼中的有機質研究進展，湛江
海洋大學學報20 (1)：74~78

郭守國(1998)，寶玉石學教程，北京：科學出版社

郭守國(2004)，珍珠，上海：上海文化出版社

黃安林(1997)，珍珠燒燙傷膏動物實驗研究和臨床應用研究，現代
應用藥學7 (6)：48

楊美琳、李大達、朱美蘭、李堃(1994)，烏梢蛇、全蝎和珍珠中的
胺基酸成分，吉林中醫藥(5)：39

錢榮華、竹劍平(2003)，珍珠粉延緩衰老作用的實驗研究，浙江臨
床醫學5 (9)：718

潘建新、張玉英、張克平(2003)，珍珠護膚品抗皮膚衰老的實驗研
究，蘇州大學學報23 (5)：535~536

Allain, C., Poon, L., Chan, C., Richmond, W. and Fu, P. (1974). Enzymatic determination of total serum cholesterol. Clin Chem
20:470-475.

Arnao, M. B., Casas, J. L., del Rio, J. A., Acosta, M., and Garcia-Canovas,
F. (1990). An enzymatic colorimetric method for measuring naringin using 2,2''-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) in the
presence of peroxidase. Anal Biochem 185:335-338.

Bachorik, P. and Ross, J. (1995). Nutrition Cholesterol Eduction Program Recommendations for measurement of Low-Density Lipoprotein
Cholesterol: Executive Summary. Clin Chem 41:1414-1420.

Bellomo, G.., Comstock, J.P., Wen, D. and Hazelwood, R. L. (1987). Prolonged fructose feeding and aldose reductase inhibition: effect on the polyol pathway in kidneys of normal rats. Proc.Soc. Exp.Biol. Med.
186(3):348-354.

Brandt, K.D., and Muir, H. (1971). Heterogeneity of protein-
polysaccharides of porcine articular cartilage. Biochem J 123:747-755.

Brenner, S. (1974). The Genetics of Canorhabditis elegans. Genetics 77:
71-94.

Carrol, K. K. (1991). Review of clinical studies on cholesterol-lowering
response to soy protein. J. Am. Dietetic Assoc 91:820-827.

Chen, D., Duggan, C., Ganley, J. P., Kooragayala, L. M., Reden, T.B., Texada, D. E. and Langford, M. P. (2004). Expression of enterovirus 70 capsid protein VP1 in Escherichia coli. Protein expression and
purification 37:426-433.

Dinis, T. C. P., Maderia, V. M. C. and Almeida, L. M. (1994). Action of phenolic derivatives (acetaminophen, salicylate, and 5-amino salicylate) as ingibitors of membrane lipid peroxidation and as peroxyl radical
scavengers. Arch. Biochem. Biophys. 315:161-169.

Draper, H. H. and Hadley, M. (1990). Malondialdehyde determination as
index of lipid peroxidation. Methods Enzymol 186:421-431.

Gordon, T., Castelli, W., Hjortland, M., Kannel, W. and Dawber, T. (1977). High density lipoprotein as a protective factor against coronary heart
disease. Am J Med 62:707-714.

Halliwell, B., and Gutteridge, J. M. (1990). The antioxidants of human
extracellular fluids. Arch Biochem Biophys 280:1-8.

Hamvas, A., Palazzo, R., Kaiser, L., Cooper, J., Shuman, T., Velazquez, M., Freeman, B. and Schuster, D. P. (1992). Inflammation and oxygen free radical formation during pulmonary ischemia-reperfusion injury. J
Appl Physiol 72:621-628.

Hata, Y., Yamamoto, M., Ohni, M., Nakajima, K., Nakamura, Y. and Takano, T. (1996). A placebo-controlled study of the effect sour milk on
blood pressure in hypertensive subjects. Am J Clin Nutr 64:767-771.

Hirano, M., Rakwal, R., Shibato, J., Argawal, G.. K. and Jwa, N. (2006). New protein extraction/solubilization protocol for gel-gased proteomics
of Rat (female) whole brain and brain regions. Mol Cell 22(1):119-125.

Hu, M. L. (1994). Measurement of protein thiol groups and glutathione in
plasma. Methods Enzymol 233:380-385.

Ishii, N., Senoo-Matsuda, N., Miyake, K., Yasuda, K., Ishii, T., Hartman, P. S. and Furukawa, S. (2004).Coenzyme Q10 can prolong Caenorhabditis elegans lifespan by lowering oxidative stress. Mechanisms of ageing and
Development. 125:41-46.

Kawasaki, T., Seki, E., Osajima, K., Yoshida, M., Asada, K., Matsui, T. and Osajima, Y. (2000). Antihypertensive effect of valine-tyrosine, a short chain peptide derived from sardine muscle hydrolyzate, on hypertensive
subjects. J Human Hyperten 14:519-523.

Kleinveld, H. A., Demacker, P. N., De Haan, A. F. and Stalenhoef, A. F. (1993). Decreased in vitro oxidizability of low-density lipoprotein in
hypercholesterolaemic patients treated with 3-hydroxy-3-methylglutaryl-
CoA reductase inhibitors. Eur. J Clin Invest 23:289-295.

Koditschek, L., and Umbreit, W. (1969). Alpha-glycerophosphate oxidase
in Streptococcus faecium F24. J Bacteriol 98:1063-1068.

Lawrence, R.A., and Burk, R.F. (1976). Glutathione peroxidase activity in
selemium-deficient rat liver. Biochem Biophys Res Commun 71:952-958.
Lingenrt, H., Vallentin, K. and Ericksson, C. E. (1979). Measurement of antioxidative effect in model systems. J. Food Process. Preserv. 3:87-103.

Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randell, R. J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem
193:265-275.

Matsui, T., Li, C. H., Tanaka, T., Maki, T., Osajima, Y. and Matsumoto, K. (2000). Depressor effect of wheat germ hydrolysate and its novel angiotensin I-converting enzyme inhibitory peptides, IIe-Val-Tyr, and the
metabolism in rat and human plasma. Biol Pharm Bull 23(4):427-431.

Miller, N. J., Rice-Evans, C., Davies, M. J., Gopinathan, V. and Milner, A. (1993). A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates.
Clin Sci (Lond) 84:407-412.

Miyoshi, S., Kaneto, T., Yoshiazawa, T., Fukui, Y., Tanaka, F. and Maruyama, H. (1991). Hypotensive activity of enzymatic α-Zein
hydrolysate. Agric Bio Chem 55(5):1407-1408.

Nagata, Y., Tanaka, K. and Sugano, M. (1981). Further studies on the hypocholesterolemic effect of soya bean protein in rats. Br. J. Nutr. 45:
233-241.

Nakamura, M., Taniguti, Y., Yamamoto, M., Hino, K. and Manabe, M. (1997). Homogeneus assay of serum LDL-cholesterol on an automatic
analyzer. Clin Chem 43:260-261.

Oyaizu, M. (1986). Studies on products of browning reaction prepared
from glucosamine. Jpn. J. Nutr. 44:307-315.

Pryor, W.A.(1973). Free radical reactions and their importance in
biochemical systems. Fed. Proc. 32(8):1862-1869.

Raifai, N., Iannotti, E., DeAngelis, K. and Law, T. (1998). Analytical and clinical performance of a homogeneous enzymatic LDL-cholesterol assay compared with the ultracentrifugation-dexyran sulfate-Mg2+ method. Clin
Chem. 44:1242-1250.
Riesen, W. (1998). Lipid Metabolism. Clinical laboratory diagnostics Use and assessment of clinical laboratory results Frankfurt/Main: TH-Books
Verlagsgesellschaft. 167-169.

Robak, J. and Gryglewski, R. J. (1988). Flavonoids are scavengers of
superoxide anions. Biochem. Pharmacol. 37(5):837-841.

Schettler, G. (1975). Plasma lipoprotein and atherosclerosis. Arch Inst
Cardiol Mex. 45(3):335-343.

Shimada, K., Fujikawa, K., Yahara, K. and Nakamura, T. (1992). Antioxidative properties of xanthane on the autoxidation of soybean oil in
cyclodextrin emulsion. J. Agric. Food Chem. 40:945-948.

Stiernagle, T. (1999). C.elegans: A practical approach. Hope I. A., 51-67.

Triantis, T. M., Yannakopoulou, E., Nikokavoura, A., Dimotikali, D. and Papadopoulos, K. (2007). Chemiluminescent studies on the antioxidant
activity of amino acids. Anal Chim Acta 591:106-111.

Trinder, P.(1969).Oxidase determination of plasma cholesterol as cholest-
4-en-3-one using iso-octane extraction. Ann of Clin Biochem. 18 : 64-70.

William, R. B. and Michael, W. P. (1994). Antioxidant nutrients and
protection from free radicals. Nutr Toxicol, 19-48.

Yokoyama, K., Chiba, H. and Yoshikawa, M. (1992). Peptide inhibitors for angiotensin I-converting enzyme from thermolysin digest of dried
bonito. Biosci Biotech Biochem. 56(10):1541-1545.

Zannoni, V., Lynch, M., Goldstein, S. and Sato, P. (1974). A rapid micromethod for determination of ascorbic acid in plasma and tissue.
Biochem Med. 11:41-48.