多甲氧基黃酮 (Polymethoxyflavones, PMFs) 為類黃酮的一種，在 C6-C3-C6 基本的骨架上接有兩個以上的甲氧取代基。許多研究證實此類物質具有和未被甲基化的黃酮相似的活性，如抗癌、抗病毒等，並且生物可利用率高於未被甲基化的黃酮。 5,7,3’,4’-Tetramethoxyflavone (TMF) 被發現存在於Kaempferia parviflora (黑薑) 和 Orthosiphon spicatus (貓鬚草) 等植物中。已有文獻指出 TMF具有抗真菌、抗發炎等活性。但目前對於 TMF生物可利用率之相關研究仍相當地少。本研究以大鼠模式來研究 TMF 之藥物動力學及組織分佈。藥物動力學結果顯示，以靜脈注射給予大鼠 TMF (5 mg/kg, i.v.)後，在血漿中測得之 Cmax為 1.54±0.62 μg/mL，T1/2為62.85±27.92 分鐘；管餵給予大鼠 TMF (50 mg/kg BW) 後，Cmax為 0.79±0.30μg/mL，Tmax 為190±24.50 分鐘，T1/2為273±90.2 分鐘；經計算得到 TMF的口服生物可利用率為 14.3 %，比起未被甲基化黃酮高，推測是由於結構上的甲氧基降低極性，促使膜穿透率及代謝穩定性提高。
在分佈試驗部分，於管餵 TMF (50 mg/kg BW) 2 小時後測定各個臟器中TMF的含量，以腸胃道的胃含量最高，佔餵食劑量之 43 % 以上；在管餵 8小時後則以盲腸、大腸及直腸含量較多。此外，管餵 TMF (50 mg/kg BW) 2 小時後，在腦中也可偵測到其存在。而測定組織臟器中3’-hydroxy-5,7,4’-trimethoxyflavone (M1)、4’-hydroxy-5,7,3’-trimethoxyflavone (M4)及5-hydroxy-7,3’,4’-trimethoxyflavo ne (M5) 等代謝物之含量，發現 TMF 以轉變為M1加M4代謝物為主，且以12小時為最高的時間點， 於12 小時M1+M4在所有組織臟器總量，佔管餵劑量百分比的57.47 %。
排除試驗結果顯示，在胃管餵食 TMF (50 mg/kg BW) 後的12-24小時內有較高量的 TMF由糞便排除， TMF於糞便中排除總量佔餵食劑量0.8121% 。而在尿液中以4-12小時為TMF主要排除期，佔總餵食劑量百分比0.0496%。利用 M4 、M5兩種代謝物標準品，以及酵素水解方式定量，得到尿液中 M1+M4及M5代謝物排除量佔管餵劑量百分比為50 %。並從尿液中也發現不同形式的代謝物，代謝物含量及結構有待未來進一步之探討。

Polymethoxyflavones (PMFs), a special group of flavonoids with two or more methoxy groups on benzene ring (C6-C3-C6), have demonstrated many physiological activities similar to unmethylated flavones, but with higher bioavailability. 5,7,3’,4’-Tetramethoxyflavone (TMF) is one of the major polymethoxyflavones present in Kaempferia parviflora and Orthosiphon spicatus. Several studies have shown that TMF has many bioactivities such as anti-allergic activity, antifungal and anti-inflammatory effects, but the oral bioavailability of TMF is not clear so far. Thus, the purpose of this study is to investigate the bioavailability of TMF in Sprague-Dawley rats. In pharmacokinetic study, Cmax and T1/2 were determined to be 1.54±0.62 μg/mL and 62.85±27.92 minutes, respectively, after intravenous injection of 5 mg/kg BW of TMF. After tube feeding 50 mg/kg BW of TMF, the pharmacokinetic parameters of Cmax, Tmax and T1/2 were 0.79±0.30 μg/mL, 190±24.50 minute and 273±90.2 minutes, respectively. The bioavailability of TMF is 14.3%, much higher than some flavonoids. The lipophilicity methoxy of groups on TMF structure gave higher cell membrane permeability and improved metabolic stability, thus higher oral bioavailability was observed.
In tissue distribution study, higher concentration of TMF were found in gastrointestinal tracts after tube feeding 50 mg/kg BW for 2 hour and 4 hours, stomach was shown to possess the highest content of TMF in 2 hours which contained up to 43% of dosing. TMF was also detected in brain. 3’-hydroxy-5,7,4’-trimethoxyflavone (M1), 4’-hydroxy-5,7,3’-trimethoxyflavone (M4) and 5-hydroxy-7,3’,4’-trimethoxyflavone (M5) were found to exhibit the tissue among the three metabolites of TMF, after feeding TMF. M1 plus M4 were also observed of 12 hours, after tube feeding 50 mg/kg BW TMF, M1 plus M4 reached the hightest concentration in all tissue containing up to 57 % of dosing.
In excretion study, the main fecal excretion of TMF was found during 12-24 hours, containing about 0.8121 % of dosing, after tube feeding 50 mg/kg BW TMF. The main urinary excretion of TMF was found during 4-12 hours, TMF content in urine was only 0.0496% of dosing. The metabolites M1 plus M4 are the major metabolites in urine, the total amount of M1, M4 and M5 can reach 50 % of dosing. There are other possible urinary metabolites of TMF which should be further identified and quantified.

中文摘要 I
Abstract III
縮寫表 V
目錄 VII
圖次 XI
表次 XIII
附錄 XV
壹、 前言 1
貳、 文獻整理 2
一、 多甲氧基類黃酮 (Polymethoxyflavone) 2
(一) 類黃酮類 (Flavonoids) 簡介 2
(二) 多甲氧基黃酮 (polymethoxyflavone, PMF) 4
(三) 多甲氧基黃酮的生理活性 6
一、 抗發炎活性： 6
二、 抗肥胖活性： 6
三、 抗癌症活性： 6
二、 5, 7, 3’, 4’ – Tetramethoxyflavone (TMF) 及其生理活性及可能代謝產物 8
(一) 5, 7, 3’, 4’ – Tetramethoxyflavone 簡介 8
(二) TMF的生理活性 9
1. 抗α-葡萄糖水解酵素 (α-glucosidase )活性 9
2. 抗發炎活性 11
3. Antiplasmodial 活性 12
4. TMF相關代謝物 13
三、 藥物動力學(Pharmacokinetic, PK) 14
(一) 藥物動力學參數 16
(二) 生物可利用率(bioavailability, BA) 18
(三) 類黃酮類的生物可利用率相關研究 20
(四) 多甲氧基黃酮之生物可利用率及藥物動力學相關研究 21
參、 研究目的與實驗架構 28
(一) Part 1-藥物動力學試驗 30
(二) Part 2-大鼠體內分佈及排除試驗 31
肆、 材料及方法 32
一、 實驗材料 32
二、 化學藥品與溶劑 32
(一) 下列藥品購買自美國 Sigma 公司 (St. Louis, MO, USA) 32
(二) 下列藥品購買自 Mallinckrodt Baker 公司 (Phillipsburg, NJ, USA) 32
(三) 其他試藥 32
三、 實驗動物及飼料 33
四、 儀器設備 33
五、 實驗方法 35
(一) 動物飼養 35
(二) 5, 7, 3’, 4’ –tetramethoxyflavone 藥物動力學參數測定 35
1. 5, 7, 3’, 4’ –tetramethoxyflavone 血漿回收率測定 35
2. 高效液相層析分析條件 36
3. 5, 7, 3’, 4’ –Tetramethoxyflavone於血漿樣品中之檢量線製作 36
4. 管灌餵食實驗 37
5. 靜脈注射實驗 37
6. 高效液相層析質譜分析條件 38
7. 藥動參數分析 39
(三) 5, 7, 3’, 4’ –tetramethoxyflavone於大鼠的吸收分佈試驗 40
1. 管灌餵食與犧牲 40
2. 動物檢體的收集與處理 40
3. 檢體萃取 40
(四) 排除試驗 42
1. 代謝籠試驗 42
2. 糞便分析 42
3. 尿液分析 43
4. 高效液相層析質譜分析條件 43
伍、結果與討論 45
一、以大鼠模式探討 5, 7, 3’, 4’-tetramethoxyflavone 之藥物動力學 45
(一) 實驗材料 5, 7, 3’, 4’-tetramethoxyflavone 及其代謝物之質譜與NMR圖譜分析 45
(二) 5,7,3’,4’-Tetramethoxyflavone 分析方法之確效 49
(三) 傳統採血法之 5, 7, 3’, 4’-tetramethoxyflavone 回收率測定 52
(四) 血漿中 TMF 之藥物動力學參數及口服生物可利用率 54
二、以大鼠模式探討 TMF之吸收與分佈 61
(一) TMF 於大鼠各組織臟器之吸收與分佈 61
(二) 代謝物 M1、M4 及 M5 於大鼠各組織臟器之吸收與分佈 74
三、以大鼠為模式探討 TMF 於尿液及糞便之排除 87
(一) TMF、M1、M4及M5以及結合態代謝物於大鼠尿液中之排除情形 87
1. TMF 於大鼠尿液中排除結果 87
2. M1、M4 及 M5 和其接合葡萄醣醛酸態及硫酸態代謝物於大鼠尿液中排除結果 91
3. TMF 於大鼠之糞便排除結果 96
四、TMF 之代謝研究 99
(一) TMF及代謝物 於大鼠肝臟中之分佈情形 103
陸、 結論 105
柒、 參考文獻 106
捌、 附錄 112

呂玟蒨。2011。以大鼠模式探討龍眼花 peoanthocyanidin A2 的生物可利用性。國立台灣大學食品科技研究所博士論文。台北。台灣
張瑋珊。2012。以大鼠模式探討橘皮素的生物可利用性及組織分佈。國立台灣大學食品科技研究所碩士論文。台北。台灣
蔡東湖。2003。大白鼠的基本手術技術與微透析應用。國立陽明大學 藥物動力學部分上課講義。台北。台灣
衛生福利部食品藥物管理署(TFDA)。2012。食品化學檢驗方法之確效規範。台北。台灣
Azuma, T., Kayano, S.-i., Matsumura, Y., Konishi, Y., Tanaka, Y., and Kikuzaki, H. (2011). Antimutagenic and alpha-glucosidase inhibitory effects of constituents from Kaempferia parviflora. Food Chemistry 125, 471-475.
Bas, E., Recio, M. C., Giner, R. M., Manez, S., Cerda-Nicolas, M., and Rios, J. L. (2006). Anti-inflammatory activity of 5-O-demethylnobiletin, a polymethoxyflavone isolated from Sideritis tragoriganum. Planta Medica 72, 136-142.
Bennett, R. N., and Wallsgrove, R. M. (1994). Secondary metabolites in plant deffense-mechanisms. New Phytologist 127, 617-633.
Cao, G. H., Sofic, E., and Prior, R. L. (1997). Antioxidant and prooxidant behavior of flavonoids: Structure-activity relationships. Free Radical Biology and Medicine 22, 749-760.
He, C. X., He, Z. G., and Gao, J. Q. (2010). Microemulsions as drug delivery systems to improve the solubility and the bioavailability of poorly water-soluble drugs. Expert Opinion on Drug Delivery 7, 445-460.
Heim, K. E., Tagliaferro, A. R., and Bobilya, D. J. (2002). Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. Journal of Nutritional Biochemistry 13, 572-584.
Kinoshita, T., and Firman, K. (1997). Myricetin 5,7,3'',4'',5''-pentamethyl ether and other methylated flavonoids from Murraya paniculata. Phytochemistry 45, 179-181.
Khaled, K. A., El-Sayed, Y. M., and Al-Hadiya, B. M. (2003). Disposition of the flavonoid quercetin in rats after single intravenous and oral doses. Drug Development and Industrial Pharmacy 29, 397-403
Koga, N., Ohta, C., Kato, Y., Haraguchi, K., Endo, T., Ogawa, K., Ohta, H., and Yano, M. (2011). In vitro metabolism of nobiletin, a polymethoxy-flavonoid, by human liver microsomes and cytochrome P450. Xenobiotica 41, 927-933.
Kunimasa, K., Kuranuki, S., Matsuura, N., Iwasaki, N., Ikeda, M., Ito, A., Sashida, Y., Mimaki, Y., Yano, M., Sato, M., Igarashi, Y., and Oikawa, T. (2009). Identification of nobiletin, a polymethoxyflavonoid, as an enhancer of adiponectin secretion. Bioorganic & Medicinal Chemistry Letters 19, 2062-2064.
Kurowska, E. A., and Manthey, J. A. (2004). Hypolipidemic effects and absorption of citrus polymethoxylated flavones in hamsters with diet-induced hypercholesterolemia. Journal of Agricultural and Food Chemistry 52, 2879-2886.
Kwan, K. C. (1997). Oral bioavailability and first-pass effects. Drug Metabolism and Disposition 25, 1329-1336.
Liversidge, G. G., and Cundy, K. C. (1995). Particle size reduction for improvement of oral bioavailability of hydrophobic drugs: I. Absolute oral bioavailability of nanocrystalline danazol in beagle dogs. International Journal of Pharmaceutics 125, 91-97.
Loon, Y. H., Wong, H. W., Yap, S. P., and Yuen, K. H. (2005). Determination of flavonoids from Orthosiphon stamineus in plasma using a simple HPLC method with ultraviolet detection. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 816, 161-166.
Lu, W.-C., Sheen, J.-F., Hwang, L. S., and Wei, G.-J. (2012). Identification of 5,7,3 '',4 ''-Tetramethoxyflavone Metabolites in Rat Urine by the Isotope-Labeling Method and Ultrahigh-Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry. Journal of Agricultural and Food Chemistry 60, 8123-8128.
Lyckander, I. M., and Malterud, K. E. (1996). Lipophilic flavonoids from Orthosiphon spicatus prevent oxidative inactivation of 15-lipoxygenase. Prostaglandins Leukotrienes and Essential Fatty Acids 54, 239-246.
Matsuda, H., Kogami, Y., Nakamura, S., Sugiyama, T., Ueno, T., and Yoshikawa, M. (2011). Structural requirements of flavonoids for the adipogenesis of 3T3-L1 cells. Bioorganic & Medicinal Chemistry 19, 2835-2841.
Mekjaruskul, C., Jay, M., and Sripanidkulchai, B. (2012). Pharmacokinetics, Bioavailability, Tissue Distribution, Excretion, and Metabolite Identification of Methoxyflavones in Kaempferia parviflora Extract in Rats. Drug Metabolism and Disposition 40, 2342-2353.
Mekjaruskul, C., Yang, Y.-T., Leed, M. G. D., Sadgrove, M. P., Jay, M., and Sripanidkulchai, B. (2013). Novel formulation strategies for enhancing oral delivery of methoxyflavones in Kaempferia parviflora by SMEDDS or complexation with 2-hydroxypropyl-beta-cyclodextrin. International Journal of Pharmaceutics 445, 1-11.
Miyata, Y., Tanaka, H., Shimada, A., Sato, T., Ito, A., Yamanouchi, T., and Kosano, H. (2011). Regulation of adipocytokine secretion and adipocyte hypertrophy by polymethoxyflavonoids, nobiletin and tangeretin. Life Sciences 88, 613-618.
Nielsen, S. E., Breinholt, V., Cornett, C., and Dragsted, L. O. (2000). Biotransformation of the citrus flavone tangeretin in rats. Identification of metabolites with intact flavane nucleus. Food and Chemical Toxicology 38, 739-746.
Okuno, Y., and Miyazawa, M. (2004). Biotransformation of sinesetin by the larvae of the common cutworm (Spodoptera litura). Biological & Pharmaceutical Bulletin 27, 1289-1292.
Onoue, S., Uchida, A., Takahashi, H., Seto, Y., Kawabata, Y., Ogawa, K., Yuminoki, K., Hashimoto, N., and Yamada, S. (2011). Development of High-Energy Amorphous Solid Dispersion of Nanosized Nobiletin, a Citrus Polymethoxylated Flavone, with Improved Oral Bioavailability. Journal of Pharmaceutical Sciences 100, 3793-3801.
Passamonti, S., Terdoslavich, M., Franca, R., Vanzo, A., Tramer, F., Braidot, E., Petrussa, E., and Vianello, A. (2009). Bioavailability of Flavonoids: A Review of Their Membrane Transport and the Function of Bilitranslocase in Animal and Plant Organisms. Current Drug Metabolism 10, 369-394.
Pietta, P. G. (2000). Flavonoids as antioxidants. Journal of Natural Products 63, 1035-1042.
Reinboth, M., Wolffram, S., Abraham, G., Ungemach, F. R., and Cermak, R. (2010). Oral bioavailability of quercetin from different quercetin glycosides in dogs. British Journal of Nutrition 104, 198-203.
Riceevans, C. A., Miller, N. J., Bolwell, G. P., Bramley, P. M., and Pridham, J. B. (1995). The Relative antioxifant activities of plant-derived polyphenolic flavonoids. Free Radical Research 22, 375-383.
RiceEvans, C. A., Miller, N. J., and Paganga, G. (1996). Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine 20, 933-956.
Sae-Wong, C., Matsuda, H., Tewtrakul, S., Tansakul, P., Nakamura, S., Nomura, Y., and Yoshikawa, M. (2011). Suppressive effects of methoxyflavonoids isolated from Kaempferia parviflora on inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells. Journal of Ethnopharmacology 136, 488-495.
Shao, X., Chen, X., Badmaev, V., Ho, C.-T., and Sang, S. (2010). Structural identification of mouse urinary metabolites of pterostilbene using liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 24, 1770-1778.
Shargel, L., Wu-Pong, S.,: Yu, Andrew B. C. (2004) In Applied Biopharmaceuties and Parmacokinetics, 5th edition: Charpter 1. Introduction to biopharmaceutics and Pharmacokinetics. Mcgraw-Hill professional Publishing, New York, NY,USA., 1,7.
Singh, S. P., Wahajuddin, Tewari, D., Patel, K., and Jain, G. K. (2011). Permeability determination and pharmacokinetic study of nobiletin in rat plasma and brain by validated high-performance liquid chromatography method. Fitoterapia 82, 1206-1214.
Takaaki YASUDA, a. Y. Y., a Hisako YABUKI,a Takahiro NAKAZAWA,a Keisuke OHSAWA,*,a, and Yoshihiro MIMAKI, b. a. Y. S. (2003). Urinary Metabolites of Nobiletin Orally Administered to Rats. Chem. Pharm. Bull. 51(12), 1426—1428.
Taylor, L. P., and Grotewold, E. (2005). Flavonoids as developmental regulators. Current Opinion in Plant Biology 8, 317-323.
Thomas, V. H., Bhattachar, S., Hitchingham, L., Zocharski, P., Naath, M., Surendran, N., Stoner, C. L., and El-Kattan, A. (2006). The road map to oral bioavailability: an industrial perspective. Expert Opinion on Drug Metabolism & Toxicology 2, 591-608.
Urso R., Blardi P., Giorgi G. (2002). A short introduction to pharmacokinetics. European Review for Medical and Pharmacological Sciences 6, 33-44.
Van Acker, S. A. B. E., Van Den Berg, D.-J., Tromp, M. N. J. L., Griffioen, D. H., Van Bennekom, W. P., Van Der Vijgh, W. J. F., and Bast, A. (1996). Structural aspects of antioxidant activity of flavonoids. Free Radical Biology and Medicine 20, 331-342.
Vichitphan S., Vichitphan K., Sirikhansaeng P. (2007) Flavonoid content and antioxidant activity of krachai-dum (Kaempferia parviflora) wine. KMITL Sci. Tech. J., 7, 97-105.
Walle, T. (2007). Methoxylated flavones, a superior cancer chemopreventive flavonoid subclass? Seminars in Cancer Biology 17, 354-362.
Walle, T. (2009). Methylation of Dietary Flavones Increases Their Metabolic Stability and Chemopreventive Effects. International Journal of Molecular Sciences 10, 5002-5019.
Walle, T. (2011). Bioavailability of resveratrol. In "Resveratrol and Health" Wiley-Blackwell, Malden., 1215, 9-15. Wiley-Blackwell, Malden.
Walle, T., Hsieh, F., DeLegge, M. H., Oatis, J. E., and Walle, U. K. (2004). High absorption but very low bioavailability of oral resveratrol in humans. Drug Metabolism and Disposition 32, 1377-1382.
Wen, X., and Walle, T. (2006). Methylated flavonoids have greatly improved intestinal absorption and metabolic stability. Drug Metabolism and Disposition 34, 1786-1792.
Wongsrikaew, N., Woo, H. C., Vichitphan, K., and Han, J. (2011). Supercritical CO2 for Efficient Extraction of Polymethoxyflavones in Kaempferia parviflora. Journal of the Korean Society for Applied Biological Chemistry 54, 1008-1011.
Xiao, H., Yang, C. S., Li, S. M., Jin, H. Y., Ho, C. T., and Patel, T. (2009). Monodemethylated polymethoxyflavones from sweet orange (Citrus sinensis) peel inhibit growth of human lung cancer cells by apoptosis. Molecular Nutrition & Food Research 53, 398-406.
Xu, J. L., Zhang, Q. Y., Zhao, L., Wang, Y., Xue, L. M., Han, T., Zheng, C. J., and Qin, L. P. (2012). Quantitative determination and pharmacokinetic study of casticin in rat plasma by liquid chromatography-mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 61, 242-246.
Yao, J., Zhou, J. P., Ping, Q. N., Lu, Y., and Chen, L. (2008). Distribution of nobiletin chitosan-based microemulsions in brain following i.v. injection in mice. International Journal of Pharmaceutics 352, 256-262.
Yasuda, T., Yoshimura, Y., Yabuki, H., Nakazawa, T., Ohsawa, K., Mimaki, Y., and Sashida, Y. (2003). Urinary metabolites of nobiletin orally administered to rats. Chemical & Pharmaceutical Bulletin 51, 1426-1428.
Yenjai, C., Prasanphen, K., Daodee, S., Wongpanich, V., and Kittakoop, P. (2004). Bioactive flavonoids from Kaempferia parviflora. Fitoterapia 75, 89-92.
Zhu, M., Chen, Y., and Li, R. C. (2000). Oral absorption and bioavailability of tea catechins. Planta Medica 66, 444-447.