臺灣博碩士論文加值系統

　　本實驗之目的乃是在探討藻褐素在培養基中的穩定性，及兩種肝細胞（BNL CL.2正常肝細胞和SK-Hep-1肝癌細胞）的吸收率。藻褐素先以絕對酒精配製成10 mM，再以酒精將藻褐素稀釋成1.0 mM（stock A）或以100％胎牛血清稀釋成1.0 mM（stock B）。取10μL Stock A加到10 mL含有10％FBS的DMEM培養基（A1），或不含FBS的DMEM中（A2）。另取10μL Stock B加到10 mL含有10％FBS的DMEM培養基（B1），或不含FBS的DMEM中（B2）。各組藻褐素的終濃度均為10μM。
　　結果顯示：培養3、6、12、24、48、72小時之後，藻褐素會因培養的時間增加而逐漸降解；其中以A2組的穩定度最好，在經過了72小時後仍有73.3%的藻褐素未被降解。而B1組的降解速度最快，於72小時培養後只有約34.9%的藻褐素未被降解。
　　在SK-Hep-1肝癌細胞吸收率方面，培養24小時後，藻褐素吸收率達到最高，其中以A2組的細胞9.81%吸收量最高，而以A1組的細胞2.10%吸收率最低。以BNL CL.2 而言，於培養24小時之後，以B1組的5.60%吸收率最高，以A1組的2.91%吸收率最低。在細胞培養過程中，HPLC分析圖出現一個不明的藻褐素代謝產物；此物質也會隨著時間的增加而累積量增加。在SK-Hep-1細胞培養中，此未知物於培養24小時之後，其累積量達最高。對BNL CL.2細胞而言，此一未知物質則是在48小時達到最高量。　　
　　此外，本研究也觀察藻褐素對細胞型態的影響；結果顯示：隨著培養時間的增加，SK-Hep-1肝癌細胞型態逐漸變形和細胞膜變得不完整，尤其在不含血清的培養基培養下，細胞的存活率很低；而BNL CL.2細胞無法在不含血清的培養基超過12小時長時間的培養，然而在含血清的培養基培養下，隨著培養時間的增加，細胞型態也會變形，並有囊泡產生的現象。
　　以上的結果顯示：以酒精配製的藻褐素在不含血清的培養基中（A2組）穩定性較好。而且SK-Hep-1細胞在A2組對藻褐素的吸收也最佳。然而在BNL CL.2細胞中，則是以胎牛血清稀釋的藻褐素在含血清的培養基中（B1）的吸收率最好。

The purpose of this study was to investigate the stability of fucoxanthin in different solvent systems and the cellular uptake in two types of liver cells. A ethanol was used first to prepare a 10 mM fucoxanthin solution, which was then further diluted to 1.0 mM in ethanol (Stock A) or in 100% FBS (Stock B). A 10 μL Stock A was added to 10 mL DMEM medium containing 10% FBS (A1) or without containing FBS (A2). Likewise, A 10 μL Stock B was added to 10 mL DMEM medium containing 10% FBS (B1) or without containing FBS (B2). The final concentration of fucoxanthin in these solvent systems was 10 μM.
We found that the fucoxanthin in the four different solvent systems degraded with increasing incubation time (3-72h). Fucoxanthin in group A2 had the best stability, with 73.3% of fucoxanthin remaining at 72 h, while fucoxanthin in group B1 had the poorest stability, with only 34.9% fucoxanthin remaining at 72 h.
Regarding cellular uptake in SK-Hep-1 cells, fucoxanthin in all aolvent systems had the highest uptake at 24 h, at which time fucoxanthin in group A2 had the highest uptake (9.81%), while group A1 had the lowest uptake (2.10%). For BNL CL.2 cells, group B1 had the highest uptake (5.6%), while group A1 had the lowest uptake (2.91%) at 24 h. In the HPLC profile, there was an unknown peak, which was suspect to be a metabolic product of fucoxanthin by cells. The concentration of this product reached the maximum level at 24 h and 48 h of incubation for SK-Hep-1 cells and BNL CL.2 cells, respectively.
In addition, we noted fucoxanthin-induced changes in cell morphology. With the increase in incubation time, SK-Hep-1 cells became deformed and had low viability, especially when incubated in the FBS-free medium (groups A2 and B2). Fucoxanthin also affected the morphology of BNL CL.2 cells by causing deformation and production of vesicles, even when FBS was present (groups A1 and B1).
In summary, this study shows that the stability was better when the fucoxanthin stock was prepared in ethanol and then added into the FBS-free medium (group A2), which also has the best cellular uptake of fucoxanthin in SK-Hep-1 cells. In BNL CL.2 cells, however, cellular uptake of fucoxanthin is better in B1 group, which was prepared in 100% FBS and then diluted in DMEM medium containing 100% FBS.

目次 I
表目錄 IV
圖目次 V
縮寫表 VII
中文摘要 VIII
英文摘要 X
壹、前言 1
貳、文獻整理 5
一、類胡蘿蔔素(CAROTENOIDS) 5
二、藻褐素(FUCOXANTHIN) 7
1、藻褐素的結構 8
2、藻褐素的代謝 8
3、藻褐素生理活性 9
三、載體運送 10
1、微粒體-Micelles 11
2、脂質小體-Liposomes 12
3、甲基-β-環狀糊-Methyl-beta-cyclodextrin(M-β-CD) 12
叁、研究動機與目的 14
一、研究動機 14
二、研究目的 15
肆、實驗架構 16
伍、材料與方法 17
一、實驗試劑 17
二、細胞選定 17
三、細胞解凍 18
四、細胞培養 18
五、細胞保存 18
六、藻褐素之載體製備 18
七、細胞存活率分析-細胞計數 19
八、穩定性分析 19
九、細胞吸收 19
十、萃取與HPLC分析 20
1、培養基的穩定性萃取 20
2、萃取細胞內的藻褐素 20
3、HPLC 分析 21
十一、統計分析 22
陸、結果與討論 24
一、藻褐素在不同系統製備中的穩定性 24
二、細胞生長曲線 36
三、SK-HEP-1 CELLS對於藻褐素的吸收 51
四、BNL CL.2. CELLS對於藻褐素的吸收 68
柒、結論 78
捌、文獻參考 79

臺灣海藻資訊網 http://www.ntm.gov.tw/seaweeds/home.asp

黃穰 & 廖婉茹 (2003) 海藻　來自海洋的保健藥草. 科學發展, 30-37.

財團法人台灣癌症基金 http://www.canceraway.org.tw/

吳純衡 & 黃培安 (2006) 海藻在保健食品、化妝品之開發. 農業生技產業季刊, No. 7.

http://dcb-carot.unibe.ch/nomen.htm

Artursson, P. & Karlsson, J. (1991) Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem Biophys Res Commun, 175, 880-885.

Asai, A., Sugawara, T., Ono, H. & Nagao, A. (2004) Biotransformation of fucoxanthinol into amarouciaxanthin A in mice and HepG2 cells: formation and cytotoxicity of fucoxanthin metabolites. Drug Metab Dispos, 32, 205-211.

Asai, A., Yonekura, L. & Nagao, A. (2008) Low bioavailability of dietary epoxyxanthophylls in humans. Br J Nutr, 1-5.

Bangalore, D.V., McGlynn, W. & Scott, D.D. (2005) Effect of beta-cyclodextrin in improving the correlation between lycopene concentration and ORAC values. J Agric Food Chem, 53, 1878-1883.

Bessler, H., Salman, H., Bergman, M., Alcalay, Y. & Djaldetti, M. (2008) In vitro effect of lycopene on cytokine production by human peripheral blood mononuclear cells. Immunol Invest, 37, 183-190.

Briviba, K., Bornemann, R. & Lemmer, U. (2006) Visualization of astaxanthin localization in HT29 human colon adenocarcinoma cells by combined confocal resonance Raman and fluorescence microspectroscopy. Mol Nutr Food Res, 50, 991-995.

Charoensit, P., Kawakami, S., Higuchi, Y. & Hashida, M. (2008) Incorporation of all-trans retinoic acid into lipoplexes inhibits nuclear factor kappaB activation mediated liver injury induced by lipoplexes in mice. J Gene Med, 10, 61-69.

Chew, B.P. & Park, J.S. (2004) Carotenoid action on the immune response. J Nutr, 134, 257S-261S.

Chew, B.P., Park, J.S., Wong, M.W. & Wong, T.S. (1999) A comparison of the anticancer activities of dietary beta-carotene, canthaxanthin and astaxanthin in mice in vivo. Anticancer Res, 19, 1849-1853.

Das, S.K., Hashimoto, T. & Kanazawa, K. (2008) Growth inhibition of human hepatic carcinoma HepG2 cells by fucoxanthin is associated with down-regulation of cyclin D. Biochim Biophys Acta, 1780, 743-749.

Das, S.K., Hashimoto, T., Shimizu, K., Yoshida, T., Sakai, T., Sowa, Y., Komoto, A. & Kanazawa, K. (2005) Fucoxanthin induces cell cycle arrest at G0/G1 phase in human colon carcinoma cells through up-regulation of p21WAF1/Cip1. Biochim Biophys Acta, 1726, 328-335.

During, A., Albaugh, G. & Smith, J.C., Jr. (1998) Characterization of beta-carotene 15,15''-dioxygenase activity in TC7 clone of human intestinal cell line Caco-2. Biochem Biophys Res Commun, 249, 467-474.

Failla, M.L., Chitchumroonchokchai, C. & Ishida, B.K. (2008) In vitro micellarization and intestinal cell uptake of cis isomers of lycopene exceed those of all-trans lycopene. J Nutr, 138, 482-486.

Ferruzzi, M.G., Lumpkin, J.L., Schwartz, S.J. & Failla, M. (2006) Digestive Stability, micellarization, and uptake of beta-carotene isomers by Caco-2 human intestinal cells. J Agric Food Chem, 54, 2780-2785.

Hosokawa, M., Kudo, M., Maeda, H., Kohno, H., Tanaka, T. & Miyashita, K. (2004) Fucoxanthin induces apoptosis and enhances the antiproliferative effect of the PPARgamma ligand, troglitazone, on colon cancer cells. Biochim Biophys Acta, 1675, 113-119.

Huang, C.S., Fan, Y.E., Lin, C.Y. & Hu, M.L. (2007) Lycopene inhibits matrix metalloproteinase-9 expression and down-regulates the binding activity of nuclear factor-kappa B and stimulatory protein-1. J Nutr Biochem, 18, 449-456.

Huang, C.S., Liao, J.W. & Hu, M.L. (2008) Lycopene inhibits experimental metastasis of human hepatoma SK-Hep-1 cells in athymic nude mice. J Nutr, 138, 538-543.

Hughes, D.A. (2001) Dietary carotenoids and human immune function. Nutrition, 17, 823-827.

Khan, D.R., Rezler, E.M., Lauer-Fields, J. & Fields, G.B. (2008) Effects of drug hydrophobicity on liposomal stability. Chem Biol Drug Des, 71, 3-7.

Kim, Y.S., Kim, N.H., Kim, H.J., Lee, J.K., Kim, S.W. & Oh, D.K. (2007) Effective production of retinal from beta-carotene using recombinant mouse beta-carotene 15,15''-monooxygenase. Appl Microbiol Biotechnol, 76, 1339-1345.

Konishi, I., Hosokawa, M., Sashima, T., Kobayashi, H. & Miyashita, K. (2006) Halocynthiaxanthin and fucoxanthinol isolated from Halocynthia roretzi induce apoptosis in human leukemia, breast and colon cancer cells. Comp Biochem Physiol C Toxicol Pharmacol, 142, 53-59.

Kotake-Nara, E., Kushiro, M., Zhang, H., Sugawara, T., Miyashita, K. & Nagao, A. (2001) Carotenoids affect proliferation of human prostate cancer cells. J Nutr, 131, 3303-3306.

Lancrajan, I., Diehl, H.A., Socaciu, C., Engelke, M. & Zorn-Kruppa, M. (2001) Carotenoid incorporation into natural membranes from artificial carriers: liposomes and beta-cyclodextrins. Chem Phys Lipids, 112, 1-10.

Lin, C.Y., Huang, C.S. & Hu, M.L. (2007) The use of fetal bovine serum as delivery vehicle to improve the uptake and stability of lycopene in cell culture studies. Br J Nutr, 98, 226-232.

Lornejad-Schafer, M.R., Lambert, C., Breithaupt, D.E., Biesalski, H.K. & Frank, J. (2007) Solubility, uptake and biocompatibility of lutein and zeaxanthin delivered to cultured human retinal pigment epithelial cells in tween40 micelles. Eur J Nutr, 46, 79-86.

Maeda, H., Hosokawa, M., Sashima, T., Takahashi, N., Kawada, T. & Miyashita, K. (2006) Fucoxanthin and its metabolite, fucoxanthinol, suppress adipocyte differentiation in 3T3-L1 cells. Int J Mol Med, 18, 147-152.

Maeda, H., Tsukui, T., Sashima, T., Hosokawa, M. & Miyashita, K. (2008) Seaweed carotenoid, fucoxanthin, as a multi-functional nutrient. Asia Pac J Clin Nutr, 17 Suppl 1, 196-199.

Martin, K.R., Loo, G. & Failla, M.L. (1997) Human lipoproteins as a vehicle for the delivery of beta-carotene and alpha-tocopherol to HepG2 cells. Proc Soc Exp Biol Med, 214, 367-373.

Maruyama, H., Tamauchi, H., Hashimoto, M. & Nakano, T. (2003) Antitumor activity and immune response of Mekabu fucoidan extracted from Sporophyll of Undaria pinnatifida. In Vivo, 17, 245-249.

Moore, A.C., Gugger, E.T. & Erdman, J.W., Jr. (1996) Brush border membrane vesicles from rats and gerbils can be utilized to evaluate the intestinal uptake of all-trans and 9-cis beta-carotene. J Nutr, 126, 2904-2912.

Nishino, H., Tsushima, M., Matsuno, T., Tanaka, Y., Okuzumi, J., Murakoshi, M., Satomi, Y., Takayasu, J., Tokuda, H., Nishino, A. & et al. (1992) Anti-neoplastic effect of halocynthiaxanthin, a metabolite of fucoxanthin. Anticancer Drugs, 3, 493-497.

O''Sullivan, S.M., Woods, J.A. & O''Brien, N.M. (2004) Use of Tween 40 and Tween 80 to deliver a mixture of phytochemicals to human colonic adenocarcinoma cell (CaCo-2) monolayers. Br J Nutr, 91, 757-764.

Pfitzner, I., Francz, P.I. & Biesalski, H.K. (2000) Carotenoid:methyl-beta-cyclodextrin formulations: an improved method for supplementation of cultured cells. Biochim Biophys Acta, 1474, 163-168.

Polyakov, N.E., Leshina, T.V., Konovalova, T.A., Hand, E.O. & Kispert, L.D. (2004) Inclusion complexes of carotenoids with cyclodextrins: 1H NMR, EPR, and optical studies. Free Radic Biol Med, 36, 872-880.

Shafaa, M.W., Diehl, H.A. & Socaciu, C. (2007) The solubilisation pattern of lutein, zeaxanthin, canthaxanthin and beta-carotene differ characteristically in liposomes, liver microsomes and retinal epithelial cells. Biophys Chem, 129, 111-119.

Sugawara, T., Baskaran, V., Tsuzuki, W. & Nagao, A. (2002) Brown algae fucoxanthin is hydrolyzed to fucoxanthinol during absorption by Caco-2 human intestinal cells and mice. J Nutr, 132, 946-951.

Sugawara, T., Kushiro, M., Zhang, H., Nara, E., Ono, H. & Nagao, A. (2001) Lysophosphatidylcholine enhances carotenoid uptake from mixed micelles by Caco-2 human intestinal cells. J Nutr, 131, 2921-2927.

Sugawara, T., Matsubara, K., Akagi, R., Mori, M. & Hirata, T. (2006) Antiangiogenic activity of brown algae fucoxanthin and its deacetylated product, fucoxanthinol. J Agric Food Chem, 54, 9805-9810.

Sun, Z. & Yao, H. (2007) The influence of di-acetylation of the hydroxyl groups on the anti-tumor-proliferation activity of lutein and zeaxanthin. Asia Pac J Clin Nutr, 16 Suppl 1, 447-452.

Sze, P. (1997) A Biology of the Algae, third edition.

Tsukui, T., Konno, K., Hosokawa, M., Maeda, H., Sashima, T. & Miyashita, K. (2007) Fucoxanthin and fucoxanthinol enhance the amount of docosahexaenoic acid in the liver of KKAy obese/diabetic mice. J Agric Food Chem, 55, 5025-5029.

Wrona, M., Korytowski, W., Rozanowska, M., Sarna, T. & Truscott, T.G. (2003) Cooperation of antioxidants in protection against photosensitized oxidation. Free Radic Biol Med, 35, 1319-1329.

Xu, X., Wang, Y., Constantinou, A.I., Stacewicz-Sapuntzakis, M., Bowen, P.E. & van Breemen, R.B. (1999) Solubilization and stabilization of carotenoids using micelles: delivery of lycopene to cells in culture. Lipids, 34, 1031-1036.

Yan, X., Chuda, Y., Suzuki, M. & Nagata, T. (1999) Fucoxanthin as the major antioxidant in Hijikia fusiformis, a common edible seaweed. Biosci Biotechnol Biochem, 63, 605-607.

Yoshiko, S. & Hoyoku, N. (2007) Fucoxanthin, a natural carotenoid, induces G1 arrest and GADD45 gene expression in human cancer cells. In Vivo, 21, 305-309.

吳純衡 & 黃培安 (2006) 海藻在保健食品、化妝品之開發. 農業生技產業季刊, No. 7.