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標題: | 金雀異黃酮與鞣花酸生物可利用率之研究 Studies on the bioavailabilities of genistein and ellagic acid |
作者: | Shang-Ta Wang 王上達 |
指導教授: | 蘇南維(Nan-Wei Su) |
關鍵字: | 金雀異黃酮,鞣花酸,生物可利用率,生物轉化,自體微乳化系統,膳食補充劑, genistein,ellagic acid,bioavailability,biotransformation,self-nanoemulsifying delivery system,dietary supplement, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | 植物性化學物質 (phytochemicals) 近年來被應用於營養醫療製劑 (nutraceuticals) 上,已有深入的研究以及廣大的市場價值。然而此類化學物質屬於植物二次代謝物 (secondary metabolism),多數水溶性不佳且生物可利用率 (bioavailability) 極低,限制了其應用性。本研究旨在針對其中兩種具有良好生理活性之化合物-金雀異黃酮 (genistein) 及鞣花酸 (ellagic acid),利用生物轉換以及劑型設計之手段,改善其生物可利用率,進而提升應用價值。研究利用本實驗室先前已發展之微生物轉換技術,將 genistein 轉化生成 genistein 7-O-phosphate (G7P)。磷酸酯化的 genistein 衍生物於先前之研究可成功提升 genistein 的溶解度達10萬倍,並且可被人體之鹼性磷酸酶 (alkaline phosphatase, ALP) 水解其磷酸酯鍵,形成 aglycone 的形式,有潛力形成 genistein 的磷酸酯前驅藥物形式。接著,我們將 G7P 自發酵液中萃取並純化,再進行一連串之藥物動力學試驗,評估其提升生物可利用率之潛力。首先,我們以不同酵素及緩衝溶液配製模擬腸液以及模擬胃液,測試 G7P 在其中之穩定性。結果顯示 G7P 於兩種模擬消化液中4 h的期間,皆無顯著降解的情形發生,顯示其可以磷酸酯衍生物的形式通過上消化道進入小腸中並保持穩定。而後我們將 G7P 進行 USP 中 paddle method 的溶離試驗,發現不論在任何溶離液中,G7P 的溶離速率皆遠高於原態的 genistein,顯示其於消化液中可良好分散,增加被腸黏膜上皮細胞吸收之機會。另外,我們探討人類 ALP 對 G7P 之水解效率,發現 G7P 比 fosphenytoin,一種被認為易被 ALP 水解的藥物,具有更高之水解速率。G7P之清除半衰期約為 fosphenytoin 的60%,顯示其具有作為磷酸酯前驅藥物之潛力。大鼠活體腸灌流試驗則證實,G7P 對小腸黏膜的有效穿透可以比 aglycone 形式的 genistein 高出一倍,預估之人體吸收比率則提升至原本的10倍。最後我們進行大鼠活體口服之藥物動力學試驗,結果顯示,G7P 於口服後30 min可達到最高血液濃度,aglycone genistein 則須經過210 min後才達到;G7P 之生物可利用率為 aglycone 形式的3.7倍,最高血液濃度則為7.2倍,顯示 G7P 作為 aglycone genistein 的磷酸酯衍生物,可加速生物體之吸收,並大幅提高生物可利用率,極具潛力作為其替代之產品。另一方面,研究針對 ellagic acid 進行食品級自體奈米乳化系統 (self-nanoemulsifying delivery system, EA-SNEDS) 之開發。經由溶解度試驗的評估後,我們找出對 ellagic acid 溶解度最高之食品級界面活性劑、輔助界面活性劑以及油脂,分別為 polysorbate 20/ polysorbate 80;PEG200/PEG400;oleic acid/(caprylic/capric) triacylglycerol。利用模擬乳化試驗建立擬三相圖 (pseudo-ternary phase diagrams),找出最佳的各乳化相組成為 polysorbate 80/PEG400/(caprylic/capric) triacylglycerol:45/45/10。以光散射儀 (light scatter) 測定其平均乳化粒徑約在120 nm左右,最高 ellagic acid 承載量為2.5 mg/mL。配製完最佳比例之乳化相後,以穿透式電子顯微鏡 (transmission electron microscope, TEM) 進行形態學探討,發現其乳化液滴顆粒之粒徑與光散射儀所測得之數值相符,顆粒呈圓球狀,大致上平均分散,有部分的輕微聚集顆粒大小約介於200-500 nm之間。溶離試驗可得知,此一乳化系統可快速溶解於一般溶離液及模擬腸液中,並且其溶離效率較未乳化之 ellagic acid 高出4-7倍,可成功提升 ellagic acid 之溶離情形。最後,經由大鼠口服藥物動力學試驗,證實此一乳化劑型可使 ellagic acid 的生物可利用率有6倍的提升,並可提升最高血液濃度達約10倍,具有發展為新型態之膳食補充劑之潛力。 Phytochemicals are low-molecular-weight organic compounds produced by plants. The growing market of phytochemicals in food, medicine and agriculture industry has led to numerous studies on its biological activities of these substances in recent years. However, many plant food phytochemicals that are poorly absorbed by humans usually undergo metabolism and rapid excretion. It is clear from in vitro and animal data that the actions of some phytochemicals are likely to be achieved only at doses much higher than those present in plasma level after ingestion. The aim of this study is to improve the oral bioavailability of two highly bioactive phytochemicals namely, genistein and ellagic acid, by using biotransformation and formulation techniques. Genistein, one of the primary bioactive agents in soybeans, has been previous revealed to convert to a water-soluble phosphate conjugate, genistein 7-O-phosphate (G7P), generated by biotransformation of Bacillus subtilis var. natto BCRC80517. We investigated the dissolution profile, intestinal permeability and oral bioavailability of genistein and G7P. G7P have firstly been stable in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) within 4 h incubation. Moreover, G7P dramatically improved the dissolution rate of genistein in various dissolution media. It also enhanced the intestinal permeability in situ, and greatly increased plasma exposure to genistein after oral administration in rats. The aqueous solubility of genistein is the absorption barrier to its oral bioavailability. G7P may be a promising and efficient alternative to genistein. On the other hand, ellagic acid is known of a predominant bioactive component in pomegranate that possesses broad health benefits. We developed a food-grade self-nanoemulsifying system to improve the dissolution and absorption of ellagic acid. Solubility assay and pseudo-ternary phase diagrams revealed suitable components for the formulation. The optimal formulation was composed of polyethylene glycol, polysorbate, caprylic/capric triacylglycerol at the ratio of 45/45/10 wt. %. With this optimal formulation and gentle stirring, a fine nanoemulsion was achieved and had mean droplet size of around 120 nm. The dissolution of ellagic acid was significantly elevated with the formulation. Rat pharmacokinetics studies showed that ellagic acid was 6.6- and 3.2-fold more bioavailable with the formulation than with aqueous suspensions and pomegranate extract, respectively. The proposed self-nanoemulsifying system to deliver ellagic acid can be a novel strategy for developing products for dietary supplements and functional foods of ellagic acid. Taking all above-mentioned results, the approaches for enhancing the bioavailability of phytochemicals provide advantages in the oral delivery strategy and improve the utilization of these bioactive substances. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77678 |
DOI: | 10.6342/NTU201703889 |
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顯示於系所單位: | 農業化學系 |
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