FLAVONOID BIOAVAILABILITY IN HUMANS--CELLULAR STUDIES

最近的流行病学研究强烈表明，黄酮类化合物是一种大的 水果、蔬菜和饮料中含有的一类植物化学物质， 在人类疾病中具有保护作用，尤其是在冠状动脉疾病中 心脏病和癌症。迅速增长的生化物质数量 研究表明，这些影响的机制似乎是合理的。然而，它并不是 明确饮食中的类黄酮，包括它们的糖苷，是否可以获得 获得建议的蜂窝作用部位；它们的生物命运 人类在本质上仍然是未知的。该项目中的研究提出了 以帮助填补这一重要的信息缺口。我们的总揽工作 假设类黄酮类的生物利用度是 细胞膜通透性、蛋白质结合和晶状体前代谢， 并且随着黄酮类化合物的化学结构的不同而明显不同。在……里面 目的1我们将重点研究人肝癌细胞系对黄酮类化合物的摄取 HEP G2和新鲜肝细胞及其作用机制(S)。这将是 包括对载体介导的转运蛋白的特定抑制剂的研究。 它还将包括测定血浆蛋白结合 黄酮类化合物及其对膜透性的影响。在《目标2》中我们将 用Hep G2细胞测定黄酮类化合物的代谢 作为人类肝脏匀浆。这将包括高效液相色谱分离 利用大气压进行代谢物和结构鉴定 化学电离(APCI)质谱仪(MS)与高场核 磁共振(核磁共振)光谱。所涉及的酶将是 也通过使用选择性化学和抗体抑制剂进行鉴定 作为异构体特异性表达系统。在目标3中，我们将确定 黄酮类物质穿越肠上皮细胞边界的能力， 使用人Caco-2细胞系，在可渗透材料上生长为单层 滤器，切除大鼠肠道作为模型。就像肝细胞一样 在目标1中，我们将确定化学结构对膜的影响 渗透性和运输机制(S)。这些研究还将 包括P-糖蛋白介导外排和新陈代谢作为因素 限制网络传输。在目标4中，我们将测试我们的体外观察 直接在体内，使用药代动力学方法。在这些研究中 我们将直接寻求确定类黄酮类的生物利用度。 糖苷及其在正常情况下对苷元的水解性 志愿者通过血浆和尿液测定。

Recent epidemiological studies strongly suggest that flavonoids, a large class of phytochemicals present in fruits, vegetables and beverages, have protective roles in human diseases, most prominently in coronary heart disease and cancer. A rapidly growing number of biochemical studies show plausible mechanisms for these effects. However, it is not clear if the dietary flavonoids, including their glycosides, can gain access to proposed cellular sites of action; their biological fate in humans remains essentially unknown. The studies in this project propose to help fill this essential information gap. Our general working hypothesis is that the bioavailability of flavonoids is a function of cell membrane penetrability, protein binding and presystemic metabolism, and varies markedly with the chemical structure of the flavonoid. In Aim 1 we will focus on flavonoid uptake by the human hepatoma cell line Hep G2 and fresh hepatocytes and the mechanism(s) involved. This will include studies of specific inhibitors of carrier-mediated transporters. It will also include determination of plasma protein binding of the flavonoids and its effect on membrane penetration. In Aim 2 we will determine the metabolism of the flavonoids, using Hep G2 cells as well as human liver homogenates. This will include HPLC isolation of metabolites and structure identification using atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) and high field nuclear magnetic resonance (NMR) spectrometry. The enzymes involved will be identified by use of selective chemical and antibody inhibitors as well as isoform-specific expression systems. In Aim 3 we will determine the ability of flavonoids to traverse the intestinal epithelial cell border, using the human Caco-2 cell line, grown as monolayers on permeable filters, and excised rat intestine, as models. As with the hepatocytes in Aim 1, we will determine the effect of chemical structure on membrane permeability and mechanism(s) of transport. These studies will also include P-glycoprotein-mediated efflux as well as metabolism as factors limiting net transport. In aim 4 we will test our in vitro observations directly in vivo, using pharmacokinetic approaches. In these studies we will in first hand seek to determine the bioavailability of flavonoid glycosides and their subsequent hydrolysis to aglycones in normal volunteers through plasma and urine determinations.