Quercetin to enhance the bioavailability and activity of green tea polyphenols

描述（由申请方提供）：根据评审员的评论和新发现，我们修改了研究设计、剂量、时间、组织处理和统计学评价。所提出的人类干预研究的目的仍然是确定槲皮素和绿色茶（GT）的组合施用是否会增加GT多酚（GTP）如表没食子儿茶素没食子酸酯（EGCG）的生物利用度并降低其代谢。限制GTP生物活性的因素是降低的生物利用度和将EGCG转化为活性降低的甲基化代谢物的广泛代谢。在我们之前的人类干预研究中，在前列腺切除术前每天喝6杯绿色茶的男性中，我们证明了GTP存在于人类前列腺组织中。然而，发现50%的EGCG是甲基化形式。槲皮素是一种在洋葱、苹果、花椰菜、浆果和茶中发现的类黄酮，是儿茶酚-O-甲基转移酶（COMT）和多药耐药转运蛋白（MRP 1和MRP 2）的天然抑制剂。我们的细胞培养研究表明，与EGCG和槲皮素的组合治疗导致4至10倍的细胞内浓度的EGCG和EGCG甲基化的显着降低，这取决于所使用的细胞系。我们在小鼠前列腺癌异种移植物研究中证明，与单独用GT或槲皮素治疗相比，GT与槲皮素的组合施用显著减小肿瘤体积，增加EGCG组织浓度并显著降低EGCG的甲基化。联合给药还显著降低了甲基转移酶和MRP 1的蛋白表达。因此，我们提出了一项安慰剂对照、双盲、随机、平行的两组人类初步干预研究，以确定GT提取物和槲皮素的消耗是否会增加血液和前列腺组织中GTP的生物利用度并降低EGCG的甲基化。我们将给予两个GT提取物胶囊与一个槲皮素胶囊（N=25）或一个安慰剂胶囊（N=15），每天两次，持续3周的男性计划进行乳房切除术。在10名参与者完成干预后，将评估肝脏毒性。在没有利耶毒性的情况下，槲皮素剂量将加倍（每日800 mg）（N=15）。将使用高效液相色谱法（HPLC）和Coularray电化学检测（ECD）分析血液和前列腺组织中的GTP、槲皮素和甲基化代谢物。为了评价所涉及的机制，将使用HPLC-ECD、真实的时间qPCR和蛋白质印迹分析来确定肿瘤和正常前列腺组织中的COMT和DNA甲基转移酶活性以及基因和蛋白质表达以及MRP 1基因和蛋白质表达和总体DNA甲基化以及红细胞中的COMT活性。本研究结果将为进一步的临床试验奠定基础，以评估槲皮素和GT在前列腺癌早期化学预防中的相互作用。

DESCRIPTION (provided by applicant): Based on the reviewers' comments and new findings we revised the study design, dose, timing, tissue processing and statistical evaluation. The objective of the proposed human intervention study remains to determine whether a combined administration of quercetin and green tea (GT) will increase the bioavailability and decrease the metabolism of GT polyphenols (GTPs) such as epigallocatechin gallate (EGCG). Factors limiting the biological activity of GTPs are the reduced bioavailability and extensive metabolism converting EGCG into methylated metabolites with decreased activity. In our previous human intervention study in men drinking 6 cups of green tea daily prior to prostatectomy we demonstrated that GTPs are present in human prostate tissue. However 50 percent of EGCG was found in methylated form. Quercetin, a flavonoid found in onions, apples, broccoli, berries and teas is a natural inhibitor of catechol-O-methyltransferase (COMT) and multidrug resistance transport proteins (MRP1 and MRP2). Our cell culture studies demonstrated that the combined treatment with EGCG and quercetin led to a 4 to 10-fold increase in intracellular concentration of EGCG and significant decrease in EGCG methylation, depending on the cell line used. We demonstrated in mouse prostate cancer xenograft studies that the administration of the combination of GT with quercetin significantly decreased tumor volume, increased EGCG tissue concentration and decreased the methylation of EGCG significantly compared to treatment with GT or quercetin alone. The combined administration also significantly decreased the protein expression of methyltransferases and MRP1. We therefore propose a placebo- controlled, double-blinded, randomized, parallel two arm human pilot intervention study to determine whether the consumption of GT extract and quercetin will increase the bioavailability of GTPs in blood and prostate tissue and decrease the methylation of EGCG. We will administer two capsules of GT extract combined with one capsule of quercetin (N=25) or with one placebo capsule (N=15) twice daily for 3 weeks to men scheduled for prostatectomy. After 10 participants will complete the intervention, liver toxicity will be evaluated. In the absence of lier toxicity the quercetin dose will be doubled (800 mg daily) (N=15). GTPs, quercetin and methylated metabolites will be analyzed in blood and prostate tissue using high performance liquid chromatography (HPLC) with coularray electrochemical detection (ECD). To evaluate the mechanism involved, COMT and DNA methyltransferase enzyme activities and gene and protein expression as well as MRP1 gene and protein expression and global DNA methylation will be determined in tumor and normal prostate tissue and COMT activity in erythrocytes using HPLC-ECD, real time qPCR and Western blot analyses. The results will lay the foundation for future clinical trials to evaluate the interaction of quercetin and GT in chemoprevention of early stages of prostate cancer.