Quercetin to enhance the bioavailability and activity of green tea polyphenols

描述（由申请人提供）：根据审稿人的意见和新发现，我们修改了研究设计、剂量、时间、组织处理和统计评价。拟制的人体干预研究的目的仍然是确定槲皮素和绿茶（GT）联合施用是否会增加生物利用度并降低GT多酚（gtp）的代谢，如表没食子儿茶素没食子酸酯（EGCG）。限制gtp生物活性的因素是生物利用度降低和EGCG转化为甲基化代谢物的广泛代谢，其活性降低。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杯绿茶的男性中我们证明了gtp存在于人类前列腺组织中。然而，50%的EGCG以甲基化形式被发现。槲皮素是一种类黄酮，存在于洋葱、苹果、西兰花、浆果和茶中，是儿茶酚- o -甲基转移酶（COMT）和多药耐药转运蛋白（MRP1和MRP2）的天然抑制剂。我们的细胞培养研究表明，EGCG和槲皮素联合处理导致细胞内EGCG浓度增加4到10倍，EGCG甲基化显著降低，这取决于所使用的细胞系。我们在小鼠前列腺癌异种移植研究中证明，与单独使用GT或槲皮素相比，GT与槲皮素联合使用可显著减少肿瘤体积，增加EGCG组织浓度，并显著降低EGCG的甲基化。联合用药也显著降低了甲基转移酶和MRP1的蛋白表达。因此，我们提出了一项安慰剂对照、双盲、随机、平行的两组人体先导干预研究，以确定摄入GT提取物和槲皮素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。我们将给予两粒GT提取物联合一粒槲皮素胶囊（N=25）或一粒安慰剂胶囊（N=15），每天两次，持续3周。10名受试者完成干预后，进行肝毒性评估。在没有肝毒性的情况下，槲皮素剂量将增加一倍（每天800毫克）（N=15）。gtp、槲皮素和甲基化代谢物将在血液和前列腺组织中使用高效液相色谱（HPLC）和科勒勒电化学检测（ECD）进行分析。为了评估其机制，我们将采用HPLC-ECD、real - time qPCR和Western blot分析，检测肿瘤和正常前列腺组织中COMT和DNA甲基转移酶活性、基因和蛋白表达以及MRP1基因和蛋白表达和整体DNA甲基化，以及红细胞中COMT活性。本研究结果将为今后评价槲皮素与GT在早期前列腺癌化学预防中的相互作用的临床试验奠定基础。

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.