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Quercetin is a flavonoid, its glycosides and aglycone are found in significant amounts in several plants and dietary supplements. Because of the high presystemic biotransformation of quercetin, mainly its conjugates appear in circulation. As has been reported in previous studies, quercetin can interact with several proteins of pharmacokinetic importance. However, the interactions of its metabolites with biotransformation enzymes and drug transporters have barely been examined. In this study, the inhibitory effects of quercetin and its most relevant methyl, sulfate, and glucuronide metabolites were tested on cytochrome P450 (CYP) (2C19, 3A4, and 2D6) enzymes as well as on organic anion-transporting polypeptides (OATPs) (OATP1A2, OATP1B1, OATP1B3, and OATP2B1) and ATP (adenosine triphosphate) Binding Cassette (ABC) (BCRP and MRP2) transporters. Quercetin and its metabolites (quercetin-3′-sulfate, quercetin-3-glucuronide, isorhamnetin, and isorhamnetin-3-glucuronide) showed weak inhibitory effects on CYP2C19 and 3A4, while they did not affect CYP2D6 activity. Some of the flavonoids caused weak inhibition of OATP1A2 and MRP2. However, most of the compounds tested proved to be strong inhibitors of OATP1B1, OATP1B3, OATP2B1, and BCRP. Our data demonstrate that not only quercetin but some of its conjugates, can also interact with CYP enzymes and drug transporters. Therefore, high intake of quercetin may interfere with the pharmacokinetics of drugs.

Quercetin is an abundant flavonoid in nature and is used in several dietary supplements. Although quercetin is extensively metabolized by human enzymes and the colonic microflora, we have only few data regarding the pharmacokinetic interactions of its metabolites. Therefore, we investigated the interaction of human and microbial metabolites of quercetin with the xanthine oxidase enzyme. Inhibitory effects of five conjugates and 23 microbial metabolites were examined with 6-mercaptopurine and xanthine substrates (both at 5 μM), employing allopurinol as a positive control. Quercetin-3′-sulfate, isorhamnetin, tamarixetin, and pyrogallol proved to be strong inhibitors of xanthine oxidase. Sulfate and methyl conjugates were similarly strong inhibitors of both 6-mercaptopurine and xanthine oxidations (IC50 = 0.2–0.7 μM); however, pyrogallol inhibited xanthine oxidation (IC50 = 1.8 μM) with higher potency vs. 6-MP oxidation (IC50 = 10.1 μM). Sulfate and methyl conjugates were approximately ten-fold stronger inhibitors (IC50 = 0.2–0.6 μM) of 6-mercaptopurine oxidation than allopurinol (IC50 = 7.0 μM), and induced more potent inhibition compared to quercetin (IC50 = 1.4 μM). These observations highlight that some quercetin metabolites can exert similar or even a stronger inhibitory effect on xanthine oxidase than the parent compound, which may lead to the development of quercetin–drug interactions (e.g., with 6-mercaptopurin or azathioprine).

A fluorescence-based enzymatic microplate intracellular glucose assay was designed and fully validated. The method was tested in a hepatocellular cancer cell line (HepG2). Our novel one-step extraction reagent gave stable cell lysates for glucose, adenosine triphosphate (ATP), and total protein determination from the same sample. Limit of detection for glucose was 0.13 µM (26 pmol/well), which is superior to commercially available glucose assays. Both intra- and interday assay imprecision in HepG2 cultures were less than 12% coefficient of variance (CV). In cell lysates spiked with glucose, recovery at two levels varied between 83.70% and 91.81%, and both linearity and stability were acceptable. HepG2 cells treated with agents affecting glucose uptake/metabolism (phloretin, quercetin, quercetin-3′-sulfate, NaF, 3-bromopyruvate, NaN3, oligomycin A, ochratoxin A, cytochalasin B, and anti-GLUT1 antibody) showed dose-dependent changes in glucose and ATP levels without total protein (cell) loss. Finally, we performed flow cytometric glucose uptake measurement in the treated cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose fluorescent glucose analog. Glucose uptake did not always mirror the intracellular glucose levels, which most likely reflects the differences between the two methodologies. However, interpreting data obtained by both methods and taking ATP/protein levels at the same time, one can get information on the mode of action of the compounds.

Some polyphenols have been shown to inhibit, at physiological levels, the VEGF-induced VEGF receptor-2 signaling that causes angiogenesis, allegedly by direct interaction with VEGF and reducing the binding to its receptor VEGFR2. Surface plasmon resonance was used to measure the parameters of binding between VEGF and polyphenols as well as the nature of the interactions by assessing the effect of physico-chemical changes in the solution. CD spectrometry was used to determine any change in the secondary structure of the protein upon binding. The kinetic parameters (ka, kd, and KD) that characterise the binding to VEGF were measured for both inhibitor and non-inhibitor polyphenolic molecules. The effect of changes in the physico-chemical conditions of the solution where the binding occurred indicated that the nature of the interactions between VEGF and EGCG was predominantly of a hydrophobic nature. CD studies suggested that a change in the secondary structure of the protein occurred upon binding. Direct interaction and binding between VEGF and polyphenol molecules acting as inhibitors of the signaling of VEGFR2 has been measured for the first time. The binding between VEGF and EGCG seemed to be based on hydrophobic interactions and caused a change in the secondary structure of the protein.

This is the first report describing an analytical method for quantitative analysis of two naturally occurring sulphur compounds, S-methyl-l-cysteine (SMC) and S-methyl-l-cysteine sulfoxide (SMCSO), in human body fluids using isotope-labelled internal standards and liquid chromatography-mass spectrometry (LC-MS)/MS techniques. This method was validated according to the guideline of the Royal Society of Chemistry Analytical Methods Committee. It offers significant advantages including simple and fast preparation of human biological samples. The limits of detection of SMC were 0.08 µM for urine and 0.04 µM for plasma. The limits of detection of SMCSO were 0.03 µM for urine and 0.02 µM for plasma. The calibration curves of all matrices showed linearity with correlation coefficients r2 > 0.9987. The intra and inter day precisions in three levels of known concentrations were >10% and >20%, respectively. The quantification accuracy was 98.28 ± 5.66%. The proposed method would be beneficial for the rapid and accurate determination of the SMC and SMCSO in human plasma and urine samples using by isotope labelled internal standards.

Oranges are rich sources of flavonoids that are bioactive and may protect against age-related diseases. The absorption of orange flavanones may be affected by factors such as processing and subject anthropometric variables, and the bioactivity of the absorbed phytochemicals depends on how they are metabolised during absorption. In a randomised cross-over study, twenty subjects consumed a single portion of orange fruit (150 g) or juice (300 g) that contained the flavanones narirutin and hesperidin, and an additional 109 subjects across a broad age range (18–80 years) consumed the juice. Flavanone metabolites were measured in regularly collected samples of plasma and urine. After consumption of fruit or juice, flavanone conjugates, but not the aglycones, were detected in plasma and urine. The flavanone conjugates were shown to include the 7- and 4′-O-monoglucuronides of naringenin, the 7- and 3′-O-monoglucuronides of hesperetin, two hesperetin diglucuronides and a hesperetin sulfo-glucuronide, but no aglycones or rutinosides. Analysis of the plasma pharmacokinetic and urinary excretion data on a dose-adjusted basis indicated no difference in absorption or excretion of either flavanone between the fruit and juice matrices. In the extended urinary excretion dataset the individual variation was very large (range 0–59 % urinary yield). There was a small but significant (P < 0·05) decrease in the excretion of hesperetin (but not naringenin) with increasing age (P < 0·05), but the effects of sex, BMI and contraceptive pill use were shown not to be associated with the variation in flavanone excretion.

Epidemiological studies suggest that people who consume more than one portion of cruciferous vegetables per week are at lower risk of both the incidence of prostate cancer and of developing aggressive prostate cancer but there is little understanding of the underlying mechanisms. In this study, we quantify and interpret changes in global gene expression patterns in the human prostate gland before, during and after a 12 month broccoli-rich diet. Volunteers were randomly assigned to either a broccoli-rich or a pea-rich diet. After six months there were no differences in gene expression between glutathione S-transferase mu 1 (GSTM1) positive and null individuals on the pea-rich diet but significant differences between GSTM1 genotypes on the broccoli-rich diet, associated with transforming growth factor beta 1 (TGFbeta1) and epidermal growth factor (EGF) signalling pathways. Comparison of biopsies obtained pre and post intervention revealed more changes in gene expression occurred in individuals on a broccoli-rich diet than in those on a pea-rich diet. While there were changes in androgen signalling, regardless of diet, men on the broccoli diet had additional changes to mRNA processing, and TGFbeta1, EGF and insulin signalling. We also provide evidence that sulforaphane (the isothiocyanate derived from 4-methylsuphinylbutyl glucosinolate that accumulates in broccoli) chemically interacts with TGFbeta1, EGF and insulin peptides to form thioureas, and enhances TGFbeta1/Smad-mediated transcription. These findings suggest that consuming broccoli interacts with GSTM1 genotype to result in complex changes to signalling pathways associated with inflammation and carcinogenesis in the prostate. We propose that these changes may be mediated through the chemical interaction of isothiocyanates with signalling peptides in the plasma. This study provides, for the first time, experimental evidence obtained in humans to support observational studies that diets rich in cruciferous vegetables may reduce the risk of prostate cancer and other chronic disease. ClinicalTrials.gov NCT00535977.

High circulating HDL concentrations and measures of various HDL functions are inversely associated with cardiovascular disease (CVD) risk. Paraoxonase 1 (PON1) contributes to many of the athero-protective functions of HDL, such as promoting the reverse cholesterol transport process and reducing the levels of oxidized LDL. PON1 activities are influenced by several factors, the most important being diet and genetic polymorphisms. Reported data from randomized controlled trials have shown that anthocyanin consumption increased PON1 activity. However, the underlying molecular mechanisms by which anthocyanins increase PON1 activity are not understood. Therefore, the aim of this research was to investigate the ability of anthocyanins and their metabolites to increase PON1 gene expression and/or enzyme activities as potential mechanisms. The effect of the two predominant dietary anthocyanins and 18 of their recently identified microbial metabolites including their phase-II conjugates on PON1 gene expression was studied using a PON1-Huh7 stably-transfected cell line and reporter gene assay. The effects of these compounds on PON1 arylesterase and lactonase activities were investigated using two isoforms of the PON1 enzyme that are the phenotypes of the 192Q/R polymorphism. None of the compounds caused even modest changes in PON1 promoter activity (p ≥ 0.05). Further, none of the compounds at physiological concentrations caused any significant changes in the arylesterase or lactonase activity of either of the iso-enzymes. Cyanidin reduced the lactonase activity of the PON1-R192R enzyme at high concentrations (−22%, p < 0.001), but not at physiologically achievable concentrations. In conclusion, none of the data reported here support the notion that anthocyanins or their metabolites affect PON1 transactivation or enzyme activities.

Prostate cancer has become the most common form of non-cutaneous (internal) malignancy in men, accounting for 26% of all new male visceral cancer cases in the UK. The aetiology and pathogenesis of prostate cancer are not understood, but given the age-adjusted geographical variations in prostate cancer incidence quoted in epidemiological studies, there is increasing interest in nutrition as a relevant factor. In particular, foods rich in phytochemicals have been proposed to reduce the risk of prostate cancer. Epidemiological studies have reported evidence that plant-based foods including cruciferous vegetables, garlic, tomatoes, pomegranate and green tea are associated with a significant reduction in the progression of prostate cancer. However, while there is well-documented mechanistic evidence at a cellular level of the manner by which individual dietary components may reduce the risk of prostate cancer or its progression, evidence from intervention studies is limited. Moreover, clinical trials investigating the link between the dietary bioactives found in these foods and prostate cancer have reported varied conclusions. Herein, we review the plant bioactives for which there is substantial evidence from epidemiological and human intervention studies. The aim of this review is to provide important insights into how particular plant bioactives (e.g., sulfur-containing compounds, carotenoids and polyphenols) present in commonly consumed food groups may influence the development and progression of prostate cancer.

Opioid analgesics are commonly used for acute low back pain and neck pain, but supporting efficacy data are scarce. We aimed to investigate the efficacy and safety of a judicious short course of an opioid analgesic for acute low back pain and neck pain. OPAL was a triple-blinded, placebo-controlled randomised trial that recruited adults (aged ≥18 years) presenting to one of 157 primary care or emergency department sites in Sydney, NSW, Australia, with 12 weeks or less of low back or neck pain (or both) of at least moderate pain severity. Participants were randomly assigned (1:1) using statistician-generated randomly permuted blocks to guideline-recommended care plus an opioid (oxycodone–naloxone, up to 20 mg oxycodone per day orally) or guideline-recommended care and an identical placebo, for up to 6 weeks. The primary outcome was pain severity at 6 weeks measured with the pain severity subscale of the Brief Pain Inventory (10-point scale), analysed in all eligible participants who provided at least one post-randomisation pain score, by use of a repeated measures linear mixed model. Safety was analysed in all randomly assigned eligible participants. The trial was registered with the Australian New Zealand Clinical Trials Registry (ACTRN12615000775516). Between Feb 29, 2016, and March 10, 2022, 347 participants were recruited (174 to the opioid group and 173 to the placebo group). 170 (49%) of 346 participants were female and 176 (51%) were male. 33 (19%) of 174 participants in the opioid group and 25 (15%) of 172 in the placebo group had discontinued from the trial by week 6, due to loss to follow-up and participant withdrawals. 151 participants in the opioid group and 159 in the placebo group were included in the primary analysis. Mean pain score at 6 weeks was 2·78 (SE 0·20) in the opioid group versus 2·25 (0·19) in the placebo group (adjusted mean difference 0·53, 95% CI –0·00 to 1·07, p=0·051). 61 (35%) of 174 participants in the opioid group reported at least one adverse event versus 51 (30%) of 172 in the placebo group (p=0·30), but more people in the opioid group reported opioid-related adverse events (eg, 13 [7·5%] of 174 participants in the opioid group reported constipation vs six [3·5%] of 173 in the placebo group). Opioids should not be recommended for acute non-specific low back pain or neck pain given that we found no significant difference in pain severity compared with placebo. This finding calls for a change in the frequent use of opioids for these conditions. Australia's National Health and Medical Research Council, University of Sydney Faculty of Medicine and Health, and ReturnToWorkSA.