Overestimation of flavonoid aglycones as a result of the ex vivo deconjugation of glucuronides by the tissue β-glucuronidase

Abstract

Flavonoid glucuronides are the main circulating metabolites of flavonoids in humans and animals. There has been a growing interest in the biological function of glucuronides. In order to differentiate biological activity and to assess efficacy it is essential to accurately determine the levels of flavonoid aglycone and metabolic conjugate in vivo. Many organs and body fluids of humans and animals exhibit β-glucuronidase against flavonoid glucuronides. Studies have shown that β-glucuronidase within the tissues hydrolyzes glucuronides to their aglycones during the tissue extraction, leading to artificially higher reported tissue levels of aglycone than actual in vivo concentrations. The aims of this study were to estimate the extent by which the aglycones were overestimated and to investigate the use of saccharo-1,4-lactone, a β-glucuronidase inhibitor, to block the ex vivo hydrolysis of flavonoid glucuronides. Our data demonstrate that in mouse liver tissues and human tumor xenografts levels of quercetin and methylated quercetin aglycones could be over-estimated by 7 fold. The inhibition of deconjugation of quercetin and baicalein glucuronides by saccharo-1,4-lactone is dose-dependent. The amount of saccharo-1,4-lactone used to produce optimal inhibition of the enzyme activity is in the range of 15 – 24 μmol per gram of liver tissue. The use of β-glucuronidase inhibitor blocks the ex vivo deconjugation resulting in an accurate estimation of tissue levels of aglycone and conjugate. Our study described here can be extended to other animal models and human studies with different types of substrates of β-glucuronidase.

Dietary Quercetin and Kaempferol: Bioavailability and Potential Cardiovascular-Related Bioactivity in Humans

“…Flavonol metabolism in body tissues is not well understood. An in vitro study suggested that β-glucuronidase enzyme found in body tissues hydrolyzes the conjugated metabolites producing aglycones [49]. Flavonol metabolites are excreted by urinary and biliary elimination.…”

Incomplete Hydrolysis of Curcumin Conjugates by β‐Glucuronidase: Detection of Complex Conjugates in Plasma

“…Also, polyphenols may undergo ex vivo hydrolysis or secondary transformation during the hydrolysis process, further contributing to analytical variability. [16,17] Thus, when the goal is to determine overall exposure to a polyphenol, it is preferred to conduct a direct analysis of all possible plasma metabolites, that is, without hydrolysis. Such analyses, however, are often not practical and difficult to perform.…”

“…While simplifying the analyte profile, this practice also introduces novel sources of variability: Whether hydrolysis of plasma conjugates is complete is difficult to assess in the absence of direct analysis of the remaining parent conjugates. Also, polyphenols may undergo ex vivo hydrolysis or secondary transformation during the hydrolysis process, further contributing to analytical variability …”

Beta-Glucuronidase Inhibition by Constituents of Mulberry Bark

“…It is well-known that natural compounds from folk medicines or medicinal plants are still primary sources for discovering lead compounds or drug candidates [21]. A wide range of natural compounds with diverse scaffolds (e.g., quercetin, eugenol, and emodinol) have been reported with EcGUS inhibition activity over the past few decades [1,22], which inspired us to find more effective EcGUS inhibitors from folk medicines. Our preliminary screening found that the crude extract of Mulberry bark (also named Sang Bai Pi in Chinese) displayed relatively potent EcGUS inhibition with an IC 50 value of 0.68 µg/ml (Fig.…”