Investigation of the factors responsible for the low oral bioavailability of alizarin using a sensitive LC‐MS/MS method: In vitro, in situ, and in vivo evaluations

Abstract

Alizarin (1,2-dihydroxyanthraquinone) is an anthraquinone reddish dye widely used for painting and textile dyeing. As the biological activity of alizarin has recently attracted increasing attention from researchers, its therapeutic potential as complementary and alternative medicine is of interest. However, no systematic research has been conducted on the biopharmaceutical and pharmacokinetic aspects of alizarin. Therefore, this study aimed to comprehensively investigate the oral absorption and intestinal/hepatic metabolism of alizarin using a simple and sensitive tandem mass spectrometry method developed and validated in-house. The present method for the bioanalysis of alizarin has merits, including a simple pretreatment procedure, small sample volume, and adequate sensitivity. Alizarin exhibited pHdependent moderate lipophilicity and low solubility with limited intestinal luminal stability. Based on the in vivo pharmacokinetic data, the hepatic extraction ratio of alizarin was estimated to be 0.165-0.264, classified as a low level of hepatic extraction. In an in situ loop study, considerable fractions (28.2%-56.4%) of the alizarin dose were significantly absorbed in gut segments from the duodenum to ileum, suggesting that alizarin may be classified as the Biopharmaceutical Classification System class II. An in vitro metabolism study using rat and human hepatic S9 fractions revealed that glucuronidation and sulfation, but not NADPHmediated phase I reactions and methylation, are significantly involved in the hepatic metabolism of alizarin. Taken together, it can be estimated that the fractions of oral alizarin dose unabsorbed from the gut lumen and eliminated by the gut and liver before reaching the systemic circulation are 43.6%-76.7%, 0.474%-36.3%, and 3.77%-5.31% of the dose, respectively, resulting in a low oral bioavailability of 16.8%. Therefore, the oral bioavailability of alizarin depends primarily on its chemical degradation in the gut lumen and secondarily on first-pass metabolism.

A High-Performance Liquid Chromatography with Photodiode Array Detection Method for Simultaneous Determination of Three Compounds Isolated from Wikstroemia ganpi: Assessment of the Effects on Cytochrome P450-Mediated Metabolism In Vitro and In Vivo

“…Several factors might have contributed to these findings. One of the causes may be low bioavailability due to chemical degradation of phytochemicals and the intestinal/hepatic first pass [ 38 , 41 , 49 , 50 ], and plasma and liver concentrations of inhibitors may be insufficient for the IC 50 obtained in vitro CYP inhibition studies [ 51 , 52 ]. Consequently, a single oral dose of WGM did not have significant effects on the pharmacokinetics of buspirone in rats, suggesting that WGM cannot function as an inhibitor of CYP3A-mediated metabolism in vivo.…”

“…Dose–response curves assessed metabolic inhibition of BUS in the presence of 1 μM BUS and 10 different concentrations (0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, and 100 μM) of 7-methoxyluteolin-5- O -glucoside, pilloin 5- O -β- d -glucopyranoside or rutarensin. The mixture without substrate and inhibitor was incubated for 5 min at 37 °C, after which the substrate and inhibitor were added to initiate the enzyme reaction [ 38 , 39 ]. After incubating BUS (reaction time: 30 min), DIC and DEX (reaction time: 60 min) at 37 °C at a speed of 500 oscillations/min, a sample was obtained from the reaction mixture and mixed with cold acetonitrile containing an internal standard (IS) to terminate the reaction.…”