3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.

[UPLC-Q-TOF-MS/MS analysis on prototypes components and metabolites of effective fractions of Polygonum orientale flower in rat serum and urine].

Ultra performance liquid chromatography coupled with time-of-flight mass spectrometry( UPLC-Q-TOF-MS/MS) method was applied to analyze the prototypes and metabolites of the effective components of Polygonum orientale in SD rat serum and urine. The separation was performed on Agilent Eclipse Plus C_(18) column( 2. 1 mm×100 mm,1. 8 µm),with 0. 1% formic acid solution( A)-acetonitrile( B) as the mobile phase for gradient elution. Mass spectrometry data of biological samples were obtained under positive and negative electrospray ion mode. By comparing chromatogram differences between blank samples and drug treatment samples,prototype components and metabolites of the effective components of P. orientale extract were identified. The results showed that 12 metabolites were detected in serum and 26 metabolites in urine( including cross-components) of rats. The main metabolic pathways included hydrogenation,hydroxylation,glucuronidation,sulfation reaction,and methylation-glucuronidation,etc. The method established in this study was reliable and effective for studying the metabolic characteristics of the effective components of P. orientale in rats,and it can provide a reference for further studies on therapeutic material basis of this herb.

Metabolite profiling and quantification of phytochemicals of Tianma-Gouteng granule in human and rat urine using ultra high performance liquid chromatography coupled with tandem mass spectrometry.

Tianma-Gouteng granule has been used for the treatment of hypertension, headache, and stroke in China. However, the metabolism of Tianma-Gouteng granule has not been clear. In the present study, an ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry method has been developed for rapid identification of 35 prototypes and 43 metabolites in human and rat urine after single oral administration of Tianma-Gouteng granule. The results showed that glucuronidation and sulfation were the main metabolic pathways for flavonoids, alkaloids, iridoidic glycosides, anthraquinones, phenols, and stilbenes that were found in Tianma-Gouteng granule. Moreover, a validated ultra high performance liquid chromatography coupled with tandem mass spectrometry method was applied for the quantification of 14 compounds in rat urine after an oral administration of Tianma-Gouteng granule (2.5 g/kg). During 0-48 h after dosing, the cumulative excretion rates of nine prototype components were 53% for gastrodin, 0.07∼1.6% for geniposide, baicalin and baicalein, wogonoside, rhynchophylline and isorhynchophylline, leonurine, and emodin, indicating that urinary excretion is the major way for gastrodin to eliminate from the body. This study provides a comprehensive understanding of metabolism and excretive kinetics of Tianma-Gouteng granule in human and/or rat, and helpful information for screening of its active components in vivo and clinical application.

In vitro metabolism of selected bioactive compounds of Eurycoma longifolia root extract to identify suitable markers in doping control.

Eurycoma longifolia Jack (Tongkat Ali, Simaroubaceae) is a medicinal plant endemic to South-East Asia. For centuries, different parts of the plant have been used as a natural remedy to treat fever, hypertension, or sexual insufficiency. Today, Eurycoma longifolia preparations are commercially available and advertised to enhance athletic performance and muscle strength. Several studies have demonstrated a testosterone-boosting effect that might be caused by the release of free testosterone from the sex-hormone-binding globulin. To date, many phytochemical constituents of Eurycoma longifolia root extracts have been identified and physiological effects have been examined, while studies on their biotransformation and monitoring are still lacking. Within this study, eurycomalide C, eurycomalactone, 5,6-dehydro-eurycomalactone, longilactone, 14,15ß-dihydroklaieanone, 11-dehydroklaieanone, 9-hydroxycanthin-6-one, and 9-methoxycanthin-6-one isolated from E. longifolia root were incubated with liver microsomes. Respective metabolites were analyzed by liquid chromatography-tandem (high-resolution) mass spectrometry. The compounds were chosen based on their potential androgenic effects (estimated by in vitro assays), their concentrations in plant extracts, and presumptive metabolic pathways. Hydroxylated phase I metabolites were only observed for 5,6-dehydro-eurycomalactone, 11-dehydroklaieanone, 9-hydroxycanthin-6-one, and 9-methoxycanthin-6-one. Moreover, an O-demethylated metabolite of 9-methoxycanthin-6-one was found. Besides, the glucuronide of 9-hydroxycanthin-6-one was detected after in vitro glucuronidation using liver microsomes. The in vitro generated metabolites were comparable to that detected in urine and serum after a single ingestion of either 9-methoxycanthin-6-one or an Eurycoma longifolia root extract. Hence, 9-methoxycanthin-6-one, its glucuronide, and the glucuronide of its O-demethylated biotransformation product are proposed to be the most suitable targets for detection of 9-methoxycanthin-6-one or Tongkat Ali application in urine and serum.

The Pocket-4-Life project, bioavailability and beneficial properties of the bioactive compounds of espresso coffee and cocoa-based confectionery containing coffee: study protocol for a randomized cross-over trial.

BACKGROUND: Coffee is an important source of bioactive compounds, including caffeine, phenolic compounds (mainly chlorogenic acids), trigonelline, and diterpenes. Several studies have highlighted the preventive effects of coffee consumption on major cardiometabolic diseases, but the impact of coffee dosage on markers of cardiometabolic risk is not well understood. Moreover, the pool of coffee-derived circulating metabolites and the contribution of each metabolite to disease prevention still need to be evaluated in real-life settings. The aim of this study will be to define the bioavailability and beneficial properties of coffee bioactive compounds on the basis of different levels of consumption, by using an innovative experimental design. The contribution of cocoa-based products containing coffee to the pool of circulating metabolites and their putative bioactivity will also be investigated. METHODS: A three-arm, crossover, randomized trial will be conducted. Twenty-one volunteers will be randomly assigned to consume three treatments in a random order for 1 month: 1 cup of espresso coffee/day, 3 cups of espresso coffee/day, and 1 cup of espresso coffee plus 2 cocoa-based products containing coffee twice per day. The last day of each treatment, blood and urine samples will be collected at specific time points, up to 24 hours following the consumption of the first product. At the end of each treatment the same protocol will be repeated, switching the allocation group. Besides the bioavailability of the coffee/cocoa bioactive compounds, the effect of the coffee/cocoa consumption on several cardiometabolic risk factors (anthropometric measures, blood pressure, inflammatory markers, trimethylamine N-oxide, nitric oxide, blood lipids, fasting indices of glucose/insulin metabolism, DNA damage, eicosanoids, and nutri-metabolomics) will be investigated. DISCUSSION: Results will provide information on the bioavailability of the main groups of phytochemicals in coffee and on their modulation by the level of consumption. Findings will also show the circulating metabolites and their bioactivity when coffee consumption is substituted with the intake of cocoa-based products containing coffee. Finally, the effect of different levels of 1-month coffee consumption on cardiometabolic risk factors will be elucidated, likely providing additional insights on the role of coffee in the protection against chronic diseases. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03166540 . Registered on May 21, 2017.

Surfactant-coated graphitized multiwalled carbon nanotubes as the pseudostationary phase in electrokinetic chromatography for the analysis of phytochemical compounds in biological fluids.

This report describes the use of surfactant-coated graphitized multiwalled carbon nanotubes (SC-GMWNTs) as a novel pseudostationary phase in CE with diode array detection for the determination of phenolic acids and tanshinones in herbal and urine samples. Several parameters influencing the separation were studied, such as the concentrations of SDS, GMWNTs, and isopropanol; choice of carbon nanotubes; sodium borate content; and buffer pH. The results revealed that the presence of SC-GMWNTs in buffer enhanced the separation efficiency for the target analytes relative to conventional micelles due to the strong interaction between the surface of the GMWNTs and the target compounds. Under the optimum conditions, the method showed good linearity, with correlation coefficients higher than 0.9950. LODs were in the range of 0.71-3.10 µg/mL. Furthermore, satisfactory separations were achieved with good recovery values in the range of 89.97 and 103.30% when 10 mM borate, 30 mM SDS, 10% isopropanol, and 6 µg/mL SC-GMWNTs were introduced into the buffer solution.

Sample preparation for liquid chromatographic analysis of phytochemicals in biological fluids.

INTRODUCTION: Natural products have been used traditionally for the treatment and prevention of diseases for thousands of years and are nowadays consumed as dietary supplements and herbal medicine. To ensure the safe and effective use of these herbal products, information about bioavailability of active compounds in plasma or target tissues should be provided via validated analytical methods combined with appropriate sampling methods. OBJECTIVE: To provide comprehensive and abridged information about sample preparation methods for the quantification of phytochemicals in biological samples using liquid chromatography analysis. METHODS: Sample pre-treatment procedures used in analytical methods for in vivo pharmacokinetic studies of natural compounds or herbal medicines were reviewed. These were categorised according to the biological matrices (plasma, bile, urine, faeces and tissues) and sample clean-up processes (protein precipitation, liquid-liquid extraction and solid-phase extraction). RESULTS: Although various kinds of sample pre-treatment methods have been developed, liquid-liquid extraction is still widely used and solid-phase extraction is becoming increasingly popular because of its efficiency for extensive clean up of complex matrix samples. However, protein precipitation is still favoured due to its simplicity. CONCLUSION: Sample treatment for phytochemical analysis in biological fluids is an indispensable and critical step to obtain high quality results. This step could dominate the overall analytical process because both the duration of the process as well as the reliability of the data depend in large part on its efficiency. Thus, special attention should be given to the choice of a proper sample treatment method that targets analytes and their biomatrix.