Comparative Analyses of Bioavailability, Biotransformation, and Excretion of Nobiletin in Lean and Obese Rats.

Nobiletin, one of the prevalent polymethoxyflavones in citrus peels, was reported to possess various health benefits. We conducted the excretion study and pharmacokinetics study of nobiletin via oral administration and intravenous injection and 15 day consecutive dosing study using the high fat diet-induced obese rats and their lean counterparts. By comparing the demethylated metabolite profiles in the urine and feces, gut microbiota demonstrated greater biotransformation activity on nobiletin than the host. The absolute oral bioavailability of nobiletin in lean (22.37% ± 4.52%) and obese (18.67% ± 4.80%) rats has a negligible statistically significant difference (P > 0.05). However, a higher extent of demethylated metabolites was found in the feces and plasma of obese rats than lean rats (P < 0.05). Moreover, the consecutive dosing of nobiletin might lead to a higher extent of demethylated metabolites in the plasma and in feces. These results suggested that gut microbiota played important roles in nobiletin metabolism.

Pharmacokinetics, tissue distribution and excretion study of Oroxylin A, Oroxylin A 7-O-glucuronide and Oroxylin A sodium sulfonate in rats after administration of Oroxylin A.

Oroxylin A (OA), as a natural flavonoid extracted from the root of Scutellaria baicalensis Georgi, is a candidate drug with multiple pharmacological activities. However, pharmacokinetic studies of OA have rarely been reported up to now. The present study aim to conduct a systemic evaluation on the pharmacokinetics, tissue distribution and excretion of OA in rats, with quantification of both OA and its two metabolites, Oroxylin A 7-O-glucuronide (OG) and Oroxylin A sodium sulfonate (OS) by the sensitive and rapid UPLC-MS/MS methods. The results show that OA was rapidly eliminated in vivo after a single-dose (2 mg/kg) i.v. administration of OA. The relative bioavailability of OA in all three i.g. administration groups (40, 120, and 360 mg/kg) were <2%. The AUC0-t values of OA, OG, and OS in rats show an apparent dose-proportionality. OA, OG, and OS all underwent a rapid and widespread tissue distribution after i.g. administration (120 mg/kg) of OA. Except for stomach and intestine, the major distribution tissues of OA and its two metabolites in rats were liver, kidney, respectively. And OA was more widely distributed in tissue than its metabolites. After i.g. administration (120 mg/kg) of OA, it was mainly excreted from the feces, and OG mainly excreted from bile and urine, while OS almost free of excretion. This work present a comprehensive pharmacokinetics information for further investigation of OA and its two metabolites.

Association between different dietary polyphenol subclasses and the improvement in cardiometabolic risk factors: evidence from a randomized controlled clinical trial.

AIMS: Due to their different chemical structures and metabolism, polyphenol subclasses may have specific impact on cardiometabolic risk factors. Our aim was to evaluate whether the intake of different polyphenol subclasses is associated with clinical outcomes beneficially improved by polyphenols in a nutritional trial performed by our group (postprandial lipid response, glucose homeostasis, early insulin secretion and oxidative stress). METHODS: The present study is a secondary analysis of a nutritional intervention study with a diet naturally rich in polyphenols. The data are derived from 78 participants at high cardiovascular risk who completed the ETHERPATH trial. The associations between variations in polyphenol subclasses (phenolic acids, anthocyanidins, flavones, flavan-3-ols, flavonols and flavanones) and clinical outcomes beneficially influenced by polyphenols were firstly explored by Spearman's correlation. Thereafter, adjustment for gender, age and body mass index (BMI) was run. Linear regression analysis was used to assess the class of polyphenols that best predicted the outcome. RESULTS: Flavanone intake was inversely correlated with postprandial lipid response, whereas flavone intake was related to postchallenge glucose response. Anthocyanidins and flavan-3-ols associated positively with early insulin secretion. The decrease in urinary isoprostanes correlated with anthocyanidins, flavan-3-ols and flavonols. Correlations did not change after adjustment for gender, age, and BMI. Linear regression analysis showed an independent association between flavonols and urinary isoprostanes, whereas early insulin secretion was mainly associated with flavan-3-ols intake. CONCLUSIONS: The results of this study show that a polyphenol-rich diet may have a pleiotropic effect on cardiometabolic risk factors thanks to the specific action of different polyphenol subclasses.

Identification of 3',4'-Dimethoxy Flavonol-3-ß-d-Glucopyranoside Metabolites in Rats by Liquid Chromatography-Electrospray Ionization Ion Trap Mass Spectrometry.

A method using liquid chromatography-electrospray ionization ion trap mass spectrometry was established for the identification of metabolites in feces, urine and bile in rats after oral administration of 3',4'-dimethoxy flavonol-3-ß-d-glucopyranoside (abbreviated DF3G). Seven metabolites in rat feces, urine and bile were firstly identified on the basis of their MS fragmentation behaviors. Three metabolites were identified in the feces, 6 in the urine and 2 in the bile, which suggested that demethylation, deglycosylation and deglycosylation followed by glucuronide conjugation were the major metabolic pathways for DF3G in vivo. Hydrolyzation might be the first step in the absorption and metabolism of DF3G. The possible metabolic pathway was proposed for the first time. The established method was simple, reliable and sensitive, revealing that it could be used to rapidly screen and identify the structures of metabolites of DF3G to better understand its metabolism in vivo.

Investigation of 6-O-methyl-scutellarein metabolites in rats by ultra-flow liquid chromatography/quadrupole-time-of-flight mass spectrometry.

Context Scutellarin (1) has been widely used in China to treat acute cerebral infarction and paralysis induced by cerebrovascular diseases. However, scutellarin (1) has unstable metabolic characteristics. Objective The metabolic profile of 6-O-scutellarein was studied to determine its metabolic stability in vivo. Materials and methods In this study, a method of UFLC/Q-TOF MS was used to study the 6-O-methyl-scutellarein metabolites in rat plasma, urine, bile and faeces after oral administration of 6-O-methyl-scutellarein (3). One hour after oral administration of 6-O-methyl-scutellarein (3) (34 mg/kg), approximately 1 mL blood samples were collected in EP tubes from all groups. Bile, urine and faeces samples were collected from eight SD rats during 0-24 h after oral administration. The mass defect filtering, dynamic background subtraction and information dependent acquisition techniques were also used to identify the 6-O-methyl-scutellarein metabolites. Results The parent compound 6-O-methyl-scutellarein (3) was found in rat urine, plasma, bile and faeces. The glucuronide conjugate of 6-O-methyl-scutellarein (M1, M2), diglucuronide conjugate of 6-O-methyl-scutellarein (M3), sulphate conjugate of 6-O-methyl-scutellarein (M4), glucuronide and sulphate conjugate of 6-O-methyl-scutellarein (M5), methylated conjugate of 6-O-methyl-scutellarein (M6) were detected in rat urine. M1, M2 and M3 were detected in rat bile. M1 was found in rat plasma and M7 was detected in faeces. Discussion and conclusion Because the parent compound 6-O-methyl-scutellarein (3) was found in rat urine, plasma, bile and faeces, we speculate that 6-O-methyl-scutellarein (3) had good metabolic stability in vivo. This warrants further study to develop it as a promising candidate for the treatment of ischemic cerebrovascular disease.

Analysis of 10 metabolites of polymethoxyflavones with high sensitivity by electrochemical detection in high-performance liquid chromatography.

Polymethoxyflavones (PMFs) have been known as a type of bioactive flavones that possess various beneficial biological functions. Accumulating evidence demonstrated that the metabolites of PMFs, that is, hydroxyl PMFs (OH-PMFs), had more potent beneficial biological effects than their corresponding parent PMFs. To facilitate the further identification and quantification of OH-PMFs in biological samples, the aim of this study was to develop a methodology for the simultaneous determination of 10 OH-PMFs using high-performance liquid chromatography (HPLC) coupled with electrochemistry detection. The HPLC profiles of these 10 OH-PMFs affected by different chromatographic parameters (different organic composition in mobile phases, the concentration of trifluoroacetic acid, and the concentration of ammonium acetate) are fully discussed in this study. The optimal condition was selected for the following validation studies. The linearity of calibration curves, accuracy, and precision (intra- and interday) at three concentration levels (low, middle, and high concentration range) were verified. The regression equations were linear (r > 0.9992) over the range of 0.005-10 µM. The limit of detection for 10 OH-PMFs was in the range of 0.8-3.7 ng/mL (S/N = 3, 10 µL injection). The recovery rates ranged from 86.6 to 108.7%. The precisions of intraday and interday analyses were less than 7.37 and 8.63% for relative standard deviation, respectively. This validated method was applied for the analysis of a variety of samples containing OH-PMFs. This paper also gives an example of analyzing the metabolites of nobiletin in mouse urine using the developed method. The transformation from nobiletin to traces of 5-hydroxyl metabolites has been discovered by this effective method, and this is the first paper to report such an association.

Identification of novel bioactive metabolites of 5-demethylnobiletin in mice.

SCOPE: Biotransformation of dietary components is important for their in vivo biological activities after oral ingestion. Herein, we investigated biotransformation of 5-demethylnobiletin (a polymethoxyflavone found in citrus fruits) in mice, and its implication in the inhibition of human colon cancer cells. METHODS AND RESULTS: Urine samples were collected from mice fed with 5-demethylnobiletin (1) and analyzed by LC-ESI-MS and HPLC coupled with an electrochemical detector. Three major metabolites were identified as 5,3'-didemethylnobiletin (2), 5,4'-didemethylnobiletin (3), and 5,3',4'-tridemethylnobiletin (4) by comparing their ESI-MS and HPLC profiles with those of authentic standards synthesized by a multistep route. Cell viability assay in human colon cancer cells demonstrated that all three metabolites of 5-demethylnobiletin, especially 5,3'-didemethylnobiletin (2), showed much stronger inhibitory effects on cancer cell growth than 5-demethylnobiletin. For example, metabolites (2-4) showed IC50 of 0.12, 5.5, and 4.2 µM in SW620 cells, respectively, while 5-demethylnobiletin at 10 µM only caused 37% inhibition after 72 h of treatment. CONCLUSION: Three novel metabolites were identified in mice after oral administration of 5-demethylniobiletin. These metabolites exhibited strong inhibitory effects against human colon cancer cells. Our results provide a first report on these bioactive metabolites and warrant further investigation on their molecular mechanism of actions.

Pharmacokinetics, bioavailability, tissue distribution, excretion, and metabolite identification of methoxyflavones in Kaempferia parviflora extract in rats.

Kaempferia parviflora (KP) is an herbal plant in the family of Zingiberaceae. KP mainly contains methoxyflavones, especially 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), and 3,5,7,3',4'-pentamethoxyflavone (PMF). The present study was designed to characterize the pharmacokinetics, including bioavailability, distribution, excretion, and identification of metabolites after administration of a KP ethanolic extract. Male rats were orally or intravenously administered a 250 mg/kg concentration of a KP extract, and blood samples were obtained at selected times to determine pharmacokinetic parameters of PMF, TMF, and DMF. For distribution and excretion studies, the organs, urine, and feces samples were collected at various times after oral administration of a larger (750 mg/kg) dose of KP extract. Methoxyflavones in the biological samples were quantified by high-performance liquid chromatography-UV, and the metabolites in urine and feces were further identified by using liquid chromatography-tandem mass spectrometry. After oral administration, concentrations of the three methoxyflavones quickly approached their maximal concentration, ranging from 0.55 to 0.88 µg/ml within 1 to 2 h after administration, and then were gradually excreted with half-lives of 3 to 6 h. The methoxyflavones showed low oral bioavailability of 1 to 4%. Three methoxyflavones were detected at their highest levels in liver followed by kidney. They were also found in lung, testes, and brain. After absorption, organ distribution, and metabolism, the components of KP were mainly eliminated through urine in the forms of demethylated, sulfated, and glucuronidated products and as demethylated metabolites in the feces. The parent compounds were found to have 0.79, 1.76, and 3.10% dose recovery in urine and 1.06, 1.77, and 0.96% dose recovery in feces for PMF, TMF, and DMF, respectively. These studies are the first to describe the pharmacokinetics of KP extract to provide the information on blood and tissue levels.

[Identification of metabolites of nobiletin in rats using ultra-performance liquid chromatography coupled with triple-quadrupole mass spectrometry].

In this study, metabolism of nobiletin in rats was studied using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). As a result, seven major metabolites were found in bile, urine and serum of rats. Three phase I products were assigned to be demethyl and di-demethyl products, and other four phase II products were assigned to be glucuronic and sulfonic conjugates. The four phase II metabolites were reported for the first time. Among the metabolites found in the present study, the glucuronic conjugates of demethyl-nobiletin played a predominant role in the metabolic pathway, indicating that its potential role for glucuronidation-related factors, such as gene polymorphism, drug-drug interaction, etc., in changing the active and toxic effect of nobiletin and that it should be paid more attention in further development.

The pharmacokinetics of C-glycosyl flavones of Hawthorn leaf flavonoids in rat after single dose oral administration.

Hawthorn leaf flavonoids (HLF) are used in the treatment of cardiovascular diseases. Various potential pharmacodynamic effects have been observed for vitexin-4''-O-glucoside (VOG) and vitexin-2''-O-rhamnoside (VOR) which are the main constituents of HLF. The aim of this study was to investigate the pharmacokinetics of VOG and VOR when a single dose of HLF was administrated orally. The levels of VOG and VOR in plasma, tissues (heart, liver, spleen, lung, kidney and brain), bile, urine and feces were measured by HPLC-UV. The results showed that VOG and VOR have the similar pharmacokinetics. Both of them were absorbed quickly into plasma with maximal plasma concentrations of VOG and VOR being reached within 0.75 h. The mean elimination half-life (t(1/2)) of VOG and VOR were 2.53 h and 2.32 h, respectively. High levels of tissue distribution of VOG and VOR were observed in liver and kidney. No VOG and VOR were detected in brain tissue. There was no long-term accumulation of VOG and VOR in rat tissues examined. The total recovery of the dose in 24 hours was 64.91% (0.70% in urine; 64.21% in feces) for VOG and 89.01% (0.72% in urine; 88.29% in feces) for VOR. The cumulative VOG and VOR excreted in bile represented 0.58% and 13.38% of the doses, respectively. VOG and VOR in HLF were not efficiently absorbed in the rodent gastrointestinal tract.

Flavanoids and pterocarpans from the bark of Erythrina fusca.

Three new isomeric flavanones, fuscaflavanones A(1) (1), A(2) (2) and B (3), together with six known flavanones, lupinifolin (4), lonchocarpol A (5), a mixture of lonchocarpols C(1) and C(2) (6a, b), and a mixture of lonchocarpols D(1) and D(2) (7a, b), five pterocarpans, sandwicensin (8), phaseollidin (9), erythrabissin I (10), and a mixture of dolichins A and B (11a, b), one chalcone, isobavachalcone (12), and one isoflavone, wighteone (13), were isolated from the bark of Erythrina fusca LOUR. Their structures were elucidated on the basis of spectroscopic data. Some isolates were tested for antiplasmodial and cytotoxic activities and it was found that 5 and 9 exhibited moderate antiplasmodial activity against Plasmodium falciparum. For cytotoxicity, compounds 1, 4, 5, 9 and 12 showed moderate to weak activity against KB, BC and NCI-H187 cells, whereas 2 exhibited only weak activity against KB cells.

Anti-inflammatory and antitumor promotional effects of a novel urinary metabolite, 3',4'-didemethylnobiletin, derived from nobiletin.

We reported previously that 3',4'-didemethylnobiletin (DDMN) is the major metabolite of nobiletin in mouse urine. In this study, we examined DDMN's molecular mechanism of action and its anti-inflammatory and antitumor properties. We demonstrated that topical application of DDMN effectively inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated transcription of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and ornithine decarboxylase (ODC) messenger RNA and protein expression in mouse skin. Pretreatment with DDMN has resulted in the reduction of TPA-induced nuclear translocation of the nuclear factor-kappa B (NF-kappaB) subunit. DDMN also reduced DNA binding by blocking phosphorylation of inhibitor kappaB (IkappaB) alpha and p65 and caused subsequent degradation of IkappaBalpha. DDMN inhibited TPA-induced phosphorylation and nuclear translocation of the signal transducer and activator of transcription 3. Moreover, DDMN suppressed TPA-induced activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt and protein kinase C that are upstream of NF-kappaB and activator protien-1. We also found that DDMN significantly inhibited TPA-induced mouse skin inflammation by decreasing inflammatory parameters. Furthermore, DDMN significantly inhibited 7,12-dimethylbenz[a]anthracene/TPA-induced skin tumor formation measured by the tumor multiplicity of papillomas at 20 weeks. Presented data for the first time reveal that DDMN is an effective antitumor agent that functions by downregulating inflammatory iNOS, COX-2 and ODC gene expression in mouse skin. It is suggested that DDMN is a novel functional agent capable of preventing inflammation-associated tumorigenesis.

Anti-inflammatory property of the urinary metabolites of nobiletin in mouse.

Nobiletin, a major component of polymethoxyflavones in citrus fruits, has a broad spectrum of health beneficial properties including anti-inflammatory and anti-carcinogenic activities. The metabolite identification of nobiletin in mouse urine has concluded that it undergoes mono-demethylation (3'- and 4'-demethylnobiletin) and di-demethylation (3',4'-didemethylnobiletin) metabolic pathway. Biological screening of nobiletin and its metabolites has revealed that the metabolites possess more potent anti-inflammatory activity than their parent compound. Therefore, this letter reports the identification of nobiletin metabolites and their anti-inflammatory activity against LPS-induced NO production and iNOS, COX-2 protein expression in RAW264.7 macrophage.

Simultaneous determination of homoeriodictyol-7-O-beta-D-Glccopyranoside and its metabolite homoeriodictyol in rat tissues and urine by liquid chromatography-mass spectrometry.

A liquid chromatographic-mass spectrometric (LC-MS) method was developed and validated for the simultaneous determination of homoeriodictyol-7-O-beta-D-glycoside (HEDT-Glc) and its active metabolite homoeriodictyol (HEDT) in rat tissues and urine. The analytes and internal standard (dihydromyricetin, IS) were detected by using negative atmospheric pressure chemical ionization mass spectrometry in selected ion monitoring (SIM) mode at m/z 464, 301 and 319 for HEDT-Glc, HEDT and IS, respectively. These compounds were eluted on a Luna reverse phase column. The mobile phase was a methanol-water mixture (70:30, v/v) containing 0.1% of formic acid at a flow rate of 0.8 ml/min. The limit of quantification (LOQ) for both HEDT-Glc and HEDT was 10 ng/ml and their limit of detection (LOD) was 1 ng/ml. Calibration curves were linear (r>0.995) over a wide range of the analytes in tissues and urine. The mean extraction recoveries were >or=75.6% for HEDT-Glc and >or=82.4% for HEDT from biological matrixes. Accuracy, expressed as the relative error, ranged from -4.0% to 3.8% for HEDT-Glc and from -2.8% to 4.7% for HEDT. The method was successfully applied to the estimation of HEDT-Glc and its metabolite HEDT in rat tissues and urine.

Comparison of supercritical fluid chromatography and liquid chromatography for the separation of urinary metabolites of nobiletin with chiral and non-chiral stationary phases.

Nobiletin (NOB), a polymethoxylated flavone found in sweet orange (Citrus sinensis) peel, is currently recognized as a promising anti-inflammatory and anti-tumor agent. It is believed that, by undergoing metabolic biotransformation in vivo, nobiletin is demethylated by hepatic P450 enzymes, yielding multiple hydroxylated metabolites. However, it has not been possible to date to separate the two demethylated nobiletin metabolites, 3'-demethyl-NOB and 4'-demethyl-NOB (regio-isomers) on reversed-phase liquid chromatography (RPLC). Additionally, both display similar mass spectrometric fragmentation, resulting in difficulties to identify the dominant metabolite. A successful separation method was developed by utilizing supercritical fluid chromatography (SFC) with chiral stationary phase. The separation was also attempted with normal-phase liquid chromatography (NPLC) in both chiral and non-chiral modes. Chromatographic separation for the two nobiletin metabolites was superior by SFC than by LC, especially using chiral stationary phase. By comparing the SFC profile of the synthesized standards, the major nobiletin metabolite in mouse urine was identified as 4'-demethyl-NOB, with the concentration of 28.9 microg/mL.

Identification of nobiletin metabolites in mouse urine.

Nobiletin, a major component of citrus polymethoxyflavones, has many potential significant health benefits. While the biological activities of nobiletin have been widely reported, its in vitro and in vivo metabolic fate has been rarely studied. To explore the biotransformation mechanism of nobiletin we conducted an investigation into its metabolic profile in mouse urine, by various analytical techniques. Due to sample amount limitations for isolating and characterizing an individual metabolite, two possible nobiletin metabolites were prepared in a similar multi-step organic synthetic route: 3'-hydroxy-5,6,7,8,4'-pentamethoxyflavone (3'-demethylnobiletin) and 4'-hydroxy-5,6,7,8,3'-pentamethoxyflavone (4'-demethylnobiletin). Normal phase (silica gel) and C(18) reverse phase chromatography, as well as liquid chromatography-mass spectrometry-mass spectrometry, were employed in the separation of 3'-demethylnobiletin and 4'-demethylnobiletin, however, without success due to the structural similarities of these mono-demethylated nobiletins. Using a chiral packed column eluted under supercritical fluid chromatography (SFC) conditions, a clear separation was achieved. Thus, by comparing the SFC profiles of metabolite mixtures with the synthesized standard compounds, the major nobiletin metabolite of mouse urine is identified as 4'-demethylnobiletin, whereas 3'-demethylnobiletin is a minor metabolite. In this study, the concentration of 4'-demethylnobiletin in mouse urine is 28.9 microg/mL.