Analysis of isoflavones and flavonoids in human urine by UHPLC

A rapid, ultra high-performance liquid chromatographic (UHPLC) method has been developed and validated for simultaneous identification and analysis of the isoflavones genistein, daidzein, glycitin, puerarin, and biochanin A, and the flavonoids (±)-catechin, (−)-epicatechin, rutin, hesperidin, neohesperidin, quercitrin, and hesperetin in human urine. Urine samples were incubated with β-glucuronidase/sulfatase. UHPLC was performed with a Hypersil Gold (50 × 2.1 mm, 1.9 μm) analytical column. Elution was with a gradient prepared from aqueous trifluoroacetic acid (0.05%) and acetonitrile. UV detection was performed at 254 and 280 nm. The calibration curves were indicative of good linearity (r2 ≥ 0.9992) in the range of interest for each analyte. LODs ranged between 15.4 and 107.0 ng mL−1 and 3.9 and 20.4 ng mL−1 for flavonoids and isoflavones, respectively. Intra-day and inter-day precision (C.V., %) was less than 3.9% and 3.8%, respectively, and accuracy was between 0.03% and 5.0%. Recovery was 70.35–96.58%. The method is very rapid, simple, and reliable, and suitable for pharmacokinetic analysis. It can be routinely used for simultaneous determination of these five isoflavones and seven flavonoids in human urine. The method can also be applied to studies after administration of pharmaceutical preparations containing isoflavones and flavonoids to humans

Intestinal Absorption and First-Pass Metabolism of Polyphenol Compounds in Rat and Their Transport Dynamics in Caco-2 Cells

<div><h3>Background</h3><p>Polyphenols, a group of complex naturally occurring compounds, are widely distributed throughout the plant kingdom and are therefore readily consumed by humans. The relationship between their chemical structure and intestinal absorption, transport, and first-pass metabolism remains unresolved, however.</p> <h3>Methods</h3><p>Here, we investigated the intestinal absorption and first-pass metabolism of four polyphenol compounds, apigenin, resveratrol, emodin and chrysophanol, using the <em>in vitro</em> Caco-2 cell monolayer model system and <em>in situ</em> intestinal perfusion and <em>in vivo</em> pharmacokinetic studies in rats, so as to better understand the relationship between the chemical structure and biological fate of the dietary polyphenols.</p> <h3>Conclusion</h3><p>After oral administration, emodin and chrysophanol exhibited different absorptive and metabolic behaviours compared to apigenin and resveratrol. The differences in their chemical structures presumably resulted in differing affinities for drug-metabolizing enzymes, such as glucuronidase and sulphatase, and transporters, such as MRP2, SGLT1, and P-glycoprotein, which are found in intestinal epithelial cells.</p> </div

The antioxidant and antiproliferative activities of methanolic extracts from Njavara rice bran

<p>Abstract</p> <p>Background</p> <p>Free radical-induced oxidative stress is the root cause for many human diseases. Naturally occurring antioxidant supplements from plants are vital to counter the oxidative damage in cells. The main objective of the present study was to characterize the antioxidant and antiproliferative potential of rice bran extracted from an important Indian rice variety, Njavara and to compare the same with two commercially available basmati rice varieties: Vasumathi, Yamini and a non medicinal variety, Jyothi.</p> <p>Methods</p> <p>Methanolic extracts of rice bran from four varieties; Vasumathi, Yamini, Jyothi and Njavara were used to study their total phenolic and flavonoid contents, <it>in vitro </it>antioxidant activities including total antioxidant activity, scavenging of nitric oxide and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical, reducing power and cytotoxic activity in C6 glioma cells. Correlation coefficient and regression analysis were done by using Sigmastat version 3.1 and Stata statistical package respectively.</p> <p>Results</p> <p>Rice bran methanolic extract from Njavara showed the highest antioxidant and cell cytotoxic properties compared to the other three rice varieties. IC₅₀values for scavenging DPPH and nitric oxide were in the range of 30.85-87.72 μg/ml and 52.25-107.18 μg/ml respectively. Total antioxidant activity and reducing power were increased with increasing amounts of the extract. Total phenolic and flavonoid contents were in the range of 3.2-12.4 mg gallic acid-equivalent (GAE)/g bran and 1.68-8.5 mg quercetin-equivalent (QEE)/g bran respectively. IC₅₀values of cytotoxic assay (MTT assay) were 17.53-57.78 μg/ml. Correlation coefficient and regression analysis of phenolic content with DPPH and NO scavenging, MTT (-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, total antioxidant assay and reducing power showed a highly significant correlation coefficient values (96-99%) and regression values (91-98%).</p> <p>Conclusion</p> <p>The results of the present study show that the crude methanolic extract from Njavara rice bran contains significantly high polyphenolic compounds with superior antioxidant activity as evidenced by scavenging of free radicals including DPPH and NO. Njavara extracts also showed highest reducing power activity, anti-proliferative property in C6 glioma cells. In conclusion, it is conceivable that the Njavara rice variety could be exploited as one of the potential sources for plant - based pharmaceutical products.</p

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Mass spectrometric methods for the analysis of chlorinated and nitrated isoflavonoids: a novel class of biological metabolites

Electrospray ionization combined with tandem mass spectrometry was applied to a study of some representative chlorinated and nitrated isoflavones-potential metabolites of isoflavones in inflammatory cells. Upon collision-induced dissociation of deprotonated [M - H](-) ions of these compounds, a number of structurally characteristic product ions were produced. The product ion analysis of 3'- and 8-chlorodaidzein in the tandom mass spectra led to ready differentiation of these isomers. 3-Nitro derivatives of both genistein and daidzein have product ions due to the losses of HNO2 and two OH groups. Chlorinated derivatives of isoflavones were detected in cell-based experiments and their structures were proposed by comparing the tandem mass spectra of their product ions with those of standards. This work provides a suitable analytical basis to aid the characterization of chlorinated and nitrated metabolites in studies in vivo and in vitro. Copyright (C) 2003 John Wiley Sons, Ltd.</p