stilbenes and formic-acid

A novel method using liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) has been optimized and established for the qualitative and quantitative analysis of ten active phenolic compounds originating from the pigeon pea leaves and a medicinal product thereof (Tongluo Shenggu capsules). In the present study, the chromatographic separation was achieved by means of a HiQ Sil C18V reversed-phase column with a mobile phase consisting of methanol and 0.1% formic acid aqueous solution. Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using the selected reaction monitoring (SRM) analysis was employed for the detection of ten analytes which included six flavonoids, two isoflavonoids and two stilbenes. All calibration curves showed excellent coefficients of determination (r(2) ≥ 0.9937) within the range of tested concentrations. The intra- and inter-day variations were below 5.36% in terms of relative standard deviation (RSD). The recoveries were 95.08-104.98% with RSDs of 2.06-4.26% for spiked samples of pigeon pea leaves. The method developed was a rapid, efficient and accurate LC-MS/MS method for the detection of phenolic compounds, which can be applied for quality control of pigeon pea leaves and related medicinal products.

Topics: Cajanus; Chromatography, Liquid; Flavonoids; Formates; Linear Models; Methanol; Plant Leaves; Reproducibility of Results; Sensitivity and Specificity; Stilbenes; Tandem Mass Spectrometry

Renal brush border membranes contain several anion exchanges that may play a role in electrolyte transport and pH regulation. To help characterize the types of exchangers present and the binding properties of these membranes, the binding of nitrate (NO3-) to highly purified rabbit kidney brush border membrane vesicles was studied. The method is based on the binding induced quadrupole relaxation of the 14N-NMR signal of nitrate [1,2]. Brush border membrane vesicles caused a relaxation of the 14N-NMR nitrate signal which could be characterized by relatively high affinity sites, KD = 6.7 +/- 1.5 mM, as well as nonspecific interactions with the membranes, KD > 150 mM. The anion transport inhibitor 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) inhibited 51 +/- 6% (n = 4) of the relaxation due to the high affinity binding sites. The DNDS inhibition could be characterized by a Ki of 10-80 microM. Both bicarbonate and formate (HCO2-) were found to partially inhibit the high affinity induced relaxation, with maximal inhibition of 37 +/- 8% (n = 3) and 30 +/- 2% (n = 3), respectively. The inhibitory effects of saturating concentrations of bicarbonate and formate were non-additive, suggesting the existence of a stilbene sensitive exchanger that can bind nitrate, as well as both bicarbonate and formate. This study indicates the usefulness of this new method for further investigation of anion exchangers on these and other membranes.

Topics: Animals; Bicarbonates; Binding Sites; Carbonic Anhydrases; Formates; Kidney; Magnetic Resonance Spectroscopy; Microvilli; Nitrates; Nitrogen Isotopes; Rabbits; Stilbenes

The coupling mechanism for Cl- and H+/OH- transport in renal brush-border vesicles was examined from intravesicular pH changes following imposed H+ and Cl- gradients. Vesicles were loaded with 6-carboxyfluorescein and exposed to H+ gradients and Cl-, gluconate, or sulfate gradients, each with and without a K+/valinomycin voltage clamp. Parallel experiments were performed with vesicles equilibrated with 10 mM HCO3- or 5 mM formate. Rate of H+/OH- transport was determined from the initial rate of change in 6-carboxyfluorescein fluorescence, vesicle buffer capacity and the relationship between fluorescence and vesicle pH. In contrast to gluconate or sulfate, Cl- caused enhanced H+/OH- transport under all conditions. This difference was eliminated with voltage clamping in the presence of gluconate, SO4(2-), or HCO3-, but not in the presence of formate. These findings were not affected by the method of preparation of the vesicles. Electrically coupled Cl-/OH- transport was not inhibited by 100 microM DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) or 100 microM DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate). SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate) was found to be a protonophore at concentrations greater than 500 microM. As a control for the method, we demonstrated amiloride inhibitable, electroneutral Na+-H+ exchange (H+ flux = 107 +/- 9 nmol/s per mg, 100 mM Na+) and electroneutral, DBDS inhibitable Cl(-)-HCO3- exchange in sealed human red blood cell ghosts. Therefore, electroneutral Cl(-)-OH- or HCO3- exchange does not measurably contribute to Cl- transport in the proximal tubule brush border. Cl(-)-formate exchange with formic acid recycling appears to be the only electroneutral coupling mechanism between Cl- and OH- transport demonstrable in renal brush-border membrane vesicles.

Topics: Animals; Cell Membrane; Chlorides; Female; Formates; Hydrogen-Ion Concentration; Hydroxides; Ion Exchange; Kidney; Microvilli; Rabbits; Stilbenes