An efficient approach for the isolation, identification and evaluation of antimicrobial plant components on an analytical scale, demonstrated by the example of Radix imperatoriae.

Using Radix imperatoriae (the root of masterwort) as an example, we describe an efficient approach for the isolation, identification and evaluation of bioactive plant components on an analytical scale. The extraction of Radix imperatoriae with ethyl acetate was enhanced by the application of ultrasound oscillations. This rhizome extract was applied to three pathogenic bacteria (Bacillus cereus, Escherichia coli, and Staphylococcus aureus) to determine its antimicrobial activity. Disk diffusion was utilized to determine susceptibility. The extract components were separated using a series of chromatography approaches (semi-preparative RP-HPLC, or RP-HPLC on an analytical scale), followed by testing. All fractions were analyzed by LC-UV-ESI-MS and 600 MHz microcoil (1)H NMR spectroscopy. Among other findings, in the fraction with the highest antibacterial activity we were able to identify oxypeucedanin and oxypeucedanin hydrate. Subsequent analysis revealed that only oxypeucedanin hydrate had antibacterial activity, whereas oxypeucedanin itself was inactive at the concentrations applied. Furthermore, oxypeucedanin hydrate appears to be largely, or exclusively, a by-product of sample preparation, since it is either not synthesized by the plant as a second metabolite or is produced by it in only very small quantities.

Electrogenic Na+-ascorbate cotransport in cultured bovine pigmented ciliary epithelial cells.

The high level of ascorbic acid (AA) in the aqueous humor of many mammals suggests an active transport of AA across the double-layered ciliary epithelium from blood to aqueous humor. We used [14C]AA to study AA uptake in bovine pigmented ciliary epithelial cells in tissue culture. We observed a 40-fold intracellular accumulation of AA, which was dependent on extracellular Na+. With labeled dehydroascorbate (DHA, the oxidized form of the vitamin) in the medium, there was a 20-fold intracellular accumulation of the label. However, the time course of DHA uptake was different compared with AA uptake and was not Na+ dependent, suggesting different transport systems for AA and DHA. AA uptake was inhibited by 1 mM phloretin and in the presence of isoascorbate. Furthermore, AA uptake was markedly reduced when intracellular Na+ was elevated by preincubation with ouabain or amphotericin B. With increasing AA concentration, Na+-dependent AA uptake exhibited first-order saturation kinetics with half-maximal uptake at 76 microM AA. Na+ dependence of AA uptake revealed a sigmoidal curve of Na+-dependent AA uptake vs. Na+ concentration with a half-maximal AA uptake at 45.4 mM Na+. The slope of the Hill plot from these data was 1.94, suggesting a transport system translocating two or more Na+ for one AA. This stoichiometry implies electrogenicity of the transporter. We, therefore, measured membrane potentials using conventional microelectrodes. Addition of 200 microM AA resulted in a depolarization of the membrane voltage by 4.9 +/- 0.5 mV (n = 22), which was absent in Na+ free medium and was markedly reduced by phloretin.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Cl-/HCO3- exchange in cultured bovine pigmented ciliary epithelium.

Many recent data indicate that transport of Cl- across the ciliary epithelium plays an important role in aqueous humor formation. We used 36Cl to investigate the pathways for Cl- transport in confluent monolayers of cultured bovine pigmented ciliary epithelial cells. Cl- uptake mainly occurred via a mechanism with typical characteristics of an anion exchanger, and could be stimulated by an outwardly directed HCO3- gradient. One mM SITS and 1 mM DIDS inhibited Cl- uptake by some 80-90%, the latter with an IC50 of about 20 microM. HCO3- stimulated Cl- uptake could be partly inhibited for furosemide and to a lesser extent by bumetanide, indicating an action of loop-diuretics on the anion exchanger. 36Cl- uptake was cis-inhibited by the halides Cl-, I- and Br-, by NO3-, formate and acetate. Inhibition of Cl- uptake by extracellular HCO3- was less effective in the absence of extracellular Na+, suggesting that not only HCO3- but also NaCO3- binds to the carrier. SO2/4-, cyclamate and gluconate did not significantly reduce Cl- uptake via the anion exchanger. DIDS-senstive Cl- uptake showed saturation kinetics with respect to the Cl- concentration with an apparent Km of 8 mM. Cl- efflux could be stimulated by external Cl- and HCO3- and was inhibited by DIDS. Thus, cultured bovine pigmented ciliary epithelial cells express a Cl-/HCO3- exchanger. A possible role of this carrier system for aqueous humor formation is discussed [corrected].

Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells.

Uptake studies with 22Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na+ transport. A large part of Na+ uptake was sensitive to amiloride, quinidine and harmaline. Na+ uptake was stimulated by intracellular acidification (using the NH+4 prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparent KM for Na+ from 38 to 86 mM without considerable changes in Vmax. In the presence of 5 mM Na+ half maximal inhibition of amiloride sensitive Na+ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na+ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H+ seemed to be competitive with a Ki of 20-40 nM. 10 microM amiloride increased the apparent KM for Na+ from 33 mM to 107 mM, while Vmax remained nearly unchanged. IC50 for amiloride was 6 microM at 5 mM Na+ and 36 microM in the presence of 150 mM Na+. Thus, amiloride behaves as a competitive inhibitor with a Ki of about 5 microM. The affinities of Na+ to the transport site (KM approximately 16 mM), to the inhibitory site for protons (KM approximately 21 mM), and to the inhibitory site for amiloride (KM approximately 26 mM) were in the same order of magnitude. In summary, we have presented evidence for the presence of a Na+/H+ exchanger in cultured bovine pigmented ciliary epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)