Detection of synergistic combinations of Baccharis extracts with terbinafine against Trichophyton rubrum with high throughput screening synergy assay (HTSS) followed by 3D graphs. Behavior of some of their components.

Forty four extracts from nine Baccharis spp. from the Caulopterae section were tested in combination with terbinafine against Trichophyton rubrum with the HTSS assay at six different ratios with the aim of detecting those mixtures that produced a ≥50% statistically significant enhancement of growth inhibition. Since an enhanced effect of a combination respective of its components, does not necessarily indicate synergism, three-dimensional (3D) dose-response surfaces were constructed for each selected pair of extract/antifungal drug with the aid of CombiTool software. Ten extracts showed synergistic or additive combinations which constitutes a 22% hit rate of the extracts submitted to evaluation. Four flavonoids and three ent-clerodanes were detected in the active Baccharis extracts with HPLC/UV/ESI-MS methodology, all of which were tested in combination with terbinafine. Results showed that ent-clerodanes but not flavonoids showed synergistic or additive effects. Among them, bacchotricuneatin A followed by bacrispine showed synergistic effects while hawtriwaic acid showed additive effects.

Phosphoinositide 3-kinase/protein kinase B signaling pathway is involved in estradiol 17ß-D-glucuronide-induced cholestasis: complementarity with classical protein kinase C.

UNLABELLED: Estradiol 17ß-D-glucuronide (E(2)17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E(2)17G-induced cholestasis. E(2)17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E(2)17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E(2)17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E(2)17G-induced cholestasis. In isolated perfused rat liver, an intraportal injection of E(2)17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [(3)H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. CONCLUSION: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E(2)17G through sustained internalization of canalicular transporters endocytosed via cPKC.

Preventive effect of silymarin against taurolithocholate-induced cholestasis in the rat.

Increased amounts of monohydroxylated bile salts (BS) have been found in neonatal cholestasis, parenteral nutrition-induced cholestasis and Byler's disease, among others. We analyzed whether the hepatoprotector silymarin (SIL), administered i.p. at the dose of 100mg/kg/day for 5 days, prevents the cholestatic effect induced by a single injection of the model monohydroxylated BS taurolithocholate (TLC, 30 micromol/kg, i.v.) in male Wistar rats. TLC, administered alone, reduced bile flow, total BS output, and biliary output of glutathione and HCO(3)(-) during the peak of cholestasis (-75, -67, -81, and -80%, respectively, P<0.05). SIL prevented partially these alterations, so that the drops of these parameters induced by TLC were of only -41, -25, -60, and -64%, respectively (P<0.05 vs. TLC alone); these differences between control and SIL-treated animals were maintained throughout the whole (120 min) experimental period. Pharmacokinetic studies showed that TLC decreased the intrinsic fractional constant rate for the canalicular transport of both sulfobromophthalein and the radioactive BS [14C]taurocholate by 60 and 68%, respectively (P<0.05), and these decreases were fully and partially prevented by SIL, respectively. SIL increased the hepatic capability to clear out exogenously administered TLC by improving its own biliary excretion (+104%, P<0.01), and by accelerating the formation of its non-cholestatic metabolite, tauromurideoxycholate (+70%, P<0.05). We conclude that SIL counteracts TLC-induced cholestasis by preventing the impairment in both the BS-dependent and -independent fractions of the bile flow. The possible mechanism/s involved in this beneficial effect will be discussed.