Pharmacokinetics and metabolism of cinnamic acid derivatives and flavonoids after oral administration of Brazilian green propolis in humans.

Brazilian green propolis (BGP) has chemical compounds from botanical origin that are mainly cinnamic acid derivatives (artepillin C, baccharin, and drupanin) and flavonoids (kaempferide and 6-methoxykaempferide). These compounds are expected to play an important role in the pharmacological activities of BGP. However, there is little known about the pharmacokinetics and metabolism of these compounds after oral administration of BGP. The aim of this study is to investigate the pharmacokinetics and metabolism of BGP components in humans. Twelve volunteers received 3 capsules containing 360 mg of BGP ethanol extract powder. Plasma samples were collected before and up to 24 h after the intake of BGP capsules. The collected plasma samples with or without hydrolysis by the deconjugating enzyme were analyzed by LC/MS/MS. After enzymatic hydrolysis, the Cmax values of artepillin C and drupanin, which were detected mainly in plasma after ingestion of BGP capsules, were 1255 ± 517 and 2893 ± 711 nM, respectively, of which 89.3% and 88.2% were found to be the phenolic glucuronide conjugate. This is the first time that the pharmacokinetics of the BGP components of human metabolites have been reported. Our results could provide useful information for the design and interpretation of studies to investigate the mechanisms and pharmacological effects of BGP.

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver.

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on expression/activity of the main DDS phase-II-metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxidation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.

Beneficial effect of spironolactone administration on ethynylestradiol-induced cholestasis in the rat: involvement of up-regulation of multidrug resistance-associated protein 2.

The effect of spironolactone (SL) administration on 17alpha-ethynylestradiol (EE)-induced cholestasis was studied, with emphasis on expression and activity of Mrps. Adult male Wistar rats were divided into the following groups: EE (5 mg/kg daily for 5 days, s.c.), SL (200 micromol/kg daily for 3 days, i.p.), EE+SL (same doses, SL administered the last 3 days of EE treatment), and controls. SL prevented the decrease in bile salt-independent fraction of bile flow induced by EE, in association with normalization of biliary excretion of glutathione. Western blot studies indicate that EE decreased the expression of multidrug resistance-associated protein 2 (Mrp2) by 41% and increased that of Mrp3 by 200%, whereas SL only affected Mrp2 expression (+60%) with respect to controls. The EE+SL group showed increased levels of Mrp2 and Mrp3 to the same extent as that registered for the individual treatments. Real-time polymerase chain reaction studies indicated that up-regulation of Mrp2 and Mrp3 by SL and EE, respectively, was at the transcriptional level. To estimate Mrp2 and Mrp3 activities, apical and basolateral excretion of acetaminophen glucuronide (APAP-glu), a common substrate for both transporters, was measured in the recirculating isolated perfused liver model. Biliary/perfusate excretion ratio was decreased in EE (-88%) and increased in SL (+36%) with respect to controls. Coadministration of rats with SL partially prevented (-53%) impairment induced by EE in this ratio. In conclusion, SL administration to EE-induced cholestatic rats counteracted the decrease in bile flow and biliary excretion of glutathione and APAP-glu, a model Mrp substrate, findings associated with up-regulation of Mrp2 expression.

Effect of spironolactone on the expression of rat hepatic UDP-glucuronosyltransferase.

Spironolactone (SL) increases the glucuronidation rate of several compounds. We analyzed the molecular basis of changes occurring in major rat liver UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform of family 2, in response to SL. UGT activity toward bilirubin, ethynylestradiol and p-nitrophenol was assayed in native and activated microsomes. Protein and mRNA levels were determined by Western and Northern blotting. The lipid composition and physicochemical properties of the microsomal membrane were also analyzed. Glucuronidation rates of bilirubin and ethynylestradiol (at both 3-OH and 17 beta-OH positions), determined in UDP-N-acetylglucosamine-activated membranes, were increased in SL group. Western blot analysis revealed increased levels of UGT1A1 and 1A5 (bilirubin and 3-OH ethynylestradiol conjugation), and 2B1 (17 beta-OH ethynylestradiol conjugation). Northern blot studies suggested transcriptional regulation by the steroid. Analysis of UGT activity in native vs. alamethicin-activated microsomes indicated increased latency, which was not associated to changes in physicochemical properties of the microsomal membrane. p-Nitrophenol glucuronidation rate and mRNA and protein levels of UGT1A6, the main isoform conjugating planar phenols, were not affected by the inducer. The data suggest transcriptional regulation of specific isoforms of hepatic UGT by SL, thus explaining previously reported increases in UGT activity toward selective substrates.