Complementary cardioprotective effects of flavonoid metabolites and terpenoid constituents of Ginkgo biloba extract (EGb 761) during ischemia and reperfusion.

Hemodynamic and electron spin resonance (ESR) analyses were performed on isolated ischemic and reperfused rat hearts to assess the cardioprotective and antioxidant effects of therapeutically relevant concentrations of Ginkgo biloba extract (EGb 761; 5, 50 or 200 microg/ml), its terpenoid constituents (ginkgolide A; 0.05 microg/ml and ginkgolide B; 0.05, 0.25 or 0.50 microg/ml), and a terpene-free fraction of EGb 761 (CP 205; 5 or 50 microg/ml). Hearts underwent 10 min of low-flow ischemia, 30 min of no-flow global ischemia, and 60 min of reperfusion. Test substances were added to the perfusion fluid during the last 10 min of control perfusion, low-flow ischemia and the first 10 min of reperfusion. A separate group of rats was treated with CP 205 (60 mg/kg/day; p.o.) for 15 days, after which the hearts were perfused with plain buffer. In ESR experiments, the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added to the perfusate to determine the effects of treatments on post-ischemic myocardial free radical generation. Results showed that in vitro exposure of hearts to EGb 761 (5 or 50 microg/ml) or to ginkgolides A and B (both at 0.05 microg/ml), or in vivo pretreatment of the rats with CP 205 delayed the onset of contracture during ischemia. The strong reperfusion-induced elevation of left ventricular end-diastolic pressure observed in untreated hearts was significantly reduced by in vitro exposure to the lowest concentrations of EGb 761, by ginkgolide A, and to a lesser extent by ginkgolide B, or by prior oral treatment with CP 205. Post-ischemic functional recovery.was significantly improved by in vivo administration of CP 205, by perfusion with 5 microg/ml of EGb 761 or with both terpenoids as compared to untreated group but in vitro CP 205 was not effective. ESR analyses revealed that DMPO-OH (the DMPO/hydroxyl radical spin-adduct) concentrations in coronary effluents were markedly decreased by all treatments, except for the lowest concentration of ginkgolide B. Perfusing 5 microg/ml EGb 761 resulted in a better inhibition of baseline DMPO-OH concentration than 5 microg/ml CP 205 (-70 % and -48 % vs. control, respectively), indicating that both terpenoid and flavonoid constituents of EGb 761 are required to produce this effect. CP 205 was significantly more efficient in reducing DMPO-OH concentration when administered in vivo than when applied in vitro, indicating that the antioxidant effect of flavonoid metabolites (formed in vivo) is superior to that of intact flavonol glycosides (present in vitro). Collectively, these findings provide the first evidence that part of the cardioprotection afforded by EGb 761 is due to a specific action of its terpenoid constituents and that this effect involves a mechanism independent of direct free radical-scavenging. Thus, the terpenoid constituents of EGb 761 and the flavonoid metabolites that are formed after in vivo administration of the extract act in a complementary manner to protect against myocardial ischemia-reperfusion injury.

Nitrone spin traps and their pyrrolidine analogs in myocardial reperfusion injury: hemodynamic and ESR implications--evidence for a cardioprotective phosphonate effect for 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide in rat hearts.

Formation of free radicals during reperfusion of the isolated ischemic heart has often been demonstrated by detecting hydroxyl radical spin adducts of the nitrone 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in coronary effluents. However, questions still remain regarding (a) whether the reported cardiovascular effects of nitrone perfusion may affect the formation of spin adducts, and (b) the primary generation of superoxide (O2.-), because of the short persistency of O2.-/DMPO spin adduct. We therefore compared the effects of perfusing 5 mM of two nitrones, DMPO and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) or the two structurally related pyrrolidines, diethyl (2-methyl-2-pyrrolidinyl) phosphonate (DEPMPH) and pyrrolidine (PyH), on postischemic functional recovery of rat hearts subjected to 10 min of low-flow ischemia, 30 min of global ischemia and 60 min of reperfusion. All compounds were added to the perfusate before ischemia, throughout low-flow ischemia and during the initial 10 min of reflow. In one additional group, hearts received DEPMPO only at reflow. Hemodynamic and in vitro ESR evidence is presented indicating that the phosphonate group of DEPMPO and DEPMPH confers these molecules with an enhanced cardioprotective efficacy, unrelated to radical scavenging, acting in synergy with the intrinsic radical trapping effects of the nitronyl group. Continuous-flow ESR spin trapping using 5.7 mM DEPMPO administered at reflow, but not before ischemia, demonstrated for the first time extended formation of O2.- in the reperfused myocardium.

Use of spin-traps during warm ischemia-reperfusion in rat liver: comparative effect on energetic metabolism studied using 31P nuclear magnetic resonance.

Detection of free radicals by electron spin resonance (ESR) proves the involvement of reactive oxygen species (ROS) in reperfused organ injuries. Spin-traps are known to ameliorate hemodynamic parameters in an isolated postischemic heart. The effects of 5 mmol/L DMPO (5,5-dimethyl-1-pyrroline-N-oxide) or DEPMPO (5-(diethlphosphoryl)-5-methyl-1-pyrroline N-oxide) on intracellular pH (pHin) and ATP level were evaluated by 31P nuclear magnetic resonance on isolated rat liver submitted to 1 hour of warm ischemia and reperfusion. At the end of the reperfusion period, during which pHin recovered to its initial value (7.16 +/- 0.03) in all groups, the ATP recovery level (expressed in percentage of initial value) was similar in controls and DEPMPO (60% +/- 5%, n = 6 and 54% +/- 4%, n = 6, respectively), but only 37% +/- 1% in DMPO-treated livers (n = 6) (p < 0.05 versus controls and p < 0.05 versus DEPMPO). Oxidative phosphorylation was not affected by an addition of nitrones on isolated mitochondria extracted from livers not submitted to ischemia-reperfusion. In contrast, mitochondria extracted at the end of the ischemia-reperfusion showed an impairment in the phosphorylation parameters, particularly in the presence of DMPO. Mass spectrum of ischemic liver perchloric acid extracts evidenced probable catabolites in treated groups. The differences in the effect of the two nitrones on energetic metabolism may be explained by the production of deleterious catabolites by DMPO as compared to DEPMPO. Even though a specific radical scavenging effect could be operative in the liver, our results indicate that catabolic effects were predominant. The absence of deleterious effects of DEPMPO in contrast to DMPO on the liver energetic metabolism was evidenced, allowing the use of DEPMPO for ESR detection.