Dalia M. Kopustinskiene

Uncoupling and antioxidant effects of ursolic acid in isolated rat heart mitochondria.

Ursolic acid (1), a pentacyclic triterpene acid, is one of the major components of certain traditional medicinal plants and possesses a wide range of biological effects, such as anti-inflammatory, antioxidative, and cytotoxic activities. Furthermore, 1, when present at 1.6-5 ng/mL concentrations in commercial herbal preparations used for patients with cardiac disorders, may also exert pro-cardiac activities. There are several indirect suggestions that the cardioprotective mechanism of ursolic acid could involve the mitochondria; however the mechanism of action is still not known. Therefore, the effects of 0.4-200 ng/mL ursolic acid (1) on the functions of isolated rat heart mitochondria oxidizing either pyruvate and malate, succinate, or palmitoyl-l-carnitine plus malate were investigated. It was found that 1 induced a statistically significant uncoupling of oxidative phosphorylation. A statistically significant decrease in H₂O₂ production in the mitochondria was observed after incubation with 5 ng/mL 1. This effect was comparable to the effectiveness of the classical uncoupler carbonyl cyanide 3-chlorophenylhydrazone. Since mild mitochondrial uncoupling has been proposed as one of the mechanisms of cardioprotection, the present results indicate that ursolic acid (1) has potential use as a cardioprotective compound.

Adenine Nucleotide Translocase Mediates the KATP-Channel-Openers-Induced Proton and Potassium Flux to the Mitochondrial Matrix

KATP channel openers have been shown to protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, although their mechanisms of action have not been fully clarified. In this study we investigated the influence of the adenine nucleotide translocase (ANT) inhibitors–carboxyatractyloside (CAT) and bongkrekic acid (BA)–on the diazoxide- and pinacidil-induced uncoupling of isolated rat heart mitochondria respiring on pyruvate and malate (6 + 6 mM). We found that both CAT (1.3 μM) and BA (20 μM) markedly reduced the uncoupling of mitochondrial oxidative phosphorylation induced by the KATP channel openers. Thus, the uncoupling effect of diazoxide and pinacidil is evident only when ANT is not fixed by inhibitors in neither the C- nor the M-conformation. Moreover, the uncoupling effect of diazoxide and pinacidil was diminished in the presence of ADP or ATP, indicating a competition of KATP channel openers with adenine nucleotides. CAT also abolished K+-dependent mitochondrial respiratory changes. Thus ANT could also be involved in the regulation of KATP-channel-openers-induced K+ flux through the inner mitochondrial membrane.

Diazoxide and Pinacidil Uncouple Pyruvate-Malate-Induced Mitochondrial Respiration

We investigated the effects of KATP channel openers diazoxide and pinacidil on the respiration rate and membrane potential (ΔΨ) of rat heart mitochondria, oxidizing pyruvate and malate. Diazoxide and pinacidil (58.8–1348.3 μM) increased the V2 (-ADP) respiration rate accordingly by 13–208% and 30–273% and decreased the ΔΨ by 2–17% and 6–55%. These effects were also similar in the respiration medium without K+. Moreover, carboxyatractyloside completely abolished diazoxide- and pinacidil-induced uncoupling, indicating a role for the mitochondrial adenine nucleotide translocase in this process.

Direct effects of K(ATP) channel openers pinacidil and diazoxide on oxidative phosphorylation of mitochondria in situ.

KATP channel openers protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, however, the protection mechanisms have not been fully clarified yet. Since the skinned fibers technique gives an opportunity to investigate an entire population of mitochondria in their native milieu, in this study we have investigated the effects of KATP channel openers pinacidil and diazoxide on the respiration rate of rat heart mitochondria in situ, oxidizing physiological substrates pyruvate and malate (6+6 mM). Respiration rates were recorded by the means of Clark-type oxygen electrode in the physiological salt solution (37°C). Our results showed that both pinacidil and diazoxide (60-1250 µM) in a concentration-dependent manner increased pyruvate-malate supported State 2 respiration rate of skinned cardiac fibers (59.1 ± 5.1 nmol O/min/mg fiber dry weight, RCI 2.6 ± 0.2, n=4) by 15-120%. Moreover, diazoxide did not affect, whereas pinacidil (60-1250 µM) decreased the State 3 respiration rate of skinned cardiac fibers (116.6 ± 13.6 nmol O/min/mg fiber dry weight, RCI 2.3 ± 0.2, n=4) by 4-27%. Thus, common effect for both KATP channel openers is uncoupling of pyruvate and malate oxidizing mitochondria in skinned cardiac fibers, whereas pinacidil under same conditions also inhibits mitochondrial respiratory chain. Since mitochondria in situ resemble to the great extent mitochondria in vivo, our results suggest that uncoupling and/or respiratory chain inhibition could play a role in the cardioprotection by KATP channel openers.

What controls the outer mitochondrial membrane permeability for ADP: facts for and against the role of oncotic pressure

In our study 10% of bovine serum albumin was added to the physiological incubation medium to mimic the oncotic pressure of the cellular cytoplasm and to test for its effect on the respiration of isolated rat heart mitochondria, saponin- or saponin plus crude collagenase (type IV)-treated heart muscle fibers and saponin-treated rat quadriceps muscle fibers. Pyruvate and malate were used as substrates. We found that albumin slightly decreased the maximal ADP-stimulated respiration rate only for saponin-treated heart muscle fibers. The apparent Km ADP of oxidative phosphorylation increased significantly, by 70-100%, for isolated heart mitochondria, saponin plus collagenase-treated heart muscle fibers and for saponin-treated quadriceps muscle fibers but remained unchanged for saponin-treated heart muscle fibers. The saponin-treated heart muscle fibers were characterized by a very high control apparent Km ADP value (234+/-24 microM ADP) compared with other preparations (14-28 microM ADP). The results suggest that in vivo the oncotic pressure is not the relevant factor causing the low outer mitochondrial membrane permeability for ADP in cardiomyocytes, in contrast to quadriceps muscle cells. It is likely that the outer mitochondrial membrane-bound protein(s) which is supposed to remain in saponin-treated heart muscle fibers is responsible for this property of the membrane.

Direct Effects of (−)-Epicatechin and Procyanidin B2 on the Respiration of Rat Heart Mitochondria

Flavonol (−)-epicatechin and its derived dimer procyanidin B2, present in high amounts in cocoa products, have been shown to exert beneficial effects on the heart and cardiovascular system; however, their mechanism of action has not been fully elucidated. We studied effects of (−)-epicatechin and procyanidin B2 on the oxidative phosphorylation of isolated rat heart mitochondria. (−)-Epicatechin and procyanidin B2 had stimulating effect (up to 30% compared to control) on substrate-driven (State 2) mitochondrial respiration. Their effect was dependent on the respiratory substrates used. (−)-Epicatechin at higher concentrations (from 0.27 µg/mL) significantly decreased (up to 15%) substrate- and ADP-driven (State 3) mitochondrial respiration in case of pyruvate and malate oxidation only. Procyanidin B2 (0.7–17.9 ng/mL) inhibited State 3 respiration rate up to 19%, the most profound effect being expressed with succinate as the substrate. (−)-Epicatechin at concentrations of 0.23 µg/mL and 0.46 µg/mL prevented loss of the cytochrome c from mitochondria when substrate was succinate, supporting the evidence of membrane stabilizing properties of this flavonol. Thus, both (−)-epicatechin and procyanidin B2 directly influenced mitochondrial functions and the observed effects could help to explain cardiometabolic risk reduction ascribed to the consumption of modest amounts of cocoa products.

Formulation and characterization of Turkish oregano microcapsules prepared by spray-drying technology

Abstract The aim of this study was optimization of spray-drying process conditions for microencapsulation of Turkish oregano extract. Different concentrations of maltodextrin and gum arabic as encapsulating agents (wall material) as well as influence of selected processing variables were evaluated. The optimal conditions were maintained on the basis of the load of main bioactive compounds – ursolic, rosmarinic acids and carvacrol – in prepared microparticles after comparison of all significant response variables using desirability function. Physicomechanical properties of powders such as flowability, wettability, solubility, moisture content as well as product yield, encapsulation efficiency (EE), density, morphology and size distribution of prepared microparticles have been determined. The results demonstrated that the optimal conditions for spray-drying mixture consisted of two parts of wall material solution and one part of ethanolic oregano extract when the feed flow rate was 40 mL/min and air inlet temperature –170 °C. Optimal concentration of wall materials in solution was 20% while the ratio of maltodextrin and gum arabic was 8.74:1.26.

The Effect of Ginkgo biloba Extract on Mitochondrial Oxidative Phosphorylation in the Normal and Ischemic Rat Heart

Free radical‐induced myocardial damage and impairment of vascular endothelium‐dependent relaxation are amongst the most important mechanisms responsible for ischemic heart injury. Ginkgo biloba leaf extract (GE) has been reported to improve blood circulation in the brain and have a beneficial impact on the cardiovascular system but its cardioprotective effects have not been elucidated yet. Therefore, this study investigated the influence of GE in 70% ethanol (1:5) administered orally to rats on the functions of isolated heart mitochondria under normal and ischemic conditions. Wistar rats were given GE or ethanol (solvent control) at a dosage of 0.32 mL/kg in drinking water for 10 and 18 days, while the control animals received untreated drinking water. Mitochondrial respiration rates were determined oxygraphically. Pyruvate and malate, succinate or palmitoyl‐l‐carnitine and malate were used as substrates.

The Role of Catechins in Cellular Responses to Oxidative Stress

Catechins are polyphenolic compounds—flavanols of the flavonoid family found in a variety of plants. Green tea, wine and cocoa-based products are the main dietary sources of these flavanols. Catechins have potent antioxidant properties, although in some cases they may act in the cell as pro-oxidants. Catechins are reactive oxygen species (ROS) scavengers and metal ion chelators, whereas their indirect antioxidant activities comprise induction of antioxidant enzymes, inhibition of pro-oxidant enzymes, and production of the phase II detoxification enzymes and antioxidant enzymes. Oxidative stress and ROS are implicated in aging and related dysfunctions, such as neurodegenerative disease, cancer, cardiovascular diseases, and diabetes. Due to their antioxidant properties, catechins may be beneficial in preventing and protecting against diseases caused by oxidative stress. This article reviews the biochemical properties of catechins, their antioxidant activity, and the mechanisms of action involved in the prevention of oxidative stress-caused diseases.

In vitro permeation studies of phenolics from horse chestnut seed gels prepared with different polyacrylic acid polymer derivatives

Abstract The aim of this study was to investigate the effects of polyacrylic acid polymers (Ultrez 10, Ultrez 20, Carbopol 980, and Carbopol 940) on the viscosity and the in vitro permeation of phenolic compounds from the gel prepared from natural horse chestnut seed extract. Experiments were performed in the presence and in the absence of peppermint oil (Mentha piperita). Our results showed that peppermint oil decreased the viscosity of the gels and permeation of phenolic compounds from all gel samples. Results show that the highest content of phenolic compounds (1.758 μg cm-2) permeated in vitro from gel based on Carbopol Ultrez 20 without peppermint oil added (p < 0.05 vs. other tested polymers).