The effect of crataegus fruit extract and some of its flavonoids on mitochondrial oxidative phosphorylation in the heart.

Crataegus (Hawthorn) fruit extracts (CE) are widely used for the treatment of various cardiovascular diseases (arrhythmias, heart failure, myocardial weakness, etc). Despite the fact that many of these diseases are associated with disturbances of the mitochondria, no data have been found on the effect of CE on their function. The aim of this study was to perform an oxygraphic investigation of the effect of CE (in concentration range from 70 ng/mL to 13.9 microg/mL of Crataegus phenolic compounds (PC)) and its several pure flavonoids on isolated rat heart mitochondria respiring on pyruvate+malate, succinate and palmitoyl-L-carnitine+malate. CE at doses under 278 ng/mL of PC had no effect on mitochondrial functions. At concentrations from 278 ng/mL to 13.9 microg/mL of PC, CE stimulated State 2 respiration by 11%-34% with all used substrates, and decreased the mitochondrial membrane potential by 1.2-4.4 mV measured with a tetraphenylphosphonium-selective electrode and H2O2 production measured fluorimetrically. Similar uncoupling effects on mitochondrial respiration were observed with several pure CE flavonoids. The highest CE concentration also slightly reduced the maximal ADP-stimulated and uncoupled respiration, which might be due to inhibition of the mitochondrial respiratory chain between flavoprotein and cytochrome c. Whether or not the uncoupling and other effects of CE on mitochondria may be realized in vivo remains to be determined.

[Antioxidant activity of tinctures prepared from hawthorn fruits and motherwort herb]. / Antioxidacní aktivita tinktur pripravených z hlohových plodu a nati srdecníku.

The paper aimed to determine and compare the antioxidant activity of two tinctures and selected flavonoids present in the tinctures. The tinctures were prepared from hawthorn fruits and motherwort herb. The group of selected flavonoids included rutin, quercetin, hyperosid, epicatechine, and procyanidin B2. Two methods were employed to determine antioxidant activities: the inhibition of peroxynitrite-induced nitration of tyrosine was measured, and the ability to quench radicals was examined by of the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Both tinctures under study were able to quench radicals and reactive forms of oxygen and nitrogen. The motherwort herb tincture was more effective, but no direct effect of phenolic compounds on the extent of antioxidant activity was demonstrated. The total content of polyphenols in the motherwort herb tincture was higher by 163 %, nevertheless antiperoxynitrite activity was higher just by 19 % (p < 0.05), and the ability to reduce DPPH differed in both tinctures in a minimal manner. Of the individual flavonoids, the substances present in the tinctures in higher concentrations contributed most to antioxidant activity. In the hawthorn fruit tincture it was epicatechine and hyperosid, whereas in the motherwort herb tincture it was rutin.

The effect of flavonoids on rat heart mitochondrial function.

In this study the effects of flavonoids (quercetin and its derivatives as rutin, hyperoside, quercitrin) on the oxidative phosphorylation in rat heart mitochondria were investigated. We found that all investigated flavonoids possessed uncoupling activity. Thus, quercetin, rutin, and quercitrin in dose-dependent manner induced a stimulation of the State 2 respiration rate by 10-110% with pyruvate + malate as substrate. The maximal stimulation of the State 2 respiration rate was obtained at 1.08 ng/ml of quercetin, 15.2 ng/ml of hyperoside and 44.4 ng/ml of rutin. Quercitrin had clearly lower effects. The State 3 respiration rate was also affected by flavonoids. Quercetin (from 1.08 ng/ml), hyperoside (from 10 ng/ml) and rutin (from 60 ng/ml) caused the decrease in State 3 respiration rate by 16-51%. We assume, that partial mitochondrial uncoupling (without affecting the State 3 respiration rate) induced by flavonoids could have a cardioprotective effect, and that mitochondria could be involved in the mechanism of this process.

Relevance of fatty acid oxidation in regulation of the outer mitochondrial membrane permeability for ADP.

The present study on saponin-treated rat heart muscle fibers has revealed a new function of the fatty acid oxidation system in the regulation of the outer mitochondrial membrane (OMM) permeability for ADP. It is found that oxidation of palmitoyl-CoA+carnitine, palmitoyl-L-carnitine and octanoyl-L-carnitine (alone or in combination with pyruvate+malate) dramatically decreased a very high value of apparent K(m) of oxidative phosphorylation for ADP. Octanoyl-D-carnitine, as well as palmitate, palmitoyl-CoA, and palmitoyl-L-carnitine were not effective in this respect, when their oxidation was prevented by the absence of necessary cofactors or blocked with rotenone. Our data suggest that oxidation, but not transport of fatty acids into mitochondria, induces an increase in the OMM permeability for ADP.

The effects of ischemia and experimental conditions on the respiration rate of cardiac fibers.

The mitochondrial respiratory parameters were measured in situ, i.e. in saponin-skinned rabbit cardiac fibers and in fibers treated with saponin + collagenase. It was found that the decrease of maximal ADP-stimulated respiration rate of saponin-skinned fibers with pyruvate + malate under the conditions of total ischemia (0.5-1.5 h) is less pronounced as compared to isolated mitochondria. Maximal succinate oxidation rate (+ADP), however, was not different from control (1 h ischemia) but it exceeded the control level when measured in the medium supplemented with cytochrome c. It was also demonstrated that treatment of fibers with collagenase alone or in combination with saponin significantly (almost 2 fold) enhanced the maximal ADP-stimulated respiration rate if compared with saponin-skinned fibers. The data obtained suggest that mitochondrial respiration in saponin-skinned rabbit cardiac fibers is not completely revealed, most probably, due to insufficient permeabilization of sarcolemma by saponin and, thus, inadequate accessibility of mitochondria to exogenous substrates, ADP in particular. These parameters can be improved by pre-treatment of fibers with collagenase.

The effect of collagenase and temperature on mitochondrial respiratory parameters in saponin-skinned cardiac fibers.

The results of a comparative study of the respiration rates of mitochondria in saponin-skinned rat cardiac fibers (SF) and in fibers treated with saponin and collagenase (SCF) suggest that only about half of the whole population of mitochondria manifest their activity in SF, in contrast to SCF, in response to extracellular substrates of oxidative phosphorylation. The apparent K(m) value for ADP with succinate as substrate, which was as high as 330 +/- 32 microM in SF in SF at 20 degrees C, decreased about 2-fold in SCF at the same temperature and in SF at 37 degrees C, and decreased further to 67 +/- 8 microM in SCF at 37 degrees C. Thus, weakening or breaking of cellular contacts by collagenase and the temperature-dependence of diffusion of substrates such as ADP, seem to be important factors that determine the respiratory activity and regulatory parameters of mitochondria in saponin-permeabilized cardiomyocytes.