Jurga Bernatoniene
email: jurgabernatoniene@yahoo.com

Daiva Majiene

Rimantas Peciura

Ale Laukeviciene

Ruta Bernatoniene

Dalia Kopustinskiene

Tauras Mekas

Arturas Kasauskas

The Effect of Ginkgo biloba extract on mitochondrial oxidative phosphorylation in the normal and ischemic rat heart

Keywords: Ginkgo biloba, heart mitochondrial respiration, ischemic injury, cardioprotection Ginkgo biloba, heart mitochondrial respiration, ischemic injury, cardioprotection ADP -adenosine 5-diphosphate, BSA -bovine serum albumin, GE -Ginkgo biloba leaf extract, ROS -reactive oxygen species

INTRODUCTION

Ischemic heart injury is the leading cause of morbidity and mortality although the molecular mechanisms responsible for it, as well as for its prevention, have not been fully clarified yet (for recent review see [START_REF] Ostadal | The past, the present and the future of experimental research on myocardial ischemia and protection[END_REF]. Free-radical induced myocardial damage is one of the main causes of the cardiovascular pathology. [START_REF] Lisa | Mitochondria and vascular pathology[END_REF]. The major targets of free radical 3 attacks are supposed to be heart mitochondria which are the main sites of cellular energy supply [START_REF] Korshunov | High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria[END_REF][START_REF] Skulachev | New data on biochemical mechanism of programmed senescence of organisms and antioxidant defense of mitochondria[END_REF]. Therefore, during investigation of myocardial damage it is very important to evaluate mitochondrial function changes responsible for 1) impaired cardiac muscle contractions and 2) cell death.

Recent studies demonstrate that herbal preparations could decrease the effects of pathogenic processes such as oxidative stress, improving rheological properties of the blood and increasing blood flow to the various organs of the body [START_REF] Addabbo | The Krebs cycle and mitochondrial mass are early victims of endothelial dysfunction: proteomic approach[END_REF][START_REF] Yapar | Protective role of Ginkgo biloba against hepatotoxicity and nephrotoxicity in uranium-treated mice[END_REF]. Gingko biloba preparations are most widely used for this purpose; however, their action was investigated mainly on brain cells and subcellular organelles [START_REF] Gertz | Review about Ginkgo biloba special extract EGb 761 (Ginkgo)[END_REF]. It was shown that Ginkgo biloba preparations, especially EGb 761 (1), improved blood flow to the brain, particularly, in the region of microcirculation, (2) inactivated toxic oxygen radicals and prevented cell damage, (3) inhibited platelet-activating factor responsible for a number of CNS pathologies [START_REF] Abdel-Kader | Stabilization of mitochondrial function by Ginkgo biloba extract (EGb 761)[END_REF][START_REF] Mahadevan | Multifaceted therapeutic benefits of Ginkgo biloba L.: chemistry, efficacy, safety, and uses[END_REF]. Furthermore, in a randomized, double-blind, placebocontrolled, parallel design trial Ginkgo biloba (EGb 761, 300 mg a day for 4 weeks) was shown to have no impact on indices on blood coagulation and no adverse bleeding effects [START_REF] Gardner | Effect of Ginkgo biloba (EGb 761) and aspirin on platelet aggregation and platelet function analysis among older adults at risk of cardiovascular disease: a randomized clinical trial[END_REF]. These broad pharmacological activities of Ginkgo biloba are predetermined by two active ingredient groups that have different functions: flavonoid fraction (quercetin, kaempferol, isorhamnetin glycosides) and terpenoid fraction (ginkgolides; bilobalide). Flavonoid fraction was demonstrated to neutralize effectively free radicals -superoxide anions, hydroxyl radicals and NO [START_REF] Oyama | Ginkgo biloba extract protects brain neurons against oxidative stress induced by hydrogen peroxide[END_REF]. Also, flavonoids influenced hemodynamic disorders, vasomotoric functions of ischemia zones, permeability of capillaries, ion regulation of cellular homeostasis, and some intracellular enzymes -phospholipases, phosphodiesterases and protein kinases [START_REF] Defeudis | Ginkgo biloba extracts and cancer: a research area in its infancy[END_REF]. Terpenoid fraction controlled platelet-activating factor, possessed vaso-relaxant properties, improved cerebral blood flow [START_REF] Kleijnen | Ginkgo biloba for cerebral insufficiency[END_REF]. In most investigations, GE rich in both flavonoid and terpenoid mixtures was used. The obtained results showed that active widely used for prevention of numerous neurological disorders (dementia, macula degeneration, tinnitus, winter depression, difficulties in concentration and learning capacity, headache, etc) [START_REF] Leistner | Ginkgo biloba and ginkgotoxin[END_REF].

EGb 761 exerted beneficial effects in endothelial dysfunction increasing production of endothelial nitric oxide. The putative mechanism for this effect was suggested to be increased endothelial nitric oxide synthase (eNOS) promoter activity and eNOS expression in vitro (Koltemann et al., 2007). Also, Ginkgo biloba EGb 761 attenuated arteriosclerotic nanoplaque formation, reduced lipoprotein a concentration, increased concentration of vasoactive substances -cAMP and cGMP [START_REF] Siegel | The importance of scavenging reactive oxygen species in anti-aging medicine[END_REF]. Recently the effect of different active ingredients of Ginkgo biloba and the biological effect of its tinctures and extracts has been intensively studied not only on the brain, but also on the heart function. Trumbeckaite with co-workers showed that GE (1:5) exerted uncoupling activity in heart mitochondria in vitro in normal conditions [START_REF] Trumbeckaite | Effect of Ginkgo biloba extract on the rat heart mitochondrial function[END_REF][START_REF] Baliutyte | Effects of Ginkgo biloba extract on heart and liver mitochondrial functions: mechanism(s) of action[END_REF]. It has been demonstrated that EGb 761, rich in bilobalides, decreased ischemic damage in mitochondria and protected the components of mitochondrial respiratory chain [START_REF] Shen | Efficiency of Ginkgo biloba extract (EGb 761) in antioxidant protection against myocardial ischemia and reperfusion injury[END_REF][START_REF] Janssens | Protection by bilobalide of the ischaemiainduced alterations of the mitochondrial respiratory activity[END_REF]. The terpenoid constituents of EGb 761 and the flavonoid metabolites, that were formed after in vivo administration of the extract, acted in a complementary manner to protect against myocardial ischemia-reperfusion injury [START_REF] Liebgott | Complementary cardioprotective effects of flavonoid metabolites and terpenoid constituents of Ginkgo biloba extract (EGb 761) during ischemia and reperfusion[END_REF]. These findings have led us to consider the possibility that GE could protect heart from injuries. However, there is very few data about the influence of GE on the energy metabolism in heart during ischemia. Therefore, the aim of this study was to investigate the effects of GE administrated per os for 10 and 18 days on rat heart mitochondrial functions under normal and ischemic conditions.

MATERIAL AND METHODS

Chemicals

Preparation of Ginkgo extract

Dried Ginkgo biloba L. leaves (Poland, Acorus Calamus) were extracted using 70% ethanol (1:5); particle size in the extract ranged from 2 to 3 mm, the production method was percolation, and the flow speed of extract -0.5 mL/min [START_REF] Bernatoniene | Technology and analysis of Ginkgo tincture[END_REF]. GE contained 3.4 ± 0.08 mg/ mL phenolic compounds. Several classes of flavonoids: quercetin -8.8 ± 2 µg/mL, hyperoside -41.2 ± 4 µg/mL, rutin -227.5 ± 8 µg/mL, and quercetrin -93.1 ± 7 µg/mL were identified in this extract by HPLC analysis [START_REF] Trumbeckaite | Effect of Ginkgo biloba extract on the rat heart mitochondrial function[END_REF].

GE administration per os to rats

Male Wistar rats weighing 250-300 g were used for our study. GE or ethanol (solvent control) were administered to rats at a dosage of 0.32 mL/kg in drinking water for 10 and 18 days, while control animals received untreated drinking water. The animals were handled according to the rules defined by the European Convention for the protection of Vertebrate Animals Used for Experimental and Other Purposes, the experiments were approved by Lithuanian State Food and Veterinary service (License No.0006).

Isolation of rat heart mitochondria

Mitochondria were isolated by differential centrifugation procedure [START_REF] Scholte | The localization of mitochondrial creatine kinase, and its use for the determination of the sidedness of submitochondrial particles[END_REF].

Hearts were excised and divided into two halves. Control heart halves were rinsed in ice-cold 0.9% KCl solution. Heart halves subjected to total ischemia, were rinsed in warm (37°C) KCl solution, placed in a humidified thermostated chamber and allowed to autolyse at 37°C for 45 min. After these procedures, the heart tissues were cut into small pieces and homogenized in a glass-Teflon homogenizer in the medium containing 160 mmol/L KCl, 10 mmol/L NaCl, 20 mmol/L Tris-HCl, 5 mmol/L EGTA and 1 mg/mL bovine serum albumin (BSA; fraction V, A4503, Sigma) (pH 7.4, 2°C). The homogenate was centrifuged at 750×g for 5 min, then the supernatant was re-centrifuged F o r P e e r R e v i e w 6 at 10 000×g for 10 min and the pellet was washed once (10 min 10 000×g) in the isolation medium without BSA, suspended in it and kept on ice. The mitochondrial protein concentration was determined by the biuret method [START_REF] Gornall | Determination of serum proteins by means of the biuret reaction[END_REF] using BSA as standard. The final mitochondrial protein concentration in all experiments was 0.5 mg/mL.

Measurement of mitochondrial respiration rate

Oxygen uptake rates were recorded at 37 o C by means of the Clark-type electrode system in a solution containing 20 mmol/L imidazole, 20 mmol/L taurine, 0.5 mmol/L dithiothreitol, 1.6 mmol/L MgCl 2 , 100 mmol/L MES, 3 mmol/L KH 2 PO 4 , 3.0 mmol/L CaK 2 EGTA, and 7.1 mmol/L K 2 EGTA (free Ca 2+ concentration -0.1 µmol/L) (pH 7.1 adjusted with KOH at 37 o C) with 6 mmol/L pyruvate + 6 mmol/L malate, 12 mmol/L succinate (+2 mmol/L amytal), and 9 µmol/L palmitoyl-L-carnitine + 0.24 mmol/L malate as substrates. Mitochondrial respiration rates were expressed as nmol O 2 /min/mg protein. The solubility of oxygen was estimated to be 211 nmol O 2 /ml [START_REF] Holtzman | Calibration of the oxygen polarograph by the depletion of oxygen with hypoxanthine-xanthine oxidase-catalase[END_REF].

Measurement of mitochondrial ROS generation

The generation of ROS in mitochondria was monitored using the Amplex Red assay for H 2 O 2 . The evaluation of ROS levels was performed at 37 °C in the same medium as for mitochondrial respiration, which was supplemented with mitochondria (0.25 mg/mL), 6 mmol/L pyruvate + 6 mmol/L malate, 10 µmol/L Amplex Red, 1 U/mL horseradish peroxidase, and 1 µmol/L antimycin A and continuously stirred. Fluorescence of the oxidized probe was measured with a Thermo Scientific fluorometer using the excitation at 544 nm and emission at 590 nm.

Statistics

Statistical analysis was performed using Prism 3.0 program (GraphPad Software Inc., La Jolla, CA, USA). The results are presented as means ± S.E. of 5 independent experiments. The data were analysed with repeated measures analysis of variance (ANOVA) followed by Tukey's multiple comparison test. p < 0.05 was taken as the level of significance.

RESULTS

In our study, mitochondrial functions were evaluated by recording mitochondrial respiration rate in the State 2 (substrate oxidation, V 2 ), State 3 (substrates + 1 mmol/L ADP, V 3 ) and State 3 in the presence of exogenous cytochrome c (32 µmol/L, V 3+Cyt c ). Pyruvate and malate, succinate or palmitoyl-L-carnitine and malate were used as substrates. Changes in V 2 reflected alterations of the permeability of the inner mitochondrial membrane to protons or ions, and, together with calculated respiratory control index (RCI = V 3 /V 2 ), allowed to determine the effectiveness of ADP phosphorylation in mitochondria. V 3 reflected the capacity of mitochondria to synthesize ATP. The degree of respiration stimulation by exogenous cytochrome c (V 3+Cyt c /V 3 ) allowed for assessing the integrity of the outer mitochondrial membrane.

First, we performed solvent control test and found that rat treatment per os with ethanol for 10 or 18 days using the same concentration as in studied GE, 0.32 mL/kg, did not influence respiration rates of isolated heart mitochondrial oxidizing all substrates used in our study (see Table ).

Fig. 1 shows that under normal non-ischemic conditions, 10 days and 18 days administration of GE did not significantly affected State 2 respiration of mitochondria oxidizing complex-I dependent substrates pyruvate and malate, but induced a 14% (p < 0.01)and 26% (p <0.001) increase in State 3 respiration rate, respectively, compared to control. 45 min ischemia had a tendency to increase State 2 respiration rate and caused a significant decrease in V 3 by 60% (p < 0.001), as well as in RCI by 64% (p < 0.001). The intactness of outer mitochondrial membrane was assessed by cytochrome c test. Cytochrome c effect was 54% higher (p < 0.001) in mitochondria isolated from ischemic hearts. When GE was administered for 10 days and 18 days, 45 min of total ischemia caused less damage to mitochondrial functions: State 3 respiration rate of isolated heart mitochondria was decreased only by 35% (p < 0.001) and 30% (p < 0.001), respectively. Treatment with GE for 10 and 18 days also decreased the damage of outer mitochondrial membrane under ischemic conditions by 17% (p < 0.001)and 16% (p < 0.001), respectively. 8 Further, we have tested the influence of GE treatment to rats on the generation of ROS by mitochondria (Fig. 2). The obtained results showed that both under normal conditions and after 45 min total ischemia 10-day treatment with GE significantly by 28% (p < 0.05) and 21% (p < 0.05) decreased H 2 O 2 production in isolated rat heart mitochondria.

In the next series of studies, the effect of GE treatment on the oxidation of FAD-specific substrate succinate was investigated under normal and ischemic conditions (Fig. 3). The results showed that after 10-day administration of GE under normal conditions mitochondrial respiration rate in State 2 and in State 3 increased by 32% (p < 0.001) and 9% (p < 0.001), respectively. After 18 days, the increase was, accordingly, 22% (p < 0.001) and 8% (p < 0.001). After 45 min of total ischemia, mitochondrial respiration rate in control rat mitochondria in State 2 decreased by 14% (p < 0.001), while in mitochondria isolated from those rats that did receive GE, respiration rate in State 2 was diminished less -by 5% (p < 0.001)and 9% (p < 0.001), respectively, after 10 and 18 days.

Respiratory rate in State 3 following total ischemia in control rat heart mitochondria decreased by 40% (p < 0.001), while in mitochondria isolated from rats that received GE for 10 and 18 days, it decreased by, accordingly, 34% (p < 0.001) and 32% (p < 0.001). GE treatment for 10 and 18 days decreased cytochrome c effect in control mitochondria from 1.5 to 1.3 (p < 0.01)and 1.2 (p < 0.001) respectively, whereas in ischemic mitochondria the effect was decreased from 1.73 to 1.53 (p < 0.001) and 1.43 (p < 0.001), indicating that GE treatment protects mitochondrial outer membrane integrity both under normal and ischemic conditions.

In further experiments the effect of GE treatment on State 2 respiration rate of mitochondria, oxidizing the main respiratory substrate of heart mitochondria -palmitoyl-L-carnitine was investigated under normal and ischemic conditions (Fig. 4). It is interesting to note that 10-days and 18-days of treatment with GE had no effect on the State 2 respiration rate under normal conditions, whereas V 3 increased by 16-25% (p < 0.001). Furthermore, 45 min ischemia had a tendency to increase V 2 in control group and significantly decreased V 3 by 52% (p < 0.001). In groups which only by 40-38% (p < 0.001) in the groups treated with GE for 10 and 18 days, respectively, under ischemic conditions. Also, addition of exogenous cytochrome c increased the State 3 respiration rate by 24-28% (p < 0.001) less in mitochondria isolated from rats treated with GE under ischemic conditions.

DISCUSSION

Cardiovascular diseases are associated with the alteration of mitochondria -main producers of ATP in the cardiac muscle cells. Numerous investigations have shown that ischemia in heart impairs mitochondrial oxidative phosphorylation (for review, see [START_REF] Piper | Mitochondrial function in the oxygen depleted and reoxygenated myocardial cell[END_REF][START_REF] Solaini | Biochemical dysfunction in heart mitochondria exposed to ischaemia and reperfusion[END_REF]. Ischemia decreases State 3 mitochondrial respiration rate [START_REF] Jennings | Structural and functional abnormalities in mitochondria isolated from ischemic dog myocardium[END_REF][START_REF] Schwartz | Biochemical and morphologic correlates of cardiac ischemia. I. Membrane systems[END_REF][START_REF] Toleikis | Functional state and the mechanisms of mitochondrial injury of ischemic heart[END_REF], increases permeability of the inner mitochondrial membrane to protons or ions in State 2 [START_REF] Toleikis | Functional state and the mechanisms of mitochondrial injury of ischemic heart[END_REF][START_REF] Piper | Mitochondrial function in the oxygen depleted and reoxygenated myocardial cell[END_REF], inhibits the activity of complexes I, II, III of mitochondrial respiratory chain [START_REF] Rouslin | Mitochondrial complexes I, II, III, IV, and V in myocardial ischemia and autolysis[END_REF][START_REF] Piper | Development of ischemia-induced damage in defined mitochondrial subpopulations[END_REF], ADP/ATP carrier [START_REF] Shug | Role of adenine nucleotide translocase in metabolic change caused by ischemia[END_REF][START_REF] Regitz | Mitochondrial damage during myocardial ischemia[END_REF][START_REF] Borutaite | Kinetic analysis of changes in activity of heart mitochondrial oxidative phosphorylation system induced by ischemia[END_REF][START_REF] Halestrap | Mitochondria and reperfusion injury of the heart--a holey death but not beyond salvation[END_REF], ATP-synthase [START_REF] Rouslin | Mitochondrial complexes I, II, III, IV, and V in myocardial ischemia and autolysis[END_REF], reduces amounts of membrane proteins and proteins located in the intermembrane space, especially cytochrome c [START_REF] Balasiavichius | Changes in the quantitative composition of cytochromes and the functional activity of heart mitochondria during ischemia[END_REF][START_REF] Piper | Development of ischemia-induced damage in defined mitochondrial subpopulations[END_REF].

Since it has been demonstrated that EGb 761, rich in bilobalides, decreases ischemic damage in heart and protects the components of mitochondrial respiratory chain [START_REF] Shen | Efficiency of Ginkgo biloba extract (EGb 761) in antioxidant protection against myocardial ischemia and reperfusion injury[END_REF][START_REF] Janssens | Protection by bilobalide of the ischaemiainduced alterations of the mitochondrial respiratory activity[END_REF], we investigated the effect of GE treatment on heart mitochondrial function under normal and ischemic conditions. The amount of GE (0.32 mL/kg) administered to the studied animals for 10 or 18 days was selected based on the recalculation from the standard therapeutical dosages used for human treatment (mL for a person with an average weight of 70 kg). Our investigations showed that in rats which received GE, the State 2 respiration in normal conditions had a tendency to increase, the most pronounced effect was observed in the case of succinate 4). In the State 2 mitochondrial respiration rate to the great extent depends on the passive proton flux through the mitochondrial inner membrane [START_REF] Groen | Quantification of the contribution of various steps to the control of mitochondrial respiration[END_REF]. Thus, this indicates that GE in vivo -similarly as previously detected in vitro [START_REF] Trumbeckaite | Effect of Ginkgo biloba extract on the rat heart mitochondrial function[END_REF] -causes a slight uncoupling of cardiac mitochondria.

Korshunov with co-workers and Starkov reported that "mild" uncoupling in mitochondria is involved in the cellular defence system against free radicals [START_REF] Korshunov | High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria[END_REF][START_REF] Starkov | Mild" uncoupling of mitochondria[END_REF], therefore in the next series we investigated the effect of ROS generation in mitochondria.

Our results showed that both under normal conditions and after 45 min total ischemia 10-day treatment with GE significantly by 28% and 21% decreased H 2 O 2 production. GE (1) directly scavenges free radicals and (2) reduces their production via mitochondria, therefore these results imply that GE may reduce mitochondrial damage by ischemia in cardiac muscle cells acting as a very strong antioxidant.

With all the studied substrates, mitochondrial respiration rate in State 3 was increased in mitochondria isolated from rats treated with GE by up to 8-25% under normal conditions, with the most pronounced effect (25%) in the case of complex I dependent substrate oxidation (pyruvate and malate as well as palmitoyl-L-carnitine and malate, Fig. 1, Fig. 4) and ~8% in the case of succinate oxidation (Fig. 3). 45 min ischemia decreased V 3 by 40-60%, also the lowest effect was observed in the case of succinate oxidation. GE treatment reduced V 3 decrease during ischemia, the most effectively in the case of complex I dependent substrate oxidation (up to 50% of decrease without GE treatment). In the State 3 complex I-dependent substrates-supported mitochondrial respiration rate depends both on the activity of mitochondrial respiratory chain and phosphorylation [START_REF] Hafner | Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the 'top-down' approach of metabolic control theory[END_REF][START_REF] Brown | Control of respiration and ATP synthesis in mammalian mitochondria and cells[END_REF], whereas in the case of succinate oxidation, ATP synthesis, transport and hydrolysis is responsible only for ~10% of respiration rate [START_REF] Borutaite | Control and kinetic analysis of ischemiadamaged heart mitochondria: which parts of the oxidative phosphorylation system are affected by ischemia?[END_REF]. Thus, our results indicate that GE treatment improves ATP synthesis in mitochondria and protects phosphorylation system from ischemic damage. In our study, the degree of respiration stimulation by exogenous cytochrome c (V 3+Cyt c /V 3 ) was used to assess the integrity of the outer mitochondrial membrane. When mitochondria were oxidizing complex I-dependent substrates under ischemic conditions, exogenous cytochrome c stimulated respiration by 60%, this stimulation was reduced by 35-50% in mitochondria isolated from GE-treated rat hearts. In the case of succinate oxidation, cytochrome c effect was more pronounced even under normal conditions, and GE also significantly reduced it implicating that GE treatment protects integrity of the outer mitochondrial membrane.

The ability of GE to reduce risk of cardiovascular events was questioned in the large recent clinical trial -the Ginkgo Evaluation of Memory (GEM) Study conducted in 2000 -2008 where GE therapy was beneficial only in the case of peripheral vascular disease but not in the case of cardiovascular or coronary heart diseases [START_REF] Kuller | Does Ginkgo biloba reduce the risk of cardiovascular events?[END_REF]. However, literature sources also list several studies demonstrating the protective effect on heart mitochondrial function of Ginkgo biloba preparations. It was reported that after 30 min of regional cardiac ischemia and 120 min of reperfusion, the control ischemic rabbit hearts demonstrated severe histological damage such as swelling and vacuolization of the mitochondria, whereas in animals after treatment with EGb 761 (10 mg/kg injected into the coronary artery), cardiac ischemia/reperfusion caused only slightly damage [START_REF] Shen | Efficiency of Ginkgo biloba extract (EGb 761) in antioxidant protection against myocardial ischemia and reperfusion injury[END_REF]. Janssens with co-workers also demonstrated that EGb 761 and one of its constituents -bilobalide -inhibited hypoxia-induced decrease in ATP content in endothelial cells in vitro [START_REF] Janssens | Protection of mitochondrial respiration activity by bilobalide[END_REF] and treatment of rats with 8 mg/kg of bilobalide protected complex I and III activities and prevented ischemia-induced decrease in State 3 respiration rate [START_REF] Janssens | Protection of mitochondrial respiration activity by bilobalide[END_REF]2000). The data of our experiments show that the administration of GE per os may lessen the ischemic damage of the heart muscle. It is likely that the protective effect of GE may be associated with the antioxidant activity of the extract.

It is noteworthy that the cardiovascular effect of EGb 761 was dose-dependent. In rats that received EGb for prolonged periods, the indexes of peripheral blood circulation were worse than in [START_REF] Tada | Longterm feeding of Ginkgo biloba extract impairs peripheral circulation and hepatic function in aged spontaneously hypertensive rats[END_REF]. Our results have shown that effects of both 10 days and 18 days rat treatment with GE were similar, indicating that prolonged treatment might not be useful for cardioprotection by GE.

In summary, GE preparation partially uncouples mitochondrial oxidation from phosphorylation and reduces generation of free radicals in the mitochondria, diminishes ischemiainduced V 3 decrease and the degree of respiration stimulation by exogenous cytochrome c. It is probable that by inhibiting free radical generation, as well as by improving ATP synthesis and preserving mitochondrial outer membrane integrity, GE protects cardiac muscle from damages caused by oxidative stress. RCI -mitochondrial respiratory control index (V 3 /V 2 ); Cyt c effect -the degree of respiration stimulation by exogenous cytochrome c (V 3+Cyt c /V 3 ). * p < 0.05 statistically significant effect of GE treatment compared to control under normal conditions, # p < 0.05 statistically significant effect of ischemia compared to control under normal conditions, ## p < 0.05 statistically significant effect of GE treatment under ischemic conditions compared to control under ischemic conditions, n = 5.

  effectively improved brain functions, therefore, Ginkgo biloba preparations are

  from Sigma-Aldrich (St. Louis, MO), except Amplex Red which was from Invitrogen (Carlsbad, CA).

  mitochondrial respiration rate in State 2 remained unchanged, whereas V 3 decreased

  ) (Fig.1, Fig.3and Fig.

  Influence of rat treatment per os with ethanol (0.32 mL/kg) on the respiration parameters of isolated heart mitochondria. Mitochondrial respiration rates were expressed as nmol O 2 /min/mg protein. V 2 -State 2 (substrates only), V 3 -State 3 (substrates + 1 mmol/L ADP), V 3+ Cyt c -State 3 in the presence of exogenous cytochrome c (32 µmol/L) respiration rate, n = 5. /L + 2 mmol/L amytal) Palmitoyl-L-carnitine and malate (9 µmol/L + 0.24 mmol/L)

Figure 1 .

 1 Figure legends:

Figure 2 .

 2 Figure 2. Effect of rat treatment per os for 10 days with Ginkgo biloba extract (GE, 0.32

Figure 3 .Figure 4 .Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 341234 Figure 3. Influence of rat treatment per os with Ginkgo biloba extract (GE, 0.32 mL/kg) on the

Control Ethanol 10 days Ethanol 18 days Control Ethanol 10 days Ethanol 18 days Control Ethanol 10 days Ethanol 18 days

	V 2 V 3	50 ± 1 185 ± 3 187 ± 2 51 ± 1 F o r 51 ± 1 186 ± 1	78 ± 2 167 ± 2 163 ± 1 76 ± 1	73 ± 1 159 ± 1	40 ± 1 130 ± 2 125 ± 3 40 ±1	40 ± 1 126 ± 2
	V 3+Cyt c 196 ± 4 195 ± 2	195 ± 1 P	247 ± 2 245 ± 1	241 ± 4	150 ± 4 148 ± 3	148 ± 3
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Acknowledgements

This work was supported by the Science Foundation of Kaunas University of Medicine.