PT104389A - COMPOUNDS CONTAINING ANTIOXIDANT FLAVONOIDS AND MITOCHONDRIAL PROTECTORS WITH NEUROPROTECTOR PROPERTIES - Google Patents

Abstract

This invention discloses assemblies containing phenols present in plant extracts with therapeutic activity against neural exchitotoxicity, oxidative stress and myocytodial dysfunction. WHETHER OR INDIVIDUALLY USED OR IN AN ASSOCIATION, QUERCETINE, CAMPFEROL, AND BIAPIGENIN SIGNIFICANTLY REDUCE NEURONAL DEATH CAUSED BY EXCITOTOXIC AGRESSIONS OR EXPOSURE TO BETA-AMYLOID PEPTIDE. FOR ANY MORE, BIAPIGENIN INTERFERTS WITH ANT COMPLEX / MITOCHONDRIAL TRANSITION PORE CONTRIBUTING TO AN INCREASED MITOCHONDRIAL CALCIUM EFFLUX, REDUCING THE CALCIUM LOAD AND CONTRIBUTING TO NEUROPROTECTION AGAINST EXCITOTOXICITY.

Description

1

COMPOSITIONS CONTAINING ANTIOXIDANT FLAVONOIDS AND MITOCHONDRIAL PROTECTORS WITH NEUROPROTECTIVE PROPERTIES "

FIELD OF THE INVENTION The present invention describes formulations containing biapigenin with neuroprotective properties against excitotoxicity and neuronal degeneration induced by the beta-amyloid protein. In addition, the invention also describes formulations containing a flavonoid blend for the same therapeutic purposes.

BACKGROUND OF THE INVENTION Excitotoxicity has been associated with an over-activation of glutamate receptors. Under prolonged activation of the N-methyl D-aspartate receptor (NMDA), ie during periods of ischemia, anoxia, neuronal degeneration (Lipton and Rosenberg 1994; Rego et al., 2001) and other neurodegenerative pathologies In the present study, it was found that the presence of calcium in the cell was associated with an increase in the amount of calcium present in the cell (Carr et al., 2004; Nicholls and Ward 2000, Vergun et al., 2001, Montai 1998, Isaev et al., 2005).

Mitochondria play a vital role in maintaining calcium homeostasis in the cell, but excessive accumulation of mitochondrial calcium may result in loss of mitochondrial transmembrane potential (A ¥ m) and decoupling of the respiratory chain, increasing the formation of oxygen free radicals and nitrogen. Mitochondrial function compromise seriously the production of adenosine triphosphate (ATP), leading to the depletion of its reserves, the failure of ionic homeostasis and the regulation of calcium concentration (Nicholls 2004; Nicholls and Budd 1998). Oxidative stress and mitochondrial calcium overload may lead to the opening of the transient permeability pore of the mitochondria, allowing the passage of solutes and large molecules into the matrix (Isaev et al., 2005; Vieira et al., 2000). The consequent increase in mitochondrial size and rupture of its outer membrane leads to loss of mitochondrial function, production of free radicals of oxygen and nitrogen that cause oxidation of membranes, proteins and nucleic acids. These processes promote the release of pro-apoptotic factors, such as cytochrome C, apoptosis-inducing factor (FIA), Smac-DIABLO and G-endonuclease, which activate effector mechanisms of cell death (Bouchier-Hayes et al.

Heiden and Thompson 1999).

Mitochondria have been seen as central protagonists in the chain of toxic events due to the over-activation of glutamate receptors, causing loss of ionic homeostasis and leading to neuronal death (Bouchier-Hayes et al., 2005c; Vander Heiden and Thompson 1999; Nicholls and Ward 2000; Duchen 2004; Kushnareva et al., 2005). The fundamental role of mitochondria in maintaining the bioenergetic reducing potential, as well as their participation in the decision of cell fate, make this organelle a valuable target for the development of new therapeutic strategies against pathologies associated with ischemic insults and neurodegenerative diseases such as Alzheimer's disease (Nicholls and Budd 1998; Bouchier-Hayes et al., 2005b; Duchen 2004; Mattson and Kroemer 2003; Won et al., 2002), since excitotoxic events are the determinants of neuronal death.

We have previously shown that extracts of flavonoid-enriched Hypericum perforatum extracts (Silva et al., 2004b) are neuroprotective against β-amyloid protein induced toxicity in primary cultures of rat hippocampal neurons (Silva et al., 2004a).

There are also references to the fact that H. perforatum is effective in an animal model of ischemia / reperfusion injury, reducing the signs of physiological and histological damage (de Paola et al., 2005). In fact, flavonoids have been identified as potentially effective in preventing cell damage resulting from cerebrovascular accidents and ischemia / reperfusion (Simonyi et al., 2005; Dajas et al., 2003; Zhao 2005). The extract of Hypericum perforatum (St. John's Wort) has been used in the treatment of nerve disorders for many years. Its application in cases of depression and psycho-autonomic disorders has increased greatly in recent years due to favorable clinical trial results.

Many papers related to the use of extracts of H. perforatum (EP599307, DE1919512, DE19714450, W09940905, US20010033872), or fractions containing hyperforins or derivatives (DE19903570, US20010020040), used in the treatment and prophylaxis of depression, dementia and others have been published. related nervous disorders.

Recently, the use of H. perforatum has been weighed in the treatment of Alzheimer's disease and amyloidoses (US0020150637).

It has also been shown that extracts of H. perforatum and certain compounds such as hypericins and hyperforins can be used as therapy directed to T-type calcium channels in various biological systems, implicated in various processes such as cerebral aging or neurodegenerative diseases ( US20030207940).

However, none of these works refer to the compounds of the present invention, namely quercetin, campperol and biapigenin, as being used in the treatment of neurological diseases, acting in particular as neuroprotective compounds. The present invention is in fact related to the neuroprotective properties of the phenolic compounds present in the extracts of H. perforatum quercetin, campferol and biapigenin against excitotoxicity, and some associations of these agents useful as therapeutics are also described.

SUMMARY OF THE INVENTION

This invention discloses flavonoid compounds (biapigenin, quercetin and campperol) with neuroprotective properties against neuronal degeneration induced by beta-amyloid protein and by excitotoxicity, acting by anti-oxidant and protective mechanisms of mitochondria, 5 retarding the failure of induced calcium homeostasis by excitotoxicity.

DESCRIPTION OF THE INVENTION The invention describes biapigenin formulations with neuroprotective properties against neuronal degeneration induced by excitotoxicity and beta-amyloid protein. Associations with biapigenin and other flavonoids for the same therapeutic purposes are also described.

The compounds described herein have anti-oxidant properties - both biapigenin and other flavonoids, namely quercetin and campferol, which act at the mitochondrial level, protecting it from calcium-induced degeneration.

The formulations described may be useful in treating or preventing neuronal degeneration caused by brain insults or neurodegenerative diseases, both in animals and humans.

Some extracts of Hypericum perforatum contain high levels of flavonoids. The three fundamental elements (quercetin, campperol and biapigenin, ΙΟμΜ each, as well as a mixture of these elements (quercetin 21μΜ, campferol Ι, ΙμΜ, biapigenin 2, βμ identific) were identified and characterized by the flavonoid compounds present in the neuroprotective fractions of Hypericum perforatum.

The mechanisms that provide neuroprotection against neuronal death by excitotoxicity (induced by 6 kainate + NMDA or by exposure to amyloid beta peptide), involving antioxidant properties (quercetin and campperol) and mitochondrial protection against calcium-induced degeneration (biapigenin). The effect of flavonoids with regard to neuroprotection and delay in calcium-induced deregulation was evaluated in hippocampal neurons of cultured rats. The anti-oxidant and mitoprotective properties of quercetin, campperol and biapigenin were evaluated in rat cortical nerve terminals and mitochondria isolated from rat brain. The first compound of the invention, biapigenin, acts by mechanisms that protect mitochondria - mitoprotection. This compound is present in the combinations of the present invention in a concentration between 0.1 and 50 μΜ.

Other combinations, within the scope of this invention, contain biapagenin and other flavonoids, such as quercetin and campperol. These compounds are also present in an individual concentration between 0.1 and 50 μΜ.

These associations show potent neuroprotective properties against excitotoxic insults in brain tissue, both anti-oxidants and preventive of calcium-induced mitochondrial degeneration.

In a preferred formulation of the present invention, their combinations should contain a concentration between 0.1 and 50 μΜ of biapigenin, quercetin and campperol, acting neurologically as neuroprotectors because of their anti-oxidant and mitoprotective mechanisms. 7

Concentrations of compounds greater than 50μΜ revealed neurotoxicity; lower than Ο, ΙμΜ did not reveal any neuroprotective effect. 1 - The phenolic compounds present in H. perforatum are neuroprotective against excitotoxicity in hippocampal neurons in culture Neuronal viability was assessed after exposure of hippocampal neurons in culture to acute excitotoxic stimulation. Feasibility decreases significantly after exposure to ΙΟΟμΜ kainate (KA) plus ΙΟΟμΜ NMDA (55 ± 1% after excitotoxic insult against 100 ± 4% in the control group). Cell death assessed after 24 hours was significantly prevented in the presence of ΙΟμΜ of quercetin, campperol or biapagenin, with viabilities of 77 ± 9%, 77 ± 12% and 87 ± 11%, respectively (Figure 1B) .

Changes in morphology were also analyzed as a sign of nervous degeneration. Immunofixation directed to microtubule-associated protein 2 (MAP-2) revealed marked dendritic degeneration after exposure to kainate and NMDA. Furthermore, the MitoTracker Red CMXRos, which accumulates in polarized mitochondria, was used to evaluate changes in mitochondrial distribution in neuronal cells. After excitotoxic insults, a large redistribution of partially depolarized mitochondria with accumulation in cytoplasmic groups was observed. Nuclear morphology was evaluated with Hoechst 33342 staining, showing very condensed nuclei in cells exposed to kainate and NMDA. Exposure to ΙΟμΜ of biapagenin partially protected neurons from the effect that cells maintained on deleterious markers of excitotoxic aggression, once exhibited fewer dendritic dystrophies, reduced mitochondrial physiology and nuclear number of cell death (Figure 1C).

2 - The phenolic compounds present in H. perforatum delayed deregulation in calcium homeostasis induced by kainate and NMDA

Intracellular calcium concentrations were monitored simultaneously with the mitochondrial transmembrane potential, using microscopic observation of each cell alone, in neurons exposed to kainate and NMDA. A protocol similar to that of the viability assays was used to find a correlation between neuroprotection by phenolic compounds, protection by delay in calcium deregulation and improvement in mitochondrial function. Figure 2A shows representative images of a control-situation (Fura-2 microfluorescence and TMRM-tetramethylrhodamine methyl ester) for the critical time periods of the experiment. The changes in the Fura-2 fluorescence (excitation index 340 / 380nm) are shown in the left panel, while in the right panel is shown the TMRM fluorescence. All images were taken in the same field at different time periods, as indicated on the left. The loss of calcium homeostasis (index 340 / 380nm, shifting to red color) correlated strongly with a significant decrease in TMRM fluorescence (fluorescence at 598nm, black shift) (r2 = 0.96, n = 6-8 independent experiments, with an average of 120 cells per field). After excitotoxic insult, a significant percentage of cells 9 lost the ability to preserve calcium homeostasis and showed a delayed calcium deregulation. By exposing the cells to ΙΟμΜ of quercetin or biapigenin, neurons were significantly protected from delayed calcium deregulation (Figure 2B), with biapagenin being particularly efficient. Campperol was the least efficient in protecting delayed calcium deregulation. 3 - Effect of H. perforatum phenolic compounds on mitochondrial bioenergy

The results obtained in neuronal cultures and described above led to the identification of mitochondria as a possible target involved in the observed neuroprotective effects. To test this hypothesis, isolated cerebral mitochondrial fractions were used to directly investigate the effect of quercetin, campperol and biapigenin on mitochondrial bioenergetics.

As can be seen in figure 3, quercetin and campperol (ΙΟμΜ) did not significantly affect either TPP capture or respiration. Incubation of cerebral mitochondria for 3 minutes with biapigenin (ΙΟμΜ) significantly affected mitochondrial depolarization and repolarization (Figure 3C-D). By itself, biapigenin did not significantly alter the electrode response to TPP or respiratory capacity, significantly decreasing breath stage 3 (30% reduction). It should be noted that breath stage 4 also increased (43%), which further contributed to the reduction of the respiratory control rate (TCR - parameter that reflects the coupling between substrate oxidation and phosphorylation of adenosine diphosphate (ADP), a decrease of 47% compared to control). Decoupled respiration was inhibited, suggesting inhibition of the respiratory chain. In addition, the ADP / O rate (reflecting the efficiency of phosphorylation) was significantly reduced by incubation with biapigenin (approximately 30%). Phosphorylation efficiency was also evaluated by time to mitochondria to phosphorylate ADP (response time significantly reduced by biapigenin (inhibition of 75% compared to control, p <0.001 - data not shown) 4 - Mitochondrial lipid peroxidation is reduced in presence of phenolic compounds derived from H. perforatum Lipid peroxidation was evaluated after exposure of isolated cerebral mitochondria to the ADP / iron oxidant pair The three compounds were able to significantly decrease the lipid peroxidation measured either by the oxygen consumption or the production of reactive substances in the presence of mitochondria (without the addition of the pro-oxidants or protective compounds), no significant peroxidation was observed, as was observed in isolated mitochondria incubated with ΙΟμΜ of the compounds. The anti-oxidant properties of the compounds present in H. perforatum were also evaluated by inducing lipid peroxidation in energy-enriched mitochondria with succinate and then exposed to ADP / iron. Loss of the overcoat as a result of loss of membrane integrity, and consequent dissipation of the proton gradient (Figure 5). Butylhydroxytoluene (BHT), known lipid peroxidation inhibitor, was used with control and completely inhibited ΔΨπ loss! contributing to the demonstration of the involvement of lipid peroxidation in this process. Quercetin and campperol were very efficient in preventing loss of ΔΨη. Energy supplementation to mitochondria preincubated with biapigenin resulted in transient hyperpolarization before the onset of peroxidation. 5 - Effects of H. perforatum-derived phenolic compounds on calcium accumulation The capacity of calcium accumulation by mitochondria was measured using two different approaches. TPP + capture was evaluated in the presence of calcium as an indirect measurement of mitochondrial calcium storage capacity (Figures 6Λ and 6B). Representative measurements are plotted in panel A, showing that TPP + capture was reduced in the presence of calcium compared to the control group (Figure 6A and 6C). Of the three compounds tested, only biapigenin was able to protect energized mitochondria from loss of ΔΨ ™ in the presence of calcium (Figure 6C). Cyclosporin A was used as a positive control, as this drug inhibits transient permeation pore opening by desensitizing the pore to calcium (Halestrap 2006) and thus enhancing the calcium-capturing ability of mitochondria. Mitochondrial calcium accumulation was also evaluated in energy-enriched mitochondria by the use of a Calcium Green-5N calcium-sensitive low-affinity probe in the assay medium. Mitochondria were supplemented with calcium energy present in the test medium, and calcium accumulation was followed by a decrease in fluorescence intensity (reflecting the decrease in calcium concentration in the medium and accumulation in the mitochondria). The graphical representation is shown in Figure 1A. In addition to the results obtained with the TPP + electrode, (APm), it was observed that biapigenin significantly decreased calcium accumulation (Figure 7B). No significant changes were observed after incubation with quercetin and campperol. On the other hand, incubation with cyclosporin A significantly increased the accumulation of mitochondrial calcium, a significantly reduced effect of biapigenin (Figure 1B). The efficiency of calcium accumulation by mitochondria as a response to an increase in cytoplasmic calcium concentration (eg during excitotoxic events) was assessed by adding ΙΟμΜ pulses of calcium to mitochondria supplemented with energy and in suspension. Figure 8 shows the graph representing the accumulation of calcium in the control group or after incubation with biapigenin (ΙΟμΜ). As can be observed, in the presence of biapigenin the mitochondria showed a lower calcium capture capacity.

6 - Biapigenin reduces the dissipation of ADP-induced transmembrane electric potential, but potentiates the ADP-induced hyperpolarization The effect of biapigenin on mitochondrial bioenergetics was assessed by the follow-up of TPP + capture in mitochondria isolated from rat brain, which represents an indirect estimate of the mitochondrial transmembrane electrical potential (AT m), and by the mitochondrial respiration, evaluated by oxygen consumption monitoring. Isolated mouse brain mitochondria (0.8 mg.ml-1) were incubated with various concentrations of biapigenin. Resting mitochondria exhibited an ATV of -180.7 ± 2.3 mV. At a concentration of ΙΟμΜ, biapigenin decreased ADP-induced depolarization (125μΜ) (67.88%; p <0.05, as compared to control) and significantly increased mitochondrial repolarization (371.7%; 0.01, as compared to the control) (Figure 9).

7 - Adenine nucleotide translocator (ANT) blockers inhibit the hyperpolarizing effect of biapigenin in the presence of ADP

The above reported results suggest that biapigenin may exert a modulatory effect on the phosphorylative system. In order to identify a possible target for biapigenin, the effects of biapigenin with known modulators of the mitochondrial phosphorylase system, ATPsintetase and the adenosine nucleotide translocator (ANT) were compared. Biapigenin-induced ADP-dependent hyperpolarization was observed to be unaffected by oligomycin (1 pg.ml-1, Figure 9D), a drug that inhibits proton flux in the F0 subunit of ATPsintetase, although the atractyloside (40 μΜ) and bongkrheic acid (ΙβμΜ), ANT inhibitors, were able to prevent the observed effect (Figure 9F and 9H, respectively).

8 - Biapigenin does not significantly affect the increase in ADP-induced respiration 14 Mitochondrial respiration was assessed by monitoring oxygen consumption in isolated mitochondria of rat brain. Mitochondria (0.8mg.ml_1) were enriched in energy with succinate (8mM). No significant differences were observed in basal respiration (basal respiration) after 3 minutes of pre-incubation with biapigenin (ΙΟμΜ), atractyloside (40μΜ), bongkréquic acid (16μΜ) or oligomycin (lpg.ml-1) . Despite the apparent effect of biapigenin on ADP-induced depolarization, the stimulation of mitochondrial respiration with ADP (stage 3) was not blocked by biapigenin. As expected, ac- chtyloside and bongkrequic acid blocked breath at stage 3. Representative graphs of control and biapigenin-treated mitochondria are shown in Figure 10A. Inhibition of ADP-stimulated and ac- tiletyl-mediated oxygen consumption was decreased in the presence of biapigenin (Figure 10B-C), an effect that was not observed with bongkreic acid (Figure 10C). 9 - Biapigenin inhibits the unbound breathing of FCCP in energized mitochondria. Maximum uncoupled respiration was monitored after addition of FCCP (ΙμΜ) to the reaction medium (representative graphs shown in Figure 11A). Biapigenin significantly decreased the stimulation of mitochondrial respiration induced by FCCP (32% reduction, p <0.05 compared to control; Figure 11A-B). The effect of biapigenin on decoupled respiration was comparable to that of atractiloside (40μΜ, Figure 11B). In contrast, although bongkrheic acid (16μΜ) per se did not affect FCCP-induced decoupled breathing, co-incubation with biapigenin (ΙΟμΜ) significantly decreased maximal FCCP-induced respiration (59%, p <0.001 compared to control ; Fig. 11B). 10 - Biapigenin potentiates induced hyperpolarization

by ATP ATPase activity was assessed by monitoring the tetraphenylphosphonate (TPP +) capture under the addition of ATP (3mM) in the presence of 2μΜ roulette, reflecting the membrane potential induced by ATP hydrolysis. Representative TPP + capture plots are shown in Figure 12A. Biapigenin (ΙΟμΜ) significantly increased ΔΔ generated by the addition of ATP (3mM) to the reaction medium (30%, p <0.01, compared to control; Figure 12A-B). Atratilystoid (40 μΜ) and bongkréchic acid (16 μΜ) significantly reduced ATP-mediated energy supplementation (12 and 30%, respectively). At the same time, the atractiloside was unable to block the effect of biapigenin, whereas bongkreic acid significantly inhibited the hyperpolarizing effect of biapigenin in the presence of ATP (Figure 12B). Oligomycin (1 pg.ml-1) also inhibited the effect of biapigenin on the generation of ATP induced AT m (Figure 12B). ATPase activity was also evaluated indirectly by monitoring pH changes in frozen and thawed mitochondria. Proton release resulting from ATP hydrolysis due to ATPase activity was followed after addition of 3 mM ATP to the mitochondria. In clear opposition to previous results, 16 ATPase activity was significantly decreased in the presence of biapigenin (16%, p <0.05 compared to control; Figure 12C). 11 - Biapigenin reduces ATPsintetase activity in intact mitochondria ATPsintetase activity was evaluated in mitochondria supplemented with energy in the presence of biapigenin, atractyloside, bongkreic acid and oligomycin. Biapigenin (ΙΟμΜ) significantly reduced ATPsintetase activity (40% reduction, p <0.01 compared to control, Figure 13B). Atracyteside (40 μΜ), bongkreic acid (16 μΜ) and oligomycin (μg ml -1) were found to be more potent inhibitors of ATPsintetase activity than biapigenin (reductions of 91, 96 and 99% respectively; p <0.001 compared to control; Figure 13A). Biapigenin (ΙΟμΜ) reduced the ADP / O rate and response time (time measurement required for recovery of ATP-induced depolarization) by 26% and 79%, respectively.

12 - Biapigenin and ANT inhibitors decrease the synthesis of ATP

To confirm a possible inhibitory effect of biapigenin on ATP synthesis, nucleotide levels of adenosine were measured after a complete phosphorylation cycle, which translated in about one minute, after addition of ADP to energy enriched mitochondria (the mean time required by control mitochondria for phosphorylation of ADP added). Biapigenin (ΙΟμΜ) significantly inhibited ATP synthesis (82% reduction; 17 p <0.001 compared to the control). (40μΜ), bongkréchic acid (ΙβμΜ) and oligomycin (lpg.ml-1) significantly inhibited ATP synthesis (reduction of 83, 95 and 94%, respectively, p <0.001, compared to the control). 13 - Biapigenin Decreases Calcium Accumulation Calcium accumulation mitochondrial capacity was evaluated in energy-enriched mitochondria by the use of a Calcium Green-5N calcium-sensitive low affinity probe. The mitochondria (0.5 mg-ml &quot; 1) were supplemented with succinate energy in the presence of 15μΜ CaCl₂ and 100æM Calcium Green-5N in the assay medium. Mitochondrial calcium accumulation was recorded following the decrease in fluorescence intensity (reflects the decrease of calcium in the medium and accumulation in mitochondria). The concentrations of biapigenin, atractyloside and bongkreic acid were adjusted to the amount of protein used, so that the data could be compared between different experimental protocols with different protein concentrations. Representative graphs are shown in Figure 14A-C. Biapigenin (ΙΟμΜ) significantly reduced calcium accumulation. After incubation with cyclosporin A (Ο, βμΜ), there was an increase in calcium accumulation capacity. Biapigenin was able to prevent the effect of cyclosporin A on calcium accumulation (Figure 14A and 14D).

To verify if the effect of biapigenin at the maximum calcium accumulation was due to a decrease in calcium uptake or an increase in efflux, the calcium efflux in mitochondria supplemented with energy was evaluated. The 18 mitochondria supplemented with succinate energy (8μΜ) were exposed to a single pulse of 40μΜ CaCl2 (Figure 15A-E, indicated by the narrow arrow). Two approaches were used to test the effect of biapigenin. At first, ΙΟμΜ of biapigenin were preincubated for 3 minutes before the calcium pulse; in the second, a pulse of biapigenin was added 1 minute after the calcium pulse (to assess a direct effect on calcium efflux Figure 15A). Pre-incubation with ΙΟμΜ of biapigenin reduced the maximum accumulation of calcium (Figure 15A), while the addition of biapigenin (ΙΟμΜ) to mitochondria with the maximum calcium capacity induced calcium efflux, as can be observed in Figure 15A. Although bongkreic acid (ΙβμΜ) increased the maximum calcium accumulation in the presence of biapigenin, it was insufficient to prevent calcium efflux induced by biapigenin (Figure 15B). 0

(40 μΜ) alone increased calcium efflux (Figure 15C) and when co-incubated with biapigenin the effect was apparently additive (Figure 15C). Cyclosporin A (Ο, βμΜ) reduced biapigenin-induced calcium efflux (Figure 15D), whereas ADP (125μΜ) plus oligomycin (lpg.ml-1) completely abolished the effects of biapigenin on calcium accumulation and efflux. 15E). A novel formulation with neuroprotective properties Taking advantage of the neuroprotective properties of the three flavonoids described in the present study (biapigenin, quercetin and campperol), we developed a new formulation with strong neuroprotective properties against amyloid beta peptide-induced neuronal degeneration (Figure 16A) and against excitotoxic insults (Figure 16B), 19 evaluated both by the morphological aspects of neuroprotection and by the viability / cell death assay using sytol3-PI. The new association described in the present work is neuroprotective and free of toxic effects on rat hippocampal neurons in culture. The composition of the new formulation was based on our previous characterization of a neuroprotective fraction isolated from extracts of Hypericum perforatum (fraction V5) in which we observed three main flavonoids: quercetin, campferol and biapigenin (Silva et al., 2004a). The most obvious advantage of the novel formulation is related to the control of the effective concentration of the individual compounds and the absence of other possible toxic contaminants present in the original V5 moiety. Neuronal death due to excitotoxicity is associated with a massive influx of calcium, loss of ionic homeostasis, and mitochondrial dysfunction, which usually precede cell death (Stout et al., 1998). Mitochondria are important organelles for calcium homeostasis, especially when there are high cytoplasmic concentrations of calcium, a result of pathological aggression or cell stress (Isaev et al., 2005; Kristian and Siesjo 1998, Nicholls 2002, Saris and Carafoli 2005, Weber 2004). ). Calcium that enters the glutamate receptors is expelled from the cell mainly by the Na + / Ca2 + - translocator (NCX) (Bano et al., 2005b), but can also be accumulated by the mitochondria present in the vicinity of membrane receptors (Peng and Greenamyre 1998; Weber 2004), possibly having an important role in the defense of the cell against the excessive increase of the cytoplasmic concentration of calcium. However, after activation of calcium-induced calpain, NCX can be cleaved by the proteolytic activity of calpainas (Bano et al., 2005a).

If the intracellular calcium concentration increases above a certain critical threshold, the mitochondria lose the ability to maintain calcium homeostasis and mitochondrial dysfunction occurs. Mitochondrial dysfunction involves the induction of a permeability transition, which leads to increased mitochondria and rupture of the outer mitochondrial membrane, releasing pro-apoptotic factors that trigger cell death processes (Bouchier-Hayes et al., 2005a Vander Heiden and Thompson 1999 ).

The phenolic compounds quercetin, campperol and biapigenin present in the extracts of H. perforatum revealed the ability to significantly protect neurons from the cultured hippocampus against an excitotoxic insult with kainate plus NMDA (FiguralB and Figure 1C). In addition, preincubation with these compounds had no apparent effect on the immediate rise in calcium due to stimulation with kainate and NMDA, suggesting no apparent effects on calcium influx or its recruitment from other intracellular stores. Microscopic observation of each cell alone provided evidence of a protective effect of the compounds under investigation, since they were able to partially protect neurons from delayed calcium deregulation (Figure 2B).

Furthermore, calcium deregulation is strongly associated with a complete loss of the mitochondrial transmembrane electrical potential, suggesting a close association between the two events. Treatment with biapigenin led to significant cell protection with early calcium deregulation and in addition, decreased the total number of cells without calcium homeostasis. These results were further confirmed by data from the cell viability assays measured 24 hours after excitotoxic aggression (kainate plus NMDA), and biapigenin significantly reduced neuronal death. Evidence from the literature is inconclusive about the role of oxidative stress as a cause or consequence of mitochondrial dysfunction in excitotoxicity. It is not certain that mitochondrial dysfunction resulting from an excess of calcium capture is responsible for an increase in the generation of oxygen free radicals (RLO), or if the generation of calcium-dependent RLO is associated with toxic mechanisms responsible for mitochondrial failure. (Kierkegaard and Costa 1995, Lafon-Cazal et al 1993, Reynolds and Hastings 1995; Weber 2004) Quercetin and campperol are considered to be good anti-oxidants (Cotelle 2001; Ishige et al., 2001 Jovanovic and Simic 2000; Rice- Evans 2001).

Previous studies have shown that both compounds are effective inhibitors of lipid peroxidation (Filipe et al., 2001; Ozgova et al., 2003 / Peng and Kuo 2003 / Schroeter et al., 2001). In agreement, we also observed that quercetin and campperol are strong inhibitors of the peroxidation of mitochondrial membranes (Figures 4 and 5). Some studies with hepatic mitochondria have pointed to quercetin as a potent inhibitor of mitochondrial respiration and a potent inhibitor of mitochondrial membrane permeability transition (Dorta et al., 2005; Santos et al., 1998). 22

However, it should be noted that the inhibitory effects reported in studies with hepatic mitochondria were observed at concentrations significantly higher than those used in our study. Quercetin and campperol have been reported to have an inhibitory effect on mitochondrial ATPase / ATPsintetase activity. Again, these effects were observed using higher concentrations of both compounds (Zen and Ramirez 2 0 0 0).

Montero and colleagues reported a significant effect of campferol on the capture of mitochondrial calcium, involving the activation of the unidirectional calcium transporter in HeLa cells; this effect was less pronounced when quercetin was tested (Montero et al., 2004). Our data resulting from the microscopic observation of each cell alone indicate that campperol is not effective in preventing delayed calcium deregulation, in contrast to quercetin which has proved to be protective. On the other hand, both compounds were significantly neuroprotective against neuronal degeneration induced by excitotoxicity, suggesting that the main mechanism behind neuroprotection may be related to anti-oxidant properties. It has recently been shown that the efficient neuroprotective compound biapigenin (now presented) has strong anti-oxidant properties (Cakir et al., 2003; Couladis et al., 2002).

The anti-oxidant properties of the compounds tested in the present study may contribute to maintain the structural and functional properties of the mitochondrial respiratory chain, essential for mitochondrial functioning and the generation of protonic force. Furthermore, we have also observed that the anti-oxidant properties of the tested compounds are effective against lipid peroxidation caused by oxidative stress induced in enriched and non-enriched mitochondria. The hyperpolarizing effect observed after incubation of mitochondria with biapigenin may contribute to the lower antioxidative capacity of this compound in energy enriched mitochondria, compared to the strong protection observed after induction of oxidative stress in non-enriched mitochondria. However, the protection conferred by biapigenin against the decline in transmembrane potential was very similar to that observed in the case of well-known transient permeation pore-opening inhibitor cyclosporin A (Halestrap 2006); On the other hand, the profiles exhibited by quercitina and campferol were much closer to BHT, a potent inhibitor of lipid peroxidation, evidencing possible differences in the neuroprotective mechanisms activated by quercetin / campperol and biapigenin.

As already mentioned, excitotoxicity is clearly associated with increased intracellular calcium concentration and compromised mitochondrial function. Seo et al. reported that high mitochondrial membrane potential and redox potential reduced intracellular calcium accumulation and neuronal death after activation of NMDA receptors. A high transmembrane potential may contribute to increased ATP synthesis, important for calcium extrusion while high redox potential may be responsible for detoxification of RLOs (Seo et al., 1999). It can be hypothesized that the hyperpolarization induced by biapigenin may also be associated with an increase in ATP production, 24 thus contributing to the increase in the extrusion of ATP and calcium. Biapigenin appears to interfere with mitochondrial phosphorylative mechanisms, significantly reducing ADP-induced depolarization and the time required to phosphorylate ADP. In addition, the adenosine diphosphate / oxygen (ADP / O) rate, which reflects the efficiency of phosphorylation, was also significantly reduced following incubation with biapigenin. The accumulation of mitochondrial calcium depends on both the rate of capture and the efflux of calcium. An increase in calcium release may be a consequence of the induction of mitochondrial transition permeability (PMT), activated by excessive calcium accumulation and oxidative stress. The sustained release of PMT is prevented in fully polarized mitochondria or in the presence of anti-oxidants (Brookes et al., 2004b; Vieira et al., 2000). Biapigenin reduces calcium accumulation capacity when evaluated directly with a fluorescent probe (Calcium Green-5N), which may explain its ability to increase the maximum membrane potential obtained in the presence of calcium. The data obtained from the accumulation of mitochondrial calcium point to an interesting effect of biapigenin, which may also help to explain the neuroprotection observed in neuronal cells. This group of results suggests that biapigenin contributes to the maintenance of mitochondrial membrane potential in the presence of calcium by a mechanism that may involve the modulation of calcium accumulation by mitochondria. This hypothesis is supported by the reduction of calcium accumulation in mitochondria incubated with cyclosporin A (therefore, with the pore of inhibited transient permeability). Another possible explanation for the phenomenon could be that biapigenin may increase the rate of calcium extrusion. These possibilities will be discussed later in this paper.

In the present study, the effect of the inhibition of the mitochondrial protein tyrosine phosphatase (PTPm) on the inhibition of the inhibitory activity of the cytochrome P450 protein such as cyclosporin A and possibly diazoxide, or indirect inhibition, by reduction of oxidative stress and / or mitoncondrial calcium overload. Cyclosporin A binds to cyclophilin D, which interacts with ANT, facilitating a calcium-induced rearrangement of this in a pore conformation (Halestrap et al., 2002). Thus, cyclosporin A increases the mitochondrial calcium accumulation capacity, inhibiting the opening of PTPm. Diazoxide targets the phosphorylative system and has been reported to be neuroprotective in in vitro neurotoxicity models (Kowaltowski et al., 2006) and in in vivo ischemia-reperfusion models (Murata et al., 2001; Teshima et al., 2003). In addition, diazoxide also inhibits the degradation of ATP during the ischemic phase, and does not have deleterious effects on normal mitochondria (Comelli et al., 2007). It may seem somewhat controversial the idea that inhibition of mitochondrial phosphorylation may result in neuroprotection, especially because under physiological conditions the cells critically need ATP synthesis. However, under stress conditions with mitochondrial function failure, such as in excitotoxic or ischemia-reperfusion events, it has been reported that inhibitors of the phosphorylative system may be neuroprotective. 26

The naturally occurring flavonoids are capable of preventing the lipid peroxidation of the mitochondria and may inhibit PTP-m opening (Santos et al., 1998). In addition, flavonoids neutralize free radicals and have anti-oxidant properties (Schroeter et al., 2000; Rice-Evans 2001), which may also contribute to the inhibitory effect of flavonoids at the opening of PTP m. These studies suggest that some flavonoids are capable of interacting with mitochondrial physiology, exerting neuroprotective actions, especially when covering the PTPm complex. Biapigenin (10 μΜ) significantly inhibited ADP-induced depolarization, and significantly increased repolarization after addition of ADP. ATPsintetase inhibitors (eg oligomycin) or ANT inhibitors (bongkrequic or atractyloside acid) also significantly inhibit ADP-induced depolarization. Oligomycin, a specific inhibitor of proton flux by the F0 subunit of ATPsintetase (Zanotti et al., 1992), was unable to block the hyperpolarizing effect of biapigenin, suggesting that ATPsintetase is not a major player in the effect of biapigenin in ADP-induced depolarization and in repolarization. In the presence of ADP (Fig. 9, F and H), the inhibitory effect of biapigenin on the presence of ADP in the presence of ADP (Fig. 9, F and H, respectively). Atractostyloid blocks ANT in a pore conformation, called c-conformation (c, to cytosolic side 27), while bongkreic acid blocks ANT in a non-pore conformation, called the m-conformation (m, to the side of the matrix ) (Dahout-Gonzalez et al., 2005). The inhibitory effect of atractyloside and bongkreic acid on the hyperpolarization induced by biapigenin, after addition of ADP, suggests that the effects of biapigenin occur at the ANT level.

As expected, increased oxygen uptake due to the addition of ADP (stage 3 of respiration) to energy-supplemented mitochondria was reduced in the presence of ANT inhibitors, atractyloside and bongkrequic acid. The effect of bongkreic acid on stage 3 of respiration was not affected by biapigenin, although the effect of atracurium was prevented by it (Figure 10C). These results support the notion that the effects of biapigenin occur at ANT level, and also indicate that they are dependent on conformation. Carbonyl-4-trifluoromethoxyphenylhydrazone (FCCP) cyanide-induced decoupling respiration was significantly inhibited in the presence of biapigenin and atraxythecin ANTto, whereas bignkrequic acid had no significant effect (Figure 11). Several authors have suggested that the mechanism of FCCP does not involve membrane translocations with the participation of ANT (Brustovetsky et al., 1990 / Andreyev et al., 20005); however, we have observed that FCCP-induced decoupling respiration was inhibited in the presence of attractyleside. Based on these data, we can not rule out a possible involvement of ANT in the effects of FCCP-mediated decoupling in the brain. Most of these previous studies used hepatic mitochondria where the ANT isoform found mostly is ANT2, while in the brain the most common isoform 28 is a ΑΝΤΙ (Dorner et al., 1999). It is possible that differences in tissue expression of specific isoforms may explain the observed differences in the effect of various molecules, including FCCP, ANT inhibitors or even biapigenin. The results also suggest that biapigenin may inhibit the respiratory chain.

It has been suggested that ANT acts as a proton channel (Brustovetsky et al., 1994; Shabalina et al., 2006). It has been proposed that ΑΝΤΙ and ANT2 isoforms are responsible for different aspects of proton conductivity: ΑΝΤΙ ΑΝΤΙ is more associated with aspects of proton basal conductivity, whereas ANT2 seems to play a role in proton decoupled conductivity (Shabalina et al., 2006) . We can only speculate that the inhibition of ANT by specific molecules, such as the atractilosídeo, bongkréquico acid or even biapigenina, can interfere with the membrane proton conductivity of mitochondria.

The inhibitory effects on ADP-induced depolarization suggest that biapigenin may decrease ADP entry into the mitochondrial matrix by ANT, although the effects of ATPsintetase activity at this time can not be excluded. The interaction of biapigenin with the phosphorylative system is in accordance with the observation that biapigenin decreases the ADP / O rate. However, breath stage 3 is not fully inhibited by biapigenin (Figure 11A), suggesting that biapigenin does not completely inhibit the influx of ADP as opposed to the inhibition of ANT by the atractytidide or bongkrequic acid. The activity of ATPsintetase was reduced by biapigenin (Figure 13B). In addition, inhibition of ATP synthesis by biapigenin (measured by high performance liquid chromatography (HPLC), by atractyloside, bongkreic acid or oligomycin was similar. Since the two methods used in this work to determine ATPsintetase activity require functionally intact mitochondria with active ANT involvement, an ANT-related biapigenin effect is also a good explanation. The effect of biapigenin on ATPsintetase activity (as assessed by variations in pH) may be associated with inhibition of ANT, reducing ADP (or Efflux of ATP) entry. However, the interaction of biapigenin with ATP synthase can not be excluded; in fact, the induction of proton flux decoupling by the F0 subunit affects ADP phosphorylation (Pietrobon et al., 1987; Bravo et al., 2001). The concept of symptomoma was recently proposed. The Ko and Chen groups showed a strong association between the ATPsintetase complex and ANT (Ko et al 2003, Chen et al., 2004), which leads to the idea that drugs whose target is any of the proteins involved in mitochondrial phosphorylation may also be able to exert indirect effects on the proteins present in this functional complex.

The results of the present study suggest that biapigenin does not inhibit the passage of ATP into the mitochondrial matrix. In addition, the hyperpolarizing effects of biapigenin on ATP-induced energy supplementation (Figure 12A) appear to be related to a decrease in the retrograde flow of protons to the matrix. This effect of biapigenin is also likely to be sensitive to ANT conformation, since the conformational state of bongkrequic acid-induced ANT was unaffected in the presence of biapigenin, whereas the ANA conformation induced by the attracty dideoxy was inhibited by biapigenin (Figure 12B). Both atractiloside and bongkreic acid bind to mutually exclusive sites (Dahout-Gonzalez et al., 2006). Atratintyloside is a non-permanent inhibitor of ADP binding to ANT (Bruni et al., 1965), whereas bongkreic acid has to cross the internal mitochondrial membrane to exert its inhibitory effects at the ADP binding sites on the matrix side. Consequently, in the presence of bongkreic acid and biapigenin, a decrease in the ADP / import of ATP from / to the matrix would occur, leading to a decrease in the availability of ATP to generate more ΔΨπι. In summary, it appears that biapigenin and atractiloside share a common target in ANT. The PTPm aperture is modulated by a number of factors, such as a high ΔΨ ™, low matrix pH or presence of adenosine nucleotides. Furthermore, under conditions that maintain the reducing status of the mitochondrial matrix (Nicotinamide adenine dinucleotide reduced (NADH) or Nicotinamide adenine dinucleotide reduced phosphate (NADPH), anti-oxidants) the opening of PTPm is inhibited (Brookes et al. et al., 2000). On the other hand, calcium and reactive oxygen substances (ROS), among other factors, are inducers of pore opening. Calcium accumulation by the mitochondria occurs at the expense of and excessive calcium capture dissipates to a level at which the PTPm gap is not inhibited, inducing the generation of ROS that cause damage to mitochondrial membrane proteins, resulting in more dissipation of ΔΨ, η in a vicious cycle (Brookes et al., 2004).

The data presented here report that the mitochondrial calcium capture capacity was reduced in the presence of biapigenin. The observation that the addition of a pulse of biapigenin to energy-enriched mitochondria and with maximum calcium loading induces calcium efflux suggests that, in the presence of calcium, biapigenin may induce the opening of PTPm. This effect would cause calcium efflux, resulting in a decrease in mitochondrial calcium accumulation, as observed (Figure 15D). When incubated with atracytoside, which induces a pore conformation, the effect of biapigenin was additive, i.e., contributed to the decrease of calcium accumulation. These results appear to be in agreement with data from the capture of TPP + and mitochondrial respiration, suggesting that the opening of PTPm can occur in the presence of biapigenin and atractilosídeo or, in this case, of biapigenina and calcium. ADP plus oligomycin or cyclosporin A, two inhibitors of PTP-Î ± opening in brain mitochondria (Brustovetsky et al., 2000; Halestrap 2006) have effectively inhibited the effects of biapigenin. The observation suggests an involvement of PTP m in the calcium efflux induced by biapigenin, possibly by the modulation of ANT function, which may include an increase of cyclophilin D binding to ANT. In fact, it has been proposed that cyclophilin D modulates ANT function, since the binding of cyclophilin D to the ANT matrix surface favors the conformational change of ANT to non-specific pore activated by calcium (Halestrap and Brenner 2003; He and Lemasters , 2002).

The present results suggest that biapigenin increases the calcium efflux of the mitochondria, possibly by inducing the transient opening of the PTPm in such a way as to allow excess calcium release and relief of mitochondrial overload. The functional effect of biapigenin on the mitochondrial calcium reported in the present study is similar to the reported effect of minocycline on the decrease of calcium uptake in brain mitochondria (Fernandez-Gomez et al., 2005). The reduction of mitochondrial calcium uptake may contribute to the preservation of mitochondrial functions, especially under stress conditions, protecting the neurons from excitotoxic cell death (Fernandez-Gomez et al., 2005; Urushianti et al., 2001, Duchen 2001).

Thus, we have shown that the phenolic compounds present in extracts of H. perforatum are neuroprotective against excitotoxicity in hippocampal neurons, involving anti-oxidant properties. In the specific case of biapigenin, we have shown that it can prevent mitochondrial dysfunction, possibly by anti-oxidant mechanisms but also involving other processes related to mitochondrial calcium homeostasis, involving the ANT function.

We propose that the interaction of biapigenin with its targets contributes to the protection of mitochondrial function, inducing the transient opening of PTPm and a reduction of mitochondrial calcium retention during excitotoxic events.

In fact, modulation of mitochondrial physiology, namely at the level of mitochondrial calcium accumulation capacity, probably plays an important role in the protection of mitochondrial function. This may be especially important under excitotoxic events when high amounts of calcium captured by energy-enriched mitochondria lead to irreversible mitochondrial depolarization and consequently to mitochondrial dysfunction. It is plausible that biapigenin, inducing the transient opening of PTPm and decreasing mitochondrial retention of calcium, is able to relieve the mitochondria of calcium accumulation, thus contributing to the maintenance of mitochondrial function under stress conditions. Finally, this would translate into a neuroprotective action that may help to resolve the calcium overload of neurons under excitotoxic events.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. A, structural formulas of the phenolic compounds used in this study: quercetin, campperol and biapigenin. B, Neuroprotection against excitotoxicity in culture of rat hippocampal neurons. Cell viability was assessed by the Syto-13 / PI life-death assay. Exposure of cultured neurons to ΙΟΟμΜ of kainate plus ΙΟΟμΜ NMDA (35 min exposure, followed by a post-exposure recovery period of 24 h), induced a considerable decrease in viability. There was significant neuroprotection with incubation with quercetin, campperol and biapigenin (10 μΜ for the three 34 compounds). C, Immunocytochemistry for MAP-2, staining with MitoTracker Red CMXRos and nuclear staining with Hoechst 33342. Exposure to ΙΟΟμΜ of kainate (KA) and ΙΟΟμΜ NMDA (35 min exposure, followed by a post-exposure recovery period of 24 h) induced (MitoTracker Red CMXRos) and nuclear condensation (arrows) were observed in the mitochondrial mitochondrial distribution (MitoTracker Red CMXRos). After excitotoxic insult, we were able to identify signs of dendritic dystrophy and loss of mitochondrial transmembrane electric potential (ΔΨ..) Note the relatively higher values of Mitotracker Red after coexposure with biapigenin (ΙΟμΜ), suggesting a ΔΨ ™ more top row - control, row of half-kaolin plus NMDA, bottom row - biapigenin plus kainate plus NMDA.

Values are reported as means ± SEM (n = 3 independent experiments); * - p &lt; 0.05, ** - p &lt; 0.01, *** - p &lt; 0.001 (compared to kainate (KA) plus NMDA).

Figure 2. Biapigenin significantly protects hippocampal neurons from delayed calcium regulation and mitochondrial potential homeostasis failure after excitotoxic insult with ΙΟΟμΜ kainate plus ΙΟΟμΜ NMDA (). A, the Figures show examples of calcium deregulation and loss of mitochondrial transmembrane potential (ΑΨ ™) in a typical control experiment, without addition of phenolic compounds, or B, after adding 10 μg of quercetin, 10 μg of campperol or 10 μg of biapigenin (□ ). Values were determined for every 5 min and averaged five to eight independent experiments. Time t '= 35 min 35 reflects the total number of cells that have lost the ability to maintain calcium homeostasis. The fluorescence intensity scales are indicated on the right side of each set of images. Biapigenin (ΙΟμΜ) was the most effective compound to prevent calcium deregulation and loss of values. The values are presented as mean ± SEM (n = 6 to 8 independent experiments); - p &lt; 0.01, *** - p &lt; 0.001 (comparing with kainate plus NMDA).

Figure 3. Changes in mitochondrial transmembrane potential (ΔΨ ™) when supplementing energy with succinate mitochondria. A, representative record for each of the conditions of the experiment: 1, control; 2, quercetin (10μΜ); 3, campferol (10μΜ); and 4, biapigenin (10μΜ). Black arrows indicate addition of 8nM succinate; White arrows indicate addition of 125μΜ of ADP. B, transmembrane mitochondrial electrical potential after energy supplementation with succinate (control, - 185 ± 4 mV); C, after ADP-induced depolarization (control, 17 + 2 mV); and D, after repolarization (control, -2 + 1 mV). Values are reported as mean ± SEM (n = 3 to 5 independent experiments); ** - p &lt; 0.01, *** p &lt; 0.001 (as compared to the control).

Figure 4. Quercetin, campperol and biapigenin significantly reduced ADP / iron-induced lipid peroxidation in isolated brain mitochondria. A, lipid peroxidation induced by 1mM ADP / 100μΜ FeS04 (), significantly reduced in the presence of quercetin (A) r campferol (▼) and biapigenin (♦) (10μΜ for all three compounds) ** - p < 0.01 for all compounds, ## - p &lt; 0.01 for campperol and biapigenin (when compared to ADP / iron 36). B, TBARS levels measured at the end of the experiment were significantly reduced in the presence of the three compounds. Values are presented as mean ± SEM (n = 3 to 4 independent experiments); * - p &lt; 0.05, ** - p &lt; 0.01 (compared to ADP / iron).

Figure 5. Changes in transmembrane mitochondrial electrical potential (ΔΨ ™) in succinate-energized mitochondria (□), induced by ADP / iron exposure. The addition of 1 mM ADP / ΙΟΟμΜ FeS04 () induced the dissipation of ΔΨ. The loss of ΔΨ ™ was significantly delayed in the presence of the compounds (ΙΟμΜ for the three compounds). Quercetin (A) and campferol (T) effectively prevent ADP / iron-induced decrease of ΔΊ,, similar to the effect observed for BHT (o) (30 μΜ).

Interestingly, biapigenin (♦) caused a significant hyperpolarization of the cerebral mitochondria and delayed the decrease in ADP / iron induced ΔΊ,, exhibiting a profile similar to cyclosporin A (0). Values are presented as mean ± SEM (n = 4 independent experiments). ## - p &lt; 0.01 for biapigenin, ### - p &lt; 0.001 for biapigenin, *** - p &lt; 0.001 (as compared to ADP / iron).

Figure 6. Capture of mitochondrial calcium assessed by TPP accumulation. Representative records of: Al and A2, controls without added calcium or after adding CaCl2 (15 μΜ), respectively; A3, cyclosporin A (Ο.βμΜ) in the presence of CaCl2 (15μΜ); B4, 10μΜ quercetin; B5, 10μg camphorol; and B6, 10μg biapigenin in the presence of CaCl2 (15μΜ). Narrow arrows - TPP additions (0.5μΜ); open arrow - addition of mitochondria (control) and CaCl2; solid arrow - addition of succinate. C, capture of TPP measured one minute after obtaining maximum load of mitochondrial calcium under energization with succinate. CsA-cyclosporin A. Values are presented as mean ± SEM (n = 3 to 5 independent experiments). * - p &lt; 0.05, ** - p &lt; 0.01, *** - p &lt; 0.001 (compared to calcium).

Figure 7. Calcium accumulation by the cerebral mitochondria evaluated by the observation of the Calcium Green-5n fluorescence. A, the Figure shows representative records of mitochondrial calcium accumulation in the control and after incubation with quercetin, campperol, biapigenin (10μΜ for the three compounds) and cyclosporin A (Ο.βμΜ). The mitochondria were energized in the presence of calcium, by the addition of 4 mM succinate. Calcium accumulation was monitored by changes in fluorescence intensity after energization. B, biapigenin significantly inhibits calcium uptake and significantly inhibits the maximal cyclosporine-mediated mitochondrial calcium uptake. Empty bars - without cyclosporin A (Ο.βμΜ); filled bars - with cyclosporin A. Values are presented as mean ± SEM (n = 3 to 5 independent experiments). * - p &lt; 0.05 (as compared to the control); ## - p &lt; 0.01 (as compared to cyclosporin A).

Figure 8. Energized mitochondria exposed to calcium pulses. Representative records of calcium accumulation in the control (□) and after incubation with biapigenin (φ) (10μΜ). was added

Pulse of 5 nM Ca2 + / mg protein (arrows) was added to suspension-energized mitochondria. At the end of the experiment, 38 (ethylenebis (oxyethylenenitrile) tetraacetic acid (EGTA). The storage capacity of mitochondrial calcium was higher for control (bottom register) compared to calcium accumulation after preincubation with biapigenin The differences between the two conditions are presented for the period in which the mitochondria exhibited a higher calcium storage capacity (inserted graph), which is evident by loss of fluorescence intensity after each calcium pulse. Values are reported as mean ± SEM (n = 3 to 4 independent experiments).

Figure 9. ANT inhibitors blog for biapigenin-mediated ADP-induced hyperpolarization. The transmembrane mitochondrial potential (ΔΨ ™) (indirectly assessed by the capture of TPP +) was monitored in isolated rat brain mitochondria energized with succinate. The representative graphs are given and the values of ΔΨχ após after repolarization (post-ADP) are presented. A - control; B-biapigenin (ΙΟμΜ); C-oligomycin (lpg.ml-1) and D-oligomycin plus biapigenin; E - atractyloside (40 μΜ) and F - atractyloside plus biapigenin; G - bongkreguic acid (16 μΜ) and H - bongkréchic acid plus biapigenin.

Figure 10. Breath stage 3 is not significantly blocked by biapigenin. Isolated rat brain mitochondria (0.8mg.ml_1) were incubated for 3 min with biapigenin (ΙΟμΜ) prior to energization with succinate (8mM). 125μg of ADP was added 1 min after energization. (A) Records representative of mitochondrial respiration for control and for pre-39 mitochondria incubated with biapigenin (ΙΟμΜ). (B) Pre-incubation with atractyloside (40μΜ) inhibited ADP-induced respiration stimulation, which was preserved in the presence of biapigenin (ΙΟμΜ). (C) Breathing was evaluated after addition of 125 μΜ of ADP to energized mitochondria. Values are presented as mean ± SEM of three to four independent experiments. ** p &lt; 0.01 (as compared to control); # p &lt; 0.05, ## p &lt; 0.01 (compared to biapigenin).

Figure 11. Biapigenin reduces FCCP-stimulated mitochondrial respiration. Isolated rat brain mitochondria (0.8mg.ml_1) were incubated for 3 min with biapigenin (10 μΜ) prior to energization. FCCP (1 μΜ) was added 1 min after energization. A) Representative graphs of mitochondrial respiration in the control and in mitochondria preincubated with ΙΟμΜ of biapigenin. B) Decoupled mitochondrial respiration was monitored by monitoring oxygen consumption after adding ΙμΜ of FCCP to energized mitochondria. Values are presented as mean ± SEM of three to four independent experiments. * p &lt; 0.05, *** p &lt; 0.001 (as compared to control); # p &lt; 0.05, ### p &lt; 0.001.

Figure 12. Effect of biapigenin on mitochondria with energy supplementation induced by ATP. It was measured by observing the capture of TPP + by mitochondria, after addition of 3 mM of ATP to the reaction medium. The biapigenin (ΙΟμΜ) was preincubated for 3 min. (A) Representative records of mitochondria with ATP-induced energization for control and biapigenin-treated mitochondria. The arrows indicate the addition of oligomycin (lpg.ml-1). (B) Biapigenin (ΙΟμΜ) significantly increased ATP-induced energization (from 142 to 185 mV, p <0.01, compared to control). Atratilostyloid (40 μΜ), bongkréchic acid (16 μΜ) and oligomycin (μg ml -1) significantly inhibited ATP-induced activation. (C) ATPase activity (assessed on injured mitochondria) was slightly but significantly decreased in the presence of biapigenin (ΙΟμΜ). Oligomycin was used as a control. Values are presented as mean ± SEM of three independent experiments. * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001 (as compared to control); ## p &lt; 0.01.

Figure 13. Effect of biapigenin on ATPsintetase activity in rat brain mitochondria (0.8mg.ml ~ x). The activity of ATPsintetase was evaluated by monitoring the pH variations in energized mitochondria by adding 125μΜ of ADP. At the end of each experiment, calibration was performed by the addition of 50nM NaOH and counter-titrated with 50nM HCl. (A) Records representative of the activity of ATP synthase for control and in biapigenin treated mitochondria. (B) ATPsintetase activity was decreased after incubation with biapigenin (10μΜ) and strongly inhibited in the presence of atractyloside (40μΜ), bongkreic acid (ΙβμΜ) or oligomycin (lpg.ml-1). Values are presented as mean ± SEM of three independent experiments. ** p &lt; 0.01, *** p &lt; 0.001 (as compared to control).

Figure 14. Capture of mitochondrial calcium. The capture and retention of mitochondrial calcium were evaluated in isolated rat brain mitochondria (0.2mg.ml'1), after energizing with 8mM of succinate (capture of calcium induced by energization). Biapigenin (10μΜ) was incubated for 3 min. Mitochondria were also incubated with: A) cyclosporin A (Ο.βμΜ) or co-incubated with biapigenin; B) bongkréquico acid (16μ) or co-incubated with biapigenin; C) atractyloside (40 μΜ) or co-incubated with biapigenin. Values are presented as mean + SEM of three to eight independent experiments. D) Maximum accumulation of mitochondrial calcium. Values are presented as mean ± SEM of three to eight independent experiments. * p &lt; 0.05 (as compared to control); # p &lt; 0.05.

Figure 15. Biapigenin reduces the accumulation of mitochondrial calcium and increases the efflux of calcium. The retention of mitochondrial calcium in energized mitochondria (0.2mg.ml_1) after addition of a calcium pulse (40 μΜ) was evaluated. Calcium present in the reaction medium ([Ca 2+] out) was evaluated by observation of Calcium Green 5-n fluorescence (100 nM); a decrease in fluorescence corresponds to accumulation of calcium in the mitochondria, while the increase in fluorescence corresponds to the efflux of mitochondrial calcium into the reaction medium. (A) Pre-incubation of mitochondria with biapigenin (10μΜ) for 3 min, decreases the maximum accumulation of calcium; the addition of a biapigenin pulse (indicated by the open arrow) induced efflux of mitochondrial calcium. (B) Bongkrheic acid (16μΜ) increased mitochondrial calcium accumulation, but did not prevent calcium efflux induced by biapigenin. (C) Pre-incubation with atractostyloid (40μΜ) induced calcium efflux, and this effect was additive with that of biapigenin. (D) Cyclosporin A (Ο, βμΜ) increased calcium accumulation and partially blocked the effect of biapigenin on calcium efflux. (E) ADP (125μΜ) plus oligomycin (lpg.ml 2) increased mitochondrial calcium accumulation. ADP plus oligomycin completely blocked the decrease in biapigenin-induced calcium accumulation and biapigenin-mediated calcium efflux. After preincubation with biapigenin, the addition of a 125μ pulso pulse of ADP (lilac arrow) increased calcium accumulation. Values are presented as mean ± SEM of two to three independent experiments.

Figure 16. A novel formulation with neuroprotective properties. We developed a new formulation with quercetin (21μΜ), campferol (Ι, ΙμΜ) and biapigenin (2.6μΜ) - V5 mixture, capable of protecting rat hippocampal neurons in culture from neurotoxicity induced by (A) beta amyloid β peptide exposure (25-35), (25μΜ) or (B) to an excitotoxic mixture of kainate - KA (ΙΟΟμΜ) plus NMDA (ΙΟΟμΜ).

DETAILED DESCRIPTION OF THE INVENTION 1. Formulations of the present invention 1.1. Formulations with biapigenin

These associations contain biapigenin between 0.1 and 50μΜ. They may also contain other substances, such as other active substances and acceptable pharmaceutical carriers. 1.2. Formulations with biapigenin, quercetin and campperol Other combinations, within the scope of this invention, contain biapigenin and other flavonoids, such as quercetin and campperol. These compounds may be present in the formulations referred to in individual concentrations of 0.1-50 μ. In addition to these substances, there may be other pharmaceutically acceptable active substances and carriers.

In a preferred formulation of this invention, there is reported an association of quercetin (21μΜ), campferol (Ι, ΙμΜ) and biapigenin (2.6μΜ). 2. Chemicals Kainate was supplied by Ocean Produce International (USA) and NMDA was supplied by Tocris (USA). Alexa Fluor conjugated antibodies, Calcium Green-5n, Fura2-AM, Hoechst 33342, Mitotracker Red CMXRos, propidium iodide, Syto-13, TMRM were supplied by Molecular Probes (USA). Cyclosporin A, proteases (Subtilisin, Carlsberg) type VIII and tetraphenylphosphonium chloride (TPP) were obtained from Sigma (Portugal). Digitonin and pluronic acid were obtained from Calbiochem (USA). Calcium Green-5n was supplied by Invitrogen (USA). The salt of monopotassium adenosine diphosphate dihydrate, magnesium triphosphate salt, atractyloside, bongkreic acid, bovine serum free of fatty acids (BSA), cyclosporin A, oligomycin, proteases (Subtilisin, Carlsberg) type VIII and tetraphenylphosphonium chloride (TPP) were obtained from Sigma (Spain). Quercetin, campperol and biapigenin were isolated by HPLC technique from extract of H. perforatum, as described in Dias et al. The purity of the three compounds was 98-99%. All other chemicals had the highest purity available on the market. 3. Neuronal cultures 44

Hippocampal neurons were dissociated from the hippocampus of embryos of E18-E19 Wistar rats after treatment with trypsin (2.0mg / ml, 15min, 37øC) and deoxyribonuclease I

(0.15 mg / ml) in Hank's solution equilibrated in free Ca2 + and Mg2 + (137 mM NaCl, 5.36 mM KCl, 0.44 mM KH2 PO4, 0.34 mM

Na2HP04.2H20, 4.16mM NaHC03, 5mM glucose, supplemented with 0.001% phenol red, 1mM pyruvate, 10mM 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES), pH 7.4). Cells were cultured in B-27 and serum-free (Gibco) supplemented neurobasal medium, containing glutamate (25μ), glutamine (0.5mM) and gentamicin (0.12mg / ml) as described previously (Silva et al. ). Cultures were maintained at 37øC in a humidified incubator with 5% CO2 / 95% air for 7 days, the time required for the maturation of hippocampal neurons. For the purposes of viability studies with Syto-13 and propidium iodide (PI) cells were coated at a density of 45x10 3 cells / cm 2 on lamellas covered with poly D lysine (0.1 mg / ml). 4. Cell Viability and Immunocytochemical Assays Neural viability was assessed with Syto-13 and the life / death propidium iodide assay after exposure of culture hippocampal neurons to kainate plus NMDA, alone or in the presence of the compounds. The structural formulas of these three compounds tested are shown in Figure 1A. The neurons were continuously exposed to 100 μl kainate plus 100 μM NMDA for 35 minutes at 37 ° C and were allowed to recover for 24 hours in conditioned medium. Syto-13 is a fluorescent green color with membrane permeability. PI is a nonpermeable red fluorescent staining that only harms cells that have lost membrane integrity - late or necrotic apoptotic (Silva et al., 2004a). Cell death resulting from the isolation and coating process represented 30% of the highly condensed and PI-positive nuclei (data not shown). Immunocytochemistry directed to the microtubule-associated protein MAP-2 was performed, and mitochondrial and nuclear morphology were evaluated with MitoTracker Red CMXRos and Hoechst 33342, respectively. Briefly, the cells were incubated for 30 min with 125 mM MitoTracker Red CMXRos, washed and fixed with paraformaldehyde (4% paraformaldehyde, 4% sucrose in phosphate buffer) for 30 min.

After washing, the cells were permeabilized with 0.2% Triton X-100, blocked with 3% bovine albumin serum (BSA) and subsequently incubated with a primary mouse anti-MAP-2 antibody for 1 hour. After washing, the cells were incubated with goat derived Alexa Fluor 488 anti-mouse IgG conjugated secondary antibody. After washing the cells were incubated for 5 min with Hoechst 33342 and subsequently placed under glass slides using a fluorescence medium (Dako Cytomation, USA). 5. Calcium dysregulation and loss of mitochondrial membrane potential - unicellular imaging studies Calcium dysregulation and mitochondrial transmembrane potential were monitored by observation of individual cells of culture hippocampal neurons. For 40 min the cells were given 5 μg Fura-2 μg, 20 μM methyl tetramethylrhodamine ester (TMRM), 0.1% fatty acid free BSA and 0.2% pluronic acid F-127 in Krebs buffer (132 mM NaCl, 1 mM KCl , 1 mM MgCl 2, 2.5 mM CaCl 2, 10 mM glucose, 10 mM HEPES-Na, pH 7.4). Experiments took place at room temperature. The slides were washed with Krebs buffer and mounted in an infusion chamber. Images were acquired through the MetaFluor software program (Universal Imaging Corporation, ver. 5.0r7 2003) on an Axiovert 200 epi-fluorescence inverted microscope (Zeiss) equipped with Lambda DG-4 (Sutter Instrument Company) and a camera High-resolution LCD (CoolSnap HQ). Images were acquired alternately at 340, 380 and 598 nm (300 ms exposure time, 10 s between each image) using a Fura-2 / rhodamine filter. Five minutes after the start of imaging, the cells were exposed to Krebs medium containing 20nM TMRM and 100μΜ kainate plus 100μΜ NMDA. In another group of experiments, the cells were preincubated for 15 min with 10 μΜ quercetin, campperol or biapigenin (after 40 minutes exposure to the Krebs buffer chemicals and before the start of the experiment). After this preincubation period, the compounds were present for the remainder of the experiment. Appropriate controls were performed using Krebs perfused cells. Most cells did not lose calcium homeostasis for the duration of the experiment (40 minutes). 6. Mitochondrial respiratory current and mitochondrial transmembrane electrical potential Mitochondria of male Wistar rats (8 weeks old) were isolated using a method previously described and used to evaluate mitochondrial transmembrane electrical potential (ΔΨ ™) and respiration ( Moreira et al., 2002). ΔΔ ™ was monitored by evaluating the transmembrane distribution of the tetraphenylphosphonium (TPP +) lipophilic cation with a selective TPP-47 electrode and a calomel electrode as reference (Kamo et al., 1979). The potential difference between the selective electrode and the reference electrode was measured with an electrometer and continuously recorded on a Kipp &amp; Zonen. The reactions were carried out at 30øC inside a chamber with magnetic stirring in one ml of medium (100mM sucrose, 100mM KCl, 2mM KH2PO4, ΙΟμΜ EGTA, 5mM HEPES, pH 7.4, supplemented with 2μΜ rotenone) and containing 3μΜ TPP-C1 (Moreira et al., 2002a; Oliveira et al., 2004a). Mitochondria (0.8 mg / ml) were incubated for 3 min with 10 μΜ quercetin, campperol or biapigenin. The reactions were triggered with the addition of 8 mM succinate to the suspension mitochondria. After reaching a stable distribution of TPP (plateau), 125μΜ of ADP was added and the changes of A ¥ m were recorded. Appropriate controls were carried out without addition of compounds, but always respecting the 3 min interval used in the test conditions. Oxygen consumption of the isolated mitochondria was recorded polarographically with a Clark oxygen electrode attached to a recording apparatus in a closed, thermostatic, water-jacketed, and magnetically-stirred closed chamber (Estabrook 1967). Stage 3 respiration is defined as the consumption of oxygen in the presence of substrate and ADP, while stage 4 respiration is defined as the consumption of oxygen after consumption of ADP. Mitochondrial respiration was not altered by the presence of TPP (data not shown). The RCR values obtained were in agreement with the expected values for cerebral mitochondria and referred to earlier (Moreira et al., 2005). 7. Mitochondrial lipid peroxidation The extent of lipid peroxidation was directly evaluated in non-energized mitochondria by the formation of thiobarbituric acid reactive substances (TBARS) and by oxygen consumption independent of the respiratory chain oxygen consumption of isolated mitochondria exposed to ADP plus iron; and indirectly through the monitoring of changes in the energized mitochondria exposed to the oxidant pair ADP plus iron. In summary, lipid peroxidation of mitochondrial membranes (0.8mg / ml) was evaluated by observing oxygen consumption as previously reported (Ferreira et al., 1999), with minor alterations, or by evaluating the decrease of mitochondria energized with 8mM succinate) (Abreu et al., 2000) after exposure to 1 mM ADP plus ΙΟΟμΜ of iron for 10 or 15 min at 30 ° C. To evaluate the effect of the phenolic compounds, the mitochondria were preincubated for 3 minutes with ΙΟμΜ of quercetin, campperol or biapigenin. At minute 10 (time of the end of the reaction) samples were taken to assess the extent of lipid peroxidation in non-energized mitochondria by measuring the formation of TBARS. Butylhydroxytoluene (ΒΗΤ, 30μ Utiliz) was used as a positive control of the anti-oxidant effect against lipid peroxidation induced by the ADP / iron pair. The storage capacity of mitochondrial calcium was assessed indirectly through the mitochondrial accumulation of TPP in the presence of calcium (Oliveira et al., 2004b). The mitochondria (0.8 mg / ml) were incubated with 15μΜ of calcium in the presence or absence of the compounds for 3 minutes, and then energized with 8mM of succinate. The 49 changes in were recorded with a selective TPP electrode for 10 min. The TPP accumulation was calibrated for all conditions tested by the addition of pulses of 0.5 μΜ TPP (final concentration in the system was 3μΜ TPP). The values presented represent TPP accumulation 1 min after the maximum generation. In some trials, the value of was determined according to previous references (Oliveira et al., 2004b). Calcium uptake was evaluated by Calcium Green-5n fluorescence with adequate calibration in the presence of mitochondria (0.2 mg / ml) and rotenone, with pulses of 2.5 μΜ calcium each. The reactions occurred at 30øC in a quartz cuvette with 2 ml of reaction medium and under magnetic stirring. The changes in fluorescence intensity were followed by a Perkin Elmer LS SOB fluorimeter (excitation at 506 nm, emission at 531 nm, 5 nm slit) after supplementation of energy with 4 mM succinate (Oliveira et al., 2003). When stable fluorescence was reached, FCCP was added to assess the release of calcium caused by mitochondrial depolarization and, consequently, the amount of calcium accumulated in the mitochondria due to the transmembrane electric potential; calcium capture in the mitochondria evaluated by this method was almost always close to 90-95% of total calcium capture (data not shown). Calcium capture was also evaluated after addition of 10μΜ of calcium pulses to energized mitochondria. The pulses were added every 30s and the intensity of the fluorescence was monitored. The effect of the compounds (preincubated for 3 min) on the number of pulses supported by mitochondria before mitochondrial function failure was also analyzed. Compounds were added after energization with succinate. EGTA was added after a final stable fluorescence state had been reached.

When described,

Calcium retention was assessed by fluorescence of Calcium Green-5n. Appropriate calibration was performed with pulses of 7nmol Ca 2+ / mg protein, added to the mitochondria (0.2 mg / ml suspension). Reactions occurred at 30øC in a quartz cuvette with 2 ml of reaction medium ( 100mM sucrose, 100mM KCl, 2mM KH2PO4, ΙΟμΜ EGTA, 5mM HEPES, pH 7.4, supplemented with 2μΜ rotenone) under magnetic stirring.The changes in fluorescence intensity were controlled with a Perkin Elmer LS 50B fluorimeter (excitation at 506 nm , emission at 531 nm, 5 nm of emission slits and excitation), and calcium capture was evaluated upon energization with 4 mM succinate. When stable fluorescence was reached, FCCP was added to assess the total calcium release due to mitochondrial depolarization and, consequently, the amount of calcium accumulated due to mitochondrial transmembrane electric potential. Calcium capture in the mitochondria evaluated with this method was 90-95% of the total calcium Calcium efflux was evaluated in the energized mitochondria after the addition of a single calcium pulse (42nmol Ca2 + / mg protein) added 3 minutes after the addition of mitochondria to the reaction medium. The effect of biapigenin (10 μΜ) and other products tested on calcium homeostasis was evaluated after 3 minutes of incubation. Appropriate controls were carried out to evaluate potential interferences of biapigenin with the fluorescence probe, at low or high concentrations of calcium. No interference was observed in the conditions used for the experiment. When described, biapigenin or other products were added after the calcium pulse to assess possible effects on calcium efflux. EGTA (40μΜ) was added after reaching the final state of stable fluorescence. 9. ATPase Activity ATPase activity was determined by monitoring the proton production resulting from the ATP hydrolysis, according to the potentiometric method. Reactions were run at 30øC in open chamber with magnetic stirring in 2 ml of reaction medium (100 mM sucrose, 100 mM KCl, 2 mM KH2 PO4, 0.5 mM HEPES-K, λm EGTA, pH 7.3). Mitochondria (0.8mg.ml_1) were incubated for 3 min with ΙΟμΜ of biapigenin. Reactions were triggered by the addition of 2mM ATP-Mg to the suspension mitochondria, and changes in pH were recorded for 5 min. Addition of oligomycin at the end of the experiment completely abolished proton release. The appropriate controls were carried out without any addition of compounds. The pH changes were followed continuously with a pH meter, with the electrode inserted into the reaction medium under magnetic stirring. The pH variations were recorded on a Perkin-Elmer (Model 56) pH-linked apparatus via a basal voltage compensation circuit. Internal calibration was carried out at the end of each experiment, with the addition of suitable amounts (600nmol) of NaOH, and counter-titrated by addition of the same amount of HCl (due to NaOH instability in solution). 10. Activity of ATPsintetase Activity of ATPsintetase was measured by monitoring the pH variations associated with ATP synthesis by the potentiometric method described above. The reactions were run at 30øC in open chamber with magnetic stirring in 2mL of the reaction medium without HEPES (100mM sucrose, 100mM KCl, 2mM KH2PO4, λmΜ EGTA, pH 7.3). Mitochondria (0.8 mg.rnL &quot; 1) were incubated for 3 min with ΙΟμΜ of biapigenin. The reactions were triggered with the addition of 8 mM succinate to the suspension mitochondria. One minute after the energisation, 125μΜ of ADP was added and the pH changes were recorded. Appropriate controls were carried out without addition of compounds. The internal calibration was done at the end of each experiment, with the addition of suitable amounts of HCl (5nmol). 11. Adenosine nucleotide quantification Adenosine nucleotides were recovered with an acidic extraction procedure and separated by reverse phase liquid chromatography as described above with some minor changes. All extraction procedures were carried out at 0-4 ° C to minimize nucleotide degradation. Adenosine nucleotides were extracted from succinate-energized brain mitochondria incubated only with biapigenin, or in combination with other products, after a complete cycle of phosphorylation (1 minute after total phosphorylation of ADP). 300μl of the reaction medium was withdrawn into an eppendorf tube with oligomycin and 0.5M ice-cold perchloric acid (HC104). The mixture was then centrifuged (14,000 rpm at 4øC for 5 min). The granules were stored at -80 ° C for protein quantification (with a BioRad protein test). The supernatant was recovered and the pH adjusted to 6.5 with ice cold 2.5M KOH in 1.5M KH2PO4 and centrifuged (14,000 rpm at 4øC for 2 min). The new supernatant obtained was recovered with extreme caution to avoid the soluble permanganate salts produced, and stored at -80øC for further chromatographic analysis.

Samples were centrifuged at 14,000 rpm for 5 min before being injected into the HPLC system. The concentration of adenosine nucleotides (ATP, ADP and AMP) in each sample was determined by HPLC on a Gilson-Asted system, consisting of a model 305 pump connected to a computer with a 506C interface and Gilson 715 HPLC controller software. Briefly, the adenosine nucleotides were separated into a Lichrospher 100 RP-18 (5ym, 125mm) Merck (Darmstadt, Germany), protected by a Lichrospher 100 RP-18 (5 μm, 4 mm) column. In each of the tests an isocratic elution was used with 100 mM potassium phosphate buffer (pH 6.0) and 1% methanol for 8 min at a flow rate of 1.1 ml / min. The determination was made with a Gilson UVf model 116 detector at 254 nm. The nucleotide concentration was determined after determination of normal nucleotide solutions under identical conditions, and the chromatograms were subsequently analyzed with Gilson software. The limit of detection for each analyte was 1-2 ppm / injection. 12. Statistical analysis

The results are reported as mean ± SE of the indicated number of experiments, usually done in triplicate or quadruplicate unless otherwise noted. Statistical relevance was determined with a one-way analysis of variance (ANOVA) for multiple comparisons, followed by a Bonferroni post-test. 54

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Lisbon, June 08, 2009

Claims (5)

A formulation characterized in that it consists of a mixture of flavonoids biapigenin, campperol and quercetin with antioxidant properties and capable of protecting mitochondrial functions. The formulation according to claim 1, wherein said flavonoids are present individually in a series of active tissue concentrations of Ο, ΙμΜ to 50μΜ. A formulation characterized by containing biapigenin and being able to protect mitochondrial function. The formulation according to claim 3, characterized in that said biapigenin is present individually in a series of active tissue concentrations of Ο, ΙμΜ to 50μΜ. The formulation according to the preceding claims characterized in that it is used as a neuroprotective in brain disorders. Lisbon, June 08, 2009