WO2009087568A2 - Compositions comprising antioxidant and mitoprotective flavonoids with neuroprotective properties - Google Patents

Abstract

The present invention discloses compositions comprising phenolic compounds present in plant extracts with therapeutic activity against neuronal excitotoxicity, oxidative stress and mitochondrial dysfunction. When used individually or in a formula composition quercetin, kaempferol and biapigenin significantly reduced neuronal death caused by excitotoxic insults or to exposure to amyloid-beta peptide. Moreover, it is also disclosed that biapigenin interferes with the ANT/mitochondrial transition pore complex contributing for enhanced mitochondrial calcium efflux, thereby reducing calcium burden and contributing for neuroprotection against excitotoxicity.

Description

Description

Title of Invention: COMPOSITIONS COMPRISING ANTIOXIDANT AND MITOPROTECTIVE FLAVONOIDS WITH

NEUROPROTECTIVE PROPERTIES

FIELD OF THE INVENTION

[1] The present invention describes formulas comprising biapigenin with neuroprotective properties against excitotoxic and amyloid beta-induced neurodegeneration. Further, the present invention also describes formulas comprising a mixture of flavonoids with the same therapeutic purposes. BACKGROUND OF THE INVENTION

[2] Excitotoxicity has been associated with excessive glutamate receptor. Under prolonged NMDA receptor activation, e.g. during ischemia, anoxia, activation leading to neuronal degeneration (Lipton and Rosenberg 1994; Rego et al. 2001; Carafoli et al. 2001) and in other neurodegenerative pathologies (Brookes et al. 2004a; Nicholls 2004; Nicholls and Budd 1998; Chen and Lipton 2006), massive calcium influx into the cell occurs, leading to failure in neuronal calcium homeostasis (Carafoli et al. 2001; Nicholls 2004; Nicholls and Budd 1998; Nicholls and Ward 2000; Vergun et al. 2001; Montal 1998; Isaev et al 2005).

[3] Mitochondria play a vital role in maintaining calcium homeostasis in the cell, but excessive mitochondrial calcium accumulation can also result in loss of mitochondrial transmembrane potential ( D Ψ_m) and uncoupling of respiratory chain, increasing the generation of oxygen and nitrogen reactive species. Impairment of mitochondrial function can compromise ATP production and, consequently, lead to depletion of ATP stores and failure of ion homeostasis, including regulation of calcium concentration (Nicholls 2004; Nicholls and Budd 1998).

[4] Oxidative stress and mitochondrial calcium overload can lead to the opening of the mitochondrial permeability transition pore, which allows the passage of solutes and large molecules into the matrix space (Isaev et al. 2005; Vieira et al. 2000). Consequently, swelling of mitochondria and rupture of the outer mitochondrial membrane occurs leading to loss of mitochondrial function, production of reactive oxygen and nitrogen species which cause oxidation of membranes, proteins and nucleic acids. These processes promote the release of pro-apoptotic factors, such as cytochrome c, apoptosis-inducing factor (AIF), Smac-DIABLO and endonuclease G, which trigger the activation of effector mechanisms of cell death (Bouchier-Hayes et al. 2005d; Vander Heiden and Thompson 1999).

[5] It has been described that mitochondria are central players in the toxic events due to overactivation of glutamate receptors, causing loss of ion homeostasis and downstream pathways 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 critical role of mitochondria in the maintenance of the bioenergetic redox status, together with the participation in the decision of cell fate, makes this organelle key target for the development of new therapeutic strategies to treat pathologies associated with ischemic insults and neurodegenerative diseases such as Alzheimer's disease (Nicholls and Budd 1998; Bouchier-Hayes et al 2005b; Duchen 2004; (Duchen 2004; Mattson and Kroemer 2003; Won et al 2002), where excitotoxic events are major players in neuronal death.

[6] Previously we have shown that Hypericum perforatum extracts enriched in flavonoids (Silva et al. 2004b) are neuroprotective against β-amyloid-induced toxicity in primary cultures of rat hippocampal neurons (Silva et al. 2004a).

[7] H. perforatum has also been reported to be effective in an animal model of ischemia and reperfusion injury, reducing physiological and histological signs of damage (de Paola et al 2005). In fact, flavonoids have been pointed as potentially effective in preventing cell damage resulting from stroke and ischemia-reperfusion (Simonyief al 2005;Dajas et al 2003;Zhao 2005).

[8] Hypericum perforatum (St. John's wort) extract has been used for the treatment of nervous disorders for many years past. Its application for depression and psycho- autonomic disorders has greatly increased over the past few years in the light of the favorable results of clinical trials.

[9] Several documents have been disclosed concerning the utilization of H. perforatum extracts (EP599307, DE1919512, DE19714450, WO9940905, US20010033872) or fractions containing hyperforins or derivatives (DE19903570, US20010020040), derived from H. perforatum, for the treatment and prophylaxis depression, dementia or other related nervous disorders.

[10] Recently, the utilization of H. perforatum was envisaged as a method for treating

Alzheimer's disease and other amyloidoses (US0020150637).

[11] Also, it is already disclosed that H. perforatum extracts and certain compounds like hypericins and hyperforins can be used as therapeutics targeted at T-type calcium channels in various biological systems to treat diseases treatable with T-type calcium blocking agents, including brain aging or neurodegenerative related diseases (US20030207940).

[12] However, none of these documents refer the compounds of the present invention, namely quercetin, kaempferol and biapigenin, to be used in treatment of neurological diseases, in particular acting as neuroprotective compounds.

[13] In fact, the present invention relates to the neuroprotective properties of phenolic compounds present in H. perforatum extracts - quercetin, kaempferol and biapigenin - against excitotoxicity and further some compositions comprising those compounds useful in therapeutics, are described. SUMMARY OF THE INVENTION

[14] The present invention describes compositions of flavonoids (biapigenin, quercetin and kaempferol) with neuroprotective properties against amyloid beta-induced and ex- citotoxic-induced neurodegeneration, acting through antioxidant and mitoprotective mechanisms, delaying excitotoxic-induced calcium homeostasis failure. DESCRIPTION OF THE INVENTION

[15] The present invention describes compositions comprising biapigenin with neuroprotective properties against excitotoxic and amyloid beta-induced neurodegeneration. Further, the present invention also describes compositions comprising biapigenin and other flavonoids with the same therapeutic purposes.

[16] The compositions described in the present invention comprise antioxidant compounds, at least biapigenin and/or other flavonoids, namely quercetin and kaempferol, which act at mitochondrial targets and protects mitochondria from calcium-induced failure.

[17] The formulas described may be useful to treat or prevent neurodegeneration caused by brain insults or neurodegeneratives diseases in animals, including humans.

[18] Some Hypericum perforatum extracts contain high levels of flavonoid compounds.

By extracting and characterizing flavonoid compounds present in neuroprotective fractions from Hypericum perforatum three key neuroprotective flavonoids (quercetin, kaempferol and biapigenin, 10 μM each), as well as a mixture of these compounds (quercetin 21 μM, kaempferol 1.1 μM, biapigenin 2.6 μM) were identified.

[19] The mechanisms underlying neuroprotection against excitotoxic neuronal death

(induced by kainate+NMDA or to amyloid beta peptide exposure) involve antioxidant properties (quercetin and kaempferol) and mitochondrial protection of calcium-induced mitochondrial failure (biapigenin).

[20] The effect of the flavonoids concerning neuroprotection and delayed-calcium deregulation was evaluated in cultured rat hippocampal neurons. Antioxidant and mitoprotective properties of quercetin, kaempferol and biapigenin were evaluated in rat cortical synaptosomes and in rat brain isolated mitochondria.

[21] Therefore, the first object of the invention is a composition comprising biapigenin, which acts via mechanisms that protect the mitochondria - mitoprotective. This compound may be present in the compositions of the present invention in a range of 0.1 and 50 μM.

[22] Other compositions, in the scope of the present invention, comprise biapigenin plus other flavonoids, such as quercetin and kaempferol. These compounds may be present in the referred compositions in an individual concentration of 0.1-50 μM.

[23] These compositions display potent neuroprotective properties against excitotoxic insults, in brain tissue, involving antioxidant properties and prevention of calcium- induced mitochondrial failure.

[24] In a preferred embodiment of the present invention, it is referred compositions comprising 0.1 to 50 μM of biapigenin, quercetin and kaempferol, acting at neurological level as neuroprotective due to its antioxidants and mitoprotective mechanisms.

[25] Compound concentrations above 50 μM reveal to be neurotoxic and below 0.1 μM no neuroprotective effects.

[26] 1 - Phenolic compounds present in H. perforatum are neuroprotective against excito- toxicity in cultured hippocampal neurons

Neuronal viability was assessed after exposure of cultured hippocampal neurons to an acute excitotoxic challenge. Viability significantly decreased after exposure to 100 μM kainate plus 100 μM NMDA (55 ± 1% following excitotoxic insult as compared to 100 ± 4% in the control). Cell death evaluated after 24 h, was significantly prevented in the presence of 10 μM quercetin, kaempferol or biapigenin, with viability of 77 ±9% , 77 ±12% and 87 ± 11% of the control, respectively (Fig. 15).

[27] We also analysed changes in morphology as a sign of neurodegeneration Im- munostaining against MAP-2 revealed a marked dendritic degeneration following exposure to kainate plus NMDA. Moreover, we took advantage of MitoTracker Red CMXRos which accumulates in polarized mitochondria, to evaluate changes in mitochondrial distribution in neuronal cells. Following excitotoxic insults a major redistribution of partially depolarised mitochondria were observed with mitochondrial accumulation in cytoplasmatic clusters. Nuclear morphology was assessed by Ηoechst 33342 staining, showing highly condensed nuclei in cells exposed to kainate plus NMDA. Exposure to 10 μM biapigenin partially protected neurons from the deleterious action of the excitotoxic aggression, since neurons showed less dendritic dystrophy, maintenance of mitochondrial physiology and reduced number of nuclear markers of cell death (Fig. 1C).

[28] 2 - Phenolic compounds present in H. perforatum delayed calcium deregulation induced by kainate plus NMDA

We monitored intracellular calcium concentrations and, simultaneously, mitochondrial transmembrane potential by using single-cell imaging of neurons exposed to kainate plus NMDA. A similar protocol to the viability assays was used aiming to find a correlation between neuroprotection by phenolic compounds, protection from delayed calcium deregulation and improvement of mitochondrial function. Fig. 2A shows representative images of a control situation (Fura-2 and TMRM imaging) for the critical time points of the experiment. Changes in Fura-2 fluorescence (excitation ratio 340/380 nm) are shown in the left panel, whereas the right panel shows TMRM fluorescence. All images were taken from the same field and obtained at different time points, as indicated in the left. Loss of calcium homeostasis (340/380 ratio shifting towards the red colour) was strongly correlated with a significant decrease in TMRM fluorescence (fluorescence at 598 nm, shift to black) (r²= 0.96, n = 6-8 independent experiments, average 120 cells per field). Following the excitotoxic insult, a significant percentage of cells lost the ability to preserve calcium homeostasis and showed delayed calcium deregulation. Exposing the cells to 10 μM quercetin or biapigenin significantly protected neurons from delayed calcium deregulation (Fig. 25), biapigenin being particularly efficient. Kaempferol was the least efficient compound in protecting from delaying calcium deregulation.

[29] 3 - Effect of H. perforatum phenolic compounds on mitochondrial bioenergetics

The results obtained in neuronal cultures and above described led us to identify mitochondria as a possible target involved in the observed neuroprotective effects. To test this hypothesis, we used isolated brain mitochondrial fractions in order to directly investigate the effect of quercetin, kaempferol and biapigenin on mitochondrial bioenergetics.

[30] As can be observed in Fig. 3 quercetin and kaempferol (10 μM) did not significantly affect mitochondrial TPP uptake and respiration. Incubation of brain mitochondria for 3 minutes with biapigenin (10 μM) significantly affected mitochondrial depolarization and repolarization (Fig. 3C-Z)). By itself biapigenin did not significantly alter the TPP electrode response and respiratory capacity, decreasing significantly state 3 respiration (30% reduction). It is noticeable that state 4 respiration was also increased (43% increase), which further contributed to the reduction of the respiratory control ratio (RCR - parameter that reflects coupling between substrate oxidation and ADP phosphorylation, 47% decrease comparatively to the control). Uncoupled respiration was also inhibited, which suggests inhibition of the respiratory chain. Moreover, the ADP/ O ratio (reflecting phosphorylative efficiency) was also significantly reduced by incubation with biapigenin (approximately 30%). The phosphorylative efficiency was also evaluated through the time required by energized mitochondria to phosphorylate ADP (lag phase), and was significantly reduced by biapigenin (75% inhibition as compared with control, p < 0.001 - data not shown).

[31] 4 - Mitochondrial lipid peroxidation is reduced in the presence of phenolic compounds from H. perforatum

Lipid peroxidation was evaluated after exposure of isolated brain mitochondria to the oxidant pair ADP/iron. The three compounds were able to significantly reduce lipid peroxidation as evaluated either by oxygen consumption and TBARS production (Fig. AA and AB, respectively). Mitochondria alone (without addition of the pro-oxidants or the protective compounds) did not undergo significant peroxidation; and the same was observed for isolated mitochondria incubated with 10 μM of the compounds.

[32] The antioxidant properties of the compounds present in H. perforatum were also evaluated by inducing lipid peroxidation in energized mitochondria. Mitochondria were energized with succinate and then exposed to ADP/iron. Loss of D Ψ_m followed, mainly as a result of loss of membrane integrity, and consequent dissipation of the protonic gradient (Fig. 5). Butylhydroxytoluene (BΗT), a well known inhibitor of lipid peroxidation was used as a control, and completely inhibited loss of D Ψ_m further supporting the involvement of lipid peroxidation in this process. Quercetin and kaempferol were very efficient in preventing the loss of D Ψ_m. Interestingly, energization of mitochondria pre-incubated with biapigenin resulted in a transient hyperpo- larization just before peroxidation started.

[33] 5 - Effects of H. perforatum phenolic compounds on mitochondrial calcium accumulation

The ability of mitochondria to accumulate calcium was assessed using two different approaches. TPP⁺ uptake was evaluated in the presence of calcium as an indirect measurement of the mitochondrial calcium loading capacity (Fig. 6A and 65). Representative recordings are depicted in panel A, showing that TPP⁺ uptake was reduced in the presence of calcium when compared to a control condition (Fig. 6A and 6C). Of the three compounds tested, only biapigenin was able to significantly protect energized mitochondria from loss of D Ψ_m in the presence of calcium (Fig. 6C). Cyclosporin A was used as a positive control, since this drug is known to inhibit the opening of the permeability transition pore, desensitising the pore to calcium (Ηalestrap 2006) and thereby increasing mitochondrial calcium uptake capacity.

[34] Mitochondrial calcium accumulation was also evaluated in energized mitochondria by the use of a low affinity calcium-sensitive probe, Calcium Green-5N in the assay medium. Mitochondria were energized with calcium present in the assay medium, and calcium accumulation was followed by decrease in fluorescence intensity (reflecting decreased calcium concentration in the medium and accumulation into mitochondria). Representative traces are shown in Fig. IA. Additionally to the results obtained with the TPP⁺ electrode ( D Ψ_ra), we observed that biapigenin significantly reduced calcium accumulation (Fig. IB). No significant changes were observed after incubation with quercetin and kaempferol. On the other hand, incubation with cyclosporin A significantly increased mitochondrial calcium accumulation, an effect that was significantly reduced by biapigenin (Fig. IB).

[35] The efficiency of mitochondria to accumulate calcium as a response to an increase in cytoplasmatic calcium concentration, (e.g., during excitotoxicity events) was evaluated by adding pulses of 10 μM calcium to energized mitochondria in suspension. Fig. 8 shows the representative traces of calcium accumulation in control or after incubation with biapigenin (10 μM). As can be observed, in the presence of biapigenin mitochondria showed a lower calcium uptake capacity.

[36] 6 - Biapigenin decreases the ADP-induced dissipation of the mitochondrial transmembrane electric potential, but potentiates ADP-induced hyperpolarization

The effect of biapigenin in mitochondrial bioenergetics was evaluated by following TPP⁺ uptake in isolated rat brain mitochondria, which is an indirect estimation of the mitochondrial transmembrane electric potential (ΔΨ_m), and by following mitochondrial respiration assessed by monitoring oxygen consumption. Isolated rat brain mitochondria (0.8 mg.ml⁴) were incubated with several concentrations of biapigenin. Resting mitochondria exhibited a ΔΨ_m of -180.7 ± 2.3 mV. At a concentration of 10 μM, biapigenin significantly reduced ADP (125 μM)-induced depolarization (by 67.8%; p < 0.05, when compared to the control) and significantly increased mitochondrial repolarization (by 371.7%; p < 0.01, when compared to the control)(Fig. 9).

[37] 7 - ANT blockers inhibit the hyperpolarizing effect of biapigenin in the presence of

ADP

The results reported above suggest that biapigenin can exert a modulatory effect in the phosphorylative system. In order to identify a possible target for biapigenin, we compared the effects of biapigenin with known modulators of the mitochondrial phosphorylative system, the ATP synthase and the adenine nucleotide translocator (ANT). We observed that biapigenin-induced ADP-dependent hyperpolarization was not affected by oligomycin (1 μg.ml-¹, Fig. 9D), a drug that inhibits proton flow through the F₀ subunit of the ATP synthase, although inhibitors of the ANT, atractyloside (40 μM) and bongkrekic acid (16 μM) were able to prevent the observed effect (Fig. 9F and 9H, respectively).

[38] 8 - Biapigenin does not significantly affect ADP-induced increase in respiration

Mitochondrial respiration was assessed by monitoring oxygen consumption in isolated rat brain mitochondria. Mitochondria (0.8 mg. ml"¹) were energized with succinate (8 mM). No significant differences were observed in state 2 respiration (substrate-driven basal respiration) after 3 min pre-incubation with biapigenin (10 μM), atractyloside (40 μM), bongkrekic acid (16 μM) or oligomycin (1 μg.ml-¹). Despite the notorious effect of biapigenin on ADP-induced depolarization, stimulation of mitochondrial respiration with ADP (state 3 respiration) was not blocked by biapigenin. As expected, atractyloside and bongkrekic acid blocked state 3 respiration. Representative recordings for control and for biapigenin-treated mitochondria are shown in Fig. 1OA. Atractyloside-mediated inhibition of ADP-stimulated oxygen consumption was decreased in the presence of biapigenin (Fig 1 OB-C), an effect which was not observed with bongkrekic acid (Fig. 10C).

[39] 9 - Biapigenin inhibits FCCP-uncoupled respiration in energized mitochondria

Maximal uncoupled respiration was monitored after FCCP (1 μM) addition to the reaction medium (representative traces are shown in Fig. HA). Biapigenin (10 μM) significantly decreased FCCP-induced stimulation of mitochondrial respiration (32% reduction, p < 0.05 when compared to control; Fig. 1 IA-B). Interestingly, the effect of biapigenin on uncoupled respiration was comparable to that of atractyloside (40 μM, Fig 11.B). In contrast, although bongkrekic acid (16 μM) per se did not affect FCCP- induced uncoupled respiration, co-incubation with biapigenin (10 μM) significantly decreased maximal respiration induced by FCCP (59%, p < 0.001 when compared to control; Fig. HB).

[40] 10 - Biapigenin potentiates ATP-induced hyperpolarization

ATPase activity was evaluated by monitoring TPP⁺ uptake upon addition of ATP (3 mM) in the presence of 2 μM rotenone, reflecting membrane potential induced by ATP hydrolysis. Representative recordings of TPP⁺ uptake are shown in Fig. 12A. Biapigenin (10 μM) significantly increased ΔΨ_m generated by addition of ATP (3 mM) to the reaction medium (30%, p < 0.01 when compared to control; Fig. 12A-B). Atractyloside (40 μM) and bongkrekic acid (16 μM) significantly reduced ATP- mediated energization (12 and 30%, respectively). Interestingly, atractyloside was unable to block the effect of biapigenin, whereas bongkrekic acid significantly inhibited the hyperpolarizing effect of biapigenin in the presence of ATP (Fig. 12B). Oligomycin (1 μg.ml"¹) also inhibited the effect of biapigenin on ATP-induced ΔΨ_ra generation (Fig. 12B).

[41] The ATP-hydrolyzing (ATPase) activity was also assessed indirectly by monitoring pH changes in freeze-thaw mitochondria. The release of protons resulting from ATP hydrolysis due to the ATPase activity was followed after addition of 3 mM ATP to mitochondria. In clear opposition with the previous results, ATPase activity was significantly reduced in the presence of biapigenin (16%, p < 0.05 when compared to control; Fig. 12C).

[42] 11 - Biapigenin reduces ATP synthase activity in intact mitochondria

The ATP synthase activity was assessed in energized mitochondria in the presence of biapigenin, atractyloside, bongkrekic acid and oligomycin. Biapigenin (10 μM) significantly reduced ATP synthase activity (40% reduction; p < 0.01 when compared to the control; Fig. 13B). Atractyloside (40 μM), bongkrekic acid (16 μM) and oligomycin (1 μg.ml-¹) were more powerful inhibitors of ATP synthase activity than biapigenin (91, 96 and 99% reduction, respectively; p < 0.001 when compared to control; Fig. 13A). Accordingly, biapigenin (10 μM) reduced the ADP/O ratio and the lag phase (measurement of time required for recovery from ADP-induced depolarization) by 26% and 79%, respectively.

[43] 12 - Biapigenin and ANT inhibitors decrease ATP synthesis

In order to confirm a possible inhibitory effect of biapigenin on ATP synthesis, we measured the levels of adenine nucleotides after a complete phosphorylative cycle; which roughly translated into one minute after ADP addition to energized mitochondria (the average time required by control mitochondria to phosphorylate added ADP). Biapigenin (10 μM) significantly inhibited ATP-synthesis (82% reduction; p < 0.001, when compared to the control). Atractyloside (40 μM), bongkrekic acid (16 μM) and oligomycin (1 μg.ml-¹) significantly inhibited ATP synthesis (83, 95 and 94% reduction, respectively; p < 0.001, when compared to the control).

[44] 13 - Biapigenin decreases calcium accumulation

The ability of mitochondria to accumulate calcium was evaluated in energized mitochondria by the use of a low affinity calcium-sensitive probe, Calcium Green-5N. Mitochondria (0.2 mg.ml-¹) were energized with succinate in the presence of 15 μM CaCl₂ and 100 nM Calcium Green-5N in the assay medium. Mitochondrial calcium accumulation was recorded by following the decrease in fluorescence intensity (reflecting decrease calcium concentration in the medium and accumulation into mitochondria). The concentration of biapigenin, atractyloside and bongkrekic acid was adjusted to the amount of protein used, so that data could be compared between different experimental protocols with different protein concentrations. Representative traces are shown in Fig. 14A-C. Biapigenin (10 μM) significantly reduced calcium accumulation. Upon incubation with cyclosporin A (0.6 μM), an increase in calcium loading capacity was observed. Interestingly, biapigenin was able to prevent the effect of cyclosporin A on calcium accumulation (Fig. 14A and 14D).

[45] In order to ascertain if the effect of biapigenin in maximal calcium accumulation was due to decreased calcium uptake or due to increased efflux, we evaluated calcium efflux in energized mitochondria. Mitochondria energized with succinate (8 mM) were exposed to a single pulse of 40 μM CaCl₂ (Fig. 15A-E, indicated by full arrow). Two approaches were used to test the effect of biapigenin. In the first one, 10 μM biapigenin was pre-incubated for 3 minutes previous to the calcium pulse; in a second approach, a pulse of biapigenin was added 1 minute after the calcium pulse (to assess a direct effect in calcium efflux - Fig. 15A).

[46] Pre-incubation with 10 μM biapigenin reduced maximal calcium accumulation (Fig. 15A), whereas addition of biapigenin (10 μM) to calcium-loaded mitochondria induced calcium efflux as can be observed in Fig. 15A. Although bongkrekic acid (16 μM) increased maximal calcium accumulation in the presence of biapigenin, it was insufficient to prevent biapigenin-induced calcium efflux (Fig. 15B). Atractyloside (40 μM) alone increased calcium efflux (Fig. 15C) and when co-incubated with biapigenin the effect was apparently additive (Fig. 15C). Moreover, cyclosporin A (0.6 μM) reduced biapigenin-induced calcium efflux (Fig. 15D), whereas ADP (125 μM) plus oligomycin (1 μg.ml-¹) completely abolished the effects of biapigenin in calcium accumulation and efflux (Fig. 15E).

[47] 14. A new formulation with neuroprotective properties

Taking advantage of the neuroprotective properties of the three flavononoids described in the present work (biapigenin, quercetin and kaempferol), we developed a new formulation with strong neuroprotective properties against amyloid beta induced neurodegeneration (Fig. 16A) and against excitotoxic insults (Fig. 16B), as evaluated by both the morphological aspects of neuroprotection and cell viability life/cell death assay using sytol3-PI assay.

[48] The new formulation described in the present work is neuroprotective and is devoid of toxic effects in rat cultured hippocampal neurons. The composition of the new formulation was based on our previous characterization of a neuroprotective fraction isolated from Hypericum perforatum extracts (fraction V5) in which we determined three main flavonoids: quercetin, kaempferol and biapigenin (Silva et al., 2004a). The most obvious advantage of the new formulation is related with the control of the effective concentration of individual compounds and lack of other possible toxic contaminants present in the original V5 fraction.

[49] Neuronal death due to excitotoxicity is associated with massive calcium influx, loss of ionic homeostasis and mitochondrial dysfunction, usually preceding cell death (Stout et al. 1998). Mitochondria are important organelles for calcium homeostasis, especially under elevated cytoplasmatic calcium concentration resulting from pathological insults or stress conditions (Isaev et al. 2005;Kristian and Siesjo 1998;Nicholls 2002;Saris and Carafoli 2005;Weber 2004). Calcium entering through glutamate receptors is primarily extruded by the NaVCa²⁺ exchanger - NCX (Bano et al. 2005b), but it can also be accumulated by mitochondria present in the vicinity of membrane receptors (Peng and Greenamyre 1998;Weber 2004), possibly playing a major role in the cellular defence to excessive rise in cytoplasmatic calcium concentration. However, following calcium-induced calpain activation, NCX can be cleaved by the proteolytic activity of calpains (Bano et al. 2005a).

[50] The intracellular calcium concentration rises above a critical threshold, mitochondria lose the ability to maintain calcium homeostasis and mitochondrial dysfunction occurs. Mitochondrial dysfunction involves the induction of the permeability transition, which can cause mitochondrial swelling and rupture of the outer mitochondrial membrane, releasing pro-apoptotic factors which then trigger processes of cell death (Bouchier-Hayes et al. 2005a; Vander Heiden and Thompson 1999). [51] The phenols quercetin, kaempferol and biapigenin present in H. perforatum extracts were able to significantly protect cultured hippocampal neurons against an excitotoxic insult with kainate plus NMDA (Fig. IB and Fig. IQ. Moreover, pre-incubation with these compounds had no apparent effect in the immediate calcium rise due to stimulation with kainate plus NMDA, suggesting no apparent effects on calcium influx or its recruitment from intracellular stores. Single cell calcium imaging studies supplied evidence for a protective effect of the compounds under investigation, since they were able to partially protect the neurons from delayed calcium deregulation (Fig. 2B).

[52] Furthermore, calcium deregulation was strongly associated with a complete loss of mitochondrial transmembrane electric potential, suggesting a close association between the two events. Treatment with biapigenin afforded a significant protection to cells suffering early calcium deregulation, and, furthermore, decreased the total number of cells losing calcium homeostasis. These results were further supported by the data from the cell viability assays measured 24 hours after the excitotoxic aggression (kainate plus NMDA), where biapigenin significantly reduced neuronal death.

[53] Evidences from the literature are not conclusive about the role of oxidative stress as a cause or a consequence of mitochondrial dysfunction in excitotoxicity. It is uncertain whether mitochondrial dysfunction resulting from excessive calcium uptake is responsible for increased generation of reactive oxygen species (ROS), or if calcium- dependent ROS generation is associated with toxic mechanisms responsible for mitochondrial failure (Kiedrowski and Costa 1995;Lafon-Cazal et al. 1993;Reynolds and Hastings 1995;Weber 2004). Quercetin and kaempferol are considered good antioxidants (Cotelle 2001;Ishige et al. 2001 ;Jovano vie and Simic 2000;Rice-Evans 2001).

[54] Previous studies have reported that both compounds are effective inhibitors of lipid peroxidation (Filipeef al. 2001;Ozgova et al. 2003;Peng and Kuo 2003;Schroeter et al 2001). In accordance, we also observed that quercetin and kaempferol are strong inhibitors of peroxidation of mitochondrial membranes (Fig. 4 and 5). Some studies with liver mitochondria indicated 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). However, it must be stressed that the inhibitory effects reported in the studies with liver mitochondria were observed for concentrations significantly higher than those used in our study. Quercetin and kaempferol were reported to have an inhibitory effect in the mitochondrial ATPase / ATP synthase activity. Again, these effects were observed using higher concentrations of both compounds (Zheng and Ramirez 2000).

[55] Montero and colleagues reported a significant effect of kaempferol in mitochondrial calcium uptake, involving activation of the mitochondrial calcium uniporter in HeLa cells; such effect was less pronounced when quercetin was tested (Montero et al. 2004). Our data from single cell calcium imaging studies indicates that kaempferol was not effective in preventing delayed calcium deregulation, in contrast to quercetin that was shown to be protective. On the other side, both compounds were significantly neuroprotective against excitotoxicity-induced neuronal degeneration, suggesting that the main mechanism underlying neuroprotection might be related with antioxidant properties. Accordingly, the efficient neuroprotective compound biapigenin (now reported) has recently been shown to be endowed with strong antioxidant properties (Cakir et al. 2003;Conforti et al. 2002;Couladis et al. 2002).

[56] The antioxidant properties of the tested compounds in the present study can contribute to maintain the structural and functional properties of mitochondrial respiratory chain, essential for mitochondrial function and generation of the proton- motive force. Moreover, we also observed that the antioxidant properties of the tested compounds were efficient against lipid peroxidation caused by oxidative stress induced in non-energized but also in energized mitochondria. The hyperpolarizing effect observed after incubation of mitochondria with biapigenin can contribute to the lower antioxidant efficiency of this compound in energized mitochondria, comparatively to the strong protection observed after induction of oxidative stress in non-energized mitochondria. However, and interestingly, the protection afforded by biapigenin against transmembrane potential decay was very similar to that observed in the case of cyclosporin A, a well known inhibitor of the opening of the permeability transition pore (Halestrap 2006); while on the other hand, the profiles exhibited by quercetin and kaempferol were much closer to that of BHT, a known potent inhibitor of lipid peroxidation, highlighting possible differences in neuroprotective mechanisms triggered by quercetin/kaempferol or by biapigenin.

[57] As mentioned above, excitotoxicity is closely associated with increased intracellular calcium concentration and compromised mitochondrial function. Seo et al. reported that increased mitochondrial membrane potential and redox potential reduced the accumulation of intracellular calcium and neuronal death following the activation of the NMDA receptors. Increased transmembrane potential may contribute to increase ATP synthesis important for calcium extrusion, whereas increased redox potential can account for the detoxification of ROS (Seo et al. 1999). One could hypothesize that the hyperpolarization induced by biapigenin might also be associated with increased production of ATP, therefore contributing to increase ATP and calcium extrusion. Moreover, biapigenin appears to interfere with the mitochondrial phosphorylative mechanisms, significantly reducing the ADP-induced depolarization and the time required to phosphorylate added ADP. Additionally, the ADP/0 ratio, which reflects the efficiency of phosphorylation, was also significantly reduced after incubation with biapigenin.

[58] Mitochondrial calcium accumulation depends on both the rate of calcium uptake and release. Enhanced calcium release can be a consequence of the induction of the mitochondrial permeability transition (MPT), triggered by excessive calcium accumulation and oxidative stress. The sustained opening of MPT is prevented in fully polarized mitochondria or in the presence of antioxidants (Brookes et al. 2004b;Vieira et al. 2000). Biapigenin reduced mitochondrial calcium loading capacity when evaluated directly with a fluorescent probe (Calcium Green-5N), which may explain why it was able to increase the maximal membrane potential attained in the presence of calcium. The data obtained from mitochondrial calcium accumulation points to an interesting effect of biapigenin, which might also help explain the observed neuroprotection in neuronal cells. This group of results suggests that biapigenin contributes to the maintenance of mitochondrial membrane potential in the presence of calcium through a mechanism that can involve modulation of calcium accumulation by mitochondria. This hypothesis is further supported by the reduction in calcium accumulation by mitochondria incubated with cyclosporin- A (thus with the permeability transition pore inhibited). Another possible explanation for the phenomenon could be that biapigenin can increase the rate of calcium extrusion. These possibilities will be futher discussed in the present document.

[59] Novel therapeutic strategies against several forms of neuronal or cardiac injury

(Belisle and Kowaltowski 2002; Kristal et al. 2004; Wu et al. 2006; Halestrap et al. 2007 ) may involve direct inhibition of mPTP by agents such as cyclosporin A and, possibly, diazoxide, or indirect inhibition, by reducing oxidative stress and/or mitochondrial calcium overload. Cyclosporin A binds to cyclophilin D, which is known to interact with the ANT facilitating a calcium-induced rearrangement of the ANT into a pore-forming conformation (Halestrap et al. 2002). Therefore, cyclosporin A increases mitochondrial calcium loading capacity by inhibiting mPTP opening. Diazoxide targets the phosphorylative system and it has been reported to be neuroprotective in in vitro models of neurotoxicity (Kowaltowski et al. 2006) and in in vivo models of ischemia- reperfusion (Murata et al. 2001; Teshima et al. 2003). Moreover, diazoxide also inhibits ATP degradation during the ischemic phase, while it has no deleterious effects in normal mitochondria (Comelli et al. 2007). It might seem somehow controversial the idea that inhibition of mitochondrial phosphorylation can result in neuroprotection, especially because under physiological conditions cells critically require ATP synthesis. However, under stressful conditions, with impairment of mitochondrial function, such as in excitotoxic or ischemia-reperfusion events, it has been described that inhibitors of the phosphorylative system can be neuroprotective. [60] Naturally occurring flavonoids are able to prevent mitochondrial lipid peroxidation and can inhibit mPTP opening (Santos et al. 1998). Moreover, flavonoids are endowed with free radical scavenging and antioxidant properties (Schroeter et al. 2000; Rice- Evans 2001), which can also contribute to the inhibitory effect of flavonoids towards mPTP opening. These studies suggest that some flavonoids are able to interact with mitochondrial physiology, exerting neuroprotective actions, especially when able to target the mPTP complex.

[61] Biapigenin (10 μM) significantly inhibited ADP-induced depolarization, and significantly increased repolarization following ADP addition. Inhibitors of the ATP synthase (e.g. oligomycin), or inhibitors of the ANT (bongkrekic acid or atractyloside) also significantly inhibited ADP-induced depolarization.

[62] Oligomycin, a specific inhibitor of proton flow through the F₀ subunit of the ATP synthase (Zanotti et al. 1992), was unable to block the hyperpolarizing effect of biapigenin, which suggests that the ATP synthase is not a major player on the effect caused by biapigenin on ADP-induced depolarization and repolarization.

[63] Atractyloside (40 μM) and bongkrekic acid (16 μM), two specific inhibitors of the

ANT (Dahout-Gonzalez et al. 2005), were able to inhibit the hyperpolarizing effect of biapigenin in the presence of ADP (Fig. 9F and H, respectively). Atractyloside blocks the ANT in a pore-forming conformation, termed c-conformation (c, for cytosolic side); whereas, bongkrekic acid blocks the ANT in a non-pore forming conformation, termed m-conformation (m, for matrix side) (Dahout-Gonzalez et al. 2005). The inhibitory effect of atractyloside and bongkrekic acid on the hyperpolarization induced by biapigenin, after ADP addition, suggests that the effects of biapigenin occur at the ANT level.

[64] As expected, increased oxygen consumption due to ADP addition (state 3 respiration) to energized mitochondria was reduced in the presence of the inhibitors of the ANT, atractyloside and bongkrekic acid. Interestingly, the effect of bongkrekic acid on state 3 respiration was not affected by biapigenin; whereas, the inhibitory effect of atractyloside on state 3 respiration was prevented by biapigenin (Fig. 10C). Taken together, these results further support the notion that the effects of biapigenin occur at the ANT level, and also indicate that they are conformational-dependent.

[65] Uncoupled respiration induced by FCCP was significantly inhibited in the presence of both biapigenin and atractyloside, whereas bongkrekic acid had no significant effect (Fig. 11). Several authors suggested that FCCP mechanism does not involve membrane translocation with the participation of the ANT (Brustovetsky et al, 1990; Andreyev et al. 20005); however we found that FCCP-induced uncoupled respiration was inhibited in the presence of atractyloside. Based on the present data we cannot discard a possible involvement of the ANT in the uncoupling effects mediated by FCCP in brain. The majority of these previous studies were performed using liver mitochondria, where the major ANT isoform found is the ANT2; whereas in brain, the most common isoform is ANTl (Dorner et al. 1999).

[66] It is possible that differences in tissue expression of a specific isoform may account for observed differences in the effect of several molecules including FCCP, ANT inhibitors or even biapigenin. The results also suggest that biapigenin may also inhibit the respiratory chain.

[67] Interestingly, the ANT has been suggested to act as a proton channel (Brustovetsky et al. 1994; Shabalina et al 2006). It has been proposed that the ANTl and ANT2 are responsible for different aspects of proton conductance: the ANTl is more associated with aspects of basal proton conductance, whereas the ANT2 seems to play a significant role in uncoupled proton conductance (Shabalina et al. 2006). It is, therefore, tentative to speculate that inhibition of the ANT by specific molecules, such as atractyloside, bongkrekic acid or even biapigenin, may interfere with mitochondrial membrane proton conductance.

[68] The inhibitory effects in ADP-induced depolarization suggest that biapigenin can decrease the entry of ADP into the mitochondrial matrix through the ANT, although effects on the ATP synthase activity cannot be excluded at this point. Interaction of biapigenin with the phosphorylation system agrees with the observation that biapigenin decreases the ADP/O ratio. Interestingly, state 3 respiration is not entirely inhibited by biapigenin (Fig. 1 IA), suggesting that biapigenin does not completely inhibit ADP influx as opposed to ANT inhibition with atractyloside or bongkrekic acid.

[69] The ATP synthase activity was significantly reduced by biapigenin (Fig. 13B).

Moreover, inhibition of ATP synthesis by biapigenin (as measured by HPLC) by atractyloside or by bongkrekic acid or oligomycin was similar. Since the two methods used in this work to determine the ATP synthase activity require functionally intact mitochondria with active ANT participation, an ANT-related effect of biapigenin is still a good explanation. The effect of biapigenin in ATP synthase activity (assessed by pH variations) can be associated with the inhibition of the ANT, reducing ADP entry (or ATP efflux). However, interaction of biapigenin on the ATP synthase can not be excluded; in fact, induction of decoupling of proton flow through the F₀ subunit is known to affect ADP phosphorylation (Pietrobon et al. 1987; Bravo et al. 2001).

[70] Also, the concept of synthasome has been recently proposed. The groups of Ko and Chen showed a strong association between the ATP synthase complex and the ANT (Ko et al. 2003; Chen et al. 2004), which raises the idea that drugs targeting either of the proteins involved in mitochondrial phosphorylation could also be able to exert indirect effects in the proteins present in this functional complex.

[71] The results in the present study suggest that biapigenin does not inhibit ATP translocation into the mitochondrial matrix. Moreover, the hyperpolarizing effects of biapigenin on ATP-induced energization (Fig. 12A) seem to be related with a decrease of proton back flow into the matrix. Interestingly, this effect of biapigenin is also likely ANT-conformation sensitive, since bongkrekic acid-induced conformational state of ANT was not affected in the presence of biapigenin, whereas ANT in atractyloside- induced conformation was inhibited by biapigenin (Fig. 12B).

[72] Atractyloside and bongkrekic acid bind to mutually exclusive sites

(Dahout-Gonzalez et al. 2006). Atractyloside is a non-permeant inhibitor of ADP binding to the ANT (Bruni et al. 1965), whereas bongkrekic acid must cross the mitochondrial inner membrane to exert its inhibitory effects in ADP binding sites in the matrix side. Consequently, in the presence of bongkrekic acid plus biapigenin a decrease in ADP export/ATP import from/into the matrix would occur and, therefore, may lead to a decrease in ATP availability for further ΔΨ_m generation. In summary, it seems that biapigenin and atractyloside share a common target in the ANT.

[73] The opening of the mPTP is modulated by several factors such as high ΔΨ_m, low matrix pH or the presence of adenine nucleotides. Moreover, under conditions that maintain the reducing status of the mitochondrial matrix (NADH or NADPH, antioxidants) the opening of the mPTP is inhibited (Brookes et al. 2004; Vieira et al. 2000). On the other hand, calcium and ROS, among other factors, are known inducers of pore opening. Calcium accumulation by mitochondria occurs at the expense of ΔΨ_m and excessive calcium uptake dissipates ΔΨ_m to a level at which mPTP opening is not inhibited, inducing ROS generation, which ends up by causing damage to mitochondrial membrane proteins, resulting in further dissipation of ΔΨ_m in a vicious cycle (Brookes et al. 2004).

[74] The data here presented describe that mitochondrial calcium uptake capacity was reduced in the presence of biapigenin. The observation that addition of a pulse of biapigenin to calcium-loaded energized mitochondria induces calcium efflux suggests that, in the presence of calcium, biapigenin can induce mPTP opening. This effect would cause calcium efflux resulting in a decrease in mitochondrial calcium accumulation, as observed (Fig. 15D). When incubated with atractyloside, which induces a pore-forming conformation, the effect of biapigenin was additive, i.e. a contribution towards decreased calcium accumulation. These results seem to be in accordance with data from TPP⁺ uptake and mitochondrial respiration, suggesting that opening of the mPTP can occur in the presence of biapigenin and atractyloside or, in this case, of biapigenin and calcium.

[75] ADP plus oligomycin or cyclosporin A, two inhibitors of mPTP opening in brain mitochondria (Brustovetsky et al. 2000; Halestrap 2006), efficiently inhibited the effects of biapigenin. The observation suggests an involvement of the mPTP in biapigenin- induced calcium efflux, possibly by modulating ANT function, which may include increased cyclophilin D binding to the ANT. In fact, it has been proposed that cy- clophilin D modulates ANT function, as binding of cyclophilin D to the matrix surface of the ANT favors calcium-triggered ANT-conformational change to a non-specific pore (Halestrap and Brenner 2003; He and Lemasters, 2002).

[76] Taken together, the present results suggest that biapigenin increases calcium efflux from mitochondria, possible by inducing transient mPTP opening in such a way that allows the release of excessive calcium and relief of mitochondrial burden. The functional effect of biapigenin in mitochondrial calcium reported in the present paper is closely similar to the reported effect of minocycline in decreasing calcium uptake in brain mitochondria (Fernandez-Gomezeϊ al. 2005; Mansoone? al. 2007).

[77] Decreased mitochondrial calcium uptake may contribute to preserve mitochondrial functions, especially under stressful conditions, and protect neurons from excitotoxic cell death (Fernandez-Gomeze? al. 2005; Dubinsky et al. 2004; Stout et al. 1998; Urushitani et al. 2001; Duchen 2001).

[78] Therefore, it is shown that phenolic compounds present in H. perforatum extracts are neuroprotective against excitotoxicity in cultured hippocampal neurons involving antioxidant properties. Moreover, in the specific case of biapigenin we show that it may prevent mitochondrial dysfunction possibly by antioxidant mechanisms but also involving other processes related with mitochondrial calcium homeostasis, involving the ANT function.

[79] We propose that the interaction of biapigenin with its targets contributes to protection of mitochondrial function, by inducing transient mPTP opening and a decrease in mitochondrial calcium retention during excitotoxic events.

[80] In fact, modulation of mitochondrial physiology, namely at the level of mitochondrial calcium loading capacity, is likely to play an important role in the protection of mitochondrial function. This may be especially relevant under excitotoxic events, where high amounts of calcium taken up by energized mitochondria lead to irreversible mitochondrial depolarization and, consequently, to mitochondrial dysfunction. It seems plausible that biapigenin, by inducing transient mPTP opening and decreasing mitochondrial calcium retention, would be able to relieve mitochondria from the calcium burden, therefore contributing for maintenance of mitochondrial function under stressful conditions. Ultimately, this would translate into a neuroprotective action that may help neurons in dealing with calcium overload under excitotoxic events.

[81] BRIEF DRESCRIPTION OF THE DRAWINGS

Figure 1. A, Structural formulas of the phenolic compounds used in the present study: quercetin, kaempferol and biapigenin. B, Neuroprotection against excitotoxicity in cultured rat hippocampal neurons. Cell viability was evaluated by the live/death assay Syto-13/PI. Exposure of cultured neurons to 100 μM kainate plus 100 μM NMDA (35 min exposure, followed by a 24h post-exposure recovery period), induced a significant decrease in viability. Significant neuroprotection was afforded by incubation with quercetin, kaempferol and biapigenin (10 μM for the three compounds). C, Immunocy- tochemistry for MAP-2, staining with MitoTracker Red CMXRos and nuclear staining with Hoechst 33342. Exposure to 100 μM kainate plus 100 μM NMDA (35 min exposure, followed by 24h post-exposure recovery period) induced major changes in neuronal morphology (assessed by microtubule-associated protein MAP-2), in mitochondrial physiology/subcellular distribution (MitoTracker Red CMXRos) and nuclear condensation (arrow). Following excitotoxic insult, signs of dendritic network dystrophy and loss of mitochondrial transmembrane electric potential ( D Ψ_m) were identified. Notice the relatively higher labelling of Mitotracker Red following co- exposure with biapigenin (10 μM), which suggests a higher D Ψ_m. Top row - control; middle row - kainate plus NMDA; bottom row - biapigenin plus kainate plus NMDA. Values are presented as mean + SEM (n = 3 independent experiments); * - p < 0.05, **

- p < 0.01, *** - p < 0.001 (comparatively to kainate plus NMDA).

[82] Figure 2. Biapigenin significantly protected hippocampal neurons from delayed calcium regulation and failure of mitochondrial potential homeostasis, following excitotoxic insult with 100 μM kainate plus 100 μM NMDA ( W ). A , Figures show examples of calcium deregulation and loss of mitochondrial transmembrane potential ( D Ψ_m) in a typical control experiment, without adding phenolic compounds, or B following addition of 10 μM quercetin, 10 μM kaempferol or 10 μM biapigenin ( D ). Values were determined for each 5 min and represent the mean of five to eight independent experiments. Time t' = 35 min reflects the total number of cells that lost the ability to maintain calcium homeostasis. Scales of fluorescence intensity are indicated at the right of each set of images. Biapigenin (10 μM) was the most effective compound in preventing calcium deregulation and loss of D ψ_m. Values are presented as mean ± SEM (n = 6 to 8 independent experiments); ** - p < 0.01, *** - p < 0.001 (comparatively to kainate plus NMDA).

[83] Figure 3. Changes in mitochondrial transmembrane potential ( D Ψ_m) upon energization of mitochondria with succinate. A, Representative recording for each experimental conditions: 1, control; 2, quercetin (10 μM); 3, kaempferol (10 μM); and 4, biapigenin (10 μM). Black arrows indicate addition of 8 nmoles succinate; white arrows indicate addition of 125 μmoles ADP. B, Mitochondrial transmembrane electric potential upon energization with succinate (control, - 185 + 4 mV); C,after ADP induced depolarization (control, 17 + 2 mV); and D, upon repolarization (control, - 2 ± 1 mV). Values are presented as mean + SEM (n = 3 to 5 independent experiments); **

- p < 0.01, *** - p < 0.001 (comparatively to the control). [84] Figure 4. Quercetin, kaempferol and biapigenin significantly reduced lipid peroxidation induced by ADP/iron in isolated brain mitochondria. A, Lipid peroxidation induced by 1 mM ADP/ 10O m M FeSO₄ ( ■ ) was significantly reduced in the presence of quercetin ( A) , kaempferol ( ▼ ) and biapigenin ( ♦ ) (10 m M for all three compounds) ** - p < 0.01 for all compounds, ## - p < 0.01 for kaempferol and biapigenin (when compared with ADP/iron). B, Levels of TBARS measured at the end of the experiment were significantly reduced in the presence of the three compounds. Values are shown as mean ± SEM (n = 3 to 4 independent experiments); * - p < 0.05, ** - p < 0.01 (comparatively to ADP / iron).

[85] Figure 5. Changes in mitochondrial transmembrane electric potential ( D Ψ_m) in mitochondria energized with succinate ( D ), induced by exposure to ADP/iron. Addition of 1 mM ADP / 10O m M FeSO₄ ( ■ ) induced dissipation of DY_m . Loss of DY_m was significantly delayed in the presence of the compounds (10 m M for all three compounds) . Quercetin ( A) and kaempferol ( T ) efficiently prevent the drop in DY_n, induced by ADP/iron, similarly to the effect observed for BHT ( o ) (30 μM). Interestingly, biapigenin ( ♦ ) caused a significant hyperpolarization of brain mitochondria and delayed the ADP/iron-induced decay in DY_m , and exhibited a similar profile to cyclosporin A ( O ). Values are presented as mean ± SEM (n = 4 independent experiments). ## - p < 0.01 for biapigenin, ### - p < 0.001 for biapigenin, *** - p < 0.001 (comparatively to ADP / iron).

[86] Figure 6 . Mitochondrial calcium uptake evaluated by following TPP accumulation.

Representative recordings of: Al and A2, controls without added calcium or after addition of CaCl₂ (15 μM), respectively; A3, cyclosporin A (0.6 m M) in the presence of CaCl₂ (15 μM); B4, 1O m M quercetin; 55, 10 μM kaempferol; and B6, 10 μM biapigenin in the presence of CaCl₂ (15 μM). Thin arrows - additions of TPP (0.5 m M); open arrow - addition of mitochodria (control) and CaCl₂; full arrow - addition of succinate. C, TPP uptake measured one minute after maximal mitochondrial calcium loading upon energization with succinate. CsA - cyclosporin A. Values are shown as mean ± SEM (n = 3 to 5 independent experiments). * - p < 0.05, ** - p < 0.01, *** - p < 0.001 (comparatively to calcium).

[87] Figure 7. Calcium accumulation by brain mitochondria evaluated by following

Calcium Green-5N fluorescence. A, Figure shows representative recordings of mitochondrial calcium accumulation in control and after incubation with quercetin, kaempferol, biapigenin (10 μM for the three compounds) and cyclosporin A (0.6 μM). Mitochondria were energized in the presence of calcium, by addition of 4 mM succinate. Calcium accumulation was monitored by changes in fluorescence intensity following energization. B, Biapigenin significantly inhibits calcium uptake and also significantly inhibits cyclosporin-mediated maximal mitochondrial calcium uptake. Empty bars - without cyclosporin A (0.6 μM); full bars - with cyclosporin A. Values are presented as mean ± SEM (n = 3 to 5 independent experiments). * - p < 0.05 (comparatively to control); ## - p < 0.01 (comparatively to cyclosporin A).

[88] Figure 8 . Energized mitochondria exposed to pulses of calcium. Representative recordings of calcium accumulation in control ( D) and after incubation with bi- apigenin ( ♦ ) (10 μM). Pulses of 5 nmoles Ca²⁺/mg protein were applied (arrows) to energized mitochondria in suspension. At the end of the experiment, EGTA was added. Mitochondrial calcium loading capacity was higher for the control (lower recording), comparatively to calcium accumulation after pre-incubation with biapigenin for 3 min (upper recording). Loss of fluorescence reflects mitochondrial calcium loading capacity. Differences between the two conditions are shown for the time period where mitochondria exhibited higher calcium loading capacity (insert graph), as shown by loss of fluorescence intensity after each calcium pulse. Values are shown as mean ± SEM (n = 3 to 4 independent exp eriments).

[89] Figure 9. Inhibitors of the ANT block the biapigenin-mediated ADP-induced hyper- polarization. Mitochondrial transmembrane potential (ΔΨ_m) (indirectly evaluated by TPP+ uptake) was monitored in isolated rat brain mitochondria energized with succinate. Representative traces are shown; values of ΔΨ_m after repolarization (post-ADP) are indicated. A - control; B - biapigenin (10 μM); C - oligomycin (1 μ g.ml"¹) and D - oligomycin plus biapigenin; E - atractyloside (40 μM) and F - atractyloside plus biapigenin; G - bongkrekic acid (16 μM) and H - bongkrekic acid plus biapigenin.

[90] Figure 10. State 3 respiration is not significantly blocked by biapigenin. Isolated rat brain mitochondria (0.8 mg.ml-¹) were incubated for 3 min with biapigenin (10 μM), before energization with succinate (8 mM). Addition of 125 μM ADP was performed 1 min after energization. (A) Representative recordings of mitochondrial respiration for a control, and for mitochondria pre-incubated with biapigenin (10 μM). (B) Preincubation with atractyloside (40 μM) inhibited ADP-induced stimulation of respiration, which was preserved in the presence of biapigenin (10 μM). (C) Respiration was evaluated after addition of 125 μM ADP to energized mitochondria. Values are presented as mean ± SEM from three to four independent experiments. ** p < 0.01 (comparatively to control); # p < 0.05, ## p < 0.01 (comparatively to biapigenin).

[91] Figure 11. Biapigenin reduces mitochondrial respiration stimulated by FCCP.

Isolated rat brain mitochondria (0.8 mg.mr-¹) were incubated for 3 min with biapigenin (10 μM) before energization. FCCP (1 μM) addition was performed 1 min after energization. A) Representative traces of mitochondrial respiration in the control and in mitochondria pre-incubated with 10 μM biapigenin. B) Mitochondrial uncoupled respiration was assessed by monitoring oxygen consumption after addition of 1 μM FCCP to energized mitochondria. Values are presented as mean ± SEM from three to four independent experiments. * p < 0.05, *** p < 0.001 (comparatively to control); # p < 0.05, ### p < 0.001.

[92] Figure 12. Effect of biapigenin in ATP-induced mitochondrial energization.ΔΨ_m was measured by monitoring TPP⁺ uptake by mitochondria upon addition of 3 mM ATP to the reaction medium. Biapigenin (10 μM) was pre-incubated for 3 min. (A) Representative recordings of ATP-induced mitochondrial energization for a control and bi- apigenin-treated mitochondria. Arrows indicate addition of oligomycin (1 μ g.ml"¹)- (B) Biapigenin (10 μM) significantly increased ATP-induced energization (from 142 to 185 mV; p < 0.01, when compared with control). Atractyloside (40 μM), bongkrekic acid (16 μM) and oligomycin (1 μ g.ml"¹) significantly inhibited ATP-induced energization. (C) ATPase activity (assessed in disrupted mitochondria) was slightly, but significantly, decreased in the presence of biapigenin (10 μM). Oligomycin was used as a control. Values are presented as mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (comparatively to control); ## p < 0.01.

[93] Figure 13. Effect of biapigenin in the ATP synthase activity in rat brain mitochondria

(0.8 mg.ml"¹). ATP synthase activity was assessed by monitoring pH variations in energized mitochondria after addition of 125 μM ADP. At the end of each experiment, calibration was performed by adding 50 nmol NaOH and counter-tittered with 50 nmol HCl. (A) Representative recordings of ATP synthase activity for control and bi- apigenin-treated mitochondria. (B) ATP synthase activity was decreased after incubation with biapigenin (10 uM) and robustly inhibited in the presence of atractyloside (40 μM), bongkrekic acid (16 μM) or oligomycin (1 μ g.ml-¹). Values are presented as mean ± SEM from three independent experiments. ** p < 0.01, *** p < 0.001 (comparatively to control).

[94] Figure 14. Mitochondrial calcium uptake. Mitochondrial calcium uptake and retention were evaluated in isolated rat brain mitochondria (0.2 mg.ml"¹) after energization with 8 mM succinate (energization driven calcium uptake). Biapigenin (10 μM) was incubated for 3 min. Mitochondria were also incubated with: A) cyclosporin A alone (0.6 μM SHAPE \* MERGEFORMAT ) or co-incubated with biapigenin; B) bongkrekic acid alone (16 μM) or co-incubated with biapigenin; C) atractyloside alone (40 μM) or co-incubated with biapigenin. Values are presented as mean + SEM from three to eight independent experiments. D) Maximal mitochondrial calcium accumulation. Values are presented as mean ± SEM from three to eight independent experiments. * p < 0.05 (comparatively to control); # p < 0.05.

[95] Figure 15. Biapigenin reduces mitochondrial calcium accumulation and increases calcium efflux. Mitochondrial calcium retention was evaluated in energized mito- chondria (0.2 mg.ml"¹) upon addition of a calcium pulse (40 μM). Calcium present in the reaction medium ([Ca²⁺]_out) was assessed by monitoring Calcium Green 5-N (100 nM) fluorescence; decrease in fluorescence corresponds to calcium accumulation by mitochondria, whereas increased fluorescence corresponds to mitochondrial calcium efflux into the reaction medium. (A) Pre-incubation of mitochondria with biapigenin (10 μM), for 3 min, decreased maximal calcium accumulation; addition of a pulse of biapigenin (indicated by the open arrow) induced mitochondrial calcium efflux. (B) Bongkrekic acid (16 μM) increased mitochondrial calcium accumulation, but did not prevent biapigenin-induced calcium efflux. (C) Pre-incubation with atractyloside (40 μM) induced calcium efflux, and this effect was additive with biapigenin. (D) Cyclosporin A (0.6 μM) increased calcium accumulation and partially blocked the effect of biapigenin in calcium efflux. (E) ADP (125 μM) plus oligomycin (1 μg.ml"¹) increased mitochondrial calcium accumulation. ADP plus oligomycin completely blocked biapigenin-induced decrease in calcium accumulation and biapigenin- mediated calcium efflux. Following pre-incubation with biapigenin, addition of a pulse of 125 μM ADP (purple arrow) increased calcium accumulation. Values are presented as mean ± SEM from two to three independent experiments.

[96] Figure 16. A new formulation with neuroprotective properties. We developed a new formulation containing quercetin (21 μM), kaempferol (1.1 μM) and biapigenin (2.6 μM) able to protect cultured rat hippocampal neurons to neurotoxicity induced by amyloid beta peptide 25-35 exposure (25 μM) (A) or to an excitotocic mixture of kainate (100 μM) plus NMDA (100 μM). DETAILED DISCRIPTION OF THE INVENTION

[97] 1. Compositions of the present invention

1.1 Compositions with biapigenin - These compositions comprise biapigenin in a range of 0.1 and 50 μM. Besides, they also may comprise other substances, such as other active substances and vehicles pharmaceutically acceptable.

1.2 Compositions with biapigenin, quercetin and kaempferol - Other compositions, in the scope of the present invention, comprise biapigenin plus other flavonoids, such as quercetin and kaempferol . These compounds may be present in the referred compositions in an individual concentration of 0.1-50 μM. Besides, they also may comprise other substances, such as other active substances and vehicles pharmaceutically acceptable.

[98] In a preferred embodiment of the present invention, it is referred a composition comprising quercetin (21 μM), kaempferol (1.1 μM) and biapigenin (2.6 μM) .

[99] 2. Chemicals

Kainate was supplied by Ocean Produce International (USA) and NMDA was supplied by Tocris (USA). [100] 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).

[101] Cyclosporin A, protease (Subtilisin, Carlsberg) type VHI and tetraphenylphosphonium-chloride (TPP) were obtained from Sigma (Portugal).

[102] Digitonin and pluronic acid were obtained from Calbiochem (USA).

[103] Calcium Green-5N was supplied by Invitrogen (USA). Adenoside diphosphate monopotassium salt dihydrate, and triphosphate magnesium salt, atractyloside, bongkrekic acid, bovine serum albumin fatty acid free (BSA), cyclosporin A, oligomycin, protease (Subtilisin, Carlsberg) type Vm and tetraphenylphosphonium- chloride (TPP) were obtained from Sigma (Spain).

[104] Quercetin, kaempferol and biapigenin were isolated by preparative HPLC from an H. perforatum extract, as described in Dias et al. 1998.

[105] Purity for all three compounds was 98-99%. All the other chemicals were of the highest grade of purity commercially available.

[106] 3. Neuronal cultures

Hippocampal neurons were dissociated from hippocampi of E 18-El 9 Wistar rat embryos, after treatment with trypsin (2.0 mg/ml, 15 min, 37°C) and deoxyri- bonuclease I (0.15 mg/ml) in Ca²⁺ and Mg²⁺ free Hank's balanced solution (137 mM NaCl, 5.36 mM KCl, 0.44 mM KH₂PO₄, 0.34 mM Na₂HPO₄.2H₂O, 4.16 mM NaHCO₃, 5 mM glucose, supplemented with 0.001% phenol red, 1 mM pyruvate, 10 mM HEPES, pH 7.4). The cells were cultured in B-27 supplemented serum-free Neurobasal medium (Gibco), containing glutamate (25 μM), glutamine (0.5 mM) and gentamicin (0.12 mg/ml), as described previously (Silva et al. 2001). Cultures were kept at 37 ⁰C in a humidified incubator in 5% CO₂ / 95% air, for 7 days, the time required for maturation of hippocampal neurons. For viability studies with Syto-13 and propidium iodide (PI) cells were plated at a density of 45x10³ cell/cm² on poly-D-lysine-coated (0.1 mg/ml) coverslips.

[107] 4. Cell viability and immunocytochemical assays

Neuronal viability was assessed by using the Syto-13 and propidium iodide live/ death assay after exposure of cultured hippocampal neurons to kainate plus NMDA, alone or in the presence of the compounds. The structural formulas of the three compounds tested are shown in Fig. IA. Neurons were exposed continuously to 100 μM kainate plus 100 μM NMDA, for 35 minutes at 37⁰C, and left to recover for 24 hours in conditioned medium. Syto-13 is a green fluorescent membrane-permeable dye. PI is a non-permeable red fluorescent dye which only stains cells that lost membrane integrity - late apoptotic or necrotic (Silva et al. 2004a). Cell death resulting from the isolation procedure and plating accounted for 30% highly condensed PI- positive nuclei (data not shown). Immunocytochemistry was performed for mi- crotubule-associated protein MAP-2, and mitochondrial morphology and nuclear morphology were evaluated by using MitoTracker Red CMXRos and Hoechst 33342, respectively. Briefly, cells were incubated for 30 min with 125 nM MitoTracker Red CMXRos, washed and fixed with paraformaldehyde (4% paraformaldehyde, 4% sucrose in phosphate buffer) for 30 min.

[108] After washing, cells were permeabilized with 0.2% Triton X-100, blocked with 3% bovine serum albumin (BSA) and then incubated with primary antibody mouse anti MAP-2 for 1 hour. After washing, cells were incubated with the conjugated secondary antibody Alexa Fluor 488 Goat Anti-Mouse IgG. After washing, cells were incubated for 5 min with Hoechst 33342 and then mounted in glass coverslips using fluorescence mounting medium (Dako Cytomation, USA).

[109] 5. Calcium deregulation and loss of mitochondrial membrane potential - single cell imaging assays

Calcium deregulation and mitochondrial transmembrane potential were monitored by single-cell imaging of cultured hippocampal neurons. Cells were loaded for 40 min with 5 μM Fura-2/AM, 20 nM tetramethylrhodamine methyl 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 MgCl₂, 2.5 mM CaCl₂, 10 mM glucose, 10 mM HEPES-Na, pH 7.4). Experiments were carried out at room temperature. The coverslips were rinsed with Krebs buffer and mounted in a perfusion chamber. Image acquisition was performed using the MetaFluor software (Universal Imaging Corporation, ver. 5.0r7 2003) in an Axiovert 200 epi-fluorescence inverted microscope (Zeiss), equipped with a Lambda DG-4 (Sutter Instrument Company) and a high-resolution LCD-camera (CoolSnap HQ). Image acquisition was performed alternately at 340, 380 and 598 nm (300 ms exposure time, 10s between acquisitions), using a Fura-2/rhodamine filter. Five minutes after starting image acquisition, cells were exposed to Krebs medium containing 20 nM TMRM and 100 μM kainate plus 100 μM NMDA. In another set of experiments cells were pre-incubated for 15 min with 10 μM of quercetin, kaempferol or biapigenin (after the loading period and prior to the beginning of the experiment). After this pre-incubation period, the compounds were present for the remaining period of the experiment. Adequate controls were performed using cells perfused with Krebs. The majority of cells did not lost calcium homeostasis for the time of the experiment (40 minutes).

[110] 6. Mitochondrial respiratory chain and mitochondrial transmembrane electric potential assays

Brain mitochondria were isolated from male Wistar rats (8 weeks old), using a method previously described and used to evaluate mitochondrial transmembrane electric potential ( D Ψ_m) and respiration (Moreira et al. 2002). The D Ψ_m was monitored by evaluating transmembrane distribution of the lipophilic cation tetraphenylphosphonium ion (TPP⁺) using a TPP-selective electrode with a calomel electrode as reference (Kamo et al. 1979). The difference in potential between the selective electrode and the reference electrode was measured with an electrometer and recorded continuously in a Kipp and Zonen recorder. Reactions were carried out at 30 ⁰C in a chamber with magnetic stirring in 1 ml of medium (100 mM sucrose, 100 mM KCl, 2 mM KH₂PO₄, 10 μM EGTA, 5 mM HEPES, pH 7.4, supplemented with 2 μM rotenone) and containing 3 μM TPP-Cl (Moreira et al. 2002a;Oliveira et al. 2004a). Mitochondria (0.8 mg/ml) were incubated for 3 min with 10 μM quercetin, kaempferol or biapigenin. The reactions were started by adding 8 mM succinate to mitochondria in suspension. After reaching a steady-state distribution of TPP (plateau), 125 μM ADP was added and alterations in D Ψ_m recorded. Adequate controls were performed without addition of compounds, but respecting the same 3 min lag phase used in the test conditions. Oxygen consumption of isolated mitochondria was monitored polaro- graphically with a Clark oxygen electrode connected to a suitable recorder in a 1 ml thermostatic, water-jacketed closed chamber with magnetic stirring (stabrook 1967). State 3 respiration is defined as the consumption of oxygen in the presence of substrate and ADP, whereas state 4 respiration is defined as the consumption of oxygen after ADP consumption. Mitochondrial respiration was not altered by the presence of TPP (data not shown). RCR values obtained were in accordance with the expected values for brain mitochondria and previously reported (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 species (TBARS) and by respiratory chain-independent oxygen consumption of isolated mitochondria exposed to ADP plus iron; and indirectly by monitoring changes in D Ψ_m of energized mitochondria exposed to the oxidant pair ADP plus iron. Briefly, 1 ipid peroxidation of mitochondrial membranes (0.8 mg/ml) was assessed by monitoring oxygen consumption as reported before (Ferreira et al. 1999), with minor modifications, or by evaluating the decay of D Ψ_m (mitochondria energized with 8 mM succinate) (Abreu et al. 2000), after exposure to 1 mM ADP plus 100 μM iron for 10 or 15 min at 30⁰C. For evaluation of the effect of phenolic compounds, mitochondria were pre-incubated for 3 minutes with 10 μM quercetin, kaempferol or biapigenin. At minute 10 (time point for the end of reaction) samples were taken for evaluation of the extent of lipid peroxidation in non-energized mitochondria by measuring TBARS formation. Butylhy- droxytoluene (BHT, 30 μM) was used as a positive control of antioxidant effect against ADP/iron-induced lipid peroxidation. [112] 5. Mitochondrial calcium loading capacity, calcium uptake and calcium efflux

Mitochondrial calcium loading capacity was assessed indirectly by measuring mitochondrial TPP uptake in the presence of calcium (Oliveira et al. 2004b). Mitochondria (0.8 mg/ml) were incubated with 15 μM calcium in the presence or absence of the compounds for 3 minutes, and then energized with 8 mM succinate. Changes in D Ψ_m were recorded using a TPP-selective electrode for 10 min. TPP uptake was calibrated for all conditions tested, by adding pulses of 0.5 μM TPP (final concentration in the system was 3 μM TPP). Values shown represent TPP uptake 1 min after generation of maximal D Ψ_m. In some assays, the value of D ψ_m was determined according to previous references (Oliveira et al. 2004b). Calcium uptake was assessed by following Calcium Green-5N fluorescence with appropriate calibration in the presence of mitochondria (0.2 mg/ml) and rotenone with pulses of 2.5 μM calcium each. Reactions occurred at 30⁰C in a quartz cuvette with 2 ml reaction medium and under magnetic stirring. Changes in fluorescence intensity were monitored using a fluorimeter Perkin Elmer LS 50B (excitation at 506 nm, emission at 531 nm, 5 nm slit) after energization with 4 mM succinate (Oliveira et al. 2003). When steady state fluorescence was achieved, FCCP was added to evaluate calcium release caused by mitochondrial depolarization and, therefore, the amount of calcium that was accumulated in mitochondria due to transmembrane electric potential; calcium uptake into mitochondria assessed with this method was always near 90-95% of the total calcium uptake (data not shown). Calcium uptake was also evaluated upon addition of pulses of 10 μM calcium to energized mitochondria. Pulses were added each 30 s and fluorescence intensity was monitored. The effect of the compounds (pre-incubated for 3 min) on the number of pulses supported by mitochondria before failure in mitochondrial calcium accumulation was also evaluated. Compounds were added after energization with succinate. EGTA was added after achieving final steady-state fluorescence.

[113] Calcium retention was assessed by following Calcium Green-5N fluorescence. Appropriate calibration was performed with pulses of 7 nmol Ca²⁺/mg protein, added to mitochondria (0.2 mg/ml) in suspension. Reactions occurred at 30⁰C in a quartz cuvette with 2 ml reaction medium (100 mM sucrose, 100 mM KCl, 2 mM KH₂PO₄, 10 μM EGTA, 5 mM HEPES, pH 7.4, supplemented with 2 μM rotenone) under .magnetic stirring. Changes in fluorescence intensity were monitored using a fluorimeter Perkin Elmer LS 50B (excitation at 506 nm, emission at 531 nm, 5 nm emission and excitation slits), and calcium uptake was assessed after energization with 4 mM succinate. When steady state fluorescence was achieved, FCCP was added to evaluate total calcium release due to mitochondrial depolarization and, therefore, the amount of calcium that was accumulated due to mitochondrial transmembrane electric potential. Calcium uptake into mitochondria assessed with this method was 90-95% of the total calcium uptake (data not shown). Calcium efflux was evaluated in energized mitochondria upon addition of a single calcium pulse (42 nmol Ca²⁺/mg protein) added 3 minutes after addition of mitochondria to reaction medium. The effect of biapigenin (10 uM) or drugs tested in calcium homeostasis was evaluated after 3 minutes incubation. Adequate controls were performed in order to assess possible interferences of biapigenin with the probe fluorescence, under either low or high calcium concentrations. No interference was observed for the experimental conditions used. When described, biapigenin or drugs were added after the calcium pulse to evaluate possible effects in calcium efflux. EGTA (40 μM) was added after achieving final steady-state fluorescence.

[114] 9. ATPase activity

The ATPase activity was determined by following the production of protons resulting from ATP hydrolysis, in accordance with the potentiometric method. Reactions were carried out at 30⁰C in an open chamber with magnetic stirring in 2 ml of reaction medium (100 mM sucrose, 100 mM KCl, 2 mM KH₂PO₄, 0.5 mM HEPES- K, 10 μM EGTA, pH 7.3). Mitochondria (0.8 mg.ml-¹) were incubated for 3 min with 10 μM biapigenin. Reactions were started by adding 2 mM ATP-Mg to mitochondria in suspension, and changes in pH were recorded for 5 min. The addition of oligomycin at the end of the experiment completely abolished proton release. Adequate controls were performed without addition of compounds. The pH changes were continuously monitored with a pH meter, with the electrode inserted in the reaction medium under magnetic stirring. pH variations were registered in a Perkm-Elmer recorder (Model 56) connected to the pH set through a circuit of compensation of basal- voltage. Internal calibration was performed at the end of each experiment, with the addition of adequate (600 nmol) amounts of NaOH, and counter-titrated by addition of the same amount of HCl (due to instability of NaOH in solution).

[115] 10. ATP-synthase activity

The ATP-synthase activity was measured by following the pH variations associated to ATP-synthesis, through the use of the potentiometric method described previously . Reactions were carried out at 30⁰C in an open chamber with magnetic stirring in 2 mL of reaction medium without HEPES (100 mM sucrose, 100 mM KCl, 2 mM KH₂PO₄, 10 μM EGTA, pH 7.3). Mitochondria (0.8 mg.mL-¹) were incubated for 3 min with 10 μM biapigenin. Reactions were started by the addition of 8 mM succinate to mitochondria in suspension. One minute after energization, 125 μM ADP was added and changes in pH were recorded. Adequate controls were performed without addition of compounds. Internal calibration was performed at the end of each experiment, with the addition of adequate amounts of HCl (5 nmol).

[116] -^i. Adenylate nucleotide quantification Adenylate nucleotides were recovered by using an acidic extraction procedure and separated by reverse-phase liquid chromatography, as described previously with some minor modifications. All the extraction procedures were carried out at 0-4⁰C to minimize nucleotides degradation. Adenylate nucleotides were extracted from succinate-energized brain mitochondria incubated with either biapigenin alone, or in combination with other drugs, after a complete phosphorylation cycle (1 minute after total ADP phosphorylation). From the reaction medium, 300 μl were removed to an eppendorf tube containing oligomycin plus ice-cold 0.5M perchloric acid (HClO₄). The mixture was then centrifuged (14,000 rpm at 4⁰C, for 5 min). The pellets were stored at -80⁰C for protein quantification (using BioRad protein assay). The supernatant was recovered and the pH set to 6.5 with ice-cold 2.5 M KOH in 1.5 M KH₂PO₄ and centrifuged (14,000 rpm at 4°C, for 2 min). The new obtained supernatant was recovered with extreme caution, in order to avoid the soluble permanganate salts produced, and stored at -80⁰C for further chromatographic analysis.

[117] Samples were centrifuged at 14,000 rpm for 5 min before injecting into the HPLC system. Adenine nucleotides (ATP, ADP and AMP) concentration in each sample was determined by HPLC in a Gilson-Asted system, consisting of a Pump model 305 coupled to a computer, by an interface system model 506C and Gilson Software 715 HPLC Controller. In brief, adenine nucleotides were separated on a Lichrospher 100 RP- 18 (5 μm, 125 mm) from Merck (Darmstadt, Germany), protected by a guard column Lichrospher 100 RP- 18 (5 μm, 4 mm). During each run, an isocratic elution with 100 mM potassium phosphate buffer (pH 6.0) and 1% methanol was performed for 8 min, at a flow rate of 1.1 ml/min. Detection was performed using a Gilson UV detector Model 116, at 254 nm. The concentration of the nucleotides was determined after running standard nucleotide solutions in the same conditions, and chromatograms were later analyzed with Gilson software. The detection limit for each analyte was 1-2 pmol/injection.

[118] 12. Statistical analysis

Results are presented as means ± SE of the indicated number of experiments, usually run in triplicate or quadruplicate unless otherwise stated. Statistical significance was determined by using the one-way ANOVA test for multiple comparisons, followed by Bonferroni's Post-test.

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Claims

Claims

[Claim 1] A composition comprising a mixture of the flavonoids bi- apigenin, kaempferol and quercetin with antioxidant properties and able to protect mitochondrial functions.

[Claim 2] The composition of claim 1 wherein the said flavonoids are individually present in a range of tissue-active concentration from 0.1 uM to 50 uM.

[Claim 3] A composition comprising biapigenin able to protect mitochondrial function.

[Claim 4] The composition of claim 3 wherein the said biapigenin is individually present in a range of tissue-active concentration from 0.1 μM to 50 uM.

[Claim 5] The compositions of the previous claims to be used as neuroprotective to brain pathologies.