Effect of Naturally Occurring Flavonoids on Lipid Peroxidation and Membrane Permeability Transition in Mitochondria

Free Radical Biology & Medicine, Vol. 24, No. 9, pp. 1455–1461, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00

PII S0891-5849(98)00003-3

Original Contribution EFFECT OF NATURALLY OCCURRING FLAVONOIDS ON LIPID PEROXIDATION AND MEMBRANE PERMEABILITY TRANSITION IN MITOCHONDRIA ANTONIO C. SANTOS,* S´ERGIO A. UYEMURA,* JOA˜ O L. C. LOPES,† JOSE´ N. BAZON,† F´ABIO E. MINGATTO,† and CARLOS CURTI† *Departamento de Ana´lises Clı´nicas, Toxicolo´gicas e Bromatolo´gicas; and †Departamento de Fı´sica e Quı´mica, Faculdade de Cieˆncias Farmaceˆuticas, Universidade de Sa˜o Paulo, Ribeira˜o Preto, Sa˜o Paulo, Brazil (Received 2 June 1997; Revised 25 August 1997; Re-revised 24 November 1997; Accepted 23 December 1997)

Abstract—The ability of eight structurally related naturally occurring flavonoids in inhibiting lipid peroxidation and mitochondrial membrane permeability transition (MMPT), as well as respiration and protein sulfhydryl oxidation in rat liver mitochondria, was evaluated. The flavonoids tested exhibited the following order of potency to inhibit ADP/ Fe(II)-induced lipid peroxidation, estimated with the thiobarbituric acid assay: 39-O-methyl-quercetin . quercetin . 3,5,7,39,49-penta-O-methyl-quercetin . 3,7,39,49-tetra-O-methyl-quercetin . pinobanksin . 7-O-methyl-pinocembrin . pinocembrin . 3-O-acyl-pinobanksin. MMPT was estimated by the extent of mitochondrial swelling induced by 10 mM CaCl2 plus 1.5 mM inorganic phosphate or 30 mM mefenamic acid. The most potent inhibitors of MMPT were quercetin, 7-O-methyl-pinocembrin, pinocembrin, and 3,5,7,39,49-penta-O-methyl-quercetin. The first two inhibited in parallel the oxidation of mitochondrial protein sulfhydryl involved in the MMPT mechanism. The most potent inhibitors of mitochondrial respiration were 7-O-methyl-pinocembrin, quercetin, and 39-O-methyl-quercetin while the most potent uncouplers were pinocembrin and 3-O-acyl-pinobanksin. In contrast 3,7,39,49-tetra-O-methyl-quercetin and 3,5,7,39,49-penta-O-methyl-quercetin showed the lowest ability to affect mitochondrial respiration. We conclude that, in general, the flavonoids tested are able to inhibit lipid peroxidation on the mitochondrial membrane and/or MMPT. Multiple methylation of the hydroxyl substitutions, in addition to sustaining good anti-lipoperoxidant activity, reduces the effect of flavonoids on mitochondrial respiration, and therefore, increases the pharmacological potential of these compounds against pathological processes related to oxidative stress. © 1998 Elsevier Science Inc. Keywords—Flavonoids, Mitochondria, Rat liver, Mitochondrial respiration, Lipid peroxidation, Mitochondrial membrane permeability transition, Protein sulfhydryl oxidation, Free radicals

INTRODUCTION

protective effect of flavonoids against membrane lipoperoxidative damage has been well established, and seems to depend both on their structure and ability to interact with and penetrate the lipid bilayers.5–7 However, certain flavonoids cause a respiratory burst in isolated mitochondria and undergo auto-oxidation, generating reactive oxygen species.1,8,9 Mitochondria are the most important intracellular source of reactive oxygen species. Nearly 90% of the oxygen consumed by mammals is delivered to the mitochondria, where an electron reduction to H2O by the respiratory chain produces a proton electrochemical gradient on the inner membrane utilized for ATP synthesis. Nearly 4% of this oxygen is incompletely reduced, generating reactive oxygen species that, under normal con-

Flavonoids are a class of naturally occurring benzo-gpyrone derivatives widely distributed among plants. Individual differences within each subclass are determined by variation in number and arrangement of the hydroxyl groups, as well as by the nature and extent of alkylation. Flavonoids have multiple biological activities including potent anti-allergic, anti-inflammatory, and antiviral actions, which may result, at least in part, from their antioxidant and free radical-scavenging abilities.1– 4 The Address correspondence to: Carlos Curti, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Av. Cafe´, s/n°, 14040-903 Ribeira˜o Preto, Sa˜o Paulo, Brazil; Tel: 0055-016-6332107; Fax: 0055-016-633-1092; E-Mail: [email protected]. 1455

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importance of hydroxyl substitutions and alkylation in flavonoid structure. MATERIALS AND METHODS

Chemicals Quercetin was obtained from an ethanol extract of the aerial parts of Lychnophora ericoides Mart. (Asteraceae) by column chromatography over silica-gel. Similarly, a hexane/ethyl acetate extract of the aerial parts of L. brunioides Mart. was used to obtain pinocembrin, 7-Omethyl-pinocembrin, pinobanksin and 3-O-acyl-pinobanksin, and a hexane/ethyl acetate extract of the aerial parts of L. salicifolia Mart. was used to obtain 39-Omethyl-quercetin, 3,7,39,49-tetra-O-methyl-quercetin and 3,5,7,39,49-penta-O-methyl-quercetin. Flavonoids were dissolved in dimethylsulfoxide. The amount of solvent added had no effect on the assays. All other chemicals were reagent grade. The identification and purity of the flavonoids were defined by 1H and 13C NMR spectroscopy. Fig. 1. Structures of the flavonoids used in this study.

ditions, are scavenged by the antioxidant defenses of the organelle (for a review, see ref. 11). During pathological processes in which these radicals accumulate, the mitochondrial membrane may undergo lipid peroxidation and/or an increase in permeability known as mitochondrial membrane permeability transition (MMPT).12 This phenomenon is dependent on the opening of an unselective pore elicited by calcium plus inducer agents such as prooxidants, Pi, or uncouplers of oxidative phosphorylation. It is increasingly apparent that MMPT is a key event in the course of a variety of toxic, hypoxic and oxidative forms of cell injury, as well as apoptosis (for reviews see refs. 13–16). Therefore, agents that inhibit the MMPT, as well as lipid peroxidation on the mitochondrial membrane, may be of high pharmacological potential. In the present study we evaluated the ability of eight structurally related naturally occurring flavonoids (Fig. 1) to inhibit ADP/Fe(II)-induced lipid peroxidation on the mitochondrial membrane and MMPT, by determining TBA-reactive substances and by monitoring mitochondrial swelling, respectively. The most effective inhibitors of MMPT were assayed for their ability to inhibit protein sulfhydryl oxidation. Effects on mitochondrial respiration, as a parameter of mitochondrial function, were also evaluated. A possible structure-activity relationship is discussed, with emphasis on the

Isolation of rat liver mitochondria Male Wistar rats weighing approximately 200 g were sacrificed by cervical dislocation. The liver (10 –15 g) was immediately excised, sliced in a medium (50 ml) containing 250 mM sucrose, 1 mM EGTA, and 10 mM Hepes-KOH, pH 7.4, and homogenized three times in a Potter-Elvehjem homogenizer for 15 s at 1 min intervals. Mitochondria were isolated as previously described,17 with slight modifications. The homogenate was centrifuged at 770 3 g for 5 min, and the resulting supernatant was centrifuged at 9,800 3 g for 10 min. The pellet was suspended in 10 ml of medium containing 250 mM sucrose, 0.3 mM EGTA, and 10 mM Hepes-KOH, pH 7.4, and centrifuged at 4,500 3 g for 15 min. The final mitochondrial pellet was suspended in 1 ml of medium containing 250 mM sucrose and 10 mM Hepes-KOH, pH 7.4. The suspension was used within 2 h. All procedures were performed at 4°C. Solutions were prepared with glass-distilled deionized water. Mitochondrial protein was determined by the biuret reaction.18 Each experiment was repeated using at least three different mitochondrial preparations, and the results are from representative experiments or are reported as means 6 SEM. The Mann-Whitney test was used for statistical evaluation of the data. Oxygen consumption assay Oxygen consumption by mitochondria was measured polarographically using an oxygraph equipped with a

Effect of flavonoids on mitochondria

Clark-type oxygen electrode (Gilson Medical Electronics, Middleton, WI, USA), and the respiratory parameters were determined as previously described.19 The respiratory substrates, 5 mM potassium succinate 1 1 mg/ml rotenone or 5 mM potassium glutamate 1 5 mM potassium malate, were incubated in a respiration medium (1.5 ml final volume) containing 125 mM sucrose, 65 mM KCl, 10 mM potassium phosphate, 0.5 mM EGTA, and 10 mM Hepes-KOH, pH 7.4, at 30°C. One mg mitochondrial protein/ml was used, and state 3 respiration was initiated by the addition of 0.5 mmol ADP. Lipid peroxidation assay FeCl2 (75 mM) and ADP (0.5 mM) were incubated with 1 ml of mitochondrial suspension (2 mg protein) containing 4 mM potassium phosphate, 250 mM sucrose, 2 mM potassium succinate, 6 mM rotenone, 20 mM atractyloside, and 10 mM Hepes-KOH, pH 7.4, with shaking, for 30 min, at 30°C. TBA-reactive compounds were measured after mixing the mitochondrial suspension with 2 ml of TBA medium containing 250 mM HCl, 15% trichloroacetic acid, 3 mM TBA, and 0.1% BHT. The color was allowed to develop in the dark for 12 h and absorbance was measured at 533 nm. The amount of MDA was calculated from e 5 1.49 3 105 M21.20,21 Mitochondrial swelling assay Mitochondria (0.4 mg protein) were incubated in 1.5 ml of medium containing 125 mM sucrose, 65 mM KCl, 2 mM potassium succinate, 5 mM rotenone, and 10 mM Hepes-KOH, pH 7.4, at 30°C, and changes in absorbance were monitored at 540 nm. Reactions were initiated by the addition of 10 mM CaCl2 plus flavonoids followed 2 min later by 1.5 mM potassium phosphate or 30 mM mefenamic acid (final concentrations). Alternatively, mitochondria (0.4 mg protein) were incubated in 1.5 ml of medium containing 125 mM sucrose, 65 mM KCl, and 10 mM Hepes-KOH, pH 7.4, at 30°C, and reactions were initiated by the addition of flavonoids followed 2 min later by 0.3 mM tert-butyl hydroperoxide plus 0.5 mM CaCl2 (final concentrations). Protein sulfhydryl oxidation assay After 15 min incubation under the swelling assay conditions (see above), mitochondria (0.4 mg protein) were treated with perchloric acid (7% final concentration) in order to precipitate proteins, and centrifuged at 4,500 3 g for 5 min. The pellet was suspended with 100 ml of 7% perchloric acid, supplemented with 1 ml of water, and centrifuged at 4,500 3 g for 5 min. The final

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Table 1. Concentrations of Flavonoids Inducing a Minimum Measurable Inhibition of State 3 and Stimulation of State 4 Respiration in Isolated Rat Liver Mitochondria Respiring with Succinate State 3 (mM)

State 4 (mM)

50 25 100 100 25 25 .100 .100

25 .100 50 25 .100 100 .100 100

Pinocembrin 7-O-methyl-pinocembrin Pinobanksin 3-O-acyl-pinobanksin Quercetin 39-O-methyl-quercetin 3,7,39,49-Tetra-O-methyl-quercetin 3,5,7,39,49-Penta-O-methyl-quercetin

Rat liver mitochondria (1.5 mg protein) were incubated for 2 min with the flavonoids in a respiration medium (1.5 ml final volume) containing 125 mM sucrose, 65 mM KCl, 10 mM potassium phosphate, 0.5 mM EGTA, and 10 mM Hepes-KOH, pH 7.4, at 30°C, before respiration was initiated by the addition of 5 mM potassium succinate 1 1 mg/ml rotenone. State 3 respiration was initiated by the addition of 0.5 mmol ADP. Control values for rate of state 3 and state 4 respiration: 127 and 20 ngAtO2/ min, respectively; for RCR, 6.35; and for ADP/O, 2.0.

pellet was suspended with 0.2 ml of 10% Triton X-100, and supplemented with 0.8 ml of water. An aliquot of 0.2 ml of 500 mM potassium phosphate, pH 7.6, was added to 0.8 ml of the suspension. The amount of sulfhydryl groups was determined from e 5 13,600 M21, using the difference in absorbance at 412 nm before and 5 min after the addition of DTNB (0.2 mM final concentration) corrected for the absorbance of DTNB.22 RESULTS

Effects of flavonoids on mitochondrial respiration Table 1 shows the concentrations of flavonoids that affect succinate-supported respiration in isolated rat liver mitochondria. The most potent inhibitors of respiration were 7-O-methyl-pinocembrin, quercetin and 39-Omethyl-quercetin, and the most potent uncouplers were pinocembrin and 3-O-acyl-pinobanksin, as demonstrated by concentrations inducing a minimum measurable inhibition of state 3 or stimulation of state 4 respiration, respectively. The lowest ability to affect mitochondrial respiration (state 3 and state 4) was shown by 3,5,7,39,49tetra-O-methyl-quercetin and 3,5,7,39,49-penta-O-methyl-quercetin. The effects of flavonoids on glutamate plus malate-supported respiration were similar to those observed for succinate oxidation (results not shown) Effects of flavonoids on lipid peroxidation Figure 2 shows the concentration-response curves for the inhibitory effects of flavonoids on lipid peroxidation in isolated rat liver mitochondria, estimated by the

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Effects of flavonoids on MMPT Figure 3 shows the effects of flavonoids on mitochondrial swelling in isolated rat liver mitochondria, as an evaluation of MMPT. The order of potency in inhibiting the swelling induced by Pi was quercetin . 7-O-methylpinocembrin . 3,5,7,39,49-penta-O-methyl-quercetin . pinocembrin . 3-O-acyl-pinobanksin . pinobanksin . 3,7,39,49-tetra-O-methyl-quercetin . 39-O-methyl-quercetin. For mitochondrial swelling induced by Mef the order of potency was quercetin . pinocembrin . 7-Omethyl-pinocembrin . 3,5,7,39,49-penta-O-methyl-quercetin . 39-O-methyl-quercetin . 3-O-acyl-pinobanksin . pinobanksin . 3,7,39,49-tetra-O-methyl-quercetin. As can be observed, pinocembrin, 7-O-methyl-pinocembrin, quercetin and 3,5,7,39,49-penta-O-methyl-quercetin were the most potent swelling inhibitors when both Pi and Mef were used as inducers and were selected for evaluation of the effects on protein sulfhydryl oxidation in mitochondria. When tert-butyl hydroperoxide plus CaCl2 were used as inducers, in non-energized mitochondria, the extent of swelling was inhibited by approximately 40 and 50% in the presence of 50 mM pinocembrin and 3,5,7,39,49-penta-O-methyl quercetin, respectively, but no substantial inhibition was observed in the

Fig. 2. Concentration-response curves for the inhibitory effects of flavonoids on lipid peroxidation in isolated rat liver mitochondria. Two mg mitochondrial protein were incubated for 30 min (with shaking) with the flavonoids in the presence of 75 mM FeCl2, 0.5 mM ADP, 4 mM potassium phosphate, 250 mM sucrose, 2 mM potassium succinate, 6 mM rotenone, 20 mM atractyloside, and 10 mM Hepes-KOH, pH 7.4, at 30°C (1 ml final volume). TBA-reactive compounds were determined as described in Materials and Methods, and are expressed as fractions of control (1.72 6 0.084 nmol/mg protein). (a) Pinocembrin, (b) 7-O-methyl-pinocembrin, (c) pinobanksin, (d) 3-O-acyl-pinobanksin, (e) quercetin, (f) 39-O-methyl-quercetin, (g) 3,7,39,49-tetra-Omethyl-quercetin, (g) 3,5,7,39,49-penta-O-methyl-quercetin.

amount of TBA-reactive compounds accumulated 30 min after the mitochondria were incubated with ADP/ Fe(II). The IC50 values and/or profiles of the concentration-response curves indicate the following order of potency: 39-O-methyl-quercetin . quercetin . 3,5,7,39,49penta-O-methyl-quercetin . 3,7,39,49-tetra-O-methylquercetin . pinobanksin . 7-O-methyl-pinocembrin . pinocembrin . 3-O-acyl-pinobanksin. It is interesting to note that methylation of the hydroxyl substitutions in the flavonoid structure change the profile of concentrationresponse curves from a straight to a curved shape.

Fig. 3. Representative recordings showing the effect of flavonoids (50 mM) on the swelling of isolated rat liver mitochondria induced by 1.5 mM potassium phosphate–Pi (A), or 30 mM mefenamic acid–Mef (B), in a medium (1.5 ml final volume) containing 125 mM sucrose, 65 mM KCl, 2 mM potassium succinate, 5 mM rotenone, and 10 mM HepesKOH, pH 7.4, at 30°C. The reactions were initiated by the addition of 10 mM CaCl2 plus: (a) pinocembrin, (b) 7-O-methyl-pinocembrin, (c) pinobanksin, (d) 3-O-acyl-pinobanksin, (e) quercetin, (f) 39-O-methylquercetin, (g) 3,7,39,49-tetra-O-methyl-quercetin, (h) 3,5,7,39,49-pentaO-methyl-quercetin. *Flavonoids inhibiting swelling at 25 mM; †flavonoids inhibiting swelling at 100 mM.

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Table 2. Content of Protein Sulfhydryl Groups in Isolated Rat Liver Mitochondria after 15 min Incubation with Flavonoids in the Swelling Assay Conditions

Pi Mef

Control

Pinocembrin

7-O-Methyl-pinocembrin

Quercetin

3,5,7,39,49-Penta-O-methyl-quercetin

73.4 6 3.5 71.2 6 1.7

78.1 6 3.5 72.3 6 5.2

100.0 6 7.0* 77.9 6 1.7*

105.3 6 5.2* 78.8 6 1.7*

73.4 6 1.7 74.6 6 3.5

Rat liver mitochondria (0.4 mg protein) were incubated for 15 min with the flavonoids (50 mM) in 1.5 ml of medium containing 125 mM sucrose, 65 mM KCl, 2 mM potassium succinate, 5 mM rotenone, 10 mM CaCl2, and 10 mM Hepes-KOH, pH 7.4, at 30°C, in the presence of 1.5 mM potassium phosphate (Pi) or 30 mM mefenamic acid (Mef) (swelling assay conditions). The amount of protein sulfhydryl groups was determined as described in Materials and Methods, and is expressed as nmols/mg protein. The content of protein sulfhydryl groups of untreated mitochondria was 95.0 6 12.2 nmols/mg protein. * Significantly different from controls, p , .05.

presence of 7-O-methyl pinocembrin and quercetin (results not shown).

Effects of flavonoids on protein sulfhydryl oxidation Table 2 shows the effects of the most effective swelling inhibitors on content of protein sulfhydryl groups in isolated rat liver mitochondria under the swelling assay conditions, as an evaluation of protein sulfhydryl oxidation. Only 7-O-methyl-pinocembrin and quercetin significantly inhibited the protein sulfhydryl oxidation induced by Pi or Mef, and the inhibitory effect was more prominent for the first inducer. DISCUSSION

The present results show that the flavonoids tested, in general, are able to inhibit lipid peroxidation on the mitochondrial membrane and/or mitochondrial membrane permeability transition. This is of pharmacological relevance because both events are implicated in several pathological processes related to oxidative stress. The mitochondrial membrane, besides being the main intracellular source of reactive oxygen species, is particularly susceptible to the action of these radicals, that may impair mitochondrial function due lipid peroxidation and/or MMPT. In spite of these protective effects, various flavonoids tested within the same concentration range that affected lipid peroxidation and MMPT (about 25 mM), also inhibited and/or uncoupled mitochondrial respiration. This evidence is consistent with previous reports on certain flavonoids.9,23–25 While a clear relationship between inhibition of respiration and structure of the flavonoids tested is not evident, for the uncoupling effect the hydroxyl substitutions at positions 5 and 7 seem to be required, as is the case for pinocembrin and 3-O-acylpinobanksin. These groups might confer an electron withdrawing power, but more than two hydroxyls might decrease the lipophilic nature of the molecules; both

molecular properties associated with uncoupling of mitochondrial respiration. According to Bors et al.,26 the following structural groups are important determinants for the radical-scavenging and/or antioxidant potential of flavonoids: the O-dihydroxy (catechol) structure in the B ring, the 2,3double bond in conjugation with a 4-oxo function, and the additional presence of both 3- and 5-hydroxyl groups. Our results on the efficiency of flavonoids in inhibiting lipid peroxidation on the mitochondrial membrane are partially consistent with these criteria. Hence, the three less potent lipid peroxidation inhibitors, namely pinocembrin, 7-O-methyl-pinocembrin and 3-O-acyl-pinobanksin lack these groups, and pinobanksin, showing intermediate potency, only satisfies the requirement of 3and 5-hydroxyl substitutions. The other more potent inhibitors satisfy at least one of these requirements and, except for quercetin, present in addition methyl substitutions in the structure. The 3-hydroxyl group in combination with a 2,3 double bond, present in quercetin and 39-O-methylquercetin, is known to improve antioxidant efficiency,4 and this may be the reason why these flavonoids were the most potent inhibitors of lipid peroxidation. On the other hand, previous reports have shown that the 3-hydroxyl group, which is highly suceptible to oxidation, functions as a chelating group, although Fe(II) chelation has been proposed to play a role in the inhibition of lipid peroxidation only by less active scavengers.4 This might explain the anti-lipoperoxidant activity of pinobanksin despite the lack of structural requirements for good scavenging activity. However, the concept of chelation by flavonoids is still controversial27,28 and so the possibility that antilipoperoxidant activity of the other flavonoids tested includes this mechanism can not be ruled out. 7-Hydroxy-flavone has been proposed to be a potent inhibitor of xanthine oxidase implicated in the generation of reactive oxygen species.7 Our results do not support this observation since flavonoids with 7-hydroxyl sub-

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stitutions, such as pinocembrin and 3-O-acyl-pinobanksin, were poor inhibitors of lipid peroxidation. A recent study has reported that flavonoids could behave as both antioxidants and prooxidants and that hydroxyl substitutions activate while methylation inactivates both effects.28 This seems not to be totally true with respect to the effect of methylation on anti-lipoperoxidant activity in our assay system. Thus, depending on the nature and extent of methylation of the hydroxyl substitutions, the anti-lipoperoxidant activity of flavonoids on the mitochondrial membrane was sustained or even increased. This evidence is supported by comparing quercetin and 39-O-methyl-quercetin, as well as 3,7,39,49-tetra-O-methyl-quercetin and 3,5,7,39,49-penta-O-methyl-quercetin. Although quercetin satisfies all structural requirements for a high antioxidant activity,4,7,25 39-O-methyl-quercetin, which carries a methyl group in the catechol moiety, was a more potent anti-lipoperoxidant. The same correlation of efficiency can be observed concerning the last two flavonoids. One hypothesis is that solubilization in the mitochondrial membrane due to an increase in the lipophilic nature of molecules is implicated. In this regard, the effectiveness of protection of flavonoids against lipid peroxidation has been proposed to depend on their orientation in biomembranes, and consequently on the partition coefficients in the lipid phase.3,5,6 A fact apparently supporting this hypothesis is that the dose-response curves for flavonoids carrying methyl groups reveal a tendency to saturation that might be due to limitations in the incorporating capacity of the mitochondrial membrane. There is no clear relationship between the ability of the flavonoids tested to inhibit MMPT and their structural features or effects on lipid peroxidation in mitochondria. This is not surprising because different mechanisms are known to account for MMPT inhibition, in the same way that MMPT induction may involve more than one still unknown mechanism. The lack of a relationship with respect to the effect on lipid peroxidation is expected because this process does not participate in the MMPT induced by mefenamic acid,29 and participates only partially when the inducer is Pi at 1.5 mM.30 With regard to the relationship between the mitochondrial respiration and the MMPT inhibiting abilities of the flavonoids, our results suggest that while respiratory chain inhibition may account for MMPT inhibition by 7-O-methyl-pinocembrin and quercetin, effective inhibitors of respiration, another mechanism seems predominate concerning MMPT inhibition by pinocembrin and especially 3,5,7,39,49-penta-O-methyl-quercetin, not so effective respiration inhibitors. Accordingly, the first two inhibited only swelling in energized mitochondria, while the latter two inhibited also swelling induced by tertbutyl hydroperoxide plus CaCl2, a condition in which interference of mitochondrial respiration is absent.31 In-

terestingly, only the most effective respiratory chain inhibitors prevented significantly the mitochondrial sulfhydryl oxidation in parallel to mitochondrial swelling inhibition. In conclusion, the present results show that polyhydroxy-substituted flavonoids have a high anti-lipoperoxidant activity on the mitochondrial membrane, but also a high ability to affect mitochondrial respiration; these flavonoids exhibit also a high prooxidant activity.8,28 On the other hand, multiple methylation of the hydroxyl substitutions, in addition to sustaining good anti-lipoperoxidant activity, reduces the effect of flavonoids on mitochondrial respiration. These results suggest that multiple methylation of the hydroxyl groups increases the pharmacological potential of flavonoids against pathological processes related to oxidative stress. Accordingly, 3,5,7,39,49-penta-O-methyl-quercetin presents a high anti-lipoperoxidant activity and inhibits MMPT without a substantial effect on mitochondrial respiration. A hypothesis is that methylation, that increases lipophilic nature of molecules, increases availability of flavonoids in the mitochondrial membrane.

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ABBREVIATIONS

BHT—2,6-tert-butyl-4-methylphenol DTNB—5,59-dithiobis-(2-nitrobenzoic acid) EGTA—ethylene glycol bis(b-aminoethyl ether)-N,N,N9,N9tetraacetic acid Hepes—N-(2-Hydroxyethyl)piperazine-N9-(2-ethanesulfonic acid) Mef —mefenamic acid MDA—malondialdehyde MMPT—mitochondrial membrane permeability transition Pi—inorganic phosphate TBA—thiobarbituric acid