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Effect of Stryphnodendron adstringens (barbatimão) on energy metabolism in the rat liver
Toxicology Letters 143 (2003) 55 /63 www.elsevier.com/locate/toxlet
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Effect of Stryphnodendron adstringens (barbatima˜o) on energy metabolism in the rat liver Marcelo Alessandro Rebecca a, Emy Luiza Ishii-Iwamoto b, Ana Maria KelmerBracht b, Silvana Martins Caparroz-Assef a, Roberto Kenji Nakamura Cuman a, Clairce Luzia Salgueiro Pagadigorria b, Joa˜o Carlos Palazzo de Mello c, Adelar Bracht b, Ciomar Aparecida Bersani-Amado a,* a
Laboratory of Inflammation, Department of Pharmacy and Pharmacology, University of Maringa´, Avenida Colombo, 5790, CEP-87020-900 Maringa´-Pr, Brazil b Laboratory of Liver Metabolism, Department of Biochemistry, University of Maringa´-UEM, Maringa´-Pr, Brazil c Laboratory of Pharmacognosy, Department of Pharmacy and Pharmacology, University of Maringa´-UEM, Maringa´-Pr, Brazil Received 6 September 2002; received in revised form 30 December 2002; accepted 30 December 2002
Abstract The action of a barbatima˜o extract on hepatic energy metabolism was investigated using isolated mitochondria and the perfused rat liver. In mitochondria the barbatima˜o extract inhibited respiration in the presence of ADP and succinate. Stimulation occurred, however, after ADP phosphorylation (state IV respiration). The ADP/O and respiratory control ratios were reduced. The activities of succinate-oxidase, NADH-oxidase and the oxidation of ascorbate were inhibited. The ATPase of intact mitochondria was stimulated, but the ATPases of uncoupled and disrupted mitochondria were inhibited. In perfused livers the extract caused stimulation of oxygen consumption, inhibition of gluconeogenesis and stimulation of glycolysis. Glucose release due to glycogenolysis was stimulated shortly after the introduction of the extract, but inhibition gradually developed as the infusion was continued. Apparently the barbatima˜o extract impairs the hepatic energy metabolism by three mechanisms: (1) uncoupling of oxidative phosphorylation, (2) inhibition of mitochondrial electron transport, and (3) inhibition of ATP-synthase. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Stryphnodendron adstringens ; Barbatima˜o ; Liver; Mitochondria; Energy metabolism
1. Introduction
* Corresponding author. Tel.: /55-44-261-4523; fax: /5544-263-6231. E-mail address: [email protected] (C.A. Bersani-Amado).
Stryphnodendron adstringens (Martius), Coville, Leguminosae, popularly known as barbatima˜o , is a medicinal plant which grows abundantly in the central savannas of Brazil. Decoctions or infusions of the crude plant are traditionally used by the native population of Brazil in the treatment of
0378-4274/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-4274(03)00065-1
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leukorrhea, diarrhoea and as an anti-inflammatory agent (Santos et al., 1987; Panizza et al., 1988). A preliminary pharmacological study of this plant has demonstrated that the aqueous extract of the bark has a significant wound healing effect (Neves et al., 1992a,b). It has also been demonstrated that the aqueous extract of Stryphnodendron adstringens has anti-inflammatory and analgesic properties and that it protects the gastric mucous membranes (Bersani-Amado et al., 1996; Lima et al., 1998; Audi et al., 1999). Recent investigations in our laboratory have indicated that the oral effective doses of the aqueous extract in animals for anti-ulcerogenic and for antiinflammatory effects vary between 200 and 800 mg/kg and that the mean oral lethal dose (LD50) is 2.7 g/kg (Rebecca et al., 2002). The administration of 800 and 1600 mg/kg over a long period, however, was toxic to the animals. Decrease in body weight, thymic involution and increase of plasma glucose and aspartate aminotransferase (AST) levels were found after a 30-day treatment period. AST is found in the cytoplasm and mitochondria of many cells, but its concentration is higher in liver, heart muscle and skeletal muscle cells (Chaves and Silva, 1998; Latha et al., 1998). Higher plasmatic AST levels indicate, thus, damage of some of those organs. In addition, change in plasmatic glucose levels and decrease in weight gain is likely to reflect impaired cell metabolism. These questions led us to investigate the possible action of barbatima˜o extracts on liver cell energy metabolism. As a first approach we have measured the respiratory activity and several membranebound enzymatic activities in isolated mitochondria. In order to facilitate an extrapolation to the condition of living cells, we have also measured parameters strictly related to energy metabolism in the isolated perfused liver, e.g. oxygen consumption, gluconeogenesis and glycogenolysis.
2. Methods and materials 2.1. Plant material The stem bark of S. adstringens (Martius) Coville, Leguminosae, was collected in Sa˜o Jer-
oˆnimo da Serra-Pr (23843?7.8ƒS, 50845?23.5ƒW at an altitude of 926 m), Parana´ State, Brazil, in October 1995. A voucher specimen was deposited at the Herbarium of the Biology Department under number HUM-3800. Air-dried stem bark was extracted with Me2CO /H2O (7:3, 29 l) according to Foo and Porter (1978), Cork and Krockenberger (1991) and de Mello et al. (1999). The extract was filtered, evaporated under reduced pressure, and lyophilized. The lyophilized extract was stored at /20 8C until further use. Tests were done with the total lyophilized extract dissolved in water just before use. 2.2. Animals Male Wistar rats weighing 200 and 250 g and fed with a standard laboratory diet (Purina† ) and water ad lib were used. The protocol for these experiments was accepted and approved by the Animal Ethics Committee of the University of Maringa´. 2.3. Isolation of mitochondria Rat liver mitochondria were isolated by differential centrifugation in a mannitol /sucrose medium, according to Voss et al. (1961). Freeze / thawing disrupted mitochondria were used for the assay of membrane-bound enzymes. Intact mitochondria were frozen in liquid nitrogen and thawed rapidly at 37 8C. This procedure was repeated three times. The resulting disrupted mitochondria were maintained at 0/4 8C for use. 2.4. Determination of oxygen consumption, ADP/O ratio and respiratory control ratio (RC) Oxygen consumption by intact mitochondria was measured polarographically using an incubation medium containing 5 mM disodium phosphate, 10 mM Tris /HCl (pH 7.4), 0.2 mM EDTA, 10 mM potassium chloride, 0.25 M mannitol and 50 mg% fatty acid /free bovine serum albumin. The following substrates were added to the incubation medium when required: succinate (12.5 mM), a-ketoglutarate (12.5 mM) and ADP (125 or 250 mM). The ADP/O ratio was calculated accord-
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ing to Chance and Williams (1955) and it represents the amount of ADP added to the incubation system divided by the amount of oxygen consumed during the active phase of respiration (state III respiration). The respiratory control (RC) ratio is given by the rate of active respiration which follows ADP addition divided by the rate of respiration after ADP exhaustion (state IV respiration; Chance and Williams, 1955). In this and in other experiments with isolated mitochondria, the extracts were added to the incubation medium in the concentration range between 25 and 500 mg/ml. After a preincubation with mitochondria for a period of 2 min, the reactions were initiated by the addition of specific substrates.
2.5. ATPase activity The ATPase activity was assayed by measuring phosphate release according to Pullman et al. (1960). When intact mitochondria were used as enzyme source, the reaction medium contained: 0.2 M sucrose, 12 mM Tris /HCl (pH 7.4), 50 mM KCl and, when required, 200 mM 2,4-dinitrophenol. When disrupted mitochondria were incubated, the medium contained 20 mM Tris /HCl (pH 7.4) and 0.15 M KCl. The reaction was started by the addition of 5 mM ATP and stopped, after 20 min of incubation at 37 8C, by the addition of ice-cold 5% trichloroacetic acid. Phosphate was measured as described by Fiske and Subbarow (1925).
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2.7. Determination of protein contents Protein contents of the mitochondrial suspensions were measured using the method of Lowry et al. (1951). The standard was bovine serum albumin.
2.8. Liver perfusion The non-recirculating perfusion technique described by Scholz and Bu¨cher (1965) was used. For the surgical procedure, the animals were anesthetized by i.p. injection of sodium pentobarbital (50 mg/kg). The perfusion fluid was Krebs/Henseleitbicarbonate buffer, pH 7.4 (Krebs and Henseleit, 1932), saturated with an oxygen/carbon dioxide mixture (95/5%). The fluid was pumped through a temperature-regulated (37 8C) membrane oxygenator prior to entering the liver via a cannula inserted in the portal vein. The perfusion flow was constant in each individual experiment and it was adjusted between 30 and 35 ml/min, depending on the liver weight. Samples of the effluent perfusion fluid were collected at 2-min intervals and analyzed for glucose, lactate and pyruvate. When glycogen catabolism was measured, the livers of fed rats were used in the experiments. Livers from 24-h fasted rats were used for the measurement of gluconeogenesis. When required the barbatima˜o extract was dissolved in the perfusion fluid for a final concentration of 100 mg/l.
2.9. Analytical 2.6. Membrane-bound enzymatic activities Freeze /thawing disrupted mitochondria were used as enzyme source for assaying membranebound enzymatic activities. NADH-oxidase and succinate-oxidase activities were assayed polarographically using a 20 mM Tris /HCl (pH 7.4) medium (Singer, 1974). A polarographic assay was also run with TMPD (N ,N ,N ?,N ?-tetramethyl-pphenylenediamine) plus ascorbate as substrates. The reactions were started by the addition of 12.5 mM NADH, 12.5 mM succinate or 0.2 mM TMPD/5 mM ascorbate.
Lactate and pyruvate were measured by standard enzymatic methods, using lactate dehydrogenase (Gutmann and Wahlefeld, 1974; Czok and Lamprecht, 1974). Glucose was measured by an enzymatic-colorimetric method using glucose-oxidase (Bergmeyer and Bernt, 1974). The oxygen concentration in the venous perfusate was monitored continuously employing a Teflon-shielded platinum electrode. Metabolic rates were calculated from the input /output differences and the total flow rates and were referred to the wet weight of the liver.
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2.10. Treatment of data The statistical significance of the differences between parameters was evaluated by means of Student’s t -test or Student /Newman /Keuls test. The latter was applied after submitting the data to variance analysis. The results are mentioned in the text as the P values; P B/0.05 was adopted as a criterion of significance.
3. Results 3.1. Effect of the aqueous extract of barbatima˜o on oxygen consumption in isolated mitochondria Oxygen consumption by intact mitochondria was measured using FAD-dependent (succinate) and NAD -dependent (a-ketoglutarate) substrates in the presence of exogenously added ADP (state III respiration) or after ADP exhaustion (state IV respiration). The aqueous extract of the barbatima˜o stem bark was added to the incubation medium for final concentrations between 25 and 250 mg/ml. The results are illustrated by Fig. 1. When succinate was the substrate (Fig. 1A) the rates of oxygen consumption in the absence of ADP (state IV respiration) were increased in the concentration range of 25/125 mg/ml. At the concentration of 125 mg/ml the rates of oxygen consumption were 92.2% (P /0.01) above the basal ones (absence of barbatima˜o extract). In the presence of ADP, the barbatima˜o extract caused a dose-dependent inhibition of oxygen consumption (state III respiration). When a-ketoglutarate was used as a substrate (Fig. 1B), state IV respiration was also progressively increased in the presence of the barbatima˜o extract. Maximal stimulation was found at the concentration of 125 mg/ml (/189.5%, P /0.01). The changes in state III respiration were less pronounced than those observed in the presence of succinate. Table 1 shows the effect of the barbatima˜o extract on the ADP/O ratios and the respiratory control ratios (RC). The respiratory control ratios (RC) and the ADP/O ratios were decreased by the extract with both substrates, a-ketoglutarate and
Fig. 1. Effects of the total extract of barbatima˜o on the respiratory activity of isolated rat liver mitochondria. Mitochondria (0.25 /2.5 mg/ml) were added to the reaction medium in the closed vessel of the oxygraph (temperature, 37 8C). The reaction was initiated by the addition of succinate (A) or aketoglutarate (B) and the oxygen consumption was followed polarographically during approximately 5 min. After this time 0.25 /0.5 nmols of ADP were added. Rates of oxygen consumption were computed from the slopes of the polarographic records. Each data point is the mean9/S.E.M. of 11 (succinate) and five (a-ketoglutarate) independent experiments. *, P B/ 0.05, ANOVA with Newman /Keuls test.
succinate. At concentrations above 125 mg/ml no reliable estimation of the ADP/O ratios was possible. 3.2. Effects of the barbatima˜o extract on the activities of membrane-bound enzymes The effects of the barbatima˜o extract on succinate-oxidase, NADH-oxidase and TMPD-ascorbate oxidation, measured in disrupted mitochondria, are shown in Fig. 2. The addition of the barbatima˜o extract caused inhibition of all enzymatic activities in a dose-dependent manner.
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Table 1 Effects of the total extract of barbatima˜o on the ADP/O ratios and the respiratory control coefficients (RC) in rat liver mitochondria Extract (mg/ml)
0.0 25.0 62.5 125.0 187.5 250.0
a-Ketoglutarate (n/5)
Succinate (n /8) ADP/O
RC
ADP/O
RC
1.489/0.20 1.389/0.16 1.329/0.24 1.239/0.24* / /
3.879/0.82 3.179/0.61** 2.279/0.34** 1.399/0.24** 1.109/0.09** 1.00**
2.159/0.23 2.279/0.22 2.039/0.25 1.779/0.20* / /
2.559/0.19 2.349/0.13 1.859/0.19** 1.229/0.13** 1.049/0.09** 1.00**
Mitochondria (0.25 /2.5 mg/ml) were incubated in reaction medium as described in Section 2. Oxygen consumption was recorded polarographically. ADP (250 or 500 nmols) was added at appropriate times. The respiratory control coefficients (RC) and ADP/O ratios were calculated according to Chance and Williams (1955). *, P B/0.05; **, P B/0.01, ANOVA with Newman /Keuls test.
Fig. 2. Effects of the total barbatima˜o extract on several membrane-bound enzymatic activities in rat liver mitochondria. Panel A: NADH-oxidase, succinate-oxidase and TMPD-ascorbate oxidation, measured with freeze /thawing disrupted mitochondria, incubated at 37 8C in reaction medium as described in Section 2. Panel B: ATPase activity of coupled, uncoupled and disrupted mitochondria, incubated at 37 8C in reaction medium as described in Section 2. Each assay point represents the mean of 8 /11 experiments and the bars are S.E.M. *, P B/ 0.05, ANOVA with Newman /Keuls test.
The calculated mean inhibitory doses (ID50) were 96.39/7.8 mg/ml for succinate-oxidase, 78.69/5.4 mg/ml for NADH-oxidase and 156.39/12.5 mg/ml for TMPD-ascorbate oxidation. Fig. 2B shows results of the effects of the barbatima˜o extract on the ATPase activity measured in intact mitochondria either in the absence (coupled mitochondria) or in the presence of 2,4dinitrophenol (uncoupled mitochondria) and in freeze /thawing disrupted mitochondria. The actions of the barbatima˜o extract were different in each preparation. The ATPase activity of coupled mitochondria was increased, with maximal stimulation at a concentration of 125 mg/ml (/282.8%, P /0.001). A tendency toward inhibition at higher concentrations, however, was evident: at a concentration of 500 mg/ml the ATPase activity stimulation was reduced to 64% of the control. In the case of uncoupled mitochondria, the barbatima˜o extract caused a small stimulation at low concentrations. Maximal stimulation (31%) was found at a concentration of 62.5 mg/ml (P / 0.01). Higher concentrations, however, were inhibitory. Inhibition was 41% at 500 mg/ml (P / 0.001). When disrupted mitochondria were used as the enzyme source, the barbatima˜o extract caused only inhibition of the ATPase activity. An inhibition of 72% was found at a concentration of 500 mg/ml (P /0.001).
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3.3. Effects of the barbatima˜o extract on glycogen catabolism and oxygen consumption in the liver of fed rats In order to verify if the effects found in isolated mitochondria manifest themselves in intact cells and also in order to evaluate further the potential toxicity of the extract to the liver, we have investigated the action of the barbatima˜o extract on oxygen consumption, glycogenolysis, glycolysis
and gluconeogenesis. Fig. 3A shows the results of experiments performed with livers from fed rats. These livers were perfused with substrate-free perfusion fluid, in a open system. As shown in Fig. 3A, under these conditions, the livers release glucose, lactate and pyruvate as a result of glycogen catabolism (Scholz and Bu¨cher, 1965). The introduction of 100 mg/l of barbatima˜o extract at 20 min of perfusion produced changes in all parameters. Oxygen consumption, lactate and pyruvate production were increased. When the infusion of the barbatima˜o extract was interrupted at 50 min, the metabolic fluxes did not return to the rates before infusion (basal rates). The barbatima˜o extract exerted complex effects on glucose release. A rapid increase in glucose release was observed at 2 /3 min of infusion, followed by a progressive decrease, with a minimum around 40 min. After this minimum, the rates of glucose release tended to increase gradually until the terminus of the infusion. After termination of the infusion, glucose release increased rapidly and remained elevated during the subsequent 20 min. At this time, glucose release was nearly 112% above the basal values (P /0.01). 3.4. Effects of the barbatima˜o extract on oxygen consumption and gluconeogenesis in the liver from fasted rats
Fig. 3. Effects of the total extract of barbatima˜o on metabolic fluxes in perfused livers isolated from fed (A) and fasted (B) rats. Livers from fed or 24-h fasted rats were perfused with Krebs/Henseleit-bicarbonate buffer (pH 7.4), as described in Section 2. In the experiments with fed rats (panel A), the barbatima˜o extract (100 mg/l) was infused at 20 /50 min, as indicated by the horizontal bar. In the experiments with fasted rats (panel B), lactate (2 mM) and pyruvate (0.2 mM) were infused at 20 /76 min and the barbatima˜o extract (100 mg/l) at 36 /56 min. The effluent perfusate was sampled in 2-min intervals and analyzed for glucose, lactate and pyruvate in livers from fed rats and for glucose in livers from fasted rats. Each experimental point is the mean9/S.E.M. of six experiments.
Fig. 3B illustrates the effects of the barbatima˜o extract on oxygen consumption and glucose production due to lactate plus pyruvate in perfused livers from fasted rats. Both the glycogen stores and the levels of endogenous gluconeogenic substrates in livers from 24-h fasted rats are minimal. In consequence, glucose production depends almost entirely from the exogenously supplied substrates. As expected, during the time period before lactate (2.0 mM) plus pyruvate (0.2 mM) infusion (1 and 16 min of the time scale in Fig. 3B), glucose release was minimal. The introduction of gluconeogenic substrates immediately increased glucose release and oxygen consumption. The infusion of the barbatima˜o extract caused a rapid and transient increase in oxygen consumption, followed by a gradual decrease to values around those measured before the infusion. Glucose release, on the
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other hand, decreased progressively. At the end of the barbatima˜o extract infusion, glucose release was reduced by 46% (P /0.001) when compared with the rates measured before the infusion. The interruption of the infusion was followed by a recovery of glucose release to values still 12.6% below those measured before the infusion of the barbatima˜o extract.
4. Discussion The results of the present work reveal that the total aqueous extract of S. adstringens (barbatima˜o) has complex effects on liver metabolism. The data obtained with isolated mitochondria indicate that the barbatima˜o extract impairs oxidative phosphorylation by at least three modes of action: (1) uncoupling of oxidative phosphorylation; (2) inhibition of electron flow in the respiratory chain; (3) inhibition of the ATP-synthase complex. The uncoupling action is indicated by the effects on mitochondrial respiration and ATPase activity, namely stimulation of state IV respiration (irrespective of the substrate), decrease of the respiratory control ratio, decrease of the ADP/O ratio and increase of ATP hydrolysis in intact coupled mitochondria. The stimulatory effect on ATP hydrolysis was absent when the latter was measured in disrupted mitochondria, indicating that the integrity of the mitochondrial membrane is essential for the stimulatory action. All these effects are found when mitochondria are incubated with uncouplers such as 2,4-dinitrophenol and FCCP (Hopfer et al., 1968; Hanstein, 1976). Inhibition of electron flow in the respiratory chain was revealed by the inhibitory action on NADH, succinate and TMPD-ascorbate oxidation in disrupted mitochondria. Even in intact coupled mitochondria, inhibition of state IV respiration was visible at higher concentrations of the barbatima˜o extract with both succinate or a-ketoglutarate as substrates. The exact site of the action on the electron transport chain cannot be inferred from the available data. However, two different ID50-values were found: one for NADH- and succinate-oxidase and a much higher one for TMPD-ascorbate oxidation. This finding suggests
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the existence of at least two sites of action: a step between the quinone pool and complex III and another step between cytochrome c and oxygen. Alternatively, the components of the barbatima˜o extract could be acting directly on dehydrogenases, such as NADH-dehydrogenase and succinate dehydrogenase. Further experiments are required to clarify this point. The effects of the barbatima˜o extract on the ATPase activity of different mitochondrial preparations indicate the third probable mode of action. Whereas in intact coupled mitochondria the barbatima˜o extract was able to stimulate the ATPase activity, inhibition was the dominant effect in 2,4-dinitrophenol-uncoupled mitochondria as well as in freeze/thawing-disrupted mitochondria. This set of observations excludes a direct action of the barbatima˜o extract on the ADP/ATP exchange system. A direct inhibition of the ATP synthase complex seems to be, thus, the most probable mechanism of action. The rat liver perfusion experiments provided a clear indication that the barbatima˜o extract is also active on intact cells. Infusion of the barbatima˜o extract caused activation of oxygen consumption either in livers from fed or fasted rats. It is unlikely that this activation represents an increase in the energy demands of the liver, because there was in parallel a significant reduction in gluconeogenesis. Gluconeogenesis from lactate plus pyruvate is a metabolic pathway strictly dependent on mitochondrial ATP production. Oxygen consumption stimulation seems to be, thus, due to an uncoupling of oxidative phosphorylation, which reduces the net rates of ATP production. In agreement with this interpretation, the barbatima˜o extract stimulated glycolysis (lactate plus pyruvate production) in livers from fed rats. Activations of glycolysis and glycogenolysis are expected responses to diminished mitochondrial ATP production and is a general characteristics of the action of electron flow blockers and uncouplers (Nascimento et al., 1992; Constantin et al., 1995). The effects of the barbatima˜o extract on glucose release from glycogen were complex and cannot be explained only by its action on mitochondria. Activation was in fact observed in the first minutes of the infusion. However, glucose release de-
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creased progressively during the rest of the infusion period, a phenomenon that was followed by a posterior increase when the infusion was interrupted. We have, so far, no explanation for these phenomena. Changes in liver hemodynamics or in enzymatic systems implicated in glucose release from glycogen are possibilities that deserve attention in future work. The inhibitory action of the barbatima˜o extract on hepatic energy metabolism could be responsible, partly at least, for its reported toxicity in animals (Rebecca et al., 2002). An impairment of oxidative phosphorylation may cause changes in membrane integrity, favoring enzyme leakage (Hess, 1963). Moreover, another consequence may be a perturbation of biosynthetic pathways, which could be contributing to the reported decrease in body weight gain. A further question which arises is one regarding the nature of the active substances implicated in the effects. The precise identity of the active principles of S. adstringens is not known until now. However, tannins are substances which could be considered at least as a starting point. The plant contains a large quantity of condensed tannins, varying between 10 and 37%, depending on the place and season of the year in which the sample has been collected (Teixeira et al., 1990; de Mello et al., 1996). It has been demonstrated that tannins and related compounds exert several biological and pharmacological activities, such as bactericidal, antiviral, molluscicidal and antihelmintic actions, antihepatotoxic action, inhibition of xanthine oxidase and mono-amino oxidase and inhibition of glucosyltransferases (Haslam, 1996). It is believed that these properties derive from their antioxidant and radical scavenging activities, and from their ability to form complexes with other macromolecules, such as proteins and polysaccharides, and metal ions (Haslam, 1996). In vitro tests have demonstrated that some tannins inhibit the activities of several enzymes, including both NADH-dehydrogenase and succinate-oxidase of rat liver mitochondria (Nishizawa et al., 1983; Konishi et al., 1993; Konishi and Tanaka, 1999). Our data are in agreement with both observations, because the barbatima˜o extract was able to inhibit both NADH-oxidase and succinate-oxidase activ-
ities. Based on such data, it seems reasonable to suggest that the tannins are implicated, partly at least, in the metabolic effects reported in this work. Evidently, this speculation does not exclude, but rather emphasizes, the necessity of isolation and identification of the major active principles of S. adstringens, with subsequent studies on their mechanisms of action.
Acknowledgements The authors are grateful to Jailson Araujo Dantas and Aparecida P.M. Hermoso for their technical assistance. This work was supported by the Programa Nacional de Nu´cleos de Exceleˆncia (PRONEX), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).
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M.A. Rebecca et al. / Toxicology Letters 143 (2003) 55 /63 plants: insights from investigations on Eucalyptus leaves. J. Chem. Ecol. 17, 123 /134. Czok, R., Lamprecht, W., 1974. Pyruvate, phosphoenolpyruvate and D-glycerate 2-phosphate. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1446 /1451. de Mello, J.C.P., Petereit, F., Nahrstedt, A., 1996. Flavan-3-ols and prodelphinidins from Stryphnodendron adstringens . Phytochemistry 41, 807 /813. de Mello, J.C.P., Petereit, F., Nahrstedt, A., 1999. A new dimeric prodelphinidin from Stryphnodendron adstringens . Phytochemistry 51, 1105 /1107. Fiske, C.H., Subbarow, Y., 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66, 375 /400. Foo, L., Porter, L.J., 1978. Prodelphinidin polymers: definition of structural units. J. Chem. Soc. Perkin Trans. 1, 1186 / 1190. Gutmann, I., Wahlefeld, A.W., 1974. L(/) lactate determination with lactate dehydro-genase and NAD . In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 1464 /1466. Hanstein, W., 1976. Uncoupling of oxidative phosphorylation. Biochim. Biophys. Acta 456, 129 /148. Haslam, E., 1996. Natural polyphenols (vegetable tannins) as drugs and medicines: possible modes of action. J. Nat. Prod. 59, 205 /215. Hess, B., 1963. Biochemistry and biology of plasma enzymes. In: Enzymes in Blood Plasma. Academic Press, New York, p. 48. Hopfer, U., Lehninger, A.L., Thompson, T.E., 1968. Protonic conductance across phospholipid bilayer membranes induced by uncouplers of oxidative phosphorylation. Proc. Natl. Acad. Sci. (USA) 59, 484 /490. Konishi, K., Tanaka, T., 1999. Inhibitory effects of tannins on the NADH dehydrogenase activity of bovine heart mitochondrial complex I. Biol. Pharm. Bull. 22, 240 /243. Konishi, K., Adashi, H., Ishigaki, N., Kanamura, Y., Adashi, I., Tanaka, T., Nishioka, I., Nonaka, G., Horikoshi, I., 1993. Inhibitory effects of tannins on NADH dehydrogenases of various organisms. Biol. Pharm. Bull. 16, 716 / 718. Krebs, H.A., Henseleit, H., 1932. Untersuchungen u¨ber die Harnstoffbildung im Tier-ko¨rper. Z. Physiol. Chem. 210, 33. Latha, R.M., Geentha, T., Varalakshmi, P., 1998. Effect of Vernonia cinerea Less. Flower extract in adjuvant-induced arthritis. Gen. Pharmacol. 31, 601 /606. Lima, J.C.S., Martins, D.T.O., de Souza, P.T., Jr, 1998. Experimental evaluation of stem bark of Stryphnodendron adstringens (Mart.) Coville for antiinflammatory activity. Phytother. Res. 12, 218 /220.
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Lowry, O.H., Rosebrough, N.J., Farr, A.C., Randal, R.L.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265 /275. Nascimento, E.A., Kelmer-Bracht, A.M., Bracht, A., IshiiIwamoto, E.L., 1992. Activation of hepatic glycogenolysis by non-steroidal anti-inflammatories is independent of Ca2 . Pharmacol. Commun. 3, 129 /138. Neves, M.C.L., Jorge Neto, J., Ifa, D.R., Fracasso, J.F., Lepera, E.Z.P., Silva, R.F.P., 1992a. Estudo dos efeitos farmacolo´gicos produzidos pelos extratos aquosos de hamamelis e barbatima˜o. In: Reunia˜o Anual da Federac¸a˜o de Sociedades de Biologia Experimental. Caxambu, Resumos, p. 7. Neves, M.C.L., Jorge Neto, J., Ifa, D.R., Fracasso, J.F., Lepera, E.Z.P., Silva, R.F.P., Lepera, E.Z.P., 1992b. Estudo dos efeitos farmacolo´gicos de hamamelis e barbatima˜o. In: Simpo´sio de Plantas Medicinais do Brasil. Universidade Federal do Parana´, Curitiba, Resumos, p. 12. Nishizawa, M., Yamagishi, T., Ohyama, T., 1983. Inhibitory effect of gallotannins on the respiration of rat liver mitochondria. Chem. Pharm. Bull. 31, 2150 /2152. Panizza, S., Rocha, A.B., Gecchi, R., Silva, R.A.P.de S., 1988. Stryphnodendron barbadetiman (Vellozo) Martius: teor em tanino na casca e sua propriedade cicatrizante. Rev. Cieˆncias Farmaceˆuticas 10, 101 /106. Pullman, M.E., Penefski, H.S., Datta, A., Racker, E., 1960. Purification and properties of soluble dinitrophenol-stimulated adenosine triphosphate. J. Biol. Chem. 235, 3322 / 3329. Rebecca, M.A., Ishii-Iwamoto, E.L., Grespan, R., Cuman, R.K.N., Caparroz-Assef, S.M., Palazzo de Mello, J.C., Bersani-Amado, C.A., 2002. Toxicological studies on Stryphno-dendron adstringens. J. Ethnopharmacol. 83, 101 /104. Santos, C.A.de M., Torres, K.R., Leonart, R., 1987. Plantas Medicinais (Herbarium, Flora et Scientia). Scientia et Labor, Curitiba, pp. 39. Scholz, R., Bu¨cher, T., 1965. Hemoglobin-free perfusion of rat liver. In: Chance, B., Estabrook, R.W., Willianson, J.R. (Eds.), Control of Energy Metabolism. Academic Press, New York, pp. 393 /414. Singer, T.P., 1974. Determination of the activity of succinate, NADH choline and glycero-phosphate dehydrogenase. Methods Biochem. Anal. 22, 125 /190. Teixeira, M.L., Soares, A.R., Scolforo, J.R.S., 1990. Variac¸a˜o do teor de tanino da casca de barbatima˜o (Stryphnodendron adstringens (Mart, Coville)( em 10 locais de Minas Gerais. Cieˆnc. Pra´t. Lavras 14, 229 /232. Voss, D.O., Campelo, A.P., Bacila, M., 1961. The respiratory chain and the oxidative phosphorylation of rat brain mitochondria. Biochem. Biophys. Res. Commun. 4, 48 /51.
Short communication
Effect of Stryphnodendron adstringens (barbatima˜o) on energy metabolism in the rat liver Marcelo Alessandro Rebecca a, Emy Luiza Ishii-Iwamoto b, Ana Maria KelmerBracht b, Silvana Martins Caparroz-Assef a, Roberto Kenji Nakamura Cuman a, Clairce Luzia Salgueiro Pagadigorria b, Joa˜o Carlos Palazzo de Mello c, Adelar Bracht b, Ciomar Aparecida Bersani-Amado a,* a
Laboratory of Inflammation, Department of Pharmacy and Pharmacology, University of Maringa´, Avenida Colombo, 5790, CEP-87020-900 Maringa´-Pr, Brazil b Laboratory of Liver Metabolism, Department of Biochemistry, University of Maringa´-UEM, Maringa´-Pr, Brazil c Laboratory of Pharmacognosy, Department of Pharmacy and Pharmacology, University of Maringa´-UEM, Maringa´-Pr, Brazil Received 6 September 2002; received in revised form 30 December 2002; accepted 30 December 2002
Abstract The action of a barbatima˜o extract on hepatic energy metabolism was investigated using isolated mitochondria and the perfused rat liver. In mitochondria the barbatima˜o extract inhibited respiration in the presence of ADP and succinate. Stimulation occurred, however, after ADP phosphorylation (state IV respiration). The ADP/O and respiratory control ratios were reduced. The activities of succinate-oxidase, NADH-oxidase and the oxidation of ascorbate were inhibited. The ATPase of intact mitochondria was stimulated, but the ATPases of uncoupled and disrupted mitochondria were inhibited. In perfused livers the extract caused stimulation of oxygen consumption, inhibition of gluconeogenesis and stimulation of glycolysis. Glucose release due to glycogenolysis was stimulated shortly after the introduction of the extract, but inhibition gradually developed as the infusion was continued. Apparently the barbatima˜o extract impairs the hepatic energy metabolism by three mechanisms: (1) uncoupling of oxidative phosphorylation, (2) inhibition of mitochondrial electron transport, and (3) inhibition of ATP-synthase. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Stryphnodendron adstringens ; Barbatima˜o ; Liver; Mitochondria; Energy metabolism
1. Introduction
* Corresponding author. Tel.: /55-44-261-4523; fax: /5544-263-6231. E-mail address: [email protected] (C.A. Bersani-Amado).
Stryphnodendron adstringens (Martius), Coville, Leguminosae, popularly known as barbatima˜o , is a medicinal plant which grows abundantly in the central savannas of Brazil. Decoctions or infusions of the crude plant are traditionally used by the native population of Brazil in the treatment of
0378-4274/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-4274(03)00065-1
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leukorrhea, diarrhoea and as an anti-inflammatory agent (Santos et al., 1987; Panizza et al., 1988). A preliminary pharmacological study of this plant has demonstrated that the aqueous extract of the bark has a significant wound healing effect (Neves et al., 1992a,b). It has also been demonstrated that the aqueous extract of Stryphnodendron adstringens has anti-inflammatory and analgesic properties and that it protects the gastric mucous membranes (Bersani-Amado et al., 1996; Lima et al., 1998; Audi et al., 1999). Recent investigations in our laboratory have indicated that the oral effective doses of the aqueous extract in animals for anti-ulcerogenic and for antiinflammatory effects vary between 200 and 800 mg/kg and that the mean oral lethal dose (LD50) is 2.7 g/kg (Rebecca et al., 2002). The administration of 800 and 1600 mg/kg over a long period, however, was toxic to the animals. Decrease in body weight, thymic involution and increase of plasma glucose and aspartate aminotransferase (AST) levels were found after a 30-day treatment period. AST is found in the cytoplasm and mitochondria of many cells, but its concentration is higher in liver, heart muscle and skeletal muscle cells (Chaves and Silva, 1998; Latha et al., 1998). Higher plasmatic AST levels indicate, thus, damage of some of those organs. In addition, change in plasmatic glucose levels and decrease in weight gain is likely to reflect impaired cell metabolism. These questions led us to investigate the possible action of barbatima˜o extracts on liver cell energy metabolism. As a first approach we have measured the respiratory activity and several membranebound enzymatic activities in isolated mitochondria. In order to facilitate an extrapolation to the condition of living cells, we have also measured parameters strictly related to energy metabolism in the isolated perfused liver, e.g. oxygen consumption, gluconeogenesis and glycogenolysis.
2. Methods and materials 2.1. Plant material The stem bark of S. adstringens (Martius) Coville, Leguminosae, was collected in Sa˜o Jer-
oˆnimo da Serra-Pr (23843?7.8ƒS, 50845?23.5ƒW at an altitude of 926 m), Parana´ State, Brazil, in October 1995. A voucher specimen was deposited at the Herbarium of the Biology Department under number HUM-3800. Air-dried stem bark was extracted with Me2CO /H2O (7:3, 29 l) according to Foo and Porter (1978), Cork and Krockenberger (1991) and de Mello et al. (1999). The extract was filtered, evaporated under reduced pressure, and lyophilized. The lyophilized extract was stored at /20 8C until further use. Tests were done with the total lyophilized extract dissolved in water just before use. 2.2. Animals Male Wistar rats weighing 200 and 250 g and fed with a standard laboratory diet (Purina† ) and water ad lib were used. The protocol for these experiments was accepted and approved by the Animal Ethics Committee of the University of Maringa´. 2.3. Isolation of mitochondria Rat liver mitochondria were isolated by differential centrifugation in a mannitol /sucrose medium, according to Voss et al. (1961). Freeze / thawing disrupted mitochondria were used for the assay of membrane-bound enzymes. Intact mitochondria were frozen in liquid nitrogen and thawed rapidly at 37 8C. This procedure was repeated three times. The resulting disrupted mitochondria were maintained at 0/4 8C for use. 2.4. Determination of oxygen consumption, ADP/O ratio and respiratory control ratio (RC) Oxygen consumption by intact mitochondria was measured polarographically using an incubation medium containing 5 mM disodium phosphate, 10 mM Tris /HCl (pH 7.4), 0.2 mM EDTA, 10 mM potassium chloride, 0.25 M mannitol and 50 mg% fatty acid /free bovine serum albumin. The following substrates were added to the incubation medium when required: succinate (12.5 mM), a-ketoglutarate (12.5 mM) and ADP (125 or 250 mM). The ADP/O ratio was calculated accord-
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ing to Chance and Williams (1955) and it represents the amount of ADP added to the incubation system divided by the amount of oxygen consumed during the active phase of respiration (state III respiration). The respiratory control (RC) ratio is given by the rate of active respiration which follows ADP addition divided by the rate of respiration after ADP exhaustion (state IV respiration; Chance and Williams, 1955). In this and in other experiments with isolated mitochondria, the extracts were added to the incubation medium in the concentration range between 25 and 500 mg/ml. After a preincubation with mitochondria for a period of 2 min, the reactions were initiated by the addition of specific substrates.
2.5. ATPase activity The ATPase activity was assayed by measuring phosphate release according to Pullman et al. (1960). When intact mitochondria were used as enzyme source, the reaction medium contained: 0.2 M sucrose, 12 mM Tris /HCl (pH 7.4), 50 mM KCl and, when required, 200 mM 2,4-dinitrophenol. When disrupted mitochondria were incubated, the medium contained 20 mM Tris /HCl (pH 7.4) and 0.15 M KCl. The reaction was started by the addition of 5 mM ATP and stopped, after 20 min of incubation at 37 8C, by the addition of ice-cold 5% trichloroacetic acid. Phosphate was measured as described by Fiske and Subbarow (1925).
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2.7. Determination of protein contents Protein contents of the mitochondrial suspensions were measured using the method of Lowry et al. (1951). The standard was bovine serum albumin.
2.8. Liver perfusion The non-recirculating perfusion technique described by Scholz and Bu¨cher (1965) was used. For the surgical procedure, the animals were anesthetized by i.p. injection of sodium pentobarbital (50 mg/kg). The perfusion fluid was Krebs/Henseleitbicarbonate buffer, pH 7.4 (Krebs and Henseleit, 1932), saturated with an oxygen/carbon dioxide mixture (95/5%). The fluid was pumped through a temperature-regulated (37 8C) membrane oxygenator prior to entering the liver via a cannula inserted in the portal vein. The perfusion flow was constant in each individual experiment and it was adjusted between 30 and 35 ml/min, depending on the liver weight. Samples of the effluent perfusion fluid were collected at 2-min intervals and analyzed for glucose, lactate and pyruvate. When glycogen catabolism was measured, the livers of fed rats were used in the experiments. Livers from 24-h fasted rats were used for the measurement of gluconeogenesis. When required the barbatima˜o extract was dissolved in the perfusion fluid for a final concentration of 100 mg/l.
2.9. Analytical 2.6. Membrane-bound enzymatic activities Freeze /thawing disrupted mitochondria were used as enzyme source for assaying membranebound enzymatic activities. NADH-oxidase and succinate-oxidase activities were assayed polarographically using a 20 mM Tris /HCl (pH 7.4) medium (Singer, 1974). A polarographic assay was also run with TMPD (N ,N ,N ?,N ?-tetramethyl-pphenylenediamine) plus ascorbate as substrates. The reactions were started by the addition of 12.5 mM NADH, 12.5 mM succinate or 0.2 mM TMPD/5 mM ascorbate.
Lactate and pyruvate were measured by standard enzymatic methods, using lactate dehydrogenase (Gutmann and Wahlefeld, 1974; Czok and Lamprecht, 1974). Glucose was measured by an enzymatic-colorimetric method using glucose-oxidase (Bergmeyer and Bernt, 1974). The oxygen concentration in the venous perfusate was monitored continuously employing a Teflon-shielded platinum electrode. Metabolic rates were calculated from the input /output differences and the total flow rates and were referred to the wet weight of the liver.
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2.10. Treatment of data The statistical significance of the differences between parameters was evaluated by means of Student’s t -test or Student /Newman /Keuls test. The latter was applied after submitting the data to variance analysis. The results are mentioned in the text as the P values; P B/0.05 was adopted as a criterion of significance.
3. Results 3.1. Effect of the aqueous extract of barbatima˜o on oxygen consumption in isolated mitochondria Oxygen consumption by intact mitochondria was measured using FAD-dependent (succinate) and NAD -dependent (a-ketoglutarate) substrates in the presence of exogenously added ADP (state III respiration) or after ADP exhaustion (state IV respiration). The aqueous extract of the barbatima˜o stem bark was added to the incubation medium for final concentrations between 25 and 250 mg/ml. The results are illustrated by Fig. 1. When succinate was the substrate (Fig. 1A) the rates of oxygen consumption in the absence of ADP (state IV respiration) were increased in the concentration range of 25/125 mg/ml. At the concentration of 125 mg/ml the rates of oxygen consumption were 92.2% (P /0.01) above the basal ones (absence of barbatima˜o extract). In the presence of ADP, the barbatima˜o extract caused a dose-dependent inhibition of oxygen consumption (state III respiration). When a-ketoglutarate was used as a substrate (Fig. 1B), state IV respiration was also progressively increased in the presence of the barbatima˜o extract. Maximal stimulation was found at the concentration of 125 mg/ml (/189.5%, P /0.01). The changes in state III respiration were less pronounced than those observed in the presence of succinate. Table 1 shows the effect of the barbatima˜o extract on the ADP/O ratios and the respiratory control ratios (RC). The respiratory control ratios (RC) and the ADP/O ratios were decreased by the extract with both substrates, a-ketoglutarate and
Fig. 1. Effects of the total extract of barbatima˜o on the respiratory activity of isolated rat liver mitochondria. Mitochondria (0.25 /2.5 mg/ml) were added to the reaction medium in the closed vessel of the oxygraph (temperature, 37 8C). The reaction was initiated by the addition of succinate (A) or aketoglutarate (B) and the oxygen consumption was followed polarographically during approximately 5 min. After this time 0.25 /0.5 nmols of ADP were added. Rates of oxygen consumption were computed from the slopes of the polarographic records. Each data point is the mean9/S.E.M. of 11 (succinate) and five (a-ketoglutarate) independent experiments. *, P B/ 0.05, ANOVA with Newman /Keuls test.
succinate. At concentrations above 125 mg/ml no reliable estimation of the ADP/O ratios was possible. 3.2. Effects of the barbatima˜o extract on the activities of membrane-bound enzymes The effects of the barbatima˜o extract on succinate-oxidase, NADH-oxidase and TMPD-ascorbate oxidation, measured in disrupted mitochondria, are shown in Fig. 2. The addition of the barbatima˜o extract caused inhibition of all enzymatic activities in a dose-dependent manner.
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Table 1 Effects of the total extract of barbatima˜o on the ADP/O ratios and the respiratory control coefficients (RC) in rat liver mitochondria Extract (mg/ml)
0.0 25.0 62.5 125.0 187.5 250.0
a-Ketoglutarate (n/5)
Succinate (n /8) ADP/O
RC
ADP/O
RC
1.489/0.20 1.389/0.16 1.329/0.24 1.239/0.24* / /
3.879/0.82 3.179/0.61** 2.279/0.34** 1.399/0.24** 1.109/0.09** 1.00**
2.159/0.23 2.279/0.22 2.039/0.25 1.779/0.20* / /
2.559/0.19 2.349/0.13 1.859/0.19** 1.229/0.13** 1.049/0.09** 1.00**
Mitochondria (0.25 /2.5 mg/ml) were incubated in reaction medium as described in Section 2. Oxygen consumption was recorded polarographically. ADP (250 or 500 nmols) was added at appropriate times. The respiratory control coefficients (RC) and ADP/O ratios were calculated according to Chance and Williams (1955). *, P B/0.05; **, P B/0.01, ANOVA with Newman /Keuls test.
Fig. 2. Effects of the total barbatima˜o extract on several membrane-bound enzymatic activities in rat liver mitochondria. Panel A: NADH-oxidase, succinate-oxidase and TMPD-ascorbate oxidation, measured with freeze /thawing disrupted mitochondria, incubated at 37 8C in reaction medium as described in Section 2. Panel B: ATPase activity of coupled, uncoupled and disrupted mitochondria, incubated at 37 8C in reaction medium as described in Section 2. Each assay point represents the mean of 8 /11 experiments and the bars are S.E.M. *, P B/ 0.05, ANOVA with Newman /Keuls test.
The calculated mean inhibitory doses (ID50) were 96.39/7.8 mg/ml for succinate-oxidase, 78.69/5.4 mg/ml for NADH-oxidase and 156.39/12.5 mg/ml for TMPD-ascorbate oxidation. Fig. 2B shows results of the effects of the barbatima˜o extract on the ATPase activity measured in intact mitochondria either in the absence (coupled mitochondria) or in the presence of 2,4dinitrophenol (uncoupled mitochondria) and in freeze /thawing disrupted mitochondria. The actions of the barbatima˜o extract were different in each preparation. The ATPase activity of coupled mitochondria was increased, with maximal stimulation at a concentration of 125 mg/ml (/282.8%, P /0.001). A tendency toward inhibition at higher concentrations, however, was evident: at a concentration of 500 mg/ml the ATPase activity stimulation was reduced to 64% of the control. In the case of uncoupled mitochondria, the barbatima˜o extract caused a small stimulation at low concentrations. Maximal stimulation (31%) was found at a concentration of 62.5 mg/ml (P / 0.01). Higher concentrations, however, were inhibitory. Inhibition was 41% at 500 mg/ml (P / 0.001). When disrupted mitochondria were used as the enzyme source, the barbatima˜o extract caused only inhibition of the ATPase activity. An inhibition of 72% was found at a concentration of 500 mg/ml (P /0.001).
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3.3. Effects of the barbatima˜o extract on glycogen catabolism and oxygen consumption in the liver of fed rats In order to verify if the effects found in isolated mitochondria manifest themselves in intact cells and also in order to evaluate further the potential toxicity of the extract to the liver, we have investigated the action of the barbatima˜o extract on oxygen consumption, glycogenolysis, glycolysis
and gluconeogenesis. Fig. 3A shows the results of experiments performed with livers from fed rats. These livers were perfused with substrate-free perfusion fluid, in a open system. As shown in Fig. 3A, under these conditions, the livers release glucose, lactate and pyruvate as a result of glycogen catabolism (Scholz and Bu¨cher, 1965). The introduction of 100 mg/l of barbatima˜o extract at 20 min of perfusion produced changes in all parameters. Oxygen consumption, lactate and pyruvate production were increased. When the infusion of the barbatima˜o extract was interrupted at 50 min, the metabolic fluxes did not return to the rates before infusion (basal rates). The barbatima˜o extract exerted complex effects on glucose release. A rapid increase in glucose release was observed at 2 /3 min of infusion, followed by a progressive decrease, with a minimum around 40 min. After this minimum, the rates of glucose release tended to increase gradually until the terminus of the infusion. After termination of the infusion, glucose release increased rapidly and remained elevated during the subsequent 20 min. At this time, glucose release was nearly 112% above the basal values (P /0.01). 3.4. Effects of the barbatima˜o extract on oxygen consumption and gluconeogenesis in the liver from fasted rats
Fig. 3. Effects of the total extract of barbatima˜o on metabolic fluxes in perfused livers isolated from fed (A) and fasted (B) rats. Livers from fed or 24-h fasted rats were perfused with Krebs/Henseleit-bicarbonate buffer (pH 7.4), as described in Section 2. In the experiments with fed rats (panel A), the barbatima˜o extract (100 mg/l) was infused at 20 /50 min, as indicated by the horizontal bar. In the experiments with fasted rats (panel B), lactate (2 mM) and pyruvate (0.2 mM) were infused at 20 /76 min and the barbatima˜o extract (100 mg/l) at 36 /56 min. The effluent perfusate was sampled in 2-min intervals and analyzed for glucose, lactate and pyruvate in livers from fed rats and for glucose in livers from fasted rats. Each experimental point is the mean9/S.E.M. of six experiments.
Fig. 3B illustrates the effects of the barbatima˜o extract on oxygen consumption and glucose production due to lactate plus pyruvate in perfused livers from fasted rats. Both the glycogen stores and the levels of endogenous gluconeogenic substrates in livers from 24-h fasted rats are minimal. In consequence, glucose production depends almost entirely from the exogenously supplied substrates. As expected, during the time period before lactate (2.0 mM) plus pyruvate (0.2 mM) infusion (1 and 16 min of the time scale in Fig. 3B), glucose release was minimal. The introduction of gluconeogenic substrates immediately increased glucose release and oxygen consumption. The infusion of the barbatima˜o extract caused a rapid and transient increase in oxygen consumption, followed by a gradual decrease to values around those measured before the infusion. Glucose release, on the
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other hand, decreased progressively. At the end of the barbatima˜o extract infusion, glucose release was reduced by 46% (P /0.001) when compared with the rates measured before the infusion. The interruption of the infusion was followed by a recovery of glucose release to values still 12.6% below those measured before the infusion of the barbatima˜o extract.
4. Discussion The results of the present work reveal that the total aqueous extract of S. adstringens (barbatima˜o) has complex effects on liver metabolism. The data obtained with isolated mitochondria indicate that the barbatima˜o extract impairs oxidative phosphorylation by at least three modes of action: (1) uncoupling of oxidative phosphorylation; (2) inhibition of electron flow in the respiratory chain; (3) inhibition of the ATP-synthase complex. The uncoupling action is indicated by the effects on mitochondrial respiration and ATPase activity, namely stimulation of state IV respiration (irrespective of the substrate), decrease of the respiratory control ratio, decrease of the ADP/O ratio and increase of ATP hydrolysis in intact coupled mitochondria. The stimulatory effect on ATP hydrolysis was absent when the latter was measured in disrupted mitochondria, indicating that the integrity of the mitochondrial membrane is essential for the stimulatory action. All these effects are found when mitochondria are incubated with uncouplers such as 2,4-dinitrophenol and FCCP (Hopfer et al., 1968; Hanstein, 1976). Inhibition of electron flow in the respiratory chain was revealed by the inhibitory action on NADH, succinate and TMPD-ascorbate oxidation in disrupted mitochondria. Even in intact coupled mitochondria, inhibition of state IV respiration was visible at higher concentrations of the barbatima˜o extract with both succinate or a-ketoglutarate as substrates. The exact site of the action on the electron transport chain cannot be inferred from the available data. However, two different ID50-values were found: one for NADH- and succinate-oxidase and a much higher one for TMPD-ascorbate oxidation. This finding suggests
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the existence of at least two sites of action: a step between the quinone pool and complex III and another step between cytochrome c and oxygen. Alternatively, the components of the barbatima˜o extract could be acting directly on dehydrogenases, such as NADH-dehydrogenase and succinate dehydrogenase. Further experiments are required to clarify this point. The effects of the barbatima˜o extract on the ATPase activity of different mitochondrial preparations indicate the third probable mode of action. Whereas in intact coupled mitochondria the barbatima˜o extract was able to stimulate the ATPase activity, inhibition was the dominant effect in 2,4-dinitrophenol-uncoupled mitochondria as well as in freeze/thawing-disrupted mitochondria. This set of observations excludes a direct action of the barbatima˜o extract on the ADP/ATP exchange system. A direct inhibition of the ATP synthase complex seems to be, thus, the most probable mechanism of action. The rat liver perfusion experiments provided a clear indication that the barbatima˜o extract is also active on intact cells. Infusion of the barbatima˜o extract caused activation of oxygen consumption either in livers from fed or fasted rats. It is unlikely that this activation represents an increase in the energy demands of the liver, because there was in parallel a significant reduction in gluconeogenesis. Gluconeogenesis from lactate plus pyruvate is a metabolic pathway strictly dependent on mitochondrial ATP production. Oxygen consumption stimulation seems to be, thus, due to an uncoupling of oxidative phosphorylation, which reduces the net rates of ATP production. In agreement with this interpretation, the barbatima˜o extract stimulated glycolysis (lactate plus pyruvate production) in livers from fed rats. Activations of glycolysis and glycogenolysis are expected responses to diminished mitochondrial ATP production and is a general characteristics of the action of electron flow blockers and uncouplers (Nascimento et al., 1992; Constantin et al., 1995). The effects of the barbatima˜o extract on glucose release from glycogen were complex and cannot be explained only by its action on mitochondria. Activation was in fact observed in the first minutes of the infusion. However, glucose release de-
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creased progressively during the rest of the infusion period, a phenomenon that was followed by a posterior increase when the infusion was interrupted. We have, so far, no explanation for these phenomena. Changes in liver hemodynamics or in enzymatic systems implicated in glucose release from glycogen are possibilities that deserve attention in future work. The inhibitory action of the barbatima˜o extract on hepatic energy metabolism could be responsible, partly at least, for its reported toxicity in animals (Rebecca et al., 2002). An impairment of oxidative phosphorylation may cause changes in membrane integrity, favoring enzyme leakage (Hess, 1963). Moreover, another consequence may be a perturbation of biosynthetic pathways, which could be contributing to the reported decrease in body weight gain. A further question which arises is one regarding the nature of the active substances implicated in the effects. The precise identity of the active principles of S. adstringens is not known until now. However, tannins are substances which could be considered at least as a starting point. The plant contains a large quantity of condensed tannins, varying between 10 and 37%, depending on the place and season of the year in which the sample has been collected (Teixeira et al., 1990; de Mello et al., 1996). It has been demonstrated that tannins and related compounds exert several biological and pharmacological activities, such as bactericidal, antiviral, molluscicidal and antihelmintic actions, antihepatotoxic action, inhibition of xanthine oxidase and mono-amino oxidase and inhibition of glucosyltransferases (Haslam, 1996). It is believed that these properties derive from their antioxidant and radical scavenging activities, and from their ability to form complexes with other macromolecules, such as proteins and polysaccharides, and metal ions (Haslam, 1996). In vitro tests have demonstrated that some tannins inhibit the activities of several enzymes, including both NADH-dehydrogenase and succinate-oxidase of rat liver mitochondria (Nishizawa et al., 1983; Konishi et al., 1993; Konishi and Tanaka, 1999). Our data are in agreement with both observations, because the barbatima˜o extract was able to inhibit both NADH-oxidase and succinate-oxidase activ-
ities. Based on such data, it seems reasonable to suggest that the tannins are implicated, partly at least, in the metabolic effects reported in this work. Evidently, this speculation does not exclude, but rather emphasizes, the necessity of isolation and identification of the major active principles of S. adstringens, with subsequent studies on their mechanisms of action.
Acknowledgements The authors are grateful to Jailson Araujo Dantas and Aparecida P.M. Hermoso for their technical assistance. This work was supported by the Programa Nacional de Nu´cleos de Exceleˆncia (PRONEX), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).
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