Effect of the aqueous extract of Hyptis pectinata on liver mitochondrial respiration.
Some studies have indicated that mitochondria may be the target organelle of plants. We therefore decided to assess the effects of the aqueous extract of Hyptis pectinata leaves on liver mitochondrial respiratory function in vitro. Eight rat livers were subjected to isolation of mitochondria by differential centrifugation. In an adequate medium, the plant extract was added at different concentrations. The analyzed data were: state 3, state 4 and respiratory control ratio (RCR). H. pectinata extract caused a statistically significant decrease in state 3 (at 0.05, 0.1 and 0.2 mg/mg protein) and RCR (at 0.05, 0.1 and 0.2 mg/mg protein). Respiratory state 4 was not altered by the increasing concentrations. In conclusion, the aqueous extract of H. pectinata leaves may not injure the mitochondrial inner membrane but decreases significantly the oxidative phosphorylation.
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Keywords: Hyptis pectinata; Liver; Mitochondria; State 3; State 4; RCR; Medicinal plant
Hyptis pectinata (L.) Poit (Lamiaceae), known popularly in Brazil as "sambacaita" or "canudinho", is a herbaceous plant with opposing crossed, whole and aromatic leaves. Its flowers are small, clustered into axillary inflorescences, hermaphrodite, pentamer, strongly zygomorphous and bilabiate.
It is used popularly to treat rhinopharyngitis, nasal congestion, certain skin diseases, gastric disorders, fever and bacterial infections (Rojas et al., 1992; Malan et al., 1988). In the state of Sergipe, it has been recommended for the treatment of inflammation, pain, cancer, bacterial infections and wound healing (Bispo et al., 2001).
The essential oil of the plant contains 33 compounds. Monoterpenes are the most common (95.8%). The main constituents are p-cymene, thymol and [beta]-terpinene. Together, they correspond to 68% of the total oil (Malan et al., 1988). The presence of thymol is considered the main factor in the antiseptic property of this plant (Rojas et al., 1992).
In experimental studies, it has been disclosed that the aqueous extract of H. pectinata leaves protects the liver against injuries, stimulates its regeneration according to the concentration and has antinociceptive and antiedematogenic effects (Melo et al., 2001; Silva et al., 2002; Bispo et al., 2001). Although the mechanism of biostimulation or inhibition remains obscure, some recent studies indicate that mitochondria may be the target organelle of the plants (Janssens et al., 1999, 2000).
Considering that there are no studies defining the effects of this plant on liver and mitochondria, this research is aimed at assessing the effects of H. pectinata leaves on mitochondrial respiratory function of rat livers in vitro.
Materials and methods
Plant material and preparation of the aqueous extract
H. pectinata leaves (identified by Dr. C. Dias Silva Jr.; Voucher No. ASE 2626, deposited in the Department of Biology of Universidade Federal de Sergipe) were collected outside the blossoming period from the Live Pharmacy of Aracaju, Brazil. The leaves were dried in an oven with air renewal and circulation (model MA-037) at 37[degrees]C to complete dehydration.
Dried H. pectinata leaves were triturated in a blender until a finely granulated powder was obtained. The extract was obtained from this powder (100 g) by adding distilled water (3:10 w/v) under constant shaking for 4 h at 35[degrees]C, followed by filtration (pH 6.0). The filtrate was lyophilized (aqueous extract) and stored at 5[degrees]C, yielding 16.2 g (16.2%) of lyophilized active material. At the time of use, the extract was resuspended in distilled water at the desired concentrations.
The study was carried out on eight male Wistar albino rats (180-230 g each) obtained from the Animal Center of the Universidade de Sao Paulo. They were housed (two to a cage) under standard conditions of temperature and light (12-h light/dark cycle) and fed with a standard pellet diet and water ad libitum.
In order to perform the experiment, they were submitted to ether anesthesia and their livers were removed. After that, they were sacrificed by overdose of ether anesthesia.
Preparation of mitochondria and oxygen consumption assays
Isolation of liver mitochondria was performed by differential centrifugation as described previously (Souza et al., 1994). Mitochondrial oxygen consumption was analyzed polarographically with a home-made oxygraph equipped with a Clarck oxygen electrode, and the respiratory parameters were determined according to an established procedure (Chance and Williams, 1956). Succinate (5 mM) was used as oxidizable substrate in 1.4ml of medium with 125 mM sucrose, 65 mM KCl, 1 mM Mg[Cl.sub.2], 2 mM K[H.sub.2]P[O.sub.4], 0.1 mM EGTA and 10 mM Hepes-KOH, pH 7.4; and 2.0 mg of mitochondrial protein was used. State 3 respiration was induced with 400 nmol MgADP, and state 4 respiration (basal mitochondrial respiration) was determined after phosphorylation of additional ADP (Souza et al., 1994). The ratio between state 3 and state 4 rates (respiratory control ratio, RCR), which represents coupling between electrons transport and oxidative phosphorylation, was determined. Mitochondrial protein content was determined by the biuret method (Cain and Skilleter, 1987).
Plant extract addition
The different plant extract concentrations were added to the medium containing mitochondria. For each concentration tested, different mitochondria from the same rat were added to the oxygraph. Therefore, the mitochondria were not pooled. The concentrations were: 0.0125, 0.025, 0.05, 0.1 and 0.2 mg/mg of mitochondrial protein. "C" (control) represents the mitochondria without the plant extract.
Statistical analysis of data
All data are expressed as mean [+ or -] standard error of the mean (s.e.m.). Statistical comparisons among the different concentrations were performed using analysis of variance (ANOVA) for parametric measurements with Scheffe as post-test. Probability values of less than 0.05 were considered to be statistically significant.
As shown in Fig. 1A, mitochondrial respiratory state 3 was statistically reduced (as compared to control) when the aqueous extract of H. pectinata was used at concentrations of 0.05, 0.1 and 0.2 mg/mg protein.
State 4 of mitochondrial respiration was not changed by the increasing concentrations of the plant extract (Fig. 1B).
[FIGURE 1 OMITTED]
RCR levels disclosed a significant decrease when the H. pectinata extract was used at concentrations of 0.05, 0.1 and 0.2 mg/mg protein (Fig. 1C).
It has been reported that the aqueous extract of H. pectinata leaves protects the liver against injuries, stimulates its regeneration depending on the concentration and has antinociceptive and antiedematogenic effects (Bispo et al., 2001; Melo et al., 2001, in press; Silva et al., 2002). According to Stephenson (1956), all drugs exert a dose-dependent effect on their targets. However, this process depends on the amount of available receptors and the physical and chemical properties of the substances. This theory is applied to pharmacology. On the other hand, phytotherapy is not fully understood. It is possible that, at some concentrations, certain compounds of a plant extract could act in synergism and cause a significant effect. At lower or higher concentrations, these compounds could be antagonized by different ones. It is likely that the active principle responsible the pharmacological effect described, is not a volatile terpenoid (e.g. thymol), because the water extract was obtained from an oven dried plant material by maceration at low temperature and then lyophilized.
Although the mechanism of biostimulation or inhibition remains obscure, some recent studies indicate that mitochondria may be the target organelle of plants (Janssens et al., 1999, 2000). The critical role played by mitochondria in the maintenance of cellular energy metabolism has long been recognized. The electron transport from the oxidation of NADH and reduced FAD[H.sub.2] to [O.sub.2] is tightly coupled to the synthesis of ATP. This transport occurs through protein-bound redox centers, from complex I (NADH-coenzyme Q reductase) or II (succinate-coenzyme Q reductase) to III (coenzyme cytochrome e reductase) and then to IV (cytochrome e oxidase). The free energy released by this transport is conserved by pumping out protons in order to create an eletrochemical H gradient across the inner mitochondrial membrane. The eletrochemical potential of this gradient is then harnessed in the synthesis of ATP by complex V (ATP synthase): this process is known as oxidative phosphorylation (Jennings and Ganote, 1976; Trump et al., 1976).
In a recent work performed by our group, an antiproliferative activity of copaiba oleoresin on liver regeneration in rats was shown (Castro-e-Silva et al., 2003a). In another study from the same laboratory, liver regeneration was stimulated at 100mg/kg body weight of H. pectinata but not at 200 and 400 mg/kg body weight (Melo et al., in press). This might be explained by hepatocellular membrane receptor saturation or a possible blockage of some stage of the metabolic reaction leading to an uncoupled mitochondrial function able to cause relative decrease in liver regeneration and function, as verified in the copaiba oleoresin study (Castro-e-Silva et al., 2003a). Stimulatory and inhibitory effects are also common in biostimulation by light and plant depending on the wave length or dose, respectively (Castro-e-Silva et al., 2003a, b). This is an important question, the answer of which has not yet been fully formulated.
This question should be considered in relation to the results shown in Fig. 1. It can be observed that state 3 of mitochondrial respiration and RCR were reduced as the concentration of H. pectinata in respiration media was increased. State 3 is especially dependent on the electron transport chain, on the import of substrates into the mitochondrial matrix and on the activities of ATP synthase and adenine nucleotide translocase (Duan and Karmazym, 1989). This reduction may be explained by the effect of the inhibitory compounds of the plant extract on the existent chromophores groups in the respiratory chain or on the activity of FoF1-ATPase, as happens with oligomycin (Penefsky, 1985). In the present study, the plant, in all concentrations used, had no effect on mitochondrial oxygen consumption in state 4 respiration. State 4 represents the dissipation of the proton gradient and is in direct relationship to the coupling and phosphorylation process. Additionally, it depends on the optimal activity and integrity of the respiratory chain (complexes I, III and IV), which extrudes protons from the mitochondrial matrix toward the intermembrane space. As a consequence, it generates the proton gradient (Nicholls, 1974). The plant extract caused a decrease in RCR by depressing state 3, while state 4 was unalterable. This means that the plant may not injure the mitochondrial inner membrane, but reduces the oxidative phosphorylation significantly.
The authors are grateful to PIBIC/CNPq/UFS and FAPESP for supporting this research.
Received 20 November 2003; accepted 20 February 2004
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G.B. Melo (a), R.L. Silva (a), V.A. Melo (a), A.R. Antoniolli (b,*), M.E. Jordani Souza (c), M.C. Jordani (c), O. Castro-e-Silva Jr. (d)
(a) Department of Medicine, Universidade Federal de Sergipe, Aracaju, Brazil
(b) Department of Physiology, Universidade Federal de Sergipe, Aracaju, Brazil
(c) Laboratory of Biochemistry and Liver Transplantation, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Brazil
(d) Department of Surgery and Anatomy, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Brazil
*Corresponding author. Dept. de Fisiologia, CCBS, Lab. de Farmacologia/Bioquimica, Universidade Federal de Sergipe, CEP, 49100-000 Sao Cristovao--SE, Brazil. Tel.: +55 79 9979 1003.
E-mail addresses: firstname.lastname@example.org, email@example.com (A.R. Antoniolli).