Vol. 58, No 4/2011

Glutathione reductase (GR, E.C 1.6.4.2) is a flavoprotein that catalyzes NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). The aim of this study was to investigate in vitro effects of phenolic compounds isolated from Sideritis brevibracteata on bovine kidney GR. The Sideritis species are widely found in nature and commonly used as medicinal plants. 7-O-glycosides of 8-OH-flavones (hypolaetin, isoscutellarein and 3'-hydroxy-4'-O-methylisoscutellarein) were isolated from aerial parts of Sideritis brevibracteata. These compounds inhibited bovine kidney cortex GR in a concentration-dependent manner. Kinetic characterization of the inhibition was also performed.


INTRODuCTION
The research on natural compounds from medicinal plants and their synthetic analogues has accelerated in recent years due to their importance in drug discovery using a medicinal chemistry approach (Lee, 2010).In this study, we investigated in vitro effects of the compounds isolated from Sideritis brevibracteata P. H. Davis on glutathione reductase (GR).Sideritis L. (Lamiaceae=Labiatae) species are widely found in nature and are commonly used as medicinal plants and herbal teas in the Mediterranean region (Güvenç et al., 2005).Sideritis species contain mainly flavonoids, essential oils, diterpenes, phenylpropanoid glycosides, and iridoid glucosides (Ezer et al., 1992;Baser et al., 1997).Antibacterial, anti-inflammatory, antinociceptive, anti-oxidant, and aldose reductase inhibitory activities of compounds from this genus have been reported (Barberan et al., 1987;Güvenç et al., 2010).These biological activities are mainly attributed to the flavonoid content of this plant (Yesilada & Ezer, 1989;Rios et al., 1992;De Las Heras et al., 1994).
Glutathione reductase (GR) reduces glutathione disulfide (GSSG) to its thiol form GSH (Arscott et al., 2000).Alterations in the activity of GR may affect the cellular defense system and cause oxidative stress in the cell.Inhibition of GR decreases the GSH/GSSG ratio, and this contributes to oxidative stress and the pathogenesis of many diseases.GR is a homodimeric flavoenzyme, and cysteine, histidine and tyrosine are amino acids critical for the GR activity.The redox properties of this enzyme can be inhibited or activated by several chemicals, drugs or natural substrates (Untucht-Grau et al., 1981;Bauer et al., 2006;Krauth-Siegel et al., 1998).
Glutathione reductase (GR) is an important housekeeping enzyme for redox homeostasis both in human cells and in the causative agent of tropical malaria.Its inhibitors are usually considered as drug sensitizers: they do not display high intrinsic antimalarial or antitumoral activities when used alone, but they can enhance the effects of chloroquine or cytotoxic agents (Bauer et al., 2006).Acetylated allose containing 8-hydroxyflavone glycosides and a phenylethanoid glycoside have been isolated from aerial parts of S. brevibracteata (Güvenç et al., 2010).The present study aimed to investigate the in vitro effects of the phenolic compounds on bovine kidney cortex GR.

Materials.
Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) and oxidized glutathione (GSSG) were obtained from Sigma Chemical Co., MO, USA.
Assay of glutathione reductase.Glutathione reductase (GR) activity was determined according to the modified Stall method (Acan & Tezcan, 1989).The incubation mixture contained 100 mM sodium phosphate buffer, pH 7.4; 1 mM GSSG; 0.2 mM NADPH; and bovine kidney cortex GR.Decrease in the absorbance of NADPH at 340 nm was monitored spectrophotometrically at 37 ºC.
A unit of activity (U) was defined as the amount of enzyme that catalyzes the oxidation of 1 µmole of NADPH in 1 min under these conditions.
Purification of bovine glutathione reductase.Bovine kidney cortex GR was purified by two subsequent chromatography steps after ultracentrifugation and heat denaturation: 2',5'-ADP Sepharose 4B affinity and DEAE Sepharose Fast Flow anion exchange chromatography.All the procedures were carried out at +4 ºC.GR was purified 34 806fold with a final yield of 85 % and specific activity of 727 U/mg using this method (Tandogan & Ulusu, 2010).
Inhibition studies.Activities were measured by addition of the compounds investigated at different concentrations to the assay mixture given above for GR measurement.GR assays in the presence of the compounds were performed without enzyme-inhibitor preincubation, and the reactions were initiated by adding the enzyme to a substrate-inhibitor mixture.
Statistical analysis of kinetic data.The data were analyzed and the kinetic constants were calculated using the equations described in (Segel, 1975) by means of a nonlinear curve-fitting program of Statistica.

DISCuSSION
Natural products and their derivatives have historically been useful as a source of therapeutic agents (Koehn & Carter, 2005).Natural product structures have long been recognized to possess characteristics of high chemical diversity, biochemical specificity and molecular diversity, which make them attractive targets for the discovery of new drugs (Ajay et al., 1998;Cao & Kingston, 2009).In this study, natural products of Sideritis brevibracteata inhibited bovine kidney cortex GR.This enzyme is a significant target for the development of antimalarial and anticancer agents.Therefore, inhibition of this enzyme is an important research topic (Seefeldt et al., 2005).There is an urgent need for new, more affordable and accessible antimalarial agents.Natural products have played a dominant role in the discovery of drugs to treat human diseases (Batista et al., 2009).
In cancer therapy, inhibition of GR may be one of the important targets because this enzyme is key in the metabolism of glutathione, and its expression is often aberrant in human malignancies (Sun, 1990).Inhibition of this enzyme decreases GSH concentration in the cancer cell.Conjugation with glutathione of electrophilic xenobiotics facilitates their export from the cell and is a common detoxification mechanism, which may result in drug resistance.Therefore, a decreased GSH concentration will allow an increased effective drug concentration in the cell thus enhancing its cytotoxic effect on the cancer cell (Awasthi et al., 2009).
The phenolic compounds of S. brevibracteata inhibited bovine kidney cortex GR in a concentration-dependent manner.Compounds [1], [5] and [6] demonstrated similar inhibition kinetics, with an uncompetitive inhibition mode when GSSG was the varied substrate and a noncompetitive mode when NADPH was the varied substrate.However, compounds [2] and [4] showed different inhibition patterns.This can probably be attributed to these compounds' binding different amino acid residues or groups in the catalytic site of the enzyme.As is well known, uncompetitive inhibitors are thought to bind the enzyme-substrate complex and not the enzyme, but noncompetitive inhibition occurs when the inhibitor binds at a site away from the substrate-binding site, causing a reduction in the catalytic rate (Segel, 1975).
In conclusion, GR is a key enzyme that controls the cellular thiol-disulfide redox state in the cells, and it is essential for cellular homeostasis.During oxidative stress, intracellular GSSG accumulates, and the loss of thiol redox balance may cause deleterious consequences with regards to metabolic regulation, cellular integrity and organ homeostasis.Inhibition of GR and a disturbance in the cellular prooxidant-antioxidant balance may contribute to the genesis of many diseases.Its inhibitors have been shown to have anticancer and antimalarial activity by contributing to the reversal of drug resistance.