cistus laurifolius

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456 Male Swiss albino mice (20–25 g) were purchased from the animal breeding laboratories of Refik Saydam Central Institute of Health (Ankara, Turkey). The animals left for 2 days for acclimatization to animal room conditions were maintained on standard pellet diet and water ad libitum. The food was withdrawn on the day before the experiment, but allowed free access of water. A minimum of six animals was used in each group. Throughout the experiments, animals were processed according to the suggested international ethical guidelines for the care of laboratory animals. L. leaves were collected from Bolu, Dörtdivan in May 2002 and was identified by Prof. Dr. M. Vural from the Department of Botany, Faculty of Science, Gazi University. A voucher specimen is deposited in the Herbarium of Faculty of Pharmacy, Gazi University (GUE-2300). Some 2 kg of powdered material was extracted three times with EtOH (10 L) by stirring in a 60 C water bath for 8 days each. The combined ethanol extract was evaporated to dryness under reduced pressure to yield ‘EtOH extract’ (315 g). The EtOH extract was then resuspanded in 1500 ml of MeOH and extracted with -hexane (7 500 ml). Combined hexane extract was evaporated under reduced pressure to yield ‘Hexane fraction’ (64.8 g). MeOH was removed from the remaining solution and diluted with distilled H 2 O to 2000 ml and further fractionated by successive extractions with chloroform (7 500 ml), ethyl acetate (5 250 ml) and watersaturated -butanol (3 200 ml). The extracts as well as the remaining aqueous phase were evaporated to dryness under reduced pressure to yield the “CHCl 3 Fr.” (98.4 g), “EtOAc Fr.” (28.3 g), “BuOH Fr.” (29.7 g) and “R–H 2 O Fr.” (84.5 g), respectively. Four grams of the CHCl 3 fraction was permeated on a Sephadex LH-20 column using MeOH as eluent. Some 43 fractions of 15 ml each were collected. Fractions were compared by TLC on silica gel using CHCl 3 /MeOH (8:2) and toluene/ether (1:1) as mobile systems and combined as follows: Fr.1–13 (1.23 g), Fr.14–19 (0.72 g), Fr.20–30 (0.91 g), Fr.31–43 (1.12 g). Some additional 5.36 g of the CHCl 3 fraction were worked-up under identical conditions to obtain more material for further purification. Vacuum-chromatography of the flavonoidenriched fractions (silica gel; petroleum ether/EtOAc (1:1), (1:2) and EtOAc/MeOH (8:2) as solvent system) and reverse-phase (RP-18) chromatography using MeOH/H 2 O yielded three pure compounds: ( ) (1.23 g), ( ) (0.95 g), ( ) (0.82 g). The structure of compounds was elucidated by spectroscopic methods as quercetin-3-methyl-ether ( ), quercetin-3,7-dimethyl-ether ( ) Scheme 1. and kaempferol-3,7-dimethyl-ether ( ) (Scheme 1). The spectral data are in agreement with the reported values (Barbera et al., 1986; Guerrero et al., 2002; Smolarz et al., 2003; Stevens et al., 1995). The extract, fractions and pure compounds were suspended in 0.5% CMC in distilled water prior to oral administration to experimental animals. Test groups of mice were orally treated with EtOH extract (500 mg/kg body weight), hexane (206 mg/kg), CHCl 3 (312 mg/kg), EtOAc (90 mg/kg), -BuOH (94 mg/kg) or R-H 2 O fractions (268 mg/kg) for 7 following days, once a day by gastric gavage. The control group (untreated) and acetaminophen group (positive control) were administered in 0.5% CMC suspension for the same period. The reference drug, ascorbic acid (Vitamin C) suspended in 0.5% CMC was directly administered to animals at 100 mg/kg dose. Some 60 min after the administration of the last dose on seventh day, except the control group mice, each of the acetaminophen group and test group animals was challenged with the suspension of acetaminophen (800 mg/kg body weight) in 0.5% CMC to induce hepatic injury (Fairhurst et al., 1982). Four hours after acetaminophen administration, blood samples were withdrawn by cardiac puncture and then the mice were sacrificed by overdose of diethylether. Blood samples collected in heparinized tubes were centrifuged at 3000 (4 C) for 10 min to obtain plasma. Plasma samples were used to determine the lipid peroxide levels and the enzyme (AST, ALT) activity. Liver of each mouse was promptly removed and used to determine the tissue levels of malondialdehyde (MDA) and cellular glutathione (GSH).
457 The methodology described by Kurtel et al. (1992) was used. Briefly, 1 ml of plasma was mixed with 2.0 ml of trichloroacetic acid (TCA; 15%, w/v)–thiobarbituric acid (TBA; 0.375%)–0.25 N HCl and mixed throughly and centrifuged at 10,000 for 5 min. The supernatant was mixed with 20 l of butyl hydroxy toluene (BHT; 0.02% in 95% EtOH, w/v) to prevent further oxidation and heated for 15 min in a boiling water bath. After cooling under running water, the flocculent precipitate was removed by centrifugation at 10,000 for 5 min. The absorbance of the sample was measured at 532 nm against blank that contained all the reagents except plasma. 1,1,3,3-Tetraethoxypropan was used as standard for the curve calibration. with no homogenate. Results were expressed as tissue. mol GSH/g Biocon standard kits and DAX-48 autoanalyzer were used to measure AST and ALT activities in plasma samples according to the method of Wilkinson et al. (1972). Animals employed in the experiments were observed during 48 h and morbidity or mortality was recorded, if happens, for each group at the end of observation period. The method of Ohkawa et al. (1979) as modified by Jamall and Smith (1985) was used to determine lipid peroxidation in tissue samples. Mice were sacrified by an overdose of diethylether. The liver of each mouse was immediately excised and chilled in ice-cold 0.9% NaCl and then perfused via the portal vein with ice-cold 0.9% NaCl. After washing with 0.9% NaCl, 1.0 g of wet tissue was weighted exactly and homogenized in 9 ml of 0.25 M sucrose using a teflon homogenizer to obtain a 10% suspension. The cytosolic fraction was obtained by a two-step centrifugation first at 1000 for 10 min and then at 2000 for 30 min at 4 C. A volume of the homogenate (0.20 ml) was transferred to a vial and was mixed with 0.2 ml of a 8.1% (w/v) sodium dodecyl sulphate solution, 1.50 ml of a 20% acetic acid solution (adjusted to pH 3.5 with NaOH) and 1.50 ml of a 0.8% (w/v) solution of TBA and the final volume was adjusted to 4.0 ml with distilled water. Each vial was tightly capped and heated in boiling water bath for 60 min. The vials were then cooled under running water. Equal volumes of tissue blank or test sample and 10% TCA were transferred into a centrifuge tube and centrifuged at 1000 for 10 min. The absorbance of the supernatant fraction was measured at 532 nm in a Beckman DU 650 spectrometer. Control experiment was processed using the same experimental procedure except the TBA solution was replaced with distilled water due to the peroxidative effect of acetaminophen on tissue. Livers of acetaminophen-treated mice were used as positive control and 1,1,3,3-tetraethoxypropan was used as standard for the curve calibration. Some 200 mg of liver was homogenized in 8.0 ml of 0.02 M EDTA in an ice bath. The homogenates were kept in the ice bath until used. Aliquots of 5.0 ml of the homogenates were mixed in 15.0 ml test tubes with 4.0 ml distilled water and 1.0 ml of 50% trichloroacetic acid (TCA). The tubes were centrifuged for 15 min at approximately 3000 . Some 2.0 ml of supernatant was mixed with 4.0 ml of 0.4 M Tris–buffer, pH 8.9, 0.1 ml Ellman’s reagent [5,5 -dithiobis-(2-nitro-benzoic acid)] (DTNB) added, and the sample shaken. The absorbance was read within 5 min of the addition of DTNB at 412 nm against a reagent blank The data obtained were analyzed by one-way of variance (ANOVA) and Student–Newman–Keuls post hoc tests for the significant interrelation between the various groups using Instat computer software. < 0.05 was considered to be significant different from the control. In living systems, dietary antioxidants such as -tocopherol, ascorbic acid, carotenoids, as well as flavonoids and related phenolic compounds are suggested in protection from oxidative damage of tissues in the body and eventually for a healthy life (Haraguchi, 2001). Especially flavonoids have been shown to scavenge various reactive oxygen species and implicated as inhibitors of lipid peroxidation (Mora et al., 1990). Acetaminophen (paracetamol), a frequently used analgesic and antipyretic drug, is known to be hepatotoxic in high doses, which is primarily metabolized by sulfation and glucuronidation to unreactive metabolites, and then activated by the cytochrome P-450 system to produce liver injury. It is established that acetaminophen is bioactivated to a toxic electrophile, -acetyl- -benzoquinone imine (NAPQI), which binds covalently to tissue macromolecules, and probably also oxidizes lipids, or the critical sulphydryl groups (protein thiols) and alters the homeostasis of calcium (Lin et al., 1997). Post-mitochondrial supernatants isolated from livers of rats given a single large oral dose of acetaminophen (800 mg/kg) showed rapid rates of lipid peroxidation when incubated in vitro. Lipid peroxidation probably occurs simultaneously with the proposed covalent binding of the active metabolite of acetaminophen. Since the former process is known to cause severe and extensive membrane damage, it may be a very important factor in acetaminophen-induced liver necrosis (Fairhurst et al., 1982). As shown in Table 1, in the liver and plasma of acetaminophen-treated group, tissue and plasma lipid peroxidation levels (219.2 and 1172%) as evidenced by MDA determination increased significantly as compared to control group, however, the content of GSH in liver decreased (8.7%). Additionally, acetaminophen was found to cause several folds increases in plasma AST and ALT levels (106 and 71.6%). Ethanol (EtOH) extract of leaves administered in 500 mg/kg
Page 1: Journal of Ethnopharmacology 103 (2
Page 5 and 6: 459 levels (19.4-36.7%). The result

cistus laurifolius

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