Ethical approval of the study protocol
The study protocol was approved by an Ethics Committee of Quaid-i-Azam University for the Feeding and Care of Laboratory Animals.
Plant collection
LP was collected from Wah Cantonment in the district of Rawalpindi (Pakistan) at maturity during June 2006. It was identified and a specimen submitted at the Herbarium of Pakistan (Quaid-i-Azam University, Islamabad, Pakistan). Aerial parts of the plant (leaves, stem, flowers, seeds) were dried in the shade at room temperature for 2 weeks. They were then chopped and ground mechanically to a mesh size of 1 mm.
Preparation of plant extract
A total of 1.5 kg powder of LP was extracted with 2 L of absolute methanol in a separating funnel with refluxing for 5 h. The extract was cooled at room temperature, filtered, and evaporated under reduced pressure in a rotary evaporator. It was suspended in distilled water and fractionated with n-hexane, ethyl acetate and chloroform. The chloroform fraction of Launaea procumbens (LPCE) was dried and stored at 4°C for in-vivo studies.
HPLC of LPCE
A total of 500 mg of LPCE was extracted with 6 ml of 25% hydrochloric acid and 20 ml of chloroform for 1 h. The obtained extract was filtered to a volumetric flask. The residue was heated twice with 20 ml of chloroform for 20 min to obtain the extract. The combined extract was diluted with chloroform to 100 ml. A 5-ml portion of the solution was filtered and transferred to a volumetric flask and diluted with 10 ml of chloroform. The sample (10 μl) was injected into the HPLC apparatus. Samples were analyzed on an Agilent HPLC system (Agilent, Santa Clara, CA, USA). Separation was carried out through a 20RBAX Eclipse XDB-C18 column (5 μm; 4.6 × 150 mm, Agilent) with a ultraviolet–visible (UV–vis) Spectra-Focus detector with an autosampler. Solvent A (0.05% trifluoroacetic acid) and solvent B (0.038% trifluoroacetic acid in 83% acetonitrile (v/v) were employed with the following gradient: 0–5 min, 15% B in A, 5–10 min, 50% B in A, 10–15 min, 70% B in A. The flow rate was 1 ml/min and the injection volume was 10 μl. Six standard compounds (myricetin, catechin, vitexin, kaempferol, hyperoside, rutin) were run for comparative detection and optimized. Calibration curves were defined for each compound in the range of sample quantity 0.02–0.5 μg. All samples were assayed in triplicate. All quantitative data were evaluated using analytic software.
Animals
Thirty-six male Sprague–Dawley ratss (age, 6 weeks; 190–200 g) were provided by the National Institute of Health (Islamabad, Pakistan). They were kept in standard cages at room temperature (25 ± 3°C) with a 12-h dark–light cycle. They were allowed to consume standard laboratory food and water.
Experimental design
To study the antioxidant possessions of LP, rats were equally divided into six groups. Group 1 (control) have free access to food. Group II received 3 ml/kg of CCl4 (30% in olive oil) via the intraperitoneal route twice a week for 4 weeks. Group III received silymarin 100 mg/kg body weight (b.w.) via the oral route after 48 h of CCl4 treatment. Groups IV and V were given 100 mg/kg b.w. and 200 mg/kg b.w. LPCE, respectively, after 48 h of CCl4 treatment as described above, while group VI received only LPCE at 200 mg/kg b.w. for 4 weeks. Twenty-four hours after the last treatment, all rats were weighed and their blood collected; they were then killed. Livers were removed, weighed, and perfused in ice-cold physiological (0.9%) saline solution. Half of the liver was reated with liquid nitrogen for enzymatic and DNA-damage analyses, whereas the other portion was processed for histological analyses.
Assessment of levels of liver marker enzymes and biochemical parameters
Serum analyses of various liver marker enzymes alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (γ-GT), lactate dehydrogenase (LDH)), total cholesterol (TC), high-density lipoprotein- cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) and triglycerides (TGs) were estimated using standard AMP Diagnostic Kits (Graz, Austria).
Assessment of levels of antioxidant enzymes
Hepatic tissues were homogenized in 10 volumes of 100 mmol KH2PO4 buffer containing 1 mmol ethylenediamine tetra-acetic acid (EDTA; pH 7.4) and centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was collected and used for the assessment of antioxidant enzymes. Protein concentrations in the supernatants of liver tissue homogenates were determined using crystalline bovine serum albumin (BSA) as a standard. All chemicals used in enzymatic analyses were purchased from Sigma–Aldrich (St Louis, MO, USA).
Catalase assay
Catalase activity was determined using the method of Chance and Maehly [19] with some modifications. The reaction solution of catalase activity contained 2.5 ml of 50 mmol phosphate buffer (pH 5.0), 0.4 ml of 5.9 mmol H2O2 and 0.1 ml of hepatic supernatant. Changes in the absorbance of the reaction solution at 240 nm were determined after 1 min. One unit of catalase activity was defined as an absorbance change of 0.01 as units/min.
Superoxide dismutase (SOD) assay
The SOD activity of liver tissue was estimated using the method of Kakkar et al. [20]. he reaction mixture contained 0.1 ml of phenazine methosulfate (186 μmol), 1.2 ml of sodium pyrophosphate buffer (0.052 mmol; pH 7.0), 0.3 ml of the supernatant after centrifugation (1500 × g for 10 min followed by 10,000 × g for 15 min). The enzyme reaction was initiated by adding 0.2 ml of NADH (780 μmol) and stopped after 1 min by the addition of 1 ml of glacial acetic acid. The amount of chromogen formed was measured by recording color intensity at 560 nm. Results are expressed in units/mg protein.
Glutathione-S-transferase (GST) assay
GST activity was assayed using the method of Habig et al. [21]. The reaction mixture consisted of 1.475 ml of phosphate buffer (0.1 mol, pH 6.5), 0.2 ml of reduced glutathione (1 mmol), 0.025 ml of CDNB (1 mmol) and 0.3 ml of homogenate in a total volume of 2.0 ml. Changes in absorbance were recorded at 340 nm, and GST activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 103 M–1 cm–1.
Glutathione Reductase (GSR) assay
GSR activity was determined using the method of Carlberg and Mannervik [22]. The reaction mixture consisted of 1.65 ml of phosphate buffer: (0.1 mol; pH 7.6), 0.1 ml of EDTA (0.5 mmol), 0.05 ml of oxidized glutathione (1 mmol), 0.1 ml of nicotinamide adenine dinucleotide phosphate (NADPH) (0.1 mmol) and 0.1 ml of homogenate in a total volume of 2 ml. Enzyme activity at 25°C was estimated by measuring the disappearance of NADPH at 340 nm. It was calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 103 M–1 cm–1.
Glutathione peroxidase (GSH-px) assay
GSH-Px activity was assayed using the method of Mohandas et al. [23]. The reaction mixture consisted of 1.49 ml of phosphate buffer (0.1 mol; pH 7.4), 0.1 ml of EDTA (1 mmol), 0.1 ml of sodium azide (1 mmol), 0.05 ml of GSR (1 IU/ml), 0.05 ml of reduced glutathione (GSH; 1 mmol), 0.1 ml of NADPH (0.2 mmol), 0.01 ml of H2O2 (0.25 mmol) and 0.1 ml of homogenate in a total volume of 2 ml. The disappearance of NADPH at 340 nm was recorded at 25°C. Enzyme activity was calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 103 M–1 cm–1.
Quinone Reductase (QR) assay
The activity of QR was determined using the method of Benson et al. [24]. The 3-ml reaction mixture consisted of 2.13 ml of Tris–HCl buffer (25 mmol; pH 7.4), 0.7 ml of BSA, 0.1 ml of FAD, 0.02 ml of NADPH (0.1 mmol) and 0.l ml of homogenate. The reduction of dichlorophenolindophenol (DCPIP) was recorded at 600 nm. Enzyme activity was calculated as nmol of DCPIP reduced/min/mg protein using a molar extinction coefficient of 2.1 × 104 M–1 cm–1.
GSH assay
GSH was estimated using the method of Jollow et al. [25]. A total of 1.0 ml of homogenate was precipitated with 1.0 ml of 4% sulfosalicylic acid. Samples were kept at 4°C for 1 h and then centrifuged at 1200 × g for 20 min at 4°C. The total volume of 3.0 ml assay mixture contained 0.1 ml of a filtered aliquot, 2.7 ml of phosphate buffer (0.1 mol; pH 7.4) and 0.2 ml of DTNB (100 mmol). The yellow color that developed was read immediately at 412 nm on a SmartSpec™ Plus Spectrophotometer (Bio-Rad, Hercules, CA, USA). It was expressed as μmol GSH/g tissue.
Estimation of lipid peroxidation using levels of thiobarbituric acid reactive substances (TBARS)
The assay for lipid peroxidation was carried out using a modified method of Iqbal et al. [26]. One milliliter of 20% TCA aqueous solution and 1.0 ml of 0.67% TBA aqueous solution was added to 0.6 ml of phosphate buffer (0.1 M; pH 7.4) and 0.4 ml of homogenate sample. The reaction mixture was heated in a boiling water-bath for 20 min and then moved to a bath of crushed ice before centrifugation at 2500 × g for 10 min. The amount of TBARS formed in each of the samples was assessed by measuring the optical density of the supernatant at 535 nm using a spectrophotometer against a reagent blank. Results were expressed as nmol TBARS/min/mg tissue at 37°C using a molar extinction coefficient of 1.56 × 105 M–1 cm–1.
Nitrite assay
A nitrite assay was conducted using Griess reagent. Tissue was deproteinized using equal volumes of 0.3 mol NaOH and 5% ZnSO4, and centrifuged at 6400 × g for 20 min, and the supernatant collected. A total of 1.0 ml of Griess reagent was added into the cuvette, and the spectrophotometer blanked at 540 nm. Then 20 μl of supernatant was added in a cuvette containing Griess reagent. Nitrite concentration was calculated using a standard curve for sodium nitrite.
DNA fragmentation assay
The DNA fragmentation assay was conducted using the procedure of Wu et al. [27] with some modifications. Tissue (50 mg) was homogenized in 10 volumes of a TE solution at pH 8.0 (5 mmol Tris–HCl, 20 mmol EDTA) and 0.2% Triton X-100. A 1.0-ml aliquot of each sample was centrifuged at 27,000 × g for 20 min to separate the intact chromatin (pellet, B) from the fragmented DNA (supernatant, T). Pellet and supernatant fractions were assayed for DNA content using a freshly prepared diphenylamine (DPA) solution for the reaction. The optical density was read at 620 nm using the SmartSpec Plus Spectrophotometer. Results are expressed as % fragmented DNA using the following formula:
(1)
DNA ladder assay
DNA was isolated using the methods of Wu et al. [27] to estimate DNA damage. A 5-μg aliquot of DNA of rats was separately loaded in 1.5% agarose gel containing 1.0 μg/ml of ethidium bromide, including DNA standards (0.5 μg per well). Electrophoresis was done for 45 min at 100 V. After electrophoresis, gels were studied under a Gel Doc system and photographed using a digital camera.
Argyrophilic nucleolar organizer regions (AgNORs) counts
A silver staining technique was used according to the methods of Trere et al. [28]. Unstained fixed slides were dewaxed by dipping for 3 min in xylene and were hydrated in decreasing ethanol concentrations (90%, 70% and 50%). After drying, slides were treated with one drop of colloidal solution (2% gelatin and 1% formic acid) and two drops of 50% AgNO3 solution onto the slides and incubated at 35°C for ≈8–12 min. Progressive staining was followed under a light microscope (Dialux 20 EB, Leitz, Wetzlar, Germany) to obtain golden-colored nuclei and brown/black NORs. Then, slides were washed in distilled water, treated for 1 min with 1% sodium thiosulfate at room temperature to stop the reaction, and washed in tap water. Cells were examined under a light microscope at 100 × magnification and the number of AgNORs per cell counted.
Histopathological studies
For microscopic evaluation, liver samples were fixed in a fixative (absolute alcohol 60%, formaldehyde 30%, glacial acetic acid 10%) and embedded in paraffin, sectioned at 4-μm thickness, and subsequently stained with hematoxylin and eosin (H&E). Sections were studied under a light microscope at 40 × magnification. Slides of all treated groups were photographed and studied.
Statistical analyses
To determine the treatment effects, one-way analysis of variance was carried using SPSS 13.0 computer software (SPSS, Chicago, IL, USA). The level of significance among the various treatments was determined by least squares difference (LSD) analyses at 0.05% and 0.01% levels of probability.