Plants of Sonchus asper maturity were collected from Wah cantt District Rawalpindi (Pakistan) during the month of June, 2011. Plants were identified and a specimen was submitted vide 147 at Herbarium of Pakistan, Quaid-i-Azam University Islamabad, Pakistan. Arial parts of plant (leaves, stem, flowers and seeds) were shade dried at room temperature for two weeks, chopped, grinded mechanically of mesh size 1 mm as described by Antonio et al. .
Preparation of plant extract
1.5 kg Sonchus asper leaves powder was extracted in separatory funnel with 2.0 litre of absolute methanol with refluxing for 5 h. The extract was cooled at room temperature, filtered and evaporated under reduced pressure in rotary evaporator to Sonchus asper methanolic extract (SAME). SAME was stored at 4°C for in vivo studies .
Six week old, 30 male albino rats (190–200 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25 ± 3°C with a 12 h dark/light cycle. They were allowed to standard laboratory feed and water. The study protocol was approved by Ethical committee of Quaid-i-azam University Islamabad for laboratory animal feed and care.
To study the antioxidant effects of SAME, male albino rats were equally divided into 05 groups (6 rats). Group 1 received only raw water and free access to food materials. Group II received CCl4 3 ml/kg intraperitoneally in olive oil (Monday and Thursday). Group III and IV were given orally 100; 200 mg/kg b.w. (in DMSO), Sonchus asper methanolic extracts (SAME) after 48 h of CCl4 treatment (Wednesday and Saturday) as above. Groups V received only SAME in DMSO at a dose of 200 mg/kg b.w. (Wednesday and Saturday) . After 24 h of the last treatment, all the animals were weighted, sacrificed; collected their blood, weighted and perfuse thyroid gland in ice-cold saline solution. Half of thyroid portion were treated with liquid nitrogen for further enzymatic and DNA damage analysis while the other portion was processed for histology.
Assessment of serum thyroid hormones
Serum analysis of Various thyroid hormones’ such as T4, T3 and TSH were estimated by commercial radio amino assay kits 10227-Czch Republic (IM1447-IM3286), 10227-Czch Republic (IM1699-IM3287) and 10227-Czch Republic (IM3712-IM3713) Kit purchased from IMMUNOTECH Company respectively.
Assessment of antioxidant enzymes
70 mg of thyroid tissue were homogenized in 10 volume of 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was collected and used for the following experiments as described below. Protein concentration was determined using crystalline BSA as standard.
Catalase assay (CAT)
CAT activities were determined by the method of Chance and Maehly  with some modifications. The reaction solution of CAT activities contained: 2.5 ml of 50 mmol phosphate buffer (pH 5.0), 0.4 ml of 5.9 mmol H2O2 and 0.1 ml enzyme extract. Changes in absorbance of the reaction solution at 240 nm were determined after one minute. One unit of CAT activity was defined as an absorbance change of 0.01 as units/min.
Peroxidase assay (POD)
Activities of POD were determined by the method of Chance and Maehly  with some modifications. The POD reaction solution contained: 2.5 ml of 50 mM phosphate buffer (pH 5.0), 0.1 ml of 20 mmol guaiacol, 0.3 ml of 40 mmol H2O2 and 0.1 ml enzyme extract. Changes in absorbance of the reaction solution at 470 nm were determined after one minute. One unit of POD activity was defined as an absorbance change of 0.01 units/min.
Superoxide dismutase assay (SOD)
SOD activity of thyroid was estimated by the method of Kakkar et al. . Reaction mixture of this method contained: 0.1 ml of phenazine methosulphate (186 μmol), 1.2 ml of sodium pyrophosphate buffer (0.052 mmol; pH 7.0), 0.3 ml of supernatant after centrifugation (1500 × g for 10 min followed by 10000 × g for 15 min) of lung homogenate was added to the reaction mixture. Enzyme reaction was initiated by adding 0.2 ml of NADH (780 μmol) and stopped after 1 min by adding 1 ml of glacial acetic acid. Amount of chromogen formed was measured by recording color intensity at 560 nm. Results are expressed in units/mg protein.
Glutathione-S-transferase assay (GST)
Glutathione-S-transferase activity was assayed by the method of Habig et al. . The reaction mixture consisted of 1.475 ml phosphate buffer (0.1 mol, pH 6.5), 0.2 ml reduced glutathione (1 mmol), 0.025 ml 1-chloro-2,4-dinitrobenzene (1 mmol CDNB) and 0.3 ml of homogenate in a total volume of 2.0 ml. The changes in the absorbance were recorded at 340 nm and enzymes activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 103M-1cm-1.
Glutathione reductase assay (GR)
Glutathione reductase activity was determined by method of Carlberg and Mannervik . The reaction mixture consisted of 1.65 ml phosphate buffer: (0.1 mol; pH 7.6), 0.1 ml EDTA (0.5 mmol), 0.05 ml oxidized glutathione (1 mmol), 0.1 ml NADPH (0.1 mmol) and 0.1 ml of homogenate in a total volume of 2 ml. Enzyme activity was quantitated at 25°C by measuring disappearance of NADPH at 340 nm and was calculated as nmol NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 × 103 M-1cm-1.
Glutathione peroxidase assay (GSH-Px)
Glutathione peroxidase activity was assayed by the method of Mohandas et al. . The reaction mixture consisted of 1.49 ml phosphate buffer (0.1 mol; pH 7.4), 0.1 ml EDTA (1 mmol), 0.1 ml sodium azide (1 mmol), 0.05 ml glutathione reductase (1 IU/ml), 0.05 ml GSH (1 mmol), 0.1 ml NADPH (0.2 mmol), 0.01 ml 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 molar extinction coefficient of 6.22 × 103 M-1cm-1.
Î³-glutamyl transpeptidase assay (Î³-GT)
This was determined by the method of Orlowski and Meister  using glutamyl p-nitroanilide as substrate. The reaction mixture in a total volume of 1.0 ml contained 0.2 ml of homogenate which was incubated with 0.8 ml substrate mixture (containing 4 mmol glutamyl p-nitroanilide, 40 mmol glycylglycine and 11 mmol MgCl2 in 185 mmol Tris–HCl buffer, pH 8.25) at 37°C. Ten minutes after initiation of the reaction, 1.0 ml of 25% TCA was added and mixed to terminate the reaction. The solution was centrifuged and the supernatant fraction was read at 405 nm. Enzyme activity was calculated as nmol p-nitroaniline formed/min/mg protein using a molar extinction coefficient of 1.74 × 103 M-1cm-1.
Quinone reductase assay
The activity of quinone reductase was determined by the method of Benson et al. . The 3.0 ml reaction mixture consisted of 2.13 ml Tris–HCl buffer (25 mmol; pH 7.4), 0.7 ml BSA, 0.1 ml FAD, 0.02 ml NADPH (0.1 mmol), and 0.l ml of homogenate. The reduction of dichlorophenolindophenol (DCPIP) was recorded at 600 nm and enzyme activity was calculated as nmol of DCPIP reduced/min/mg protein using molar extinction coefficient of 2.1 × 104 M-1cm-1.
Reduced glutathione assay (GSH)
Reduced glutathione was estimated by the method of Jollow et al. . 1.0 ml sample of homogenate was precipitated with 1.0 ml of (4%) sulfosalicylic acid. The 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 filtered aliquot, 2.7 ml phosphate buffer (0.1 mol; pH 7.4) and 0.2 ml DTNB (100 mmol). The yellow color developed was read immediately at 412 nm on a SmartSpecTM plus Spectrophotometer. It was expressed as μmol GSH/g tissue.
Estimation of lipid peroxidation assay (TBARS)
The assay for lipid peroxidation was carried out following the method of Wright et al. . The reaction mixture in a total volume of 1.0 ml contained 0.58 ml phosphate buffer (0.1 mol; pH 7.4), 0.2 ml homogenate sample, 0.2 ml ascorbic acid (100 mmol), and 0.02 ml ferric chloride (100 mmol). The reaction mixture was incubated at 37°C in a shaking water bath for 1 h. The reaction was stopped by addition of 1.0 ml 10% trichloroacetic acid. Following addition of 1.0 ml 0.67% thiobarbituric acid, all the tubes were placed in boiling water bath for 20 min and then shifted to crushed ice-bath before centrifuging at 2500 × g for 10 min. The amount of TBARS formed in each of the samples was assessed by measuring optical density of the supernatant at 535 nm using spectrophotometer against a reagent blank. The results were expressed as nmol TBARS/min/mg tissue at 37°C using molar extinction coefficient of 1.56 ×105 M-1cm-1.
Hydrogen peroxide assay (H2O2)
Hydrogen peroxide (H2O2) was assayed by H2O2-mediated horseradish peroxidase-dependent oxidation of phenol red by the method of Pick and Keisari . 2.0 ml of homogenate sample was suspended in 1.0 ml of solution containing phenol red (0.28 nmol), horse radish peroxidase (8.5 units), dextrose (5.5 nmol) and phosphate buffer (0.05 mol; pH 7.0) and were incubated at 37°C for 60 min. The reaction was stopped by the addition of 0.01 ml of NaOH (10 N) and then centrifuged at 800 × g for 5 min. The absorbance of the supernatant was recorded at 610 nm against a reagent blank. The quantity of H2O2 produced was expressed as nmol H2O2/min/mg tissue based on the standard curve of H2O2 oxidized phenol red.
Nitrite assay was conducted by using Griess reagent. Tissue and serum samples were deproteinized by equal volumes of 0.3 mol NaOH and 5% ZnSO4 and centrifuged at 6400 × g for 20 min and supernatant was collected. 1.0 ml of Griess reagent was added into the cuvette and blanks the spectrophotometer at 540 nm. Then 20 μl supernatant was added in cuvette containing Griess Reagent. Nitrite concentration was calculated using a standard curve for sodium nitrite.
DNA fragmentation % assay
DNA fragmentation % assay was conducted using the procedure of Wu et al. [28
] with some modifications. The thyroid tissue (50 mg) was homogenized in 10 volumes of a TE solution pH 8.0 (5 mmol Tris–HCl, 20 mmol EDTA) and 0.2% triton X-100. 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). The pellet and supernatant fractions were assayed for DNA content using a freshly prepared DPA (Diphenylamine) solution for reaction. Optical density was read at 620nm at (SmartSpecTM Plus Spectrophotometer catalog # 170–2525) spectrophotometer. The results were expressed as amount of % fragmented DNA by the following formula;
For microscopic evaluation thyroid tissues were fixed in a fixative (Absolute alcohol 60%, Formaldehyde 30%, Glacial acetic acid 10%) and embedded in paraffin, sectioned at 4 μm and subsequently stained with hematoxylin/eosin. Sections were studied under light microscope (DIALUX 20 EB) at 40 magnifications. Slides of all the treated groups were studied and photographed. A minimum 12 fields of each section were studied and approved by pathologist without saying of its treatment nature.
To determine the treatment effects one way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% level of probability.