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Protective effect of Sonchus asper extracts against experimentally induced lung injuries in rats: A novel study
Experimental and Toxicologic Pathology 64 (2012) 725–731
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Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Protective effect of Sonchus asper extracts against experimentally induced lung injuries in rats: A novel study Rahmat Ali Khan a,b,∗ , Muhammad Rashid Khan b , Sumaira Sahreen b a b
Department of Biotechnology, University of Science and Technology, Bannu, Khyber Pukhtunkhawa, Pakistan Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 4400, Pakistan
a r t i c l e
i n f o
Article history: Received 11 October 2010 Accepted 13 January 2011 Keywords: Carbon tetrachloride Sonchus asper Lung histopathology Antioxidant Lipid peroxidation
a b s t r a c t In this study, protective effects of methanol extract (SAME) were evaluated against carbon tetrachloride induced oxidative stress in lungs. Male Sprague–Dawley rats were orally fed with various doses (100, 200 mg/kg body weight) of SAME and (50 mg/kg body weight) of rutin after 48 h of CCl4 treatment (3 ml/kg body weight, 30% in olive oil) biweekly for 4 weeks. The results showed that administration of extracts and rutin significantly restored lung contents of reduced glutathione and activities of catalase, peroxidase, superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase, quinine reductase were reduced while lipid peroxide, hydrogen peroxide, nitrite, DNA fragmentation% and activity of ␥-glutamyl transferase, increased by CCl4 , were reversed towards the control levels by the supplement of Sonchus asper extracts and rutin. Lung histopathology showed that S. asper extracts and rutin reduced the incidence of lung lesions induced by CCl4 in rats. These results suggest that S. asper fractions and rutin could protect lung against the CCl4 -induced oxidative damage in rats. © 2011 Elsevier GmbH. All rights reserved.
1. Introduction Oxidative stress describes the steady state level of oxidative damage in a cell, tissue, or organ, caused by the reactive oxygen species (ROS). Various environmental factors such as exposure to UV radiation, chemical intoxication and normal cellular activities cause production of ROS such as superoxide radical, hydrogen peroxide and hydroxyl radical. Oxidative stress is caused by an imbalance between the production of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage. Our body possesses antioxidant enzyme systems such as catalase, superoxide dismutases, peroxidases and glutathione enzyme system as well as non enzymatic antioxidant compounds like reduced glutathione, ascorbic acid and ␣-tocopherol. Disturbances in this normal redox state can cause toxic effects through the production of cell damaging peroxides and free radicals (Halliwell and Whiteman, 2004). It has been reported that good health can be maintained from the consumption of plants with high antioxidant activities (Guarrera et al., 2006). There is some evidence that bioactive compounds
∗ Corresponding author at: Department of Biotechnology, University of Science and Technology, Bannu, Khyber Pukhtunkhawa, Pakistan. Tel.: +0092928633421; fax: +0092928624987. E-mail address: Rahmatgul [email protected] (R.A. Khan). 0940-2993/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2011.01.007
and microelements from different functional foods, herbs and nutraceuticals can ameliorate or even prevent diseases (Cambie and Ferguson, 2003; Berker et al., 2009). Sonchus (Sow Thistle) constitutes food in South Africa (Afolayan and Jimoh, 2008) and Turkey (Alpinar et al., 2009) and medicinally important plant species Sonchus oleraceus, Sonchus arvensis and Sonchus asper; having beneficial bioactive compounds (Cambie and Ferguson, 2003; Alpinar et al., 2009). The food use of S. oleraceus and S. asper is justified by the high content of vitamin C, carotenoids and phenolic constituents (Afolayan and Jimoh, 2008). Extracts of these plant species also possess high antioxidant activities (Guarrera et al., 2006). Extract of S. arvensis is an integral component of an antioxidant formulation “Prolipid® ” because of high polyphenolic contents. S. asper (L.) Hill. (Spiny-leaved Sow Thistle) locally named as “Mahtari” (SA) is a common herb that grows wildly and abundantly in open fields. It is used in the treatment of cough, bronchitis, asthma (Koche et al., 2008) and in wound healing (Rehman, 2006; Hussain et al., 2008). Chemical characterization of SA has shown the presence of ionone derivatives of glycosides (Shimizu et al., 1989), flavonoids, ascorbic acid, carotenoids (Guil-Guerrero et al., 1998) and sesquiterpene lactone glycosides (Shimizu et al., 1989; Helal et al., 2000). These bioactive compounds have been shown to possess strong antioxidant and anti-inflammatory properties (Alpinar et al., 2009). Based on the traditional claims surrounding S. asper and the lack of sci-
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entific studies of its potential pharmacological properties, the objective of this study is to evaluate the protective effects of Sonchus asper (SA) against CCl4 induced oxidative stress in lungs of rats. 2. Materials and methods 2.1. Chemicals Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), CCl4 , flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) of Sigma Chemicals Co., USA were utilized in this experiment. 2.2. Plant collection Plants of S. asper (L.) Hill. at maturity were collected, identified and a specimen was submitted vide voucher # R-147 at Herbarium of Pakistan, Quaid-i-Azam University, Islamabad, Pakistan for future reference. Aerial parts of plant (leaves, stem, flowers and seeds) were shade dried, chopped and grinded of 1 mm mesh size. 2.3. Preparation of plant extract 1.5 kg powder of S. asper (SA) was extracted with 2.0 liter of absolute methanol with refluxing for 5 h, filtered and evaporated under reduced pressure to obtain crude extract of methanol (SAME), dried under reduced pressure and stored at 4 ◦ C for further use. 2.4. Animals and treatment The study protocol was approved by Ethical committee of Quaidi-Azam University, Islamabad for laboratory animal feed and care. The present study was conducted (with 96 mature male albino rats) in the laboratory of the Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan. The animals weighing 190–200 g, were divided equally into 6 groups and maintained under standard laboratory conditions (12 h light/darkness; at 25 ± 3 ◦ C) with standard animal diet and water available ad libitum. Group I remained untreated (control) while group II received olive oil intraperitoneally (Monday and Thursday) and DMSO orally (Wednesday and Saturday) at a dose of 3 ml/kg body weight. Group III was treated with CCl4 , 3 ml/kg body weight (30% in olive oil i.p. Monday and Thursday). However, group IV received rutin 50 mg/kg body weight (in DMSO) after 48 h (Wednesday and Saturday) of CCl4 treatment. Groups V and VI were administered S. asper methanolic extract at doses of 100 and 200 mg/kg body weight after 48 h (Wednesday and Saturday) of CCl4 treatment. After 24 h of the last treatment, all the animals were weighted, sacrificed; their lungs were removed, weighted and perfused in ice-cold saline solution. Half of lung tissue was treated with liquid nitrogen for further enzymatic and DNA damage analysis while the other portion was processed for histology.
used for the following experiments as described below. Protein concentration of the supernatant of lung tissue was determined by the method of Lowry et al. (1951) using crystalline BSA as standard. Catalase (CAT) and peroxidase (POD) activities were determined by the method of Chance and Maehly (1955) with some modifications. Briefly CAT activity was determined by adding H2 O2 and change in absorbance was recorded at 240 nm while POD activity was measured by using guaiacol as substrate at 470 nm. One unit of CAT and POD activity was defined as an absorbance change of 0.01 as units/min. Superoxide dismutase (SOD) activity was estimated by using phenazine methosulphate and sodium pyrophosphate buffer according to Kakkar et al. (1984). Enzyme reaction was initiated by adding NADH (780 mol) and stopped after 1 min by adding glacial acetic acid and color intensity at 560 nm was recorded. Results are expressed in units/mg tissue protein. 2.6. Assessment of lipid peroxidation enzymes Induction of lipid peroxidation by CCl4 and its protection by the S. asper extract were determined by the estimation of various enzyme activities and thiobarbituric acid reactive substance (TBARS) contents. 2.7. Glutathione-S-transferase assay (GST) Lung glutathione-S-transferase activity was determined according to Habig et al. (1974), using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. GST was measured at 340 nm using a molar extinction coefficient of 9.6 × 103 M−1 cm−1 . 2.8. Glutathione reductase assay (GSR) Glutathione reductase activity (Carlberg and Mannervik, 1975), was measured at 340 nm by using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate. An extinction coefficient of 6.22 × 103 M−1 cm−1 was used for calculation. 2.9. Glutathione peroxidase assay (GSH-Px) Glutathione peroxidase (GSH-Px) activity was measured by using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate (Mohandas et al., 1984). An extinction coefficient of 6.22 × 103 M−1 cm−1 at 340 nm was used for calculation. 2.10. -Glutamyl transpeptidase assay (-GT) ␥-GT activity was determined by the method of Orlowaski and Miester (1973) using glutamyl p-nitroanilide as substrate. 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−1 cm−1 . 2.11. Quinone reductase assay (QR) The activity of quinone reductase was determined according to Benson et al. (1980). 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−1 cm−1 .
2.5. Assessment of antioxidant enzymes 2.12. Reduced glutathione assay (GSH) Lung tissue was homogenized in 10 volume of 100 mmol KH2 PO4 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
Reduced glutathione was estimated by the method of Jollow et al. (1974) by using 1,2-dithio-bis nitro benzoic acid (DTNB) as
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Table 1 Protective effects of S. asper against CCl4 induced changes in GSH, TBARS, H2 O2 , and nitrite. Treatment
GSH (M/g tissue)
Control DMSO + olive oil 3 ml/kg CCl4 50 mg/kg rutin + CCl4 100 mg/kg SAME + CCl4 200 mg/kg SAME + CCl4
0.11 0.10 0.05 0.08 0.09 0.10
± ± ± ± ± ±
TBARS (nM/min/mg protein)
0.009++ 0.005++ 0.003** 0.005**++ 0.004**++ 0.007++
10.40 9.50 18.60 10.80 10.60 9.40
± ± ± ± ± ±
H2 O2 (nM/min/mg tissue)
0.38++ 0.40++ 0.65** 0.50++ 0.50++ 0.49++
20.40 21.30 34.16 22.80 23.64 18.81
± ± ± ± ± ±
0.68++ 0.95++ 0.62** 0.96*++ 0.99**++ 0.94++
Nitrite (M/ml) 49.50 51.40 78.30 55.51 63.70 52.30
± ± ± ± ± ±
1.20++ 0.65+ 0.86** 1.53**++ 1.01**++ 0.81++
Mean ± SE (n = 6 number). *, ** indicate significance from the control group at P < 0.05 and P < 0.01 probability level. +, ++ indicate significance from the CCl4 group at P < 0.05 and P < 0.01 probability level.
substrate. The yellow color developed was read immediately at 412 nm and expressed as mol GSH/g tissue.
2.13. Estimation of lipid peroxidation assay (TBARS) Lung thiobarbituric acid-reactive substances (TBARSs) were measured at 535 nm by using 2-thiobarbituric acid (2,6dihydroxypyrimidine-2-thiol; TBA). An extinction coefficient of 156,000 M−1 cm−1 was used for calculation according to Wright et al. (1981) as modified by Iqbal et al. (1996).
2.17. Histopathological study For microscopic evaluation lungs were fixed in a fixative (absolute ethanol 60%, formaldehyde 30%, and glacial acetic acid 10%) and embedded in paraffin, sectioned at 4 m and subsequently stained with hematoxylin/eosin. Sections were studied under light microscope (DIALUX 20 EB) at 40 and 100 magnifications. Slides of all the treated groups were studied and photographed. A minimum 12 fields of each section of lung tissues were studied and approved by pathologist without saying of its treatment nature. 2.18. Statistical analysis
2.14. Hydrogen peroxide assay (H2 O2 ) Hydrogen peroxide (H2 O2 ) was assayed by using H2 O2 mediated horseradish peroxidase-dependent oxidation of phenol red by the method of Pick and Keisari (1981) based on the standard curve of H2 O2 oxidized phenol red.
2.15. Nitrite assay Supernatant of homogenate was collected after deproteinized with NaOH and ZnSO4 and with centrifugation at 6400 × g for 20 min (Grisham et al., 1996). Griess reagent was used to blank the spectrophotometer at 540 nm and supernatant was added. Nitrite concentration was calculated using a standard curve for sodium nitrite.
2.16. DNA ladder assay DNA was isolated by using the methods of Wu et al. (2005) and Gilbert et al. (2007) to estimate DNA damages. 5 g of DNA of rats was loaded separately in 1.5% agarose gel containing 1.0 g/ml ethidium bromide including DNA standards (0.5 g per well) in Sub-Cell GT agarose gel electrophoresis system (BIO RAD). After electrophoresis (45 min) gel was studied under gel doc system and was photographed through digital camera.
To determine the treatment effects one way analysis of variance was carried by computer software SPSS 13.0. Least significance difference (LSD) at 0.05% and 0.01% level of probability was used to determine the level of significance among the various treatments. 3. Results The toxicity of CCl4 significantly decreased the lung CAT, POD, SOD, GST, GSH-Px, GSR and QR levels while increased the ␥-GT level compared to the control group (Fig. 1). An addition of rutin (50 mg/kg body weight) SA at doses of 100 mg/kg and 200 mg/kg body weight in diet reversed the CAT, POD and SOD level in lung of rats. Higher dose of SA (200 mg/kg body weight) similar to rutin restored almost completely the CAT, POD and SOD activities compared to the control level. The lung level of GST, GSH-Px, GSR, QR and ␥-GT level were partially reversed by SA fractions at 200 mg/kg body weight. Rutin also effectively lessened the influence of CCl4 on these parameters. The lung level of GSH content decreased while the TBARS, H2 O2 and nitrite levels were increased to those of control in the CCl4 -treated animals (Table 1). The induction in these parameters caused by CCl4 was diminished dose-dependently by oral addition of methanolic extract of SA. Rutin was also able to reduce the CCl4 -induced perturbations in the above markers with an effect comparable to high dose of SA fractions. GSH contents were decreased with SAME only while H2 O2 level was decreased with the administration of SAME compared to control rats.
Table 2 Protective effects of S. asper against CCl4 induced changes in GSH, TBARS, H2 O2 , and nitrite. Treatment
DNA fragmentation%
Control DMSO + olive oil 3 ml/kg CCl4 50 mg/kg rutin + CCl4 100 mg/kg SAME + CCl4 200 mg/kg SAME + CCl4
15.93 15.18 59.75 14.57 29.70 16.18
± ± ± ± ± ±
1.38++ 0.93++ 0.80**++ 0.83++ 1.42*++ 0.97++
Mean ± SE (n = 6 number). *, ** indicate significance from the control group at P < 0.05 and P < 0.01 probability level. +, ++ indicate significance from the CCl4 group at P < 0.05 and P < 0.01 probability level.
Absolute lung weight (g) 1.63 1.56 2.16 1.71 1.78 1.66
± ± ± ± ± ±
0.14++ 0.12++ 0.13** 0.16++ 0.09++ 0.13++
Relative lung weight (% to body weight) 0.0163 0.0156 0.0216 0.0171 0.0178 0.0166
± ± ± ± ± ±
0.00148++ 0.00128++ 0.00131** 0.00161++ 0.00095*++ 0.00132++
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Fig. 1. Effect of various treatments on antioxidant enzymes (A) catalase (CAT), (B) peroxidase (POD), (C) superoxide dismutase (SOD), (D) glutathione peroxidase (GSH-Px), (E) quinine reductase (QR), (F) glutathione-S-transferase (GST), (G) glutathione reductase (GSR), (H) gamma glutamyl transferase (␥-GT) in lungs of various groups in rat. I: control; II: vehicle (olive oil + DMSO); III: CCl4 3 ml/kg; IV: CCl4 + rutin 50 mg/kg; V: CCl4 + SAME 100 mg/kg; VI: CCl4 + SAME 200 mg/kg; * (P < 0.05) and ** (P < 0.01) show significance from the control group. a (P < 0.05) and b (P < 0.01) show significance from the CCl4 group. Mean ± SE (n = 06 rats).
Protective effects of S. asper against CCl4 administration in rat on lung weight and relative lung weight and body weight are shown in Table 2. Administration of CCl4 significantly increased (P < 0.01) lung weight, relative lung weight and body weight as compared to control group. Post-treatment of S. asper at lower (100 mg/kg body weight) and higher (200 mg/kg body weight) concentrations ameliorated the CCl4 intoxication and significantly reduced (P < 0.01) the lung weight, relative lung weight, as compared to CCl4 group in a concentration dependent manner. As shown in Fig. 2 CCl4 induced DNA damages. The results demonstrated that CCl4 induced DNA damages in lung tissues. The supplement of SA fractions at high dose in diet reversed the DNA damages comparable to the control levels. These effects were also demonstrated by rutin.
Treatment of CCl4 in the lungs induced the degeneration of the alveolar septa, fibrosis, disruption of the connective tissues and elastic fibers and the congestion of the blood capillaries which were also blocked with large aggregation of the blood cells. The alveolar bronchiole also showed disorganized inner epithelium and Clara cells and blockage of the air breathing passage (Table 3). Administrations of S. asper reduced the toxic effects of CCl4 and reduced injuries were observed in the lung tissues of these groups. The ameliorating effect of the extract was more pronounced at the higher dose. Most areas of the lungs showed the normal alveolar spaces, alveolar and bronchioles with minor cell degeneration, PNI, PNII but a less marked thickening was still observed in the intra-alveolar septum (Fig. 3).
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Fig. 2. Agarose gel showing DNA damage by CCl4 and preventive effect of Sonchus asper extracts in different groups (A) lungs. Lanes (from left) high molecular weight marker (M), control (1–4), CCl4 (5–8), 100 mg/kg (9, 10), 200 mg/kg (11, 12), low molecular weight marker (M).
4. Discussion In this study a novel protocol for the recovery potential of the extracts was used. We are of the opinion that for the assessment of recovery effects of pure chemical or fractions, it is more impor-
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tant to change the response and adaptation i.e., longevity of the cells rather than simply priming the cells against the insult. We also want to relate this study with human as the patients take treatments after the diagnosis of various diseases of liver, lung, cardiovascular and other organs. Based on this assumption, rats were treated with various fractions of SA (100, 200 mg/kg body weight) and rutin (50 mg/kg body weight) after 48 h when CCl4 (3 ml/kg body weight) has induced oxidative stress. This strategy might be more challenging to evaluate recovery effects as compared when toxic chemicals and extracts/chemicals have been administered simultaneously. Results obtained in this experiment revealed that CCl4 treatment significantly decreased the percent increase in body weight while absolute and relative lung weights were increased as compared to the control rats. Low increment in body weights possibly occur due to the degeneration/necrosis of body tissues while the increase in the lung weight might account for the fibrosis and inflammatory response of the organs. Administration of various fractions of SA and rutin could change the response and adaptation of the cells that possibly lead to the incremental increase of the body weight and restored lung weight comparable to control.
Table 3 Effect of S. asper on histopathology of lungs in rat. Treatment
Ab
DCT
DEF
CBC
ABC
DIEAB
PE
PF
Control DMSO + olive oil 3 ml/kg CCl4 50 mg/kg rutin + CCl4 100 mg/kg SAME + CCl4 200 mg/kg SAME + CCl4
− − +++ −/+ − −/+
− − +++ − − −
− − +++ − − −
− − +++ −/+ − −
− − +++ − −− −
− − +++ +/− −/+ −
− − +++ − − −
− − ++ − − −
−, normal; +/−, mild; +++, severe disruption. AB (alveolar breakage), DCT (degeneration of connective tissue), DEF (damages of elastic fiber), CBC (congestion of blood capillaries), ABC (aggregation of blood cells), DIEAB (disorganized inner epithelium of alveolar bronchiole), PE (pulmonary edema), PF (pulmonary fibrosis).
Fig. 3. Hematoxylin and eosin-stained sections (A) control rat lungs showing normal alveolar macrophages (AM), Clara cells (CC), and alveolar septa (AS); (B, C) CCl4 treated rats showing pulmonary alveolar fibrosis (PF), blood capillary congestion (BCC), inflammatory cells infiltration (ICI), breakages and disorganization of alveolar septa (AS) and Clara cells disorganization (CC); (D) CCl4 + 200 mg/kg SAME, showing nearly normal alveolar septa (AS), less inflammatory cells infiltration (ICI) and normal alveolar macrophages (AM) showing almost normal histology.
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Large body of evidence indicates that reactive oxygen species are important mediators of CCl4 -induced lung injury (Zakaria et al., 2004). In agreement with previous observations (Zakaria et al., 2004), CCl4 significantly decreased the activities of CAT, POD and SOD in lungs. CCl4 -induced oxidative stress decreases the activity of phase II metabolizing enzymes by depletion of GSH contents (Singh et al., 2008). Low level of GST, GSR, GSH-Px and QR indicates the severe oxidative stress and depletion of GSH in lungs with CCl4 treatment in rats. Normally, GSH is oxidized to form GSSG, which is then reduced to GSH by the NADPH-dependent glutathione reductase. In addition, glutathione peroxidase catalyzes the GSH-dependent reduction of H2 O2 and other peroxides and protects the organism from oxidative damage (Fang et al., 2002). ␥-GT enzyme has a role in the transfer of amino acids across the membranes and also in the metabolism of glutathione. Comparable results for the activity of ␥-GT with CCl4 treatment are also reported in other studies (Bhadauria et al., 2008). In addition, severity of the damage is increased under low oxygen tension in various tissues with the elevation of peroxynitrite contents (Khan and Ahmed, 2009; Khan et al., 2009). TBARS is a major reactive aldehyde resulting during the peroxidation of polyunsaturated fatty acids. It is a useful indicator showing tissue damages including a series of chain reactions (Ohkawa et al., 1979). High levels of TBARS and nitrite in lung with CCl4 treatment indicate the extensive oxidative damage in lung tissues. SA provides a dietary source of biologically active compounds that might help to prevent a wide variety of diseases (Afolayan and Jimoh, 2008). SA extract contains polyphenols, saponins and other bioactive compounds which possess antioxidant and antiinflammatory effects that can reduce the risk of various oxidative diseases. Low lipid peroxidation and increased activities of various antioxidant enzymes indicate that SA and rutin are able to protect against various pathological conditions including oxidative stress (Vardavas et al., 2006). Chemical compounds such as flavonoids and tannins have been reported to exert antioxidant activity by scavenging free radicals that cause lipid peroxidation (Alpinar et al., 2009). It has been investigated that lipid metabolites produced as a result of oxidative stress induce DNA damages (Marnett, 2000). TBARS react with DNA to form the adduct M1G; the mutagenic pirimedopurinone adduct of deoxyguanosine. In addition to free radical attack of lipids, DNA is also continuously subjected to oxidative damage. Administration of SA and rutin significantly reduced the DNA fragmentation% which was also revealed by DNA ladder assay. Histological study of the lungs showed the marked endemic variations among the treatments. In carbon tetrachloride treated group, the lung tissues were extensively damaged having destructed alveolar septa and congested blood capillaries with several inclusions in their lumen. Due to the constriction of blood capillaries, blood cells also gathered at various places in the lung tissues producing the endemic conditions. Most of the alveolar septa became hard with increased number of type II alveolar cells. Fibroblasts also increased in number and are responsible for the accumulation of collagen fibers at the junction of various alveolar walls. Alveolar macrophages present at various places in alveolar septa, were also severely or moderately damaged. Similar results have been reported in lung tissues by administration of CCl4 in rats (Zakaria et al., 2004). Administration of SA and rutin reduced the toxic effects of CCl4 and lesser injuries observed in the lung tissues of these groups. The ameliorating effects of S. asper are more pronounced at the higher dose of SA fractions. Most areas of lung at higher doses of SA fractions and rutin showed the normal alveolar spaces, alveolar and bronchioles with minor cell degeneration, normalized pneumocytes type I (PN I), pneumocytes type II (PN II) cells but a less marked thickening was still observed in the inter
alveolar septum. This further proves the ability of the extracts to prevent lung damage and preserve lung normal histopathology. 5. Conclusion Our results suggest that SA comprised of active constituents; showing protective effects against the toxic affects of CCl4 in lung of rat. Rutin and SA are able to modulate the response of cells; helping in adaptation thereby maintaining the functional integrity of tissues and organs. Further studies are required to isolate, characterize and evaluate the antioxidant components and study other pharmacological properties of the constituents. References Afolayan AJ, Jimoh FO. Nutritional quality of some wild leafy vegetables in South Africa. Int J Food Sci Nutr 2008;26:1–8. Alpinar K, Ozyurek M, Kolak U, Guclu K, Aras C, Altun M, Celik SE, Berker KI, Bektasoglu B, Apak R. Antioxidant capacities of some food plants wildly grown in Ayvalik of Turkey. Food Sci Technol Res 2009;15:59–64. Benson AM, Hunkeler MJ, Talalay P. Increase of NADPH, quinone reductase activity by dietary antioxidant: possible role in protection against carcinogenesis and toxicity. Proc Natl Acad Sci USA 1980;77:5216–20. Berker B, Kaya C, Aytac R, Satıroglu H. Homocysteine concentrations in follicular fluids are associated with poor oocyte and embryo qualities in polycystic ovary syndrome patients undergoing assisted reproduction. Hum Reprod 2009;24:2293–302. Bhadauria M, Nirala KS, Shukla S. Multiple treatment of Propolis ameliorates carbon tetrachloride induced liver injuries in rats. Food Chem Toxicol 2008;46: 2703–12. Cambie RC, Ferguson LR. Potential functional foods in the traditional Maori diet. Mutat Res 2003;523–524:109–17. Carlberg I, Mannervik EB. Glutathione level in rat brain. J Biol Chem 1975;250:4475–80. Chance B, Maehly AC. Assay of catalase and peroxidases. Methods Enzymol 1955;11:764–75. Fang YZ, Yang S, Wu G. Free radicals, antioxidants, and nutrition. Nutrition 2002;18:872–9. Gilbert MT, Haselkorn T, Bunce M, Sanchez JJ, Lucas SB, Jewell LD, Van Marck E, Worobey M. The isolation of nucleic acids from fixed, paraffin-embedded tissues, which methods are useful when? PloS ONE 2007;2(June (6)):e537. Grisham MB, Johnson GG, Lancaster JR. Quantitation of nitrate and nitrite in extracellular fluids. Methods Enzymol 1996;268:237–46. Guarrera PM, Salerno G, Caneva GL. Food, flavouring and feed plant traditions in the Tyrrhenian sector of Basilicata, Italy. J Ethnobiol Ethnomed 2006; 2:37. Guil-Guerrero JS, Gimenez-Gimenez A, Rodrguez-Garc I, Torija-Isasa ME. Nutritional composition of Sonchus species (S. asper L., S. oleraceus L., and S. tenerrimus L.). J Sci Food Agric 1998;76:628–32. Habig WH, Pabst MJ, Jakoby WB. Glutathione-S-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130–9. Halliwell B, Whiteman M. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Brit J Pharmacol 2004;142:231–55. Helal AM, Nakamura N, El-Askary H, Hattori M. Sesquiterpene lactone glucosides from Sonchus asper. Phytochemistry 2000;53:473–7. Hussain K, Shahazad A, Zia-ul-Hussnain U. An ethnobotanical survey of important wild medicinal plants of Hattar, District Haripur, Pakistan. Ethnobotanical Leaflets 2008;12:29–35. Iqbal M, Sharma SD, Zadeh HR, Hasan N, Abdulla M, Athar M. Glutathione metabolizing enzymes and oxidative stress in ferric nitrilotriacetate (Fe-NTA) mediated hepatic injury. Redox Rep 1996;2:385–91. Jollow DJ, Mitchell JR, Zampaglione N, Gillete JR. Bromobenzene induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as a hepatotoxic metabolite. Pharmacology 1974;11:151–69. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dimutase. Ind J Biochem Biol 1984;21:130–2. Khan MR, Ahmed D. Protective effects of Digera muricata (L.) Mart. on testis against oxidative stress of carbon tetrachloride in rat. Food Chem Toxicol 2009;47:1393–9. Khan MR, Rizvi W, Khan GN, Khan RA, Shaheen S. Carbon tetrachloride induced nephrotoxicity in rat: protective role of Digera muricata. J Ethnopharmacol 2009;122:91–9. Koche DK, Shirsat RP, Imran S, Nafees M, Zingare AK, Donode KA. Ethnomedicinal survey of Nigeria wild life sanctuary, District Gondia (M.S.). India-Part II. Ethnobotanical Leaflets 2008;12:532–7. Lowry OH, Rosenberg NJ, Farr AL, Randall RJ. Protein measurement with Folin Phenol reagent. J Biol Chem 1951;193:265–75. Marnett JL. Oxyradicals and DNA damage. Carcinogenesis 2000;21:361–70. Mohandas J, Marshal JJ, Duggin GG, Horvath JS, Tiller DJ. Differential distribution of glutathione and glutathione-related enzymes in rabbit kidney.
R.A. Khan et al. / Experimental and Toxicologic Pathology 64 (2012) 725–731 Possible implications in analgesic nephropathy. Biochem Pharmacol 1984;33: 1801–7. Ohkawa H, Ohishi N, Yogi K. Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. Orlowaski M, Miester A. ␥-Glutamyl cyclotransferase distribution, isozymic forms, and specificity. J Biol Chem 1973;248:2836–44. Pick A, Keisari Y. Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages—induction by multiple nonphagocytotic stimuli. Cell Immunol 1981;59:301–18. Rehman EU. Indigenous knowledge on medicinal plants, village Barali Kass and its allied areas, District Kotli Azad Jammu & Kashmir, Pakistan. Ethnobotanical Leaflets 2006;10:254–64. Shimizu S, Miyase T, Ueno A, Usmanghani K. Sesquiterpene lactone glycosides and ionone derivative glycosides from Sonchus asper. Phytochemistry 1989;28(12):3399–402.
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Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Protective effect of Sonchus asper extracts against experimentally induced lung injuries in rats: A novel study Rahmat Ali Khan a,b,∗ , Muhammad Rashid Khan b , Sumaira Sahreen b a b
Department of Biotechnology, University of Science and Technology, Bannu, Khyber Pukhtunkhawa, Pakistan Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 4400, Pakistan
a r t i c l e
i n f o
Article history: Received 11 October 2010 Accepted 13 January 2011 Keywords: Carbon tetrachloride Sonchus asper Lung histopathology Antioxidant Lipid peroxidation
a b s t r a c t In this study, protective effects of methanol extract (SAME) were evaluated against carbon tetrachloride induced oxidative stress in lungs. Male Sprague–Dawley rats were orally fed with various doses (100, 200 mg/kg body weight) of SAME and (50 mg/kg body weight) of rutin after 48 h of CCl4 treatment (3 ml/kg body weight, 30% in olive oil) biweekly for 4 weeks. The results showed that administration of extracts and rutin significantly restored lung contents of reduced glutathione and activities of catalase, peroxidase, superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase, quinine reductase were reduced while lipid peroxide, hydrogen peroxide, nitrite, DNA fragmentation% and activity of ␥-glutamyl transferase, increased by CCl4 , were reversed towards the control levels by the supplement of Sonchus asper extracts and rutin. Lung histopathology showed that S. asper extracts and rutin reduced the incidence of lung lesions induced by CCl4 in rats. These results suggest that S. asper fractions and rutin could protect lung against the CCl4 -induced oxidative damage in rats. © 2011 Elsevier GmbH. All rights reserved.
1. Introduction Oxidative stress describes the steady state level of oxidative damage in a cell, tissue, or organ, caused by the reactive oxygen species (ROS). Various environmental factors such as exposure to UV radiation, chemical intoxication and normal cellular activities cause production of ROS such as superoxide radical, hydrogen peroxide and hydroxyl radical. Oxidative stress is caused by an imbalance between the production of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage. Our body possesses antioxidant enzyme systems such as catalase, superoxide dismutases, peroxidases and glutathione enzyme system as well as non enzymatic antioxidant compounds like reduced glutathione, ascorbic acid and ␣-tocopherol. Disturbances in this normal redox state can cause toxic effects through the production of cell damaging peroxides and free radicals (Halliwell and Whiteman, 2004). It has been reported that good health can be maintained from the consumption of plants with high antioxidant activities (Guarrera et al., 2006). There is some evidence that bioactive compounds
∗ Corresponding author at: Department of Biotechnology, University of Science and Technology, Bannu, Khyber Pukhtunkhawa, Pakistan. Tel.: +0092928633421; fax: +0092928624987. E-mail address: Rahmatgul [email protected] (R.A. Khan). 0940-2993/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2011.01.007
and microelements from different functional foods, herbs and nutraceuticals can ameliorate or even prevent diseases (Cambie and Ferguson, 2003; Berker et al., 2009). Sonchus (Sow Thistle) constitutes food in South Africa (Afolayan and Jimoh, 2008) and Turkey (Alpinar et al., 2009) and medicinally important plant species Sonchus oleraceus, Sonchus arvensis and Sonchus asper; having beneficial bioactive compounds (Cambie and Ferguson, 2003; Alpinar et al., 2009). The food use of S. oleraceus and S. asper is justified by the high content of vitamin C, carotenoids and phenolic constituents (Afolayan and Jimoh, 2008). Extracts of these plant species also possess high antioxidant activities (Guarrera et al., 2006). Extract of S. arvensis is an integral component of an antioxidant formulation “Prolipid® ” because of high polyphenolic contents. S. asper (L.) Hill. (Spiny-leaved Sow Thistle) locally named as “Mahtari” (SA) is a common herb that grows wildly and abundantly in open fields. It is used in the treatment of cough, bronchitis, asthma (Koche et al., 2008) and in wound healing (Rehman, 2006; Hussain et al., 2008). Chemical characterization of SA has shown the presence of ionone derivatives of glycosides (Shimizu et al., 1989), flavonoids, ascorbic acid, carotenoids (Guil-Guerrero et al., 1998) and sesquiterpene lactone glycosides (Shimizu et al., 1989; Helal et al., 2000). These bioactive compounds have been shown to possess strong antioxidant and anti-inflammatory properties (Alpinar et al., 2009). Based on the traditional claims surrounding S. asper and the lack of sci-
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entific studies of its potential pharmacological properties, the objective of this study is to evaluate the protective effects of Sonchus asper (SA) against CCl4 induced oxidative stress in lungs of rats. 2. Materials and methods 2.1. Chemicals Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), CCl4 , flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) of Sigma Chemicals Co., USA were utilized in this experiment. 2.2. Plant collection Plants of S. asper (L.) Hill. at maturity were collected, identified and a specimen was submitted vide voucher # R-147 at Herbarium of Pakistan, Quaid-i-Azam University, Islamabad, Pakistan for future reference. Aerial parts of plant (leaves, stem, flowers and seeds) were shade dried, chopped and grinded of 1 mm mesh size. 2.3. Preparation of plant extract 1.5 kg powder of S. asper (SA) was extracted with 2.0 liter of absolute methanol with refluxing for 5 h, filtered and evaporated under reduced pressure to obtain crude extract of methanol (SAME), dried under reduced pressure and stored at 4 ◦ C for further use. 2.4. Animals and treatment The study protocol was approved by Ethical committee of Quaidi-Azam University, Islamabad for laboratory animal feed and care. The present study was conducted (with 96 mature male albino rats) in the laboratory of the Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan. The animals weighing 190–200 g, were divided equally into 6 groups and maintained under standard laboratory conditions (12 h light/darkness; at 25 ± 3 ◦ C) with standard animal diet and water available ad libitum. Group I remained untreated (control) while group II received olive oil intraperitoneally (Monday and Thursday) and DMSO orally (Wednesday and Saturday) at a dose of 3 ml/kg body weight. Group III was treated with CCl4 , 3 ml/kg body weight (30% in olive oil i.p. Monday and Thursday). However, group IV received rutin 50 mg/kg body weight (in DMSO) after 48 h (Wednesday and Saturday) of CCl4 treatment. Groups V and VI were administered S. asper methanolic extract at doses of 100 and 200 mg/kg body weight after 48 h (Wednesday and Saturday) of CCl4 treatment. After 24 h of the last treatment, all the animals were weighted, sacrificed; their lungs were removed, weighted and perfused in ice-cold saline solution. Half of lung tissue was treated with liquid nitrogen for further enzymatic and DNA damage analysis while the other portion was processed for histology.
used for the following experiments as described below. Protein concentration of the supernatant of lung tissue was determined by the method of Lowry et al. (1951) using crystalline BSA as standard. Catalase (CAT) and peroxidase (POD) activities were determined by the method of Chance and Maehly (1955) with some modifications. Briefly CAT activity was determined by adding H2 O2 and change in absorbance was recorded at 240 nm while POD activity was measured by using guaiacol as substrate at 470 nm. One unit of CAT and POD activity was defined as an absorbance change of 0.01 as units/min. Superoxide dismutase (SOD) activity was estimated by using phenazine methosulphate and sodium pyrophosphate buffer according to Kakkar et al. (1984). Enzyme reaction was initiated by adding NADH (780 mol) and stopped after 1 min by adding glacial acetic acid and color intensity at 560 nm was recorded. Results are expressed in units/mg tissue protein. 2.6. Assessment of lipid peroxidation enzymes Induction of lipid peroxidation by CCl4 and its protection by the S. asper extract were determined by the estimation of various enzyme activities and thiobarbituric acid reactive substance (TBARS) contents. 2.7. Glutathione-S-transferase assay (GST) Lung glutathione-S-transferase activity was determined according to Habig et al. (1974), using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. GST was measured at 340 nm using a molar extinction coefficient of 9.6 × 103 M−1 cm−1 . 2.8. Glutathione reductase assay (GSR) Glutathione reductase activity (Carlberg and Mannervik, 1975), was measured at 340 nm by using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate. An extinction coefficient of 6.22 × 103 M−1 cm−1 was used for calculation. 2.9. Glutathione peroxidase assay (GSH-Px) Glutathione peroxidase (GSH-Px) activity was measured by using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate (Mohandas et al., 1984). An extinction coefficient of 6.22 × 103 M−1 cm−1 at 340 nm was used for calculation. 2.10. -Glutamyl transpeptidase assay (-GT) ␥-GT activity was determined by the method of Orlowaski and Miester (1973) using glutamyl p-nitroanilide as substrate. 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−1 cm−1 . 2.11. Quinone reductase assay (QR) The activity of quinone reductase was determined according to Benson et al. (1980). 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−1 cm−1 .
2.5. Assessment of antioxidant enzymes 2.12. Reduced glutathione assay (GSH) Lung tissue was homogenized in 10 volume of 100 mmol KH2 PO4 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
Reduced glutathione was estimated by the method of Jollow et al. (1974) by using 1,2-dithio-bis nitro benzoic acid (DTNB) as
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Table 1 Protective effects of S. asper against CCl4 induced changes in GSH, TBARS, H2 O2 , and nitrite. Treatment
GSH (M/g tissue)
Control DMSO + olive oil 3 ml/kg CCl4 50 mg/kg rutin + CCl4 100 mg/kg SAME + CCl4 200 mg/kg SAME + CCl4
0.11 0.10 0.05 0.08 0.09 0.10
± ± ± ± ± ±
TBARS (nM/min/mg protein)
0.009++ 0.005++ 0.003** 0.005**++ 0.004**++ 0.007++
10.40 9.50 18.60 10.80 10.60 9.40
± ± ± ± ± ±
H2 O2 (nM/min/mg tissue)
0.38++ 0.40++ 0.65** 0.50++ 0.50++ 0.49++
20.40 21.30 34.16 22.80 23.64 18.81
± ± ± ± ± ±
0.68++ 0.95++ 0.62** 0.96*++ 0.99**++ 0.94++
Nitrite (M/ml) 49.50 51.40 78.30 55.51 63.70 52.30
± ± ± ± ± ±
1.20++ 0.65+ 0.86** 1.53**++ 1.01**++ 0.81++
Mean ± SE (n = 6 number). *, ** indicate significance from the control group at P < 0.05 and P < 0.01 probability level. +, ++ indicate significance from the CCl4 group at P < 0.05 and P < 0.01 probability level.
substrate. The yellow color developed was read immediately at 412 nm and expressed as mol GSH/g tissue.
2.13. Estimation of lipid peroxidation assay (TBARS) Lung thiobarbituric acid-reactive substances (TBARSs) were measured at 535 nm by using 2-thiobarbituric acid (2,6dihydroxypyrimidine-2-thiol; TBA). An extinction coefficient of 156,000 M−1 cm−1 was used for calculation according to Wright et al. (1981) as modified by Iqbal et al. (1996).
2.17. Histopathological study For microscopic evaluation lungs were fixed in a fixative (absolute ethanol 60%, formaldehyde 30%, and glacial acetic acid 10%) and embedded in paraffin, sectioned at 4 m and subsequently stained with hematoxylin/eosin. Sections were studied under light microscope (DIALUX 20 EB) at 40 and 100 magnifications. Slides of all the treated groups were studied and photographed. A minimum 12 fields of each section of lung tissues were studied and approved by pathologist without saying of its treatment nature. 2.18. Statistical analysis
2.14. Hydrogen peroxide assay (H2 O2 ) Hydrogen peroxide (H2 O2 ) was assayed by using H2 O2 mediated horseradish peroxidase-dependent oxidation of phenol red by the method of Pick and Keisari (1981) based on the standard curve of H2 O2 oxidized phenol red.
2.15. Nitrite assay Supernatant of homogenate was collected after deproteinized with NaOH and ZnSO4 and with centrifugation at 6400 × g for 20 min (Grisham et al., 1996). Griess reagent was used to blank the spectrophotometer at 540 nm and supernatant was added. Nitrite concentration was calculated using a standard curve for sodium nitrite.
2.16. DNA ladder assay DNA was isolated by using the methods of Wu et al. (2005) and Gilbert et al. (2007) to estimate DNA damages. 5 g of DNA of rats was loaded separately in 1.5% agarose gel containing 1.0 g/ml ethidium bromide including DNA standards (0.5 g per well) in Sub-Cell GT agarose gel electrophoresis system (BIO RAD). After electrophoresis (45 min) gel was studied under gel doc system and was photographed through digital camera.
To determine the treatment effects one way analysis of variance was carried by computer software SPSS 13.0. Least significance difference (LSD) at 0.05% and 0.01% level of probability was used to determine the level of significance among the various treatments. 3. Results The toxicity of CCl4 significantly decreased the lung CAT, POD, SOD, GST, GSH-Px, GSR and QR levels while increased the ␥-GT level compared to the control group (Fig. 1). An addition of rutin (50 mg/kg body weight) SA at doses of 100 mg/kg and 200 mg/kg body weight in diet reversed the CAT, POD and SOD level in lung of rats. Higher dose of SA (200 mg/kg body weight) similar to rutin restored almost completely the CAT, POD and SOD activities compared to the control level. The lung level of GST, GSH-Px, GSR, QR and ␥-GT level were partially reversed by SA fractions at 200 mg/kg body weight. Rutin also effectively lessened the influence of CCl4 on these parameters. The lung level of GSH content decreased while the TBARS, H2 O2 and nitrite levels were increased to those of control in the CCl4 -treated animals (Table 1). The induction in these parameters caused by CCl4 was diminished dose-dependently by oral addition of methanolic extract of SA. Rutin was also able to reduce the CCl4 -induced perturbations in the above markers with an effect comparable to high dose of SA fractions. GSH contents were decreased with SAME only while H2 O2 level was decreased with the administration of SAME compared to control rats.
Table 2 Protective effects of S. asper against CCl4 induced changes in GSH, TBARS, H2 O2 , and nitrite. Treatment
DNA fragmentation%
Control DMSO + olive oil 3 ml/kg CCl4 50 mg/kg rutin + CCl4 100 mg/kg SAME + CCl4 200 mg/kg SAME + CCl4
15.93 15.18 59.75 14.57 29.70 16.18
± ± ± ± ± ±
1.38++ 0.93++ 0.80**++ 0.83++ 1.42*++ 0.97++
Mean ± SE (n = 6 number). *, ** indicate significance from the control group at P < 0.05 and P < 0.01 probability level. +, ++ indicate significance from the CCl4 group at P < 0.05 and P < 0.01 probability level.
Absolute lung weight (g) 1.63 1.56 2.16 1.71 1.78 1.66
± ± ± ± ± ±
0.14++ 0.12++ 0.13** 0.16++ 0.09++ 0.13++
Relative lung weight (% to body weight) 0.0163 0.0156 0.0216 0.0171 0.0178 0.0166
± ± ± ± ± ±
0.00148++ 0.00128++ 0.00131** 0.00161++ 0.00095*++ 0.00132++
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Fig. 1. Effect of various treatments on antioxidant enzymes (A) catalase (CAT), (B) peroxidase (POD), (C) superoxide dismutase (SOD), (D) glutathione peroxidase (GSH-Px), (E) quinine reductase (QR), (F) glutathione-S-transferase (GST), (G) glutathione reductase (GSR), (H) gamma glutamyl transferase (␥-GT) in lungs of various groups in rat. I: control; II: vehicle (olive oil + DMSO); III: CCl4 3 ml/kg; IV: CCl4 + rutin 50 mg/kg; V: CCl4 + SAME 100 mg/kg; VI: CCl4 + SAME 200 mg/kg; * (P < 0.05) and ** (P < 0.01) show significance from the control group. a (P < 0.05) and b (P < 0.01) show significance from the CCl4 group. Mean ± SE (n = 06 rats).
Protective effects of S. asper against CCl4 administration in rat on lung weight and relative lung weight and body weight are shown in Table 2. Administration of CCl4 significantly increased (P < 0.01) lung weight, relative lung weight and body weight as compared to control group. Post-treatment of S. asper at lower (100 mg/kg body weight) and higher (200 mg/kg body weight) concentrations ameliorated the CCl4 intoxication and significantly reduced (P < 0.01) the lung weight, relative lung weight, as compared to CCl4 group in a concentration dependent manner. As shown in Fig. 2 CCl4 induced DNA damages. The results demonstrated that CCl4 induced DNA damages in lung tissues. The supplement of SA fractions at high dose in diet reversed the DNA damages comparable to the control levels. These effects were also demonstrated by rutin.
Treatment of CCl4 in the lungs induced the degeneration of the alveolar septa, fibrosis, disruption of the connective tissues and elastic fibers and the congestion of the blood capillaries which were also blocked with large aggregation of the blood cells. The alveolar bronchiole also showed disorganized inner epithelium and Clara cells and blockage of the air breathing passage (Table 3). Administrations of S. asper reduced the toxic effects of CCl4 and reduced injuries were observed in the lung tissues of these groups. The ameliorating effect of the extract was more pronounced at the higher dose. Most areas of the lungs showed the normal alveolar spaces, alveolar and bronchioles with minor cell degeneration, PNI, PNII but a less marked thickening was still observed in the intra-alveolar septum (Fig. 3).
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Fig. 2. Agarose gel showing DNA damage by CCl4 and preventive effect of Sonchus asper extracts in different groups (A) lungs. Lanes (from left) high molecular weight marker (M), control (1–4), CCl4 (5–8), 100 mg/kg (9, 10), 200 mg/kg (11, 12), low molecular weight marker (M).
4. Discussion In this study a novel protocol for the recovery potential of the extracts was used. We are of the opinion that for the assessment of recovery effects of pure chemical or fractions, it is more impor-
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tant to change the response and adaptation i.e., longevity of the cells rather than simply priming the cells against the insult. We also want to relate this study with human as the patients take treatments after the diagnosis of various diseases of liver, lung, cardiovascular and other organs. Based on this assumption, rats were treated with various fractions of SA (100, 200 mg/kg body weight) and rutin (50 mg/kg body weight) after 48 h when CCl4 (3 ml/kg body weight) has induced oxidative stress. This strategy might be more challenging to evaluate recovery effects as compared when toxic chemicals and extracts/chemicals have been administered simultaneously. Results obtained in this experiment revealed that CCl4 treatment significantly decreased the percent increase in body weight while absolute and relative lung weights were increased as compared to the control rats. Low increment in body weights possibly occur due to the degeneration/necrosis of body tissues while the increase in the lung weight might account for the fibrosis and inflammatory response of the organs. Administration of various fractions of SA and rutin could change the response and adaptation of the cells that possibly lead to the incremental increase of the body weight and restored lung weight comparable to control.
Table 3 Effect of S. asper on histopathology of lungs in rat. Treatment
Ab
DCT
DEF
CBC
ABC
DIEAB
PE
PF
Control DMSO + olive oil 3 ml/kg CCl4 50 mg/kg rutin + CCl4 100 mg/kg SAME + CCl4 200 mg/kg SAME + CCl4
− − +++ −/+ − −/+
− − +++ − − −
− − +++ − − −
− − +++ −/+ − −
− − +++ − −− −
− − +++ +/− −/+ −
− − +++ − − −
− − ++ − − −
−, normal; +/−, mild; +++, severe disruption. AB (alveolar breakage), DCT (degeneration of connective tissue), DEF (damages of elastic fiber), CBC (congestion of blood capillaries), ABC (aggregation of blood cells), DIEAB (disorganized inner epithelium of alveolar bronchiole), PE (pulmonary edema), PF (pulmonary fibrosis).
Fig. 3. Hematoxylin and eosin-stained sections (A) control rat lungs showing normal alveolar macrophages (AM), Clara cells (CC), and alveolar septa (AS); (B, C) CCl4 treated rats showing pulmonary alveolar fibrosis (PF), blood capillary congestion (BCC), inflammatory cells infiltration (ICI), breakages and disorganization of alveolar septa (AS) and Clara cells disorganization (CC); (D) CCl4 + 200 mg/kg SAME, showing nearly normal alveolar septa (AS), less inflammatory cells infiltration (ICI) and normal alveolar macrophages (AM) showing almost normal histology.
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Large body of evidence indicates that reactive oxygen species are important mediators of CCl4 -induced lung injury (Zakaria et al., 2004). In agreement with previous observations (Zakaria et al., 2004), CCl4 significantly decreased the activities of CAT, POD and SOD in lungs. CCl4 -induced oxidative stress decreases the activity of phase II metabolizing enzymes by depletion of GSH contents (Singh et al., 2008). Low level of GST, GSR, GSH-Px and QR indicates the severe oxidative stress and depletion of GSH in lungs with CCl4 treatment in rats. Normally, GSH is oxidized to form GSSG, which is then reduced to GSH by the NADPH-dependent glutathione reductase. In addition, glutathione peroxidase catalyzes the GSH-dependent reduction of H2 O2 and other peroxides and protects the organism from oxidative damage (Fang et al., 2002). ␥-GT enzyme has a role in the transfer of amino acids across the membranes and also in the metabolism of glutathione. Comparable results for the activity of ␥-GT with CCl4 treatment are also reported in other studies (Bhadauria et al., 2008). In addition, severity of the damage is increased under low oxygen tension in various tissues with the elevation of peroxynitrite contents (Khan and Ahmed, 2009; Khan et al., 2009). TBARS is a major reactive aldehyde resulting during the peroxidation of polyunsaturated fatty acids. It is a useful indicator showing tissue damages including a series of chain reactions (Ohkawa et al., 1979). High levels of TBARS and nitrite in lung with CCl4 treatment indicate the extensive oxidative damage in lung tissues. SA provides a dietary source of biologically active compounds that might help to prevent a wide variety of diseases (Afolayan and Jimoh, 2008). SA extract contains polyphenols, saponins and other bioactive compounds which possess antioxidant and antiinflammatory effects that can reduce the risk of various oxidative diseases. Low lipid peroxidation and increased activities of various antioxidant enzymes indicate that SA and rutin are able to protect against various pathological conditions including oxidative stress (Vardavas et al., 2006). Chemical compounds such as flavonoids and tannins have been reported to exert antioxidant activity by scavenging free radicals that cause lipid peroxidation (Alpinar et al., 2009). It has been investigated that lipid metabolites produced as a result of oxidative stress induce DNA damages (Marnett, 2000). TBARS react with DNA to form the adduct M1G; the mutagenic pirimedopurinone adduct of deoxyguanosine. In addition to free radical attack of lipids, DNA is also continuously subjected to oxidative damage. Administration of SA and rutin significantly reduced the DNA fragmentation% which was also revealed by DNA ladder assay. Histological study of the lungs showed the marked endemic variations among the treatments. In carbon tetrachloride treated group, the lung tissues were extensively damaged having destructed alveolar septa and congested blood capillaries with several inclusions in their lumen. Due to the constriction of blood capillaries, blood cells also gathered at various places in the lung tissues producing the endemic conditions. Most of the alveolar septa became hard with increased number of type II alveolar cells. Fibroblasts also increased in number and are responsible for the accumulation of collagen fibers at the junction of various alveolar walls. Alveolar macrophages present at various places in alveolar septa, were also severely or moderately damaged. Similar results have been reported in lung tissues by administration of CCl4 in rats (Zakaria et al., 2004). Administration of SA and rutin reduced the toxic effects of CCl4 and lesser injuries observed in the lung tissues of these groups. The ameliorating effects of S. asper are more pronounced at the higher dose of SA fractions. Most areas of lung at higher doses of SA fractions and rutin showed the normal alveolar spaces, alveolar and bronchioles with minor cell degeneration, normalized pneumocytes type I (PN I), pneumocytes type II (PN II) cells but a less marked thickening was still observed in the inter
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