Protective effects of Trifolium alexandrinum L. against lung injury induced by environmental toxin CCl4 in experimental rats


Protective effects of Trifolium alexandrinum L. against lung injury induced by environmental toxin CCl4 in experimental rats

Rahmat Ali Khan1*, Huda Mohammad Alkreathy2, Abdus Saboorshah1, Mushtaq Ahmed1 and Samiullah Khan3

1Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology Bannu, Khyber Pakhtunkhwa, Pakistan; 2Department of Pharmacology, King Abdulaziz University(KAU), Jeddah, Saudi Arabia; 3Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan


Background: In Pakistan numerous medicinal floras has used in the treatment of various human ailments. Among them Trifolium alexandrinum L. is traditionally used in the curing of disease. Presently we designed to ascertain the protective role of Trifolium alexandrinum methanolic extracts (TAME) against carbon tetrachloride (CCl4)-induced lung injury and oxidative stress in rats.

Methods: Exposure to CCl4 induces oxidative stress and causes tissue damage by the induction of CCl4 free radicals. Twenty-four male albino rats were divided equally into four groups. Rats in group I had free access to drinking water and laboratory food. Group II was treated with 1 ml/kg body weight (b.w.) CCl4 (30% in olive oil). Groups III and IV rats were fed (p.o.) 200 mg/kg b.w. TAME and 50 mg/kg b.w. silymarin after 24 h of CCl4 treatment for 2 weeks.

Results: Administration of CCl4 caused a significant (p<0.01) decrease in the activities of antioxidant enzymes (catalase, peroxidase, glutathione peroxidase, glutathione-S-transferase), and glutathione contents were decreased; however, thiobarbituric acid-reactive substances were increased (p<0.01). The alterations caused by CCl4 were significantly (p<0.01) reversed toward control levels by supplementation of TAME and silymarin.

Conclusion: These results suggest that in rats TAME and silymarin could protect the lungs against CCl4-induced oxidative damage.

Keywords: lungs; CCl4; lipid peroxidation; Trifolium alexandrinum

Citation: Food & Nutrition Research 2016, 60: 30433 -

Copyright: © 2016 Rahmat Ali Khan et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License, allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.

Received: 17 November 2015; Revised: 28 April 2016; Accepted: 5 May 2016; Published: 7 November 2016

Competing interests and funding: The authors have not received any funding or benefits from industry or elsewhere to conduct this study.

*Correspondence to: Rahmat Ali Khan, Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology Bannu, Khyber Pakhtunkhwa, Pakistan, Email:


The epithelium cells of the lungs are frequently exposed to the highest concentration of oxygen and gaseous exchanges that make them susceptible to pulmonary toxicity (1). Pulmonary toxicity is caused by the generation of ROS (highly reactive oxygen species) that alter the structure of protein, nucleic acid, and other macro molecules (2). During aerobic metabolism, in normal situations, the toxic metabolites of oxygen are formed in low quantity in lung cells (3, 4). This normal production of ROS performs an essential role in a number of physiological activities but, in excess, the generation of ROS embraces several ailments (5). Pulmonary diseases like asthma, hyperoxia, and sarcoidosis are believed to be linked with oxidative stress. Chronic pulmonary disease (CPD) is the foremost cause of death, both in the industrialized and developing countries. It is associated with a progressive decrease in lung function that ultimately results in emphysema and chronic bronchitis (6). Different environmental factors like sunlight, UV light exposure, smoking, and chemicals are the main causes of the generation of ROS (7). Carbon tetrachloride (CCl4) is a synthetic industrial chemical that induces hepatic and renal toxicity in workers as well as in experimental animals (8) via ingestion, skin absorption or intraperitoneal intoxication, or by the inhalation of CCL4 lipid peroxidation, which increases while the antioxidant defense enzymes and total tissue proteins decreases (9). CCl4 is metabolized into trichloromethyl radical (CCl3) that combines with oxygen and forms a more reactive peroxy trichloromethyl radical (OOCCl3). These free radicals produce alkoxy (R•) and peroxy radicals (ROO•) that form lipid peroxides, and cause enzymatic inactivity and tissue injury (10). CCl4 also decreases tissue proteins and the antioxidant enzymes like SOD, peroxidase (POD), and catalase (CAT) (11, 12). Oxidative stress initiates and continues a range of lung, liver, and kidney damages. Therefore, it is of the utmost importance to evaluate agents with powerful antioxidant potential against the attack of CCl4 (13). The majority of foodstuff and natural products have been reported to be excellent means of chemo-protection. Curcuma longa has been reported to have therapeutic activity due to its antioxidant potential (14, 15). Similarly, the methanolic extract of Ephorbia prostrata is a potential phototoxic and antioxidant that markedly inhibits free radicals due to the presence of flavonoids and polyphenolic compounds (16). The phytochemical assessment of Trifolium alexandrinum has revealed the presence of steroids, terpenoinds, flavonoids, and isoflavonoids (17). Hence it was selected for the present study for the evaluation of the capability of T. alexandrinum to reduce the toxicity induced by CCl4 in the lungs of albino rats.

Materials and methods


Methanol, glutathione reduced (GSH), glutathione oxidized (GSSG), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), bovine serum albumin (BSA), 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), CCl4, thiobarbituric acid (TBA), trichloroacetic acid (TCA), Folin–Ciocalteu reagent, sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, and DMSO of Sigma Aldrich Chemicals Co. were utilized in this experiment.

Plant preparation and extraction

During the maturity period, T. alexandrinum (L.) plants (leaves, stem, and flowers) grown in the north region of Bannu District, Pakistan, were collected. Then it was taxonomically identified by Prof. Abdur Rahman, serving in the Department of Botany, the Post Graduate College of Bannu. The plant extract was chopped and dried under shade for a period of one month. Then, after drying, the chopped plant was grinded into 1-mm fine powder with the help of a mechanical grinder. The powdered material (700 g) was soaked in 2.5 liter (80%) methanol for a period of 1 week at room temperature and then filtered. The filtrate was collected and concentrated with the help of a rotary evaporator at 38°C, under reduced pressure, and the crude extract was stored at 4°C for further assessment.

Animals and treatment

The study protocol was approved by the ethical committee of University of Science and Technology of Bannu, for laboratory and experimental animal feed and care. The present experiment was designed with 24 adult albino rats. The rats, weighing 200–210 g, were divided equally into four groups; kept under normal laboratory conditions on 12 h light/darkness, at room temperature; and fed standard feed and water. Group I was the control and remained untreated, while group II was treated with 1 ml/kg body weight (b.w.; 30% in olive oil i.p.) CCl4. Group III received 50 mg/kg b.w. silymarin, and group IV was treated with 200 mg/kg b.w. T. alexandrinum methanolic extracts (TAME) after 24 h of CCl4 treatment. At the end of the experiment, after 24 h of the last dose, the rats were weighed and sacrificed. Lungs were removed, washed with saline, then dried with filter paper, and weighed. Lung tissues were stored at −70°C for further enzymatic analysis.

Assessment of protein, CAT, and POD

Homogenized lung tissue (200 mg) was placed in four volumes of 0.1 mM NaH2PO4 buffer (pH 7.4) and centrifuged at 6,000×g for 30 min. The supernatant was collected and used for the analysis of antioxidant enzymes and protein. Protein was determined in the lung homogenate by the method of Lowry et al. (18), using BSA as standard. The activities of CAT and POD were carried out by the protocol of Chance and Maehly (19). In brief, the CAT activity was examined using hydrogen peroxide (H2O2). Change in absorbance was noted at 240 nm. The activity of POD was determined at 470 nm by the use of guaiacol as a substrate. One unit of enzymes (CAT and POD) was defined as an absorbance change of 0.01 as units/min.

GST assay

GST activity was determined by the method of Habig et al. (20), using CDNB as a substrate. GST was measured at 340 nm using a molar extinction coefficient of 9.6×103 M−1 cm−1.

GSH-Px assay

An assay of GSH-Px was determined by the method of Mohandas et al. (21). NADPH was used as a substrate and the absorbance measured at 340 nm. The molar coefficient was 6.22×103 M−1 cm−1.

Glutathione reductase activity

Activity of glutathione reductase in the lungs homogenate of rats was determined by the protocol of Carlberg and Mannervik (22), with some modifications. The change in absorbance was measured at 340 nm. NADPH was used as a substrate. An extinction coefficient of 6.22×103 M−1 cm−1 was used for calculations.

Estimation of TBARS

Thiobarbituric acid–reactive substances (TBARS) in lungs homogenate were estimated by the TBA method described by Wright et al. (23). Change in absorbance was noted at 535 nm. An extinction coefficient of 1.56×105 M−1 cm−1 was used for TBARS calculation.


Results of this study have shown that CCl4 toxicity significantly (p<0.01) reduced the antioxidant enzyme level of the lung compared to the control group (Table 1). The co-treatment of 50 mg/kg b.w. silymarin and TAME at doses of 200 mg/kg b.w. significantly (p<0.01) restored the activities of CAT and POD in the lungs of rats. TAME at the dose of 200 mg/kg b.w. restored completely the CAT and POD activities closely toward control level.

Table 1.  Effect of TAME on the protein and lung marker enzymes of rats
Treatment Protein (µg/mg tissue) CAT (U/min) POD (U/min)
Control 280.7±9.6++ 1.7±0.4++ 3.1±0.5++
CCl4 187.4±13.2** 0.9±0.2** 1.7±0.3**
CCl4+silymarin 277.8±10.6++ 1.5±0.3++ 3.0±0.6++
CCl4+TAME 230.5±12.3++ 1.1±0.1++ 2.7±0.5++
Mean±SEM (n=6).
**Indicate significance from the control group at p<0.01 probability level.
++Indicate significance from the CCl4 group at p<0.01 probability level.

The activities of GST and GSH-Px in the lungs of rats were significantly (p<0.01) decreased by intoxication of CCl4 that were partially reversed by the supplementation of 200 mg/kg b.w. TAME. The results shown by silymarin group also effectively (p<0.01) reduced the impact of CCl4 on these considerations as shown in Table 2.

Table 2.  Effect of TAME on GSH-Px and GST in rat lungs
Treatment GST (nmol/min/mg protein) GSH (mol/g tissue) GSH-Px (mol/g tissue)
Control 34.9±3.2++   110.8±5.3++
CCl4 23.3±2.5** 13.93±1.4++ 99.7±4.8**
CCl4+silymarin 34.5±2.7++ 8.14±0.9** 112.2±6.3++
CCl4+TAME 28.7±3.1++ 13.78±1.3++ 108.1±5.7++
Mean±SEM (n=6).
**Indicate significance from the control group at p<0.01 probability level.
++Indicate significance from the CCl4 group at p<0.01 probability level.

The contents of GSH in the lungs were significantly (p<0.01) decreased while the TBARS contents increased in the CCl4 intoxicated group as shown in Table 2 and Fig. 1, respectively. The altered level of GSH and TBARS by CCl4 was reduced by the oral addition of methanolic extract of T. alexandrinum (L.). Silymarin also reduced the CCl4-induced perturbations in the above markers.

Fig 1

Fig. 1.   Effects of TAME on TBARS in rat lungs.

The protective role of T. alexandrinum (L.) against CCl4 intoxication in rat on lungs weight and relative lungs weight and body weight are shown in Table 3. CCl4 significantly increased (p<0.01) lung weight, relative lung weight, and body weight as compared to control group treatment of 200 mg/kg b.w. T. alexandrinum (L.) concentrations ameliorated the CCl4 toxicity and reduced (p<0.01) the lung weight, relative lung weight, significantly like that of standard silymarin.

Table 3.  Effect of TAME on the body and lung weight of rats
Treatment Initial body wt (g) Final body wt (g) % increase body wt Absolute lungs wt Relative lungs wt
Control 190.3±5.7++ 198.2±5.2++ 4.2±0.7++ 1.5±0.5++ 0.75±0.05++
CCl4 185.2±6.2** 192.3±9.0** 3.8±0.5** 1.9±0.2** 0.98±0.07**
CCl4+silymarin 185.7±6.1++ 195.8±7.8++ 5.4±0.9++ 1.5±0.4++ 0.76±0.04++
CCl4+TAME 185.4±7.3++ 194.6±5.7++ 4.8±0.5++ 1.5±0.3++ 0.77±0.09++
Mean±SEM (n=6).
** Indicate significance from the control group at p<0.01 probability level.
++Indicate significance from the CCl4 group at p<0.01 probability level.


In this experiment, the recovery potential of the TAME was taken under consideration against CCl4 toxicity in the lungs of rats. The main observations in these experiments were the capability of TAME to recover the inflammatory-oxidant activity of CCl4. It is reported that CCl4 induced toxicity due to its conversion to CCl3, which is a highly reactive free radical. These CCl3 free radicals damage DNA and lipids, and contribute to the loss in protein and enzyme activity (24). Results shown in Table 3 have revealed that CCl4 intoxication in the rats significantly lowers the percent increase in body weight. The absolute weights of the rat lungs and the relative lung weights increased as compared to those of the control rats in the CCl4 group. Minimal increase in the body weights may be due to the degeneration of body tissue while the increase in the lung weights may be the effect of the inflammatory response of the rat’s lungs, as was evidenced in another study (25). Supplementations of TAME and silymarin possibly change the response and restore the changes in body and lung weights to those closer to the weights of the control group. CCl4 intoxication significantly reduces the activities of CAT, and POD in lungs, which might be due to a depletion of GSH contents (26). The low level of GST and GSH-Px correlates to oxidative stress and the depletion of GSH in the lungs resulting from the treatment of CCl4 administered to the rats. Normally, GSH is converted into GSSG, which is then reduced by glutathione reductase to GSH. TBARS is a highly reactive aldehyde formed as a result of the peroxidation of polyunsaturated fatty acids. TBARS formation is considered a sign of tissue damages (27). The high level of TBARS accumulation in the lungs, as a result of CCl4 treatment, indicates the extensive oxidative damage to lung tissues. The TAME extract contains polyphenols and flavonoids which have antioxidant and anti-inflammatory roles and reduce the risk of oxidative ailments. The reduction in the level of lipid peroxidation and the improved activity of antioxidant enzymes proved that TAME and silymarin are capable of protecting against various pathological conditions, including oxidative stress (28).


In rats, TAME significantly protects the lungs against oxidative damages, which might be due to the presence of bioactive constituents in the extract.


  1. Jackson RM. Molecular, pharmacologic, and clinical aspects of oxygen-induced lung injury. Clin Chest Med 1990; 11: 73–86. PubMed Abstract
  2. Fisher AB, Forman HJ, Glass M. Mechanisms of pulmonary oxygen toxicity. Lung 1984; 162: 255–9. PubMed Abstract | Publisher Full Text
  3. Clark JM, Lambertsen CJ. Pulmonary oxygen toxicity: a review. Pharmacol Rev 1971; 23: 37–133. PubMed Abstract
  4. Crystal RG. Oxidants and respiratory tract epithelial injury: pathogenesis and strategies for therapeutic intervention. Am J Med 1991; 91: 39S–44S. Publisher Full Text
  5. Conner EM, Grisham MB. Inflammation, free radicals, and antioxidants. Nutrition 1996; 12: 274–7. Publisher Full Text
  6. Mannino DM, Homa DM, Akinbami LJ, Ford ES, Redd SC. Chronic obstructive pulmonary disease surveillance – United States, 1971–2000. MMWR Surveill Summ 2002; 51: 1–16.
  7. Cerutti PA. Oxidant stress and carcinogenesis. Eur J Clin Invest 1991; 21: 1–5. PubMed Abstract | Publisher Full Text
  8. Abraham P, Wilfred G, Cathrine SP. Oxidative damage to lipids and proteins of the lungs, testis and kidney of rats during CCl4 intoxication. Clin Acta 1999; 289: 177–9. Publisher Full Text
  9. Daniels WM, Reiter RJ, Melchiorri D, Sewerynek E, Pablos MI, Ortiz GG. Melatonin counter acts lipid peroxidation induced by carbon tetrachloride but does not restore glucose-6-phosphatase activity. J Pineal Res 1995; 19: 1–6. PubMed Abstract | Publisher Full Text
  10. Weber LW, Boll M, Stampfl A. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol 2003; 33: 105–36. PubMed Abstract | Publisher Full Text
  11. Dogukan A, Akpolat N, Celiker H, Ilhan N, Bahcecioglu IH, Gunal AI. Protective effect of interferon-alpha on carbon tetrachloride-induced nephrotoxicity. J Nephrol 2003; 16: 81–4. PubMed Abstract
  12. Adewole SO, Salako AA, Doherty OW, Naicker T. Effect of melatonin on carbon tetrachloride-induced kidney injury in Wistar rats. Afr J Biomed Res 2007; 10: 153–64.
  13. Pradeep K, Mohan CV, Gobianand K, Karthikeyan S. Silymarin modulates the oxidant-antioxidant imbalance during diethylnitrosamine induced oxidative stress in rats. Eur J Pharmacol 2007; 560(2–3): 110–16. PubMed Abstract | Publisher Full Text
  14. Danciu C, Vlaia L, Fetea F, Hancianu M, Coricovac DE, Ciurlea SA, et al. Evaluation of phenolic profile, antioxidant and anticancer potential of two main representants of Zingiberaceae family against B164A5 murine melanoma cells. Biol Res. 2015; 48(1): 1. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  15. Antunes LMG, Darin JDC, Bianchi MLP. Effects of the antioxidants curcumin or selenium on cisplatin-induced nephrotoxicity and lipid peroxidation in rats. Pharm Res 2001; 43(2): 145–50. PubMed Abstract | Publisher Full Text
  16. Khar A, Ali AM, Pardhasaradhi BV. Induction of stress response renders human tumor cell lines resistant to curcumin-mediated apoptosis: role of reactive oxygen intermediates. Cell Stress Chap 2001; 6(4): 368–76. Publisher Full Text
  17. Khan AV, Ahmed QU, Shukla I, Khan AA. Antibacterial activity of leaves extracts of Trifolium alexandrinum Linn. against pathogenic bacteria causing tropical diseases. Asian Pac J Trop Biomed 2012; 3: 189–94. Publisher Full Text
  18. Lowry OH, Rosenberg NJ, Farr AL, Randall RJ. Protein measurement with Folin Phenol reagent. J Biol Chem 1951; 193: 265–75. PubMed Abstract
  19. Chance B, Maehly AC. Assay of catalase and peroxidases. Methods Enzymol 1955; 11: 764–75.
  20. Habig WH, Pabst MJ, Jakoby WB. Glutathione-S-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem 1974; 249: 7130–9. PubMed Abstract
  21. Mohandas J, Marshal JJ, Duggin GG, Horvath JS, Tiller DJ. Differential distribution of glutathione and glutathione-related enzymes in rabbit kidney. Possible implications in analgesic nephropathy. Biochem Pharmacol 1984; 33: 1801–7. Publisher Full Text
  22. Carlberg I, Mannervik EB. Glutathione level in rat brain. J Biol Chem 1975; 250: 4475–80.
  23. Wright JR, Colby HD, Miles PR. Cytosolic factors that affect microsomal lipid peroxidation in lung and liver. Arch Biochem Biophys 1981; 206: 296–304. Publisher Full Text
  24. Venkatesan N, Punithavathi V, Chandrakasan G. Curcumin protects bleomycin-induced lung injury in rats. Life Sci 1997; 61(6): 51–8. Publisher Full Text
  25. Khan RA, Khan MR, Sahreen S. Protective effect of Sonchus asper extracts against experimentally-induced lung injuries in rats: a novel study. Exp Toxicol Pathol 2012; 64: 725–31. doi: Publisher Full Text
  26. Singh N, Kamath V, Narasimhamurthy K, Rajini PS. Protective effects of potato peel extract against carbon tetrachloride-induced liver injury in rats. Environ Toxicol Pharmacol 2008; 26: 242–6. PubMed Abstract | Publisher Full Text
  27. Ohkawa H, Ohishi N, Yogi K. Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351–8. Publisher Full Text
  28. Vardavas CI, Majchrzak D, Wagner K-H, Elmadfa I, Kafatos A. The antioxidant and phylloquinone content of wildly grown greens in Crete. Food Chem 2006; 99: 813–21. Publisher Full Text
About The Authors

Rahmat Khan


Huda Mohammad Alkreathy
Department of Pharmacology, King Abdulaziz University(KAU), Jeddah, Saudi Arab
Saudi Arabia


Abdus Saboorshah
Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology Bannu, Khyber Pakhtunkhwa, Pakistan;

Mushtaq Ahmed
UST Bannu

Samiullah Khan

Article Metrics

Metrics Loading ...

Metrics powered by PLOS ALM

Related Content