RESEARCH ARTICLE Antifibrotic effects of D-limonene (5(1-methyl-4-[1- methylethenyl]) cyclohexane) in CCl 4 induced liver toxicity in Wistar rats Sheikh Bilal Ahmad 1 | Muneeb U. Rehman 1 | Bilques Fatima 1 | Bilal Ahmad 1 | Ishraq Hussain 1 | Sheikh Pervaiz Ahmad 2 | Adil Farooq 3 | Showkeen Muzamil 1 | Rahil Razzaq 1 | Shahzada Mudasir Rashid 1 | Showkat Ahmad Bhat 1 | Manzoor Ur Rahman Mir 1 1 Molecular Biology Lab, Division of Veterinary Biochemistry, Faculty of Veterinary Sciences & Animal Husbandry, Sheri Kashmir University of Agricultural Science & Technology (SKUAST-K), Srinagar, Jammu and Kashmir 190006, India 2 Department of Statistics, University of Kashmir, Hazratbal Srinagar, Jammu and Kashmir 190006, India 3 RAKCOPS, RAK Medical & Health Sciences University, Ras AL Khaimah, UAE-11172 Correspondence Sheikh Bilal Ahmad, Division of veterinary Biochemistry, Faculty of Veterinary Sciences & Animal Husbandry, Sheri Kashmir University of Agriculture Science & Technology (SKUAST-K), Alustang, Shuhama, Srinagar, J&K 190006, India. Email: sbilal@skuastkashmir.ac.in Abstract This study was designed to assess the potential antifibrotic effect of D-Limonene—a component of volatile oils extracted from citrus plants. D-limonene is reported to have numerous therapeutic properties. CCl 4 -intduced model of liver fibrosis in Wistar rats is most widely used model to study chemopreventive studies. CCl 4 -intoxication significantly increased serum aminotransferases and total cholesterol these effects were prevented by cotreatment with D-Limonene. Also, CCl 4 -intoxi- cation caused depletion of glutathione and other antioxidant enzymes while D-Limonene preserved them within normal values. Hydroxyproline and malondialdehyde content was increased markedly by CCl 4 treatment while D-Limonene prevented these alterations. Levels of TNF-a, TGF- b, and a-SMA were also assessed; CCl 4 increased the expression of a-SMA, NF-jB and other downstream inflammatory cascade while D-Limonene co-treatment inhibited them. Collectively these findings indicate that D-Limonene possesses potent antifibrotic effect which may be attrib- uted to its antioxidant and anti-inflammatory properties. KEYWORDS antifibrotic, CCL 4 , D-Limonene, fibrosis and liver 1 | INTRODUCTION Liver plays a primary role in the regulation of different physiological processes and it controls some vital functions, such as secretion, stor- age, and metabolism. Liver is often exposed to variety of xenobiotic and therapeutic agents. It detoxifies exogenous (toxic compounds) and endogenous (waste metabolites) substances of organisms and synthe- sizes useful agents. 1 Till now, no reports are available regarding actual curative therapy agent for liver disorder/diseases, most of the existing remedies only aid in healing or regeneration of liver. Fibrosis, a general clinical condition can be seen in different organs mostly in liver, it is often linked with the end stages of chronic inflamma- tory diseases, 2,3 Liver fibrosis is the phenomena of development of a hepatic scar due to chronic liver injury resulting from complex interrelated changes in cell population, extra cellular matrix (ECM) and cytokines. 4,5 Carbon tetrachloride (CCl 4 ) induced animal model is used to study fibrosis. Published reports show CCl 4 as xenobiotic to induce acute and chronic tissue injuries 6 through bio-activation of the phase I cyto- chrome P 450 enzymes to give rise to reactive metabolic trichloromethyl radicals (  CCl 3 ) and peroxytrichloromethyl radicals (  OOCCl 3 ). These reactive metabolites have potential to covalently bind to large molecules such as lipids, proteins, and nucleic acids. The double allylic hydrogen bonds of polyunsaturated fatty acid are vulnerable to attack by free radi- cals; CCl 4 administration leads to increase in lipoperoxide and free perox- ide radicals that are highly reactive and cause injury or necrosis. 7–9 CCl 4 Treatment generates free radicals that trigger a cascade of events result- ing in hepatic fibrosis, mimicking the oxidative stress that has a fibro- genic effect on hepatic stellate cells (HSC). 3–5 Products of lipid peroxidation (LPO) and some cytokines (proin- flammatory) like TNF-a and IL-6 together initiate hepatic pathologies Environmental Toxicology. 2017;1–9. wileyonlinelibrary.com/journal/tox V C 2017 Wiley Periodicals, Inc. | 1 Received: 6 September 2017 | Revised: 22 November 2017 | Accepted: 2 December 2017 DOI: 10.1002/tox.22523