Indian Journal of Biochemistry & Biophysics Vol. 48, December 2011, pp 380-387 Protective effect of ellagic acid and pumpkin seed oil against methotrexate-induced small intestine damage in rats Noha A El-Boghdady* Biochemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt Received 28 April 2011; revised 06 November 2011 Gastrointestinal toxicity is one of the most serious side effects in the methotrexate (MTX) treatment. This study was designed to investigate whether ellagic acid (EA) and/or pumpkin seed oil (PSO) had a protective effect on MTX-induced small intestine damage. Forty albino rats were randomized into five groups of 8 rats each. Group Ι served as a normal control group. In Group ΙΙ, MTX was administered as a single dose (20 mg/kg) intraperitoneally. Groups ΙΙΙ, ΙV and V were pre-treated respectively with either PSO (40 mg/kg), EA (10 mg/kg) or 0.2% DMSO (vehicle control) orally every day by gavage for 5 days and then they received MTX. All animals were sacrificed 5 days after the intraperitoneal injection of MTX for histopathological examination, estimation of serum prostaglandin E 2 (PGE 2 ) level, assay of tissue malondialdehyde (MDA), reduced glutathione (GSH) and nitric oxide (NO) levels and myloperoxidase (MPO), xanthine oxidase (XO) and adenosine deaminase (AD) activities. Administration of EA and/or PSO decreased the intestinal damage, PGE 2 , MDA and NO levels and MPO, XO and AD activities and increased GSH level. These results suggest that EA and PSO protect the small intestine of rats from MTX-induced damage through their antioxidant and anti-inflammatory effects and thus have potential as a promising drug in the prevention of undesired side effects of MTX. Keywords: Methotrexate, Oxidative stress, Ellagic acid, Pumpkin seed oil, Purine catabolizing enzymes, Prostaglandin E 2. Methotrexate (MTX), a folic acid antagonist is widely used as a cytotoxic chemotherapeutic agent for leukemia and other malignancies. Over the past five decades, low-dose MTX has also been used for the treatment of various inflammatory diseases, such as psoriatic and rheumatoid arthritis 1 . However, the efficacy of this agent is often limited by severe side- effects and toxic sequelae. Since cytotoxic effect of MTX is not selective for cancer cells, it also affects normal tissues that have a high rate of proliferation, including the haematopoietic cells of bone marrow and actively-dividing cells of gut mucosa. Thus, one of the major toxic effects of MTX is intestinal injury and enterocolitis 2 . The small intestinal damage induced by MTX treatment results in malabsorption and diarrhea. The malabsorption results in weight loss and disturbs the cancer chemotherapy of the patients 3 . Being a high affinity inhibitor of dihydrofolate reductase, MTX is a pro-oxidant compound that causes depletion of the dihydrofolate pool and directly affects the synthesis of thymidilate, suppressing DNA synthesis 4 . Moreover, cytosolic NAD(P)-dependent dehydrogenases 5 and NADP malic enzyme are inhibited by MTX, suggesting that the drug could decrease the availability of NADPH in the cells by inhibiting the pentose cycle enzymes 6 . Under normal conditions, NADPH is used by glutathione reductase to maintain the reduced state of cellular glutathione (GSH), which is an important cytosolic antioxidant that protects against reactive oxygen species (ROS). Considering the deleterious effects of MTX on both GSH and DNA synthesis, interest has been focused on the compounds that act as antioxidants, are capable of stimulating GSH and DNA synthesis or that have trophic effects on gut mucosa. Polyphenolic compounds are widely distributed in the vegetable kingdom and are often encountered in our daily lives, being found in tea, red wine, fruits, etc 7 . Ellagic acid (EA, Fig. 1) is a phenolic compound ______________ E-mail: nohaelbogdady@yahoo.com Tel: 02-22686509; Fax: 02-22663197 Abbreviations: AD, adenosine deaminase; DMSO, dimethyl sulphoxide; EA, ellagic acid; MDA, malondialdehyde; MPO, myloperoxidase; MTX, methotrexate; NO, nitric oxide; PGE 2 : prostaglandin E 2 ; PSO, pumpkin seed oil; ROS, reactive oxygen species; XO, xanthine oxidase. Fig. 1— Structure of ellagic acid