RESEARCH ARTICLE 2010mp04, CCC: $10 © Inventi Journals (P) Ltd Published on Web 22/06/2010, www.inventi.in Inventi Rapid: Molecular Pharmacology Vol. 1, Issue 1 [ISSN 0976-3856] INTRODUCTION Pseudomonas aeruginosa is an opportunistic pathogen [1] responsible for severe infections in patients with underlying chronic lung diseases such as chronic obstructive pulmonary disease [2] and bronchitis [3] and a major reason for morbidity and mortality in cystic fibrosis patients. [4,5] The crucial problem associated with P. aeruginosa is its ability to form alginate containing biofilms which develop anaerobic niches in the lungs of patients which minimize the penetration and affectivity of antibiotics and components of immune system such as antibodies and hence hamper the clearance of pathogen from lungs. [6] Other threats with P. aeruginosa infections include its resistance to antibiotics of many classes which may be in the form of intrinsic resistance or as acquired resistance which it acquires majorly through mutations during antibiotic treatments. [7,8] In order to prevent development of resistance during treatments and increase the rate of eradication of bacteria from lungs, dual antibiotic therapy is suggested and practiced. [9] However multidrug resistant [10,11] and panresistant strains that are resistant to all antibiotics except polymyxins have been isolated from lungs of patients. [10] This has led to the urgent requirement of new antipseudomonals. In traditional Indian folklore medicine, plant extracts have been used to alleviate many diseases. [12] In this study, two plants namely Acorus calamus and Semecarpus anacardium with different proven medicinal activities have been chosen. Ethanolic extracts of A. calamus have been shown to have 1 Department of Biochemistry & Biotechnology, Institute of Science, Nirma University, Sarkhej Gandhinagar Highway, Ahmadabad, Gujarat, India Email: sriram.seshadri@nirmauni.ac.in ; antibacterial, anti methicillin resistant staphylococcus aureus (MRSA) activity in vitro [13] and ethyl acetate and methanolic extracts have been shown to have antioxidant and glutathione and associated enzyme restoring activity in vivo in rat brain. [14] In addition to this, anti-proliferative and immunosuppressive potential of ethanolic extract of A. calamus has also been shown in vitro. [15] On the other hand, nut extracts of S. anacardium have been shown to have anti- inflammatory activity comparable to that of indomethacin [16] and it role in maintenance of the glutathione redox status by restoring the associated enzymes against oxidative stress has also been proven in vivo. [17] High oxidative stress and inflammatory conditions prevail in patients chronically infected with P. aeruginosa which cause lot of damage to lungs. [18] During literature survey, we could not find any report that showed the effects of mixture of ethanolic extracts of A. calamus and S. anacardium in chronic P. aeruginosa lung infection in vivo. This study investigates the effects of mixture of ethanolic extracts of A. calamus and S. anacardium with reference to eradication of bacteria from lungs, the inflammatory conditions and the oxidative stress and its effects on recovery and regeneration of lung tissues and of restoration of normal lung function in rats chronically infected with P. aeruginosa. MATERIALS & METHODS Animals Fifteen healthy adult female Wister rats weighing 240 to 250 g were used for the experiment. The guidance for Care and Use of Animals for Scientific and Research (Indian National Science Academy, 2000) were strictly followed. Bacterial Strains Pseudomonas aeruginosa (MTCC 741) which was used for establishment of chronic infection in rats is a clinical isolate which was obtained from Microbial Type Culture Collection, IMTECH, Chandigarh, India. P. aeruginosa was routinely grown on N-broth or N-agar. Pseudomonas Isolation Agar (Hi media) was used as a selective media for isolation and detection of P. aeruginosa from the plated lung homogenates and blood samples. [19] Rat Infection Model Streaking of cotton swabs from the nostrils of all the thirteen animals was done on Pseudomonas Isolation Agar, in order to ensure that the selected animals did not have any history of P. aeruginosa infection. For the establishment of chronic P. aeruginosa lung infection, drops of actively growing (late log phase) P. aeruginosa culture of around 0.5 x 10 8 to 2 x 10 8 CFU/ml, were put inside the nostrils of rats. 2 mL of culture was put drop wise as the drops were taken into nose by rats during respiration. Procedure was repeated for three successive days to ensure that infection has occurred. Occurrence of infection was confirmed by streaking a cotton swab of fluid coming out of running nose of the animal, following three days of infection dosage on Pseudomonas Isolation Agar. Preparation of Plant Extracts and Dosage Root extract of Acorus calamus and seed extract of Semecarpus anacardium were prepared separately. For preparation of extracts, the dried respective parts of the plants were grinded in blender to obtain a fine powder. A solute to solvent ratio of 1:10 was maintained during preparation of extracts and 5 g of solute was soxlet extracted with ethanol. Extracts were filtered to remove the particulate matter, dried by evaporation and then reconstituted in distilled water. 14.6% w/w yield was obtained for A. calamus extracts and 16.7% for S. anacardium extracts. Reconstituted extracts were mixed to give a mixture that contained both extracts in equal amounts and this mixture was given orally at final dosage of 200 mg/kg body weight/day for fifteen days. Experimental Design The experimental design for the present investigation is given in table 1. Pathological Scoring of Lungs And Histology Histopathological scoring of lungs was done according to the method of Johansen et al., [20] Evaluation of the Effect of Combination of Extracts of Acorus calamus and Semecarpus anacardium in Chronic Pseudomonas aeruginosa Lung Infection in Rats Sriram Seshadri 1 Abstract: P. aeruginosa is responsible for severe chronic infections in patients which suffer from chronic lung diseases. Current therapies for treatment infections include dual antibiotic therapies. However P. aeruginosa has developed resistance to many of these antibiotics and the microenvironment of the lung in these patients and various survival strategies of this pathogen decrease the affectivity of the antibiotics which has resulted in urgent need for new therapeutics and new approaches for treatment. In this study effect of mixture of extracts of A. calamus and S. anacardium were studied on chronic P. aeruginosa lung infections in rats. Results show eradication of bacteria from lungs of rats, decreased inflammation and oxidative stress and recovery of lung function marked by initiation of regeneration of alveolar walls. Keywords: Pseudomonas aeruginosa, chronic infections, Acorus calamus, Semecarpus anacardium