Send Orders of Reprints at reprints@benthamscience.net The Open Antimicrobial Agents Journal, 2013, 4, 1-5 1 1876-5181/13 2013 Bentham Open Open Access Inhibition of Intracellular Survival of Multi Drug Resistant Clinical Isolates of Mycobacterium tuberculosis in Macrophages by Curcumin Pramod Kumar Gupta, Savita Kulkarni* and Ramakrishna Rajan Radiation Medicine Centre, Bio-Medical Group, Bhabha Atomic Research Centre, TMH Annexe, Parel, Mumbai 400012, India Abstract: Curcuma longa commonly known as turmeric has been used in Indian Ayurvedic medicine as a constituent to treat various disorders. It is by now clear that principal curcuminoid of turmeric; curcumin, a yellow pigment, is responsi- ble for these beneficiary activities. The aim of the present study was to evaluate anti-mycobacterial effect of curcumin (CMN) on intracellular growth of MDR clinical isolates of Mycobacterium tuberculosis (MTB). Curcumin was evaluated for its efficacy to inhibit the intracellular growth of MTB H37Rv and two MDR clinical isolates in Raw 264.7 cell line us- ing CFU assay. Resazurin microtiter plate assay (REMA) was used to evaluate its direct anti-mycobacterial activity. Curcumin, though did not show direct anti-mycobacterial activity against three MTB strains, exhibited dose dependent in- hibition of intracellular growth for MTB H37Rv as well as two MDR clinical isolates. These results suggest that CMN could be a potential candidate for future, novel adjunctive anti-TB therapy. Keywords: Anti-mycobacterial activity, Curcumin, MDR, Tuberculosis. 1. INTRODUCTION Tuberculosis (TB) caused by Mycobacterium tuberculo- sis (MTB), is a public-health problem worldwide with a global mortality of 1.4 million with 350,000 deaths each year in India [1, 2]. The situation is further exacerbated with in- creasing incidence of multi-drug resistant (MDR), exten- sively drug resistant (XDR) TB and co-infection of HIV. Current drug regimens are far from effectively controling the incidence of drug resistance of MTB. Hence, the discov- ery of novel agents targeting host rather than pathogen is need of hour [3]. Since, MTB is known to alter signaling required for the production of immunostimulatory cytokines and effectors molecules in the host [4], immunomodulation beneficial to host, appears a viable strategy to control the pathogen. Curcumin (CMN- [diferuloylmethane or 1,7-bis (4- hydroxy-3-methoxy-phenyl) hepta-1, 6-diene-3, 5-dione)]) is a yellow pigment from the rhizomes of perennial herb Cur- cuma longa commonly known as turmeric. It has been shown to modulate various molecular targets in malignancy and signaling cascades involved in both innate and acquired immune response and, hence is utilized to treat various dis- orders including arthritis, cardiovascular disorders, cancers, and other pathologies [5]. In addition, CMN and largely its analogs have exhibited the antimicrobial and anti-mycobac- terial activity in nonpathogenic species [6]. *Address correspondence to this author at the Radiation Medicine Centre, Bio-Medical Group, Bhabha Atomic Research Centre, TMH Annexe, Parel, Mumbai 400012, India; Cel: +91-22-24134960; Tel: +91-9821083165; Ext: 211; Fax: +91-22-24157098; E-mail: savita.kulkarni1@gmail.com The aim of the present study was to evaluate anti- mycobacterial efficiency of CMN on intracellular growth of MDR clinical isolates of MTB inside RAW 264.7 cells at lower doses. 2. MATERIALS& METHODS 2.1. Reagents Curcumin, resazurin sodium salt and MTT were obtained from Sigma Aldrich. Cell culture media DMEM and fetal calf serum were purchased from Gibco, Life Technologies. Bacterial culture media Middlebrook 7H9, 7H11, ADC and OADC were purchased from Difco, BD biosciences. LJ tubes containing antibiotics were purchased from EOS labs, Mumbai. 2.2. Bacterial Cultures and Cell Lines M. tuberculosis clinical isolates strain-1 and strain-2 were recovered from patients in Tata memorial hospital, and KEM hospital, Mumbai (INDIA). The laboratory strain H37Rv was also included in the study. All M. tuberculosis strains were grown in Middlebrook 7H9 medium supple- mented with ADC (albumin-dextrose complex), and contain- ing 0.05% Tween 80 for 10 days 37°C with daily agitation; working stocks were prepared (10 8 bacilli/ml) stored at -70°C until use. All procedures were carried out in a Biosafety Level III (BSL III) laboratory. Murine macrophage cell line RAW 264.7 was obtained from NCCS Pune, India and main- tained in DMEM at 37°C, in an atmosphere containing 5% CO2.