REACTIVE OXYGEN SPECIES GENERATION IN THE ANTIBACTERIAL ACTIVITY OF LITSEA SALICIFOLIA LEAF EXTRACT Original Article NITUMANI KALITA* a , MOHAN CHANDRA KALITA a , MADHUCHANDA BANERJEE b a Department of Biotechnology, Gauhati University, GNB Road, Guwahati 781 014, Assam, India, b Received: 13 Apr 2016 Revised and Accepted: 20 June 2016 Department of Zoology, Midnapore College, Raja Bazar Main Rd, Medinipur, West Bengal 721101, India Email: nitumani.kalita86@gmail.com ABSTRACT Objective: The present work was carried out to investigate the antibacterial activity of Litsea salicifolia leaf extract and to study whether there is a generation of oxidative stress in its mechanism of antibacterial action. Methods: L salicifolia was screened for its antibacterial activity against the bacterial strains collected from the Microbial Type Culture Collection and gene bank (MTCC) viz. Escherichia coli MTCC 443 and Staphylococcus aureus MTCC 96. Disc diffusion method was used for screening. The preliminary screening was done with petroleum ether (PE), chloroform (CHF), methanol (MT) and aqueous (AQ) extracts of the L. salicifolia leaf. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using macro-broth dilution method. In this work, oxidative stress on bacterial cells after exposure to plant extract was measured using nitroblue tetrazolium method (NBT). Results: Experimental evidence indicated that the CHF extract is more efficient against S. aureus compared to the other extracts with MIC value of 0.076 mg/ml and MBC value of 0.4 mg/ml. Our results revealed that there was a generation of reactive oxygen species (ROS) in the treated bacterial cell cytoplasm. Transmission electron microscopy (TEM) revealed considerable damage in the cell envelope as well as morphological changes in the extract treated bacterial cells. There were also changes in DNA isolated from treated cells. Conclusion: From the present study, we can conclude that the active constituents in the plant extract contribute in cell killing involving generation of free radical-induced oxidative stress, which possibly the cause or the consequence of the alteration of some other cellular mechanisms ultimately leading to cell death. Keywords: Medicinal plant, Antibacterial activity, Oxidative stress © 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/ ) INTRODUCTION In recent years, although various antimicrobial agents have been newly introduced the increasing development of drug resistance of the infectious microbes has limited the lifespan of these new antimicrobial agents [1]. Clinical microbiologists are applying various strategies to combat drug-resistant microbes, including the search for novel antimicrobial agents from plants. Because from the recent past, plants have been an integral part of traditional medicine system and indigenous people of the world rely on plant-based medicines to cure various diseases. Besides, plant-derived antimicrobial agents are gaining popularity because of easy availability and less side effects [2]. Furthermore, antimicrobial drugs derived from plants will be new to the microbes as antimicrobial drugs of present times are mostly bacterial, or fungal origin [2] and therefore, microbes are less likely to develop resistance against plant-derived antimicrobial drugs. Plants contain a variety of secondary metabolites such as phenols, quinones, flavonoids, alkaloids, tannins, terpenoids that have been proven to possess antimicrobial activity [3-6]. In most cases, these secondary metabolites are synthesized by plants in response to plant pathogen attack. The mechanism of plant antimicrobial activity is poorly understood, although there are some reports on the probable mechanism of action of phytochemicals such as flavonoids have been reported to inhibit DNA synthesis in Proteus vulgaris [7], alkaloids like berberine have been proposed to be potent inhibitors of various enzymes like lactate and malate dehydrogenase [8]. Phenolics were thought to exert their antimicrobial effect by inhibition of enzymes through reacting with sulfhydryl groups containing compounds [9]. Quinone, a class of phytochemicals is known to provide a source of stable free radicals as well as bind to proteins irreversibly inhibiting bacterial growth [10]. Investigation of the mechanism of bacterial killing by the novel antimicrobial agents may provide a better understanding of resistance mechanisms for the advancement of future drugs. Bactericidal antibiotics have been reported to kill bacteria by a common mechanism of cell death involving the induction of ROS, which subsequently results in DNA damage, protein oxidation and lipid peroxidation [11]. Antibiotic like quinolone, a gyrase inhibitor is known to interfere with the iron regulatory pathway resulting information of ROS which eventually cause cell death [12]. Plants are known to produce activated oxygen species to fight against pathogens [13]. The main objective of this study was to find out whether ROS generated oxidative stress is responsible for bacterial killing by L. salicifolia leaf extract. Furthermore, morphological changes in bacterial cells due to plant extract treatment were also demonstrated. L. salicifolia (Roxb. ex Nees) Hook. f. commonly known as 'Dighloti' has been a part of traditional medicine systems in North East India from ancient time. The Apatani people of Ziro valley in Arunachal Pradesh are known to use the fruit of L. salicifolia in the treatment of bone fracture and stomach disorder [14]. The leaves of these plants are used in dysentery and pneumonia traditionally in Assam. L. salicifolia is cultivated for feeding Muga silk worms in North East India. Rastogi and Borthakur [15] isolated two alkaloids dicentrinone and nordicentrine from leaves of L. salicifolia. There are reports on the antibacterial activity of these two alkaloids [16]. Phytochemical screening [17] of L. salicifolia leaf extract showed the presence of flavonoids, alkaloids, terpenoids, and peptides. There are few reports on the antibacterial activity of Litsea sp. [18, 19]. There is the previous report on the insecticidal activity of L. salicifolia [20]. This is the first report on antibacterial activity and mechanism of action of L. salicifolia leaf extract. International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 8, Issue 8, 2016