MICROBICIDAL POTENTIALITY OF PURIFIED ANTHOCYANIN FROM IN VITRO CULTURE OF CLERODENDRON INFORTUNATUM L. AGAINST SELECTED PATHOGENS Original Article MEENU KRISHNAN V. G., GREESHMA MURUKAN, ASWATHY J. M., BOSCO LAWARENCE, MURUGAN K. Plant Biochemistry and Molecular Biology Laboratory, Department of Botany, University College, Trivandrum, 695034, Kerala Email: harimurukan@gmail.com * Received: 12 Apr 2017 Revised and Accepted: 20 Apr 2018 ABSTRACT Objective: Clerodendron infortunatum L. is a widely used medicinal herb over centuries for curing many skin-borne disorders. The present study was designed to validate the tribal knowledge by evaluating antimicrobial potential of purified anthocyanin extracted from in vitro cell suspension culture. Methods: The explants were inoculated on murashige and skoog (MS) medium mixed with various combinations of 2, 4-D a+BAP for callus induction. Green compact callus was initiated within 30 d from the explants on MS medium fortified with benzylaminopurine (BAP) (2.0 mg/l)+2, 4- D (0.5 mg/l). Subsequently, anthocyanin was triggered from the compact callus by subculturing in the medium containing 2, 4-D and Kinetin. Cell suspension culture was also developed. Anthocyanin production was enhanced by elicitation using salicylic acid and others. Three chromatographic methods such as solid phase extraction by Sepharose C18 column, Oasis-MCX and Amberlite XAD 7+Sephadex LH 120 sorbents were used to purify the in vitro synthesized anthocyanin from the cell cultures. HPLC and molar absorptivity assay were carried to check the purity. Antimicrobial analysis was also carried using standard protocols to check minimum inhibitory concentration (MIC) and minimum killing concentration (MKC). Results: The mean purity values obtained by high-performance thin layer chromatography (HPLC) were 90.9%±1.9, 80.60%±2.3 for Oasis MCX, Amberlite XAD-7+Sephadex LH-20 column respectively. However, the purity by molar absorptivity was found to be less. HPLC chromatogram revealed 12 fractions of anthocyanin. Inhibition zone diameter, MIC and MKC values obtained for the purified anthocyanin revealed its antimicrobial potentiality but at different levels among the selected bacteria and fungi. C. albicans, S. aureus, P. aerugenosa showed significant values followed by MRSA, E. coli and A. flavus. The results are comparable with the synthetic antibiotics. However, E. faecalis was more resistance. Mode of action was confirmed from the results of intracellular potassium leakage and bacterial membrane integrity analysis. Conclusion: Thus, the study confirms the efficacy of anthocyanin as natural antimicrobial and suggests the possibility of employing it as drugs for the treatment of infectious diseases caused by the pathogens. Keywords: Clerodendron, HPLC, MS medium, Hormones, Anthocyanin, Column chromatography, Antimicrobial © 2018 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/) DOI: http://dx.doi.org/10.22159/ijpps.2018v10i6.18649 INTRODUCTION Antibiotics are one of the most important drugs in combating pathogenic infections and to safeguard the health-related quality of human life. However, over the past many decades, these antibiotics have become less effective against many pathogens and also produce toxic reactions. Similarly, the emergence of drug-resistant bacteria coupled with resistance against the common synthetic antibiotics created health hazards among human beings. Therefore, it is essential to find out new plant-based drugs with more potentiality. Drugs derived from herbal sources play a significant role in the prevention and treatment of many common diseases. In the least developed countries, ethnic medicine becomes part of their primary healthcare systems [1]. Herbals are commonly exploited in the ethnic medicine and their curative properties are well documented. Nearly, about 61% of herbal drugs are developed based on phytochemicals and they have been effective especially against infectious disease and lifestyle diseases [2]. However, recently the rate of active novel chemical entities related to microbicidal is declining [3]. Secondary metabolites of flowering plants may give a new source of microbicidal potential with possible novel mechanisms of action [4]. The bactericidal effects of ethno medicinal plant extracts have been studied by a large number of researchers around the world. Plants possess a pool of secondary metabolites like tannins, terpenoids, alkaloids, flavonoids, glycosides, etc., which have been proved as antimicrobial under in vitro In this juncture, Clerodendron infortunatum L. of Verbenaceae has been selected. In Ayurvedic literature, the species has been described to be useful against skin disorders and its use into the treatment of hematemesis, and leucoderma has been suggested [6]. Further, their pharmacological uses as pest and disease control agents were reported. Similarly, this plant is widely used by tribal people to treat various ailments including ringworm and other fungal skin infections. No scientific data was available regarding anthocyanin, in vitro culture and its microbicidal potentiality of C. infortunatum. In this scenario, the present study was aimed to validate the ethnic knowledge of its microbicidal potential using anthocyanin. conditions. From pre-historic periods, herbal medicines have been used by common man. Medicinal potentiality of different indigenous herbals for many disorders has been documented by practitioners of traditional medicine [5]. Microbicidal powers of medicinal herbals are being increasingly reported from different parts of the earth. WHO estimates that herbal extracts or their active molecules are used as folk medicine in traditional therapies i.e., approximately 80% of the world population. Antibiotic resistance by microorganisms has increased due to their genetic ability to transmit and acquire resistance to synthetic drugs which are utilized as therapeutic agents. MATERIALS AND METHODS Plant material Clerodendron infortunatum was collected from wild habitats of Kallar region, Ponmudi hills of Western Ghats. Identity was carried by referring floras and confirmed by referring herbarium of Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI), Palode and voucher specimen was deposited in the Department of Botany, herbarium (UCB 2369). Plant material and in vitro culture Fresh excised leaves and nodes of C. infortunatum were employed as explants for culturing in the MS medium culturing [7]. The explants were surface sterilized, disinfected with teepol (5% v/v) for 20 min International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 10, Issue 6, 2018