Green synthesis of silver nanoparticles and production of medicinal compounds with anti-bacterial activity by callus cultures of Momordica charantia L. R.Indira Iyer1, H.Sowmya1,R.Pooja Yashwanthi1, C.Selvaraju2 and S.T.Santhiya1 1 Department of Genetics, Dr, ALMPGIBMS, University of Madras (Taramani Campus) ,Chennai-600113 2 National Centre for Ultrafast Processes, Dr, ALMPGIBMS, University of Madras (Taramani Campus) Chennai-600113

ABSTRACT Production of compounds of therapeutic value including lignans and rapid synthesis of silver nanoparticles with anti-bacterial activity against drug-resistant microbes was achieved with the callus cultures of Momordica charantia. This is the first report of synthesis of nanoparticles in Momordica charantia. INTRODUCTION Plant-derived natural products can counter multi - drug resistant microbes. Momordica charantia1 - anti-diabetic - anti-HIV - anti- cancer effects

Silver nanoparticles have important applications in biology and medicine Plant - mediated nanoparticle synthesis - an eco-friendly , simple technology 2 Very few reports till date on the use of plant callus cultures for nanoparticle synthesis3 Present study explores the potential of callus cultures of M.charantia - for production of bio-active molecules - rapid synthesis of silver nanoparticles with anti-bacterial activity.

MATERIALS AND METHODS ESTABLISHMENT OF CALLUS CULTURES Plant Material - zygotic embryos ( intact / fragmented) from seeds of fresh , ripe fruits cultured in MS4 based media Media Supplements (mg/L) Medium NAA BA 2,4-D Activated charcoal M1 1 - 5 250 M4 4 M5 M6 Abbreviations: BA : benzylaminopurine ; 2,4-D: 2,4 dichlorophenoxy-acetic acid ; NAA: naphthaleneacetic acid . All media had coconut milk (15%).

PHYTOCHEMICAL ANALYSIS PHENOLICS LIGNANS Callus extracted with 80% methanol and centrifuged In vitro produced biomass Supernatant + dichloromethane + water Methanolic extract Centrifuged 10 min at 1000g Phenolics Dichloromethane layer Color test Lignans TLC in chloroform: methanol(9:1)

PREPARATION OF SILVER NANOPARTICLES ANTI-BACTERIAL ACTIVITY Callus extracted with 5 vols of distilled water filtered through Whatman No.1 filter paper filtrate incubated with 9 vols of 1 mM AgNO3 for 24 h UV / Vis spectrophotometry ANTI-BACTERIAL ACTIVITY Ethanolic extract (5 / 10 / 15 μl) Silver nanoparticle suspension (15 μl) Disc diffusion assay

RESULTS AND DISCUSSION IN VITRO RESPONSES AND MEDIA Media with activated charcoal Media without activated charcoal Intact zygotic embryos Fragmented zygotic embryos Intact / fragmented zygotic embryo Germination in vitro Greening but no callusing Callusing

Fragmented explants Intact explants(X8) Responses of fragmented and intact zygotic embryo explants in M6 medium Fragmented explants Intact explants(X8) Germination showing leaves and roots (X8) Greening and initiation of callusing after two weeks (X8) b Rapid callus growth Plantlet formation e c

Massive callusing in fragmented zygotic embryo in non-charcoal media M4 one week after transfer from media with charcoal (X8) Somatic embryogenesis in greened cotyledon of zygotic embryo explants one week after transfer to M4 medium

PHYTOCHEMICAL ANALYSIS Phenolics Lignans Blue color in the ethanolic extract of the callus with Folin Phenol reagent indicating presence of phenolics and absence of coloration in the control TLC plate sprayed with methanol: sulphuric acid (1:1) and heated 2 min at 150°C . Spots with Rf value 0.23, 0.36, 0.93 and 0.97

SYNTHESIS OF SILVER NANOPARTICLES Control Callus extract Callus (1.37 g) extract incubated with 1 mM AgNO3 turns reddish brown after 2 h

Absorbance peak at 450 nm indicates the presence of silver nanoparticles in callus cultures Synthesis and stabilisation of silver nanoparticles can be attributed to phytochemicals including phenolics. First report of synthesis of nanoparticles in this species. This is significant since there are very few reports of nanoparticle synthesis by callus cultures.

ANTI- BACTERIAL ACTIVITY-Inhibition zones obtained with various organisms Organism tested Ethanolic extract(15 μl) Silver nanoparticle suspension(15 μl) ATCC E.coli - 10 mm Pseudomonas aeruginosa* ATCC Staphylococcus aureus 14 mm Staphylococcus aureus** 16 mm * metallo- beta- lactamase producing clinical isolate of P. aeruginosa ** methicillin- resistant S.aureus standard strain Ethanolic extract and silver nanoparticles exhibited antibacterial activity . Activity against drug resistant strains was also obtained. Anti-microbial effect can be attributed to phenolics.

CONCLUSION Long term callus cultures have been established from the explants of M. charantia Cultured tissues with potential to synthesize phenolics and lignans in vitro manipulations may be carried out by use of elicitors 5 to enhance the yield. Callus cultures can be sources of anti-HIV, anti- tumour compounds of this species This is the first report of nanoparticle synthesis by this species . among the very few reports of production of nanoparticles by callus cultures of plants .

Synthesis of silver nanoparticles by the callus cultures is rapid and may be attributed to its secondary metabolites. Silver nanoparticles and the ethanolic extracts of the callus showed anti - microbial activity against drug-resistant microbes indicating their therapeutic potential. Results may be utilised for the large scale production of callus - to serve as sources of medicinal compounds - for rapid synthesis of non- toxic silver nanoparticles - to combat drug resistant bacteria.

REFERENCES 1. Fang EF and Ng TB (2011).Curr. Mol. Med. 11: 417-36. 2. Kumar V and Yadav SK (2009). J Chem Technol Biotechnol. 84: 151–157. 3. Satyavani K, Gurudeeban S, Ramanathan T and Balasubramanian T (2011). J of Nanobiotechnology 9: 43-50. 4. Murashige T and Skoog F (1962 ). Physiol Plant. 15: 473-497. 5. Sarin, R. (2005). Biotechnology 4 :79-93. Acknowledgement. Prof. P. Rammurthy, Director, National Centre for Ultrafast Processes, University of Madras (Taramani Campus) Chennai-600113 is gratefully acknowledged for permission to use the spectrophotometric facilities.