Maushmi S. Kumar a,* Asim K. Pal b MBTP26 SPONGOSORITES HALICHONDRIODES AS A POTENTIAL SOURCE OF BIOACTIVE COMPOUNDS Maushmi S. Kumar a,* Asim K. Pal b a*Department of Pharmaceutical Biotechnology, Shobhaben Pratapbhai Patel School of Pharmacy and Technology, Management; SVKM’S NMIMS, Vile Parle west, Mumbai- 400056, India b Department of Fish Nutrition, Physiology and Biochemistry, Central Institute of Fisheries Education, Versova, Yari Road, Andheri west, Mumbai. Abstract In continuation of our search for drug leads from Arabian Sea sponge Spongosorites halichondriodes, we have characterized and isolated several bioactive compounds from the ethyl acetate and butanol extract fractions. Identification of bioactive compounds was based on the bioassay guided fractionation and isolation. Three alkaloids (1,2,3) and two novel steryl glycoside derivatives A-B (4,5) have been isolated by chromatographic fractionation and purification of the bioactive ethyl acetate and butanol fraction. The glycosylated steryl compounds and piperidone exhibited positive antimicrobial activities without any cytotoxic property. The structures were determined by the UV, IR, 1D NMR and high-resolution mass spectral studies like GC-MS-MS and LC-MS-MS. In addition to these, other compounds like methyl and ethyl benzoate, decanoic acid, 2,5-dichloroaniline, N- nitroso dicyclohexylamine, 7-methoxy-6-methylcoumarin were also isolated and identified using GC-MS-MS . Introduction Marine sponges are the most primitive multicellular animals containing metabolites not found in their terrestrial counterpart, in particular glycolipids having unusual long chain Δ5, 9 fatty acids (FA) sometimes with a third double bond or a bromine atom (Litchfield and Morales, 1976). In earlier work, seven monoindole derivatives were isolated from the methanol extract of marine sponge Spongosorites sp. by bioactivity-guided fractionation. Among them, five compounds were identified to be unique indole pyruvic acid derivatives (Bao et al., 2007). Bis (indole) alkaloids of topsentin and hamacanthin class were also obtained from the same sponge. Compounds of the hamacanthin class exhibited more potent antibacterial activity than topsentin class against various strains of bacteria and fungi (Oh et al., 2006). These bis indole alkaloid have also displayed fibronectin-binding property which highlights the potential of these compounds for the treatment of S. aureus infections via inhibition of sortase activity (Oh et al., 2005). 2 3 1 Methods and Materials 5 4 General experimental Procedures In this study, λmax of UV absorption was determined with the help of UVD 340 S – Photo Diode Array Detector integrated to analytical HPLC. The samples were analyzed using their fragmentation pattern of the molecular ion and the spectrum was compared with the known GC-MS library in mass bank. 1H NMR spectra were obtained from SICART, Anand recorded at 300 ºK on Bruker 500 MHz NMR spectrometers. Biological samples The sponge was collected from Khardanda (19°4'37"N 72°49'25"E) and Bandra fort (19°2'32"N 72°49'8"E) at the western part of Mumbai, India. The sample was identified as Spongosorites halichondriodes (Dendy), Class: Demospongiae; Order: Halichondria; Family: Halichondriidae by ZSI, India. Extraction and Purification of compounds The dried samples was extracted for 2-3 cycles, each with 1 L acetone per 100 g of the biomass followed by three cycles of methanol dried under vacuum. The ethyl acetate residue (300 mg) was subjected to vacuum liquid chromatography equilibrated with ethyl acetate and methanol gradient to give ten fractions. Fractions eight and ten were loaded on silica column using ethyl acetate: methanol (8:2) as an eluting solvent to give eight sub fractions. Biological Evaluation of the Compounds Antimicrobial activity was performed using disc diffusion method. Cytotoxic activity was carried out at Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Navi Mumbai using human breast cancer cell line MDA-MB-435. Figure 1. Structures of N-Methyl-2-pyrrolidone (1), 2- Pyrrolidone (2), 2, 2, 6, 6-Tetramethyl-4-piperidone (3), Glycosylated steryl derivative A (4), Glycosylated steryl derivative B (5) and other isolated from the marine sponge Spongosorites halichondriodes   Table 1. Zone of inhibition (cm) of antimicrobial activity of ethyl acetate and butanol extract fractions   Conclusions Our search for marine-sponge bioactive compounds from Arabian Sea marine sponge Spongosorites halichondriodes from Mumbai coast led us to identification of 9 known and two novel steryl glycoside derivatives. These compounds were isolated and their chemical structures were predicted using spectroscopic studies like GC-MS and proton and 13C NMR. The alkaloids belonged to pyrrole and piperidine class and the glycosylated derivatives are novel. Biological evaluation of the compounds showed that all of these compounds possessed strong antimicrobial activity with no cytotoxicity, suggesting them for development as therapeutic agents for human use in future. Results Bioactive compounds isolated from S. halichondriodes by GC-MS-MS and 1D proton NMR. The compounds isolated in pure form were N-Methyl-2-pyrrolidone (1), 2- Pyrrolidone (2) 2, 2, 6, 6-Tetramethyl-4-piperidone (3) , Glycosylated steryl derivative A (4), Glycosylated steryl derivative B (5). Fig 2: Isolation and identification of compounds from Spongosorites halichondriodes References Dr. Maushmi S. Kumar Assistant Professor, Department of Pharmaceutical Biotechnology, Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’S NMIMS University, Vile Parle west, Mumbai- 400056 Email: maushmiskumar@gmail.com Website: www.nmims.edu Phone:+91-22-42332023 Contact Litchfield, C., Morales, R.W. (1976). Are Demospongiae membranes unique in living organisms? In: Harrison FW, Cowden RR (eds.), Aspects of Sponge Biology, Academic, New York, 183. Bao, B.,  Zhang, P., Lee, Y., Hong, J., Lee, C.O., Jung J.H. (2007). Monoindole alkaloids from a marine sponge Spongosorites sp. Marine Drugs, 5(2): 31-39. Oh, K.B., Mar, W., Kim, S., Kim, J.Y., Lee T.H., Kim, J.G., Shin, D., Sim, C.J., Shin J. (2006) Antimicrobial activity and cytotoxicity of Bis(indole) alkaloids from the sponge Spongosorites sp. Bio Pharma Bull, 29 (3): 570-573. Oh, K.B., Mar, W., Kim, S., Kim, J.Y., Lee T.H., Kim, J.G., Shin, D., Sim, C.J., Shin, J. (2005). Bis (indole) alkaloids as sortase A inhibitors from the sponge Spongosorites sp. Bioorganic and Medicinal Chemistry Letters, 15: 4927-4931.