Bioactive Propensity of Macroalgae from the Andaman & Nicobar Islands

Aims: Marine macroalgae are the rich source of biologically active metabolites and potential source for development of novel biotechnological products. The present study was made to explore the metabolically active compounds from the macroalgae of the Andaman & Nicobar Islands. Methods and Material: Different solvents such as methanol (MeOH), ethyl acetate (EtoAc), butanol (BuOH) and aqueous (H2O) extracts of nine macroalgae were tested for antimicrobial, antibiofilm and cytotoxicity (brine shrimp larvae). Results: Out of the 36 extracts 27 extracts showed antimicrobial activity against the human pathogens and 14 extracts revealed antibiofilm activities. The three EtoAc extracts of Sargassum ilicifolium, MeOH extract of Sargassum sp. and MeOH extract of Padina tetrastromatica showed inhibition against 8 pathogenic bacteria. Also, aqueous extract of Padina tetrastromatica (71.82 %) and BuOH extract of Dictyosphaeria cavernosa (71.58 %) exhibited higher antibiofilm nature. The highest cytotoxic effect was exhibited by species Actinotrichia fragilis and all its four extracts significantly (P<0.01) inhibited the brine shrimp larvae, among this aqueous extract showed the lowest LC50 value, 31.7 μg/ml, followed by EtoAc extract, 89.33 μg/ml. Conclusion: It was observed that different species have different kind of bioactive nature.

saccharides inhibit activity of many bacterial species as well as viruses. 15Laminarin, a polysaccharides found in brown algae can act as a prebiotic and it has also antiviral and antibacterial properties. 16any macroalgae were widely studied for their antibacterial activity against pathogenic bacteria 6,[17][18][19][20][21] and still the investigations are going on.3][24][25][26] The brine shrimp cytotoxic assay is very useful for the isolation of biogenic compounds from plant extracts 27 and it is considered as a convenient probe for preliminary assessment of toxicity.Antifouling nature of macroalgae has also been studied widely and reports suggest that most of the marine macroalgae are potential source of antifouling compounds. 5,8,28Hence, the present study was aimed to screen the bioactive potentials including antimicrobial, antibiofilm and cytotoxic activity of macroalgae from the Andaman and Nicobar Islands.

Antibacterial assay
The antibacterial assay was performed by the disc diffusion method against 12 human pathogens viz.(5 mm Ø) was impregnated with 200 µg/disc of each extract and placed on Muller Hinton Agar plates (Hi-media, India) pre inoculated with the above human pathogens.The inoculated plates (triplicate) with extract loaded disc were incubated for 24 hrs at 37º C and the discs soaked with respective solvents used as negative control while kanamycin and streptomycin were as positive control under static condition.The assay was carried out in triplicate.The zone of inhibition which appeared around the disc after the completion of incubation period were measured in millimeter and recorded.

Anti-biofilm assay
The anti-biofilm assay was carried out as described by the modified methods of O'Toole and Kolter (1998). 29Briefly, 2 µL of aliquots (5 mg/1mL) of extract were coated to each well of a 96-well microtiter plate and 200 µL Vibrio azureus culture (18 hrs old) added to each well.Along with the test extracts respective solvent and marine broth were also added in separate wells as control.The microtiter plates with test samples were incubated at 28±2ºC for 24 hrs.After the incubation, the plates were washed with sterile water to remove planktonic Vibrio cells and the wells were stained with 200 µL of 0.1% crystal violet for 20 min.Finally, 200 µL of ethanol was added to the well to release the stain and the extent of biofilm staining was determined by measuring the absorbance of the resulting solution at 590 nm in a multimode micro plate reader (TECAN M200, Austria).All the assay were carried out in triplicate.

Brine shrimp cytotoxicity assay
The brine shrimp cytotoxic assay was performed using freshly hatched free swimming nauplii of Artemia salina.The assay system was prepared with 2 ml of filtered seawater containing specific concentration of extracts (100 to 600 µg) in a cavity block (embryo cup) with 20 nauplii.The experiment was carried out with test and control (solvent and filtered sea water) for 24 hrs, under constant illumination.The percentage of mortality was recorded after the incubation period and LC 50 of the test extract was determined using probit scale method. 30

Statistical analysis
A one -way ANOVA test was performed on the cytotoxic activity results to investigate significant differences among the effect of extracts.

DISCUSSION
The bioactive compounds produced by marine macroalgae are mostly used for its self-defence and these molecules have tremendous probabili- ties for usage in pharmaceutical industry.The present study disclosed the presence of potential bioactive compounds in the screened macroalgae and most of the species have at least one of the bioactive effects, such as antimicrobial, antibiofilm and cytotoxic effect.The four solvent extracts of all the nine macroalgae displayed different degree of antimicrobial activities against different pathogenic bacteria.Three extracts viz.EtoAc extract of Sargassum ilicifolium, MeOH extract of Sargassum sp., and MeOH extract of Padina tetrastromatica inhibited the eight pathogenic bacteria.The inhibitory effect of the methanol extract of Padina tetrastromatica also reported previously 31 and pointed out that the methanol extract (10 µl from 20 mg/1 ml) of P. tetrastromatica with the same concentration of the present study inhibited the growth of six human pathogens such as K. pneumonia, E. aerogens, M. luteus, B. subtilis, S. aureus and P. aeruginosa, while reports on ethanol extract (1 ml of aliquots) of P. tetrastromatica showed antibacterial activity against B. subtilis, E.coli, K. pneumoniae, S. typhimuruim S. aureus and P. vulgaris. 32f invertebrates.][41][42] Microfouling by bacteria can be prevented by two methods, one by bactericidal activity of natural or synthetic products or by inhibiting the biofilm formation.The bioassay study in the present work revealed that the tested fouling bacteria (Vibrio azureus) is highly sensitive to aqueous extract of P. tetrastromatica and BuOH extract of D. cavernosa (above 70% inhibition) and BuOH extract of Sargassum sp (above 50%).So it is evident that these algae possess better antibiofilm metabolites.The antibiofilm nature of Ulva reticulata, Sargassum wightii and Halimeda macroloba has also been reported. 43Recently, Yuvaraj and Arul 44 reported the antibiofilm activity of ten macroalgal extracts among this Sargassum wightii and Hypnea musiformis were prominent in inhibiting biofilm.Many of the secondary metabolites of macroalgae are well known for their cytotoxic activity.The brine shrimp cytotoxicity test has been used as a bioassay for a variety of toxic substances 45 and it is extrapolated for cell-line toxicity and anti-tumour activity as well.The cytotoxic property of plant material is due to the presence of anti-tumour compounds. 46n the present study, the higher cytotoxic profile of all solvent extracts of Actinotrichia fragilis and other macro algae Dictyosphaeria cavernosa and Padina tetrastromatica indicated the presence of high amount of cytotoxic substances.The previous inventions also reported on high cytotoxic profiles of macroalgae like Stoechospermum marginatum, S. swartzii, S. binderi, Spatoglossum asperum, Stokeyia indica and Caulerpara racemosa, 46 Acrosiphonia orientali, P. tetrastromatica and G. corticata 25 L. brandenii 26 and H. musiformis and U. fasciata. 23The Caulerpa sp. is well known for its cytotoxic effect because of compounds like caulerpin and caulerpenyne in Caulerpa cupressoides, 47 Caulerpa racemosa 48 and Caulerpa taxifolia. 49Fischel et al. 50showed that caulerpenyne caused early and marked shift into S phase followed by a blockage in the G 2 /M phase in the cell cycle.The comparatively high inhibitory activity shown by the extracts of Caulerpa peltata in this study might be due to the presence of the cytotoxic compounds caulerpenyne or caulerpin and further evaluation is required to confirm this.The extraction process of bioactive compounds were varied by researchers and the most used solvents are acetone, benzene, butanol, [51][52] ethanol, 43,53 methanol 26 and water. 19In our study an approach was made to analyse the effectiveness of the four solvents (MeOH, EtoAc, BuOH and H 2 O).The antibacterial activities of Sargassum species are widely studied and most of the reports are supporting the present study (200 µg /disc).
Rizvi 33 reported its inhibitory effects of Sargassum ilicifolium (200 µg) against Shigella dysenteriae, while Rebecca et al 34 examined 50 µl to 100 µl of 5 gm of powdered sample of Sargassum ilicifolium against human pathogen like Escherichia coli, Salmonella sp. and Klebsiella sp.Antibacterial activity of S.dentifolium and S.hystrix 35 against human pathogens and S. latifolium, 36 against shrimp pathogens were studied at 50 mg/ml concentration.Devi et al. 37 and Seenivasan et al. 38 were screened antibacterial effect of S. wightii against human pathogens using 75 and 20 µl of 5 mg crude extracts respectively.Biofilm formation (microfouling) is the initial step of biofouling followed by the attachment of macrofoulers such as spores of macroalgae and larvae

Figure1:
Figure1: Percentage of antibiofilm activity of active extracts of macroalgae.Data are mean (n = 3) ± SD and percentage of inhibition calculated with the OD values of broth and specific solvent controls.