Antibiotic Efficacy and Biochemical Composition of Ascidian, Phallusia Nigra (Savigny, 1816) from the Palk Bay, Southeast Coast of India

Indian Journal of Geo-Marine Sciences Vol. 45(6), June 2016, pp. 795-799

Antibiotic efficacy and biochemical composition of ascidian, Phallusia nigra (Savigny, 1816) from the Palk bay, Southeast coast of India

Kathirvel Iyappan & Gnanakkan Ananthan* Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, 608 502, Tamil Nadu, India. [Email: [email protected]]

Received 10 March 2015; revised 02 July 2015

An investigation was held out to examine the biochemical constituents present in the simple ascidian Phallusia nigra (P. nigra), such as protein, carbohydrate, lipids, moisture, acidity and ash content. Extractions of P. nigra (visceral) tissue sample in several solvents were assayed for antibacterial activity against foodborne pathogens. Protein content was found to be higher during the summer season in P. nigra, while the minimum quantity of lipid was recorded during monsoon season. Benzene and acetonitrile crude extracts inhibited the growth of foodborne pathogens. Chloroform extract didn't inhibit any strains.

[Keywords: P. nigra, proximate composition, mantle extraction, foodborne pathogens, Palk Bay Ascidian]

Introduction and biodeposition17. Temperate coastal areas Over the 50 years, a number of ascidian ascidian is characterized by great seasonal species have spread across a good deal of the fluctuations in environmental variables such as 1 globe . A great balance of natural compounds temperature and nutritional condition18. 2 has been pulled up from marine invertebrates . Temperature and salinity are two of the most Ascidians are conspicuous members of marine important factors affecting the distribution of fouling and benthic communities. Ascidians marine invertebrates, thus determining invasion accumulated high levels of vanadium in their success non-native ascidian species. 3 tissues which is obviously non-toxic . Reproductive and developmental processes in Ascidians are capable of producing an ascidian are known to be affected by many unfavorable or toxic condition immediately different types of environmental factors, 4 above the surface as a chemical defense . Their including salinity, temperature, food quality, soft-bodied morphology provides ascidians with photo period, and ocean hydrodynamics. High little obvious structural defense from predation. acidity due to sequestered sulfuric acid in outer Ascidians have been considered to rely on tunic tissues, or high tissue concentrations of chemical defenses against predations. It is the heavy metal vanadium15, 19 currently, there regarded as highly nutritious seafood. are insufficient data to characterize Consumption just after harvest is favored for environmental tolerances of sedans in India. the flavor as a metallic taste may become Ascidians are rich sources of compounds with evident as the product becomes less fresh. cytotoxic proprieties; these marine natural Ascidians have increasingly come to be known products can be used for the discovery and as excellent model organisms in neurobiology development of novel chemotherapeutic and endocrinology, as good as in developmental agents21. In this paper, we detail the seasonal 5-14 biology and evolutionary biology . Spicules, variation in physiological energetics and gross originally indented to provide more or less anti- biochemical composition of ascidian P. nigra predatory defenses for the marine invertebrates collected from Palk Bay, southeast coast of 15 that secrete them in their tissues . Ascidians India. Present study aims at unraveling the are characterized by a tough outer tunic made of biochemical composition of P. nigra and its 16 the polysaccharide tunicin . Large populations antibiotic potentials of ascidians can also have a negative effect on coastal aquaculture systems through phytoplankton consumption, nutrient cycling 796 INDIAN J. MAR. SCI., VOL. 45, NO. 6 JUNE 2016

Materials and Methods To estimate the moisture content, 1g of fresh Ascidian collection and extraction tissue was dried through a hot air oven at a Ascidian, Phallusia nigra (P. nigra) constant temperature of 105ºC for 24hours21. was collected regularly during four seasons in The loss of weight was taken as moisture (1–2 m depth) Palk Bay region, Southeast coast content. of India, from February 2013 to January 2014. Total protein was estimated using the Collected samples were placed in zip lock bags Biuret method of Raymont et al24. The total in coolers on the ice and transported to the carbohydrate in a dried sample was estimated laboratory for further studies. Species were spectrophotometrically following the phenol- identified through as per the standard protocol21, sulfuric acid method of Dubois et al25. 22 (Kott 1985 and 1990). Each individual was The lipid in the dried sample tissue was cleaned with the sea and sterile water by the gravimetrically estimated following the hand of any associated plant or animal material. chloroform- methanol mixture method of Folch Ascidians tissue samples were air dried for a et al26. few days after drying made into powder using a Ash content was determined mortar and pestle for biochemical analysis such gravimetrically by incinerating 1g dried sample as protein, carbohydrate and lipids. in a muffle furnace at about 55ºC for 6 hours27 and results are expressed in percentage. Biochemical analysis Ascidian (visceral) tissue extractions Antibacterial assay were based on methods given in McClintock et Antibacterial activity was determined al.23 and in Koplovitz et al.24. Frozen tissues against cultures of Bacillus cereus (MTCC were transferred to at −80 °C freezer for several 6629), Salmonella entrica (MTCC 3244), hours, then lyophilized (Freeze drier: Lark, Vibrio vulnificus (MTCC 11) using the agar Penguin) and re-weighed. The lyophilized diffusion assay method28. Three plates tissues were then extracted with different containing three wells were tested against solvent such as benzene, chloroform, ascidian sample at 1ml/well. Following acetonitrile thrice for 24 h to produce a incubation at 37ºC for 24 hours, the diameters lipophilic extract. Extracts were filtered through (mm) of the growth inhibition halos were coarse filter paper and the solvent was removed determined using a perimeter. using rotary evaporation to yield a dry lipophilic extract. Natural tissue level Results concentrations of extracts used for the Biochemical analysis biological activity. A high level of acidity (pH 6.5%) was measured on the outer surface of the tunic of The acidity of the outer surfaces of the the ascidian, P. nigra during the summer ascidian tunics was measured using standard season. While a low-level acidity (pH 2.3%) analytical pH strips. Tunic surface pH that ranged during monsoon season. measurements were first conducted by gently laying a pH strip with a broad sensitivity range Table 1. Biochemical composition of ascidian, P. of 0 to14 whole pH units onto the outer tunic nigra surface. Subsequently, higher resolution was obtained using pH strips capable of resolving pH to an accuracy of 0.3 pH units. Following

Seasons isture (%) (%) (%) measurements of the tunic surface, the tunic ic acidity Ash (%) Mo Lipids (%) Protein (%) Protein (%)

was sliced with a razor blade and the pH Tun Carbohydrate measured once again by inserting pH strips into the slice. This allowed a measurement of the Pre 4.5 69.5 11.25 6.8 1.90 1.99 monsoon internal pH of the tunic. Because the pH test Monsoon 2.03 97.8 7.1 2.3 1.03 0.14 was based on color visual discrimination, we Post 3.95 75.6 8.60 5.4 2.97 1.85 considered this a qualitative measurement and monsoon chose to round each pH measurement off at 0.5 summer 6.5 68.3 15.40 8.29 3.95 1.56 unit increments so as to be conservative.

IYAPPAN & ANANTHAN: ANTIBIOTIC EFFICACY AND BIOCHEMICAL COMPOSITION OF ASCIDIAN 797

Variations of moisture content in antimicrobial potential against foodborne species of the ascidian, P. nigranigra were ranged pathogens. fromfrom 68.3% to 97.8%. The minimum (68.3%) content was noticed in during the summer Table 2. Antibacterial activity of ascidian, P.nigra against season while maximum (97.8%) content was foodborne pathogens recorded in during monsoon season. Zone of Inhibition (mm) Food The protein of ascidian varied from pathogens Benzene chloroforme acetonitrile 7.1% to 15.40%. Maximum (15.40%) content B. cereus 10.25 - 11.5 of protein was observed in P. nigra during MTCC 6629 summer and the lowest content of protein was (7.1%) recorded on during monsoon season. Salmonella 12.55 - - entrica MTCC The carbohydrate content of ascidian, 3224 P. nigra was ranged from 2.3% to 8.9%. V.vulnfticus 11.12 - 10.12 Minimum (2.3%) was observed during MTCC 1145 monsoon season and the maximum 8.29% was observed in during the summer seaseason.son. Discussion In this study estimate the tunic acidity, protein, carbohydrate, lipid, moisture and ash contents and antibacterial assay in species of the ascidian, P. nigra. During the summer season, the acidity (6.5%), protein (15.40%) and carbohydrate (8.29%) was highly present in P. nigra while low acidity (2.03%), during pre-monsoon and protein content (7.1%) were recorded during monsoon. The water content of muscle obviously rises and this makes the flesh weak. This is the reason for the poor eating quality of spawning ascidians. Lipid content has been very low in the ascidians as compared Fig.1. Biochemical composition of ascidian, P. nigra in to protein and carbohydrate. Epelbaum et al29 seasonal wise. reported ascidians environmental tolerances

The lipid content of ascidian, P. nigra and preferences such as temperature and was ranged from 1.03 to 3.95%. The minimum salinity, generally, Botryllus schlosseri survived (1.03%) was recorded during monsoon season environmental conditions of 10-25ºC and 14- while the maximum content was observed in 38‰ , positive growth at 10-25 ºC and 20-38‰ summer season and Botrylloides violaceus tolerated Ash content in species of the ascidian, environmentalenvironmental conditions between 5-25 ºC and P. nigra was ranged from 0.14 % to 1.56%. 20-38‰, positive growth at 15-25ºC and26- 38‰. Low content lipids of ascidians have The mminimuminimum (0.14%) was recorded in during 30 monsoon season. While the maximum (1.59%) been already reported by park et al . The was recorded in during summer seasons. protein content of ascidians showed variation, these values are comparable with those previous Antibacterial assay reported. DDefensesefenses in ascidians have also been Benzene, chloroform and acetonitrile evaluated in the context of food value with some investigators suggesting ascidian tissues extract of ascidian tissue (visceral) studied in 31 this work showed antimicrobial activity against areare low in nutrients and energy . However, a some of the foodborne pathogens.pathogens. The benzene numbernumber of studies have demonstrated that the extract showed antimicrobial activity against B. nutritional value of ascidians is at or above that cereus MTCC 6629(10.25mm), Salmonella of other sessile marine benthic invertebrates such as sponges commonly consumed by entrica MTCC 3224(12.55mm) and V. 23, 32 vulnificus MTCC 1145(11.12mm). Present predators . These studies indicate that results are given in Table 1. These findings ascidians are generally high in protein and suggested that P. nigra tissue (vvisceral)isceral) has an carbohydrate and have tissue energy values ranging from 6.0 to 22.4 kJ per gram dry weight. IInn this study, the protein content was 798 INDIAN J. MAR. SCI., VOL. 45, NO. 6 JUNE 2016

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