Purification, Characterization and Antioxidant Activity of Green Seaweed Codium sp

In the present study the Codium sp. polysaccharide was extracted and partially purified in DEAE cellulose. The polysaccharide was estimated for carbohydrate, sulfate content and uronic acid. The elemental analysis of polysaccharides was analyzed for carbon, hydrogen, nitrogen and sulfur. The antibacterial activity of polysaccharide showed maximum 19 mm of inhibition zone against Bacillus cereus and 12 mm of inhibition zone against Xanthomonas sp. Free radical scavenging activity of polysaccharide from Codium sp. was assayed for total antioxidant capacity, reducing power, hydrogen peroxide scavenging activity, DPPH, ABTS, hydroxyl scavenging assay, superoxide anion radical scavenging and nitric oxide. The green seaweed Codium sp. polysaccharide showed rich sources of antibacterial and antioxidant activity.

The major polysaccharides of green algae are more heterogeneous in sugar compositions, three main groups are glucurono xylo rhamnans, glucurono xylo rhamno galactans and xylo arabino galactans. Red algae are galactans commercially known as agar and carrageenan and those of brown algae are fucans, including fucoidan, ascophyllan, sargassan and glucurono xylo fucan [3].
Polysaccharides comprise a complex group of macromolecules with a wide range of important biological properties. Marine algae are the most important source of non-animal polysaccharide. Polysaccharides from algae possess important pharmacological activities such as anticoagulant [4], antioxidant [5], anti-inflammatory, antiviral [6], antibacterial [7], antiproliferative [8], antitumoral [9], anticomplementary [10] and antiadhesive activities [11]. In recent years, algal polysaccharides especially that extracted from phaeophyta are generally known as fucoidans, as they are rich in the sugar, fucose. They have been demonstrated to play an important role as free radical scavengers and antioxidants for the prevention of oxidative damage in living organisms [12].
The algal polysaccharides play an important role as free-radical scavengers in vitro and antioxidants for the prevention of oxidative damage in living organisms. The present study was designed to study the partial purification, antibacterial and antioxidant activities of polysaccharides of Codium sp. [13].

Extraction and Partial Purification
Polysaccharide was extracted from the green seaweed Codium sp. [14]. The crude polysaccharide was further purified by DEAE-cellulose column chromatography [15].

Estimation of Total Carbohydrates
The carbohydrates content in polysaccharide was estimated by phenol sulphuric acid method [16].

Determination of Sulfate Content
Sulfate content in polysaccharides was determined by the barium chloride gelatin method [17].

Determination of Uronic Acid Content
The carbazole reaction which is the most satisfactory method for estimating uronic acid in polysaccharide was employed for quantification [18].

Analysis of Elements
The percentage content of carbon, hydrogen, nitrogen and sulfur of polysaccharides from Codium sp. was estimated by using the operating mode of PE 2400 series II CHNS/O analyzer EA1112 (CE Instrument, Italy).

Antibacterial Assay
To study the antibacterial activity of the polysaccharides, nine human clinical pathogens were selected for the present study, namely Klebsiella sp. Escherichia coli, Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Serratia sp. Citrobacter freundii and Clostridium sp. The three plant pathogens namely, Erwinia sp. Xanthomonas citri and Xanthomonas sp. Antibacterial activity of polysaccharides from Codium sp. was determined against nine clinical pathogens and two plant pathogens using paper disk assay method [19].

Antibacterial Activity of Polysaccharides against Pathogens
The Codium sp. polysaccharides showed maximum of 19 mm of inhibition zone against Bacillus cereus and maximum of 10 mm of inhibiting zone against Pseudomonas aeruginosa. The Codium sp. polysaccharides showed maximum of 15 mm of inhibition zone against Erwinia sp. and maximum of 12 mm of inhibiting zone against Xanthomonas sp. of plant pathogens.

Discussion
In present study the total carbohydrate present in the crude extracts of Codium sp. was found to be maximum in phenol sulphuric acid method (Dubois et al., 1956) 63.21 ± 0.25%. The sulfate content was found to be 9.18 ± 0.54% for Codium sp. The uronic acid was found to be 3.45 ± 0.74% for Codium sp. Similarly, Mahendran and Saravanan [15] reported total carbohydrate content (47.43%), sulfate content (12.86%) and uronic acid content (4.9%) from the polysaccharide from green seaweed C. racemosa which supports the present study.
In the present study, the antibacterial properties of Codium sp. polysaccharides against nine human and three plant pathogenic strains using tetracycline as a standard. The Codium sp. sulfated polysaccharides showed maximum of 19 mm of inhibition zone against Bacillus cereus and maximum of 10 mm of inhibiting zone against Pseudomonas aeruginosa. The Codium sp. sulfated polysaccharides showed maximum of 15 mm of inhibition zone against Erwinia sp. and maximum of 12 mm of inhibiting zone against Xanthomonas sp. of plant pathogens. Rodrigo et al. [29] reported that the crude sulfated polysaccharides from G. ornata was tested on the growth of bacteria B. subtilis, S. aureus, E. aerogens, E. coli, P. aeruginosa, S. choleraesuis and S. typhi.
The reducing properties are generally associated with the presence of reductions. Reductions were reported to be terminators of free radical chain reactions by donating a hydrogen atom. In most cases, irrespective of the stage in the oxidative chain, in which the antioxidant action is assessed, most non-enzymatic antioxidant activity is mediated by redox reactions [31]. In the present study, the sulfated polysaccharides polysaccharides from Codium sp. [(0.248 ± 0.45%) -(1.579 ± 0.32%)] and were compared with the standard ascorbic acid [(0.447 ± 0.16%) -(1.956 ± 0.24%)].
In the present study, the hydrogen peroxide scavenging activity of polysaccharides of Codium sp. is 79.34 ± 0.18%. Collen and Pedersen [32] reported that C. taxifolia when epiphytized by Lophocladia lallemandii showed increased lipid peroxidation which could be related to the increased H 2 O 2 production to compete against Lophocladia rather than a marker of oxidative damage.
In the present study, the ABTS inhibition assay for the polysaccharides from Codium sp. is 69.74 ± 0.49%. Mahendran and Saravanan [15] reported that the ABTS assay from polysaccharide of C. racemosa (73.32 ± 1.27%). In the present study, the hydroxyl scavenging assay Codium sp. is 69.74 ± 0.49%. The metal complexes thus formed cannot further react with H 2 O 2 to give a hydroxyl radical [34].
In the present study, the superoxide anion radical scavenging assay for the Codium sp. is 66.43 ± 0.27%. Mahendran and Saravanan [15] reported that the superoxide anion radical scavenging activity of polysaccharide from C. racemosa was found to be (66.17 ± 0.77 %). In the present study, the nitric oxide scavenging assay for the Codium sp. is 65.74 ± 0.18%. Mahendran and Saravanan [15] reported that the nitric oxide scavenging activity of polysaccharide of C. racemosa was found to be (40.64 ± 1.82%).

Conclusions
The present study concludes the opportunity to project the importance of marine green algae possessing antibacterial and antioxidant value since India has rich in biodiversity of marine algae. The sulfated polysaccharide extracted from Codium sp. showed higher antibacterial and antioxidant activity. Hence the future research and development of India should pay more attention for the development of drug systems from marine algae especially green algae to save the life of mankind.