Indian Journal of Marine Sciences Vol. 30, March 2001, pp. 49-52 Short Communication Blood anticoagulant activity of a green marine alga Codium dwarkense (Codiaceae, Chlorophyta) in relation to its growth stages M. Shanmugam, K. H. Mody*, R. M. Oza & B. K. Ramavat Marine Algae & Marine Environment Discipline, Central Salt & Marine Chemicals Research Institute, Bhavnagar-364 002, Gujarat, India Received 11 May 2000, revised 19 October 2000 Codium dwarkense was sampled from Porbandar from December 1998 to April 1999 and growth stages of each sample were examined. Sulphated polysaccharides (SPS) were extracted from each sample and blood anticoagulant activity was evaluated by Prothrombin Time (PT) test. Blood anticoagulant activity was minimum (CT ratio 1.75) in plants collected in vegetative stage without gametangium (December), marginal in plants with 40-50% gametangia (January and February), CT ratio 2.43 and 3.00, respectively, highest (CT ratio 3.75) in reproductive stage (March) where 100% gametangia were found. The activity again decreased (CT ratio 2.34) in lapse period with 98.3% gametangia (April). The activity was directly associated with sugar and sulphate content, which were more in the SPS from plant with 100% gametangia. Therefore, it can be concluded that biosysnthesis of anticoagulant by C. dwarkense is associated with abundance of its gametangia. Sulphated polysaccharides (SPS) are commonly found From a group of 13 green algal species belonging to in marine algae and higher animals and scarcely the family Codiaceae from Indian waters, Codium present in microbes and absent in higher plants. dwarkense and Codium tomentosum were identified Marine algal sulphated polysaccharides are reported as the most promising species. Bio-assay guided to have interesting bioactivities1 e.g. antiviral, purification of both these species yielded sulphated antitumor, hypolipidemic, blood anticoagulant arabinan as active molecular species10. This paper activities, etc. Heparin, a highly sulphated describes the blood anticoagulant activity of sulphated polysaccharide present in mammalian tissues, is used polysaccharides from different life stages of Codium as a blood anticoagulant commercially2. However, dwarkense Boergs. (Codiaceae, Chlorophyta). heparin has some disadvantages as it is extracted and Monthly samples of healthy, epiphyte free plants of purified from internal organs of higher animals C. dwarkense growing in the shallow tide pools and making its production difficult and it also exhibits puddles in the inter-tidal region on the coast of haemorrhagic like side effects2. These disadvantages Porbandar (22o 28´ N; 69o 05´ E), in the north west associated with heparin have opened up a new area of coast of India were collected during spring tide from antithrombotic research for discovering novel December, 1998 to April, 1999. Seasonal changes in anticoagulant agents. The anticoagulant activity of growth was determined by recording the mean length SPS from marine algae was first reported3 in 1936. of 10 plants in three replicates and calculating their The anticoagulant activities of marine algal SPS have standard deviation. Observations were made on recently been reviewed extensively4. Deacon-Smith fruiting behavior of C. dwarkense after taking the et al.5 have reported anticoagulant effects of British transverse sections of apical, middle and basal portion marine algae and mentioned for the first time that of the plants. Randomly ten observations on the anticoagulant activity was associated with extracts of morphology, length and breadth of vesicles and green algae. Subsequently, high molecular weight gametangia were made. Salinity, pH and seawater proteoglycans and low molecular weight sulphated temperature were recorded at the time of collection. polysaccharides from Codium fragile spp. atlanticum Samples of C. dwarkense were cleaned; quick-washed possessing blood anticoagulant activity were reported in tap water; dried in shade; powdered and, defatted and their mechanism of action was also proposed6,7. and depigmented in methanol. Defatted and Further, blood anticoagulant activity of some more depigmented sample (50g) of each collection was green algae including Codium latum were reported8,9. soaked in 20 volumes (w/v) of distilled water and kept _____________ overnight at 8 to 10oC. The extract was filtered *Corresponding author; E-mail: [email protected] through muslin cloth followed by Whatmann 541 50 INDIAN J. MAR. SCI., VOL. 30, MARCH 2001 filter paper. The extract was concentrated under performed in duplicate, and the average of the two reduced pressure (Rotavapor, Buchi RE 111, replicates was recorded. Standard blood anticoagulant Switzerland), precipitated with acetone (1:4 v/v), heparin (140.3 units/mg) was used for comparative dehydrated with acetone and dried at 40oC. The dried study. The results of the clotting tests were expressed product was dissolved in minimum distilled water, as a clotting time ratio (CT ratio). The ratio was dialysed (Sigma dialysis tubing, MWCO 12,000) and obtained by dividing the clotting time achieved with lyophilised (VirTis Freeze Dryer, USA) to obtain algal SPS included in the system by the time achieved crude sulphated polysaccharide product11. Yield of under similar conditions with normal saline solution. crude sulphated polysaccharide was calculated on dry Growth and fruiting behavior of C. dwarkense at weight basis. Ash content of SPS sample was the coast of Porbandar is given in Table 1. Codium. determined by igniting at 550oC for 6h. Total sugar dwarkense, generally grows during November to was estimated by phenol-sulphuric acid method12 and June. In December, the young plants were in protein content was estimated as per the method of vegetative stage with mean length 3.5 ± 0.5cm. In 13 Lowry et al. Sulphate content was determined January and February, mean length was 4.87 ± 1.12 cm 14 15 following Dogson & Price and Knutson & Jeans and 6.85 ± 1.99cm; 40% and 50% gamentangia were method was used for the estimation of uronic acid. All observed in apical portion of the plants respectively. the spectroscopic analyses were done on a Shimadzu Peak growth of 8.4 ± 1.94 cm mean length and 100% UV-160A UV-Visible spectrophotometer. gametangia on apical and middle portion of the plants Human blood was collected from healthy were recorded in reproductive stage (March). volunteers and normal human plasma was prepared as Marginal decline in mean length 7.4 ± 1.32 cm and follows: Blood was anticoagulated using 3.8% tri- percentage gametangial formation in apical portion sodium citrate in a polypropylene container (9 parts of (96%) was observed in lapse period (April). During blood to 1 part of tri-sodium citrate solution) and was investigation period, salinity ranged between 35- centrifuged immediately at 3000 rpm for 15 min. 37.5‰ and pH 7.27-8.454. Seawater temperature Plasma was separated and pooled. Pooled plasma was o o ranged between 18.0-26.5 C in December to stored at 4 C and it was used within 3-6 h of its February, 23.0-29.0oC in March and 26-32.0oC in collection. Algal SPS sample was prepared in normal April. saline (0.85% NaCl) solution. Freeze-dried crude SPS extracts of all samples Anticoagulant activity of algal SPS was tested were either colorless or slightly yellowish and fluffy using Prothrombin time (PT) test16 with 750μg/ml in nature. The yield varied from 1.51 to 3.46% and concentration. All clotting and control tests were ash content was ca. 25-30%. Chemical compositions Table 1 ⎯ Growth and fruiting behavior of Codium dwarkense at Porbandar Date of collection Mean length of 10 Mean (μm) of utricles Gameta- Mean (μm) of gametangia (Sample no.) plants (cm) Length Breadth ngia (%) Breadth Length 18-12-1998 3.5 ± 0.50 A: 391 ± 83.72 123 ± 30.90 Nil Nil Nil (1) B: 469 ± 70.59 141 ± 29.49 Nil Nil Nil C: 512 ±62.11 145 ± 28.23 Nil Nil Nil 17-1-1999 4.87 ± 1.12 A: 721 ± 76.39 187 ± 36.50 40 230 ± 25.0 84 ± 13.1 (2) B: 629 ± 120.4 167 ± 45.21 Nil Nil Nil C: 606 ± 73.97 174 ± 28.30 Nil Nil NIL 16-2-1999 6.85 ± 1.99 A: 570 ± 86.93 154 ± 30.67 50 235 ± 10.5 95 ± 10.0 (3) B: 663 ± 69.72 175 ± 36.77 Nil Nil Nil C 469 ± 67.86 143 ± 31.71 Nil Nil Nil 17-3-1999 8.4 ± 1.948 A: 513 ± 70.76 102 ± 27.21 100 238 ± 32.1 86 ± 14.0 (4) B: 676 ± 89.85 195 ± 51.57 100 182 ± 25.6 74 ± 26.9 C: 424 ± 99.82 147 ± 34.21 Nil Nil Nil 16-4-1999 7.4 ± .3291 A: 639 ± 68.44 166 ± 13.00 96.6 187 ± 40.1 54.0 ± 10.2 (5) B: 705 ± 91.00 180 ± 11.51 100 217 ± 30.8 76.6 ± 16.5 C: 605 ± 150.48 173 ± 05.04 Nil Nil Nil A = apical; B = middle; C = basal. SHORT COMMUNICATION 51 Table 2 ⎯ Chemical composition and blood anticoagulant activity of Codium dwarkense in relation to growth stages Sample Yield Total Sulphate Protein Uronic C H N CT ratio1 no. (%) sugar (%) (%) acid (%) (%) (%) (μg/ml) (%) (%) 500 750 1 2.11 29.46 17.17 16.20 2.80 25.31 4.94 2.13 1.77 1.75 2 3.17 26.40 19.79 16.26 2.45 25.16 5.05 1.76 1.83 2.42 3 3.02 38.24 22.37 6.98 2.56 21.23 4.52 0.60 2.00 3.00 4 3.46 39.20 26.01 6.94 2.34 21.22 4.51 0.90 2.38 3.75 5 3.00 37.37 16.94 13.96 3.08 25.21 4.95 1.86 1.10 2.34 1 Activity was measured by PT test; CT ratio of anticoagulant drug heparin (140.3 units/mg) in PT at 500μg/ml = 5.37 viz.