Indian Journal of Marine Sciences Vol. 31(1), March 2002, pp. 33-38

Screening of Codiacean algae (Chlorophyta) of the Indian coasts for blood anticoagulant activity

M. Shanmugam, K. H. Mody, B. K. Ramavat, A. Sai Krishna Murthy & A. K. Siddhanta*

Marine Algae and Marine Environment Discipline, Central Salt and Marine Chemicals Research Institute, Bhavnagar 364 002, Gujarat, India

[E-mail: salt@csir.res.in] Received 16 March 2001, revised 3 December 2001

Thirteen species of marine algae belonging to the family Codiaceae were selected from the Indian coast to study their sulphated polysaccharide (SPS) contents and their blood anticoagulant activity. The algal species studied include Codium dwarkense, C. tomentosum, C. indicum, C. geppei, C. iyengarii, C. coronatum, C. tenue, C. decorticatum, Udotea indica, U. flabellam, Halimeda tuna, H. gracilis and Avrainvillea erecta. Cold and hot water extracts were prepared from all the species and chemical compositions (e.g. total sugar, sulphate, protein and uronic acid contents) were analysed. All the SPS samples were screened for their blood anticoagulant activity (PT test). It was observed that blood anticoagulant activity (cf. Codium spp.) is generally higher with samples containing higher sugar and sulphate contents. Neutral sugar composition was analysed and active extract was found to contain relatively more arabinose. Seasonal variation in chemical composition and blood anticoagulant activity of the SPS of C. dwarkense were also studied alongside. It was observed that matured plants showed higher activity than the younger ones and arabinose was detected in higher quantity in the former. This work reveals that a few Codium species e.g. C. dwarkense, C. indicum and C. tomentosum, C. geppi produce strongly active blood anticoagulant SPS while the ones obtained from the other Indian Codiaceae species under investigation exhibit very low activity.

[ Key words: Blood anticoagulant, Codium species, Codiaceae, Udotea, Halimeda, Avrainvilliea ]

Sulphated polysaccharides (SPS) belong to the class of compounds having hemi-ester sulphate groups in their sugar units. These are commonly found in marine algae and higher animals and scarcely present in microbes and absent in higher plants. Heparin, a highly sulphated polysaccharide present in mammalian tissues, is used as blood anticoagulant both in laboratory and therapeutics. The sulphated polysaccharides from marine algae are highly diverse in nature and there exist similarities between their structure and that of heparin. The anticoagulant activity of SPS from marine algae was first reported1 in 1936. Heparin has some disadvantages. It is extracted from internal organs of higher animals and purified, hence its recovery is difficult, and it exhibits haemorrhagic side effects2.

Anticoagulant activity associated with the SPS of red and brown algae have been widely reported3-6. Heparin analogues (heparinoids) that are inhibitory to thrombin activities have been reported from Chlorophyta of Indian coasts for the first time7.

Metabolites of this group of species, in general, have been reported8 to exhibit interesting biological activities e.g. antibacterial, antiviral (including anti-HIV), cytotoxic and ichthyotoxic activities etc. In this paper we report the distribution of antithrombic sulphated polysaccharides and their chemical composition in several plants, of the family Codiaceae of Indian waters, as well as the seasonal variations in chemical composition and blood anticoagulant activity of the SPS of Codium dwarkense Boerges. for the first time.

Material and Methods Different species of algae of the family Codiaceae

(Codiales/Chlorophyta) were collected from Gujarat and Tamil Nadu coasts of India. The species belonging to the family Codiaceae are commonly found along Gujarat coast and found rarely along the Tamil Nadu coast. Avrainvillea sp. which occurs sparingly is restricted to mud covered areas of coastal Tamil Nadu. For chemical investigation and bioactivity studies, 13 Codiacean plants comprising one Avrainvillea [A. erecta, (Berk.) Gepp.], two

_________________ *Correspondent author

Indian J. Mar. Sci., Vol. 31, No. 1, March 2002

34

Halimeda spp. [Halimeda gracilis Harv. ex. J. Ag., H. tuna (Ell. et Sol.) Lamour], two Udotea spp. (Udotea indica A.& E. S. Gepp. and U. flabellum How.) and eight Codium species [Codium dwarkense Boergs, C. tomentosum (Huds.) Stackh, C. coronatum Setch., C. decorticatum (Woodw.) Harvey, C. geppi Schmidt, C. tenue Kuetz, C. iyengarii Boergs, C. indicum Dixit] were collected. Species of genera Codium, Halimeda and Udotea are found at both Gujarat and Tamil Nadu coasts. The sampling details are shown in Table 1.

Algal materials were thoroughly cleaned by removing contamination of other algae, epiphytes and animal castings and materials were quick-washed with tap water, dried in shade and powdered. Algal powders were depigmented and defatted by methanol in a Soxhlet apparatus. Cold and hot water extractions of the algal materials, testing of the extracts for anticoagulant activity [expressed as clotting time ratio (CT ratio) using heparin as standard] and chemical analysis were carried out using the procedure as reported earlier7,9. For blood anticoagulant activity studies, 2 mg/ml concentration of SPS samples was used.

Results Chemical composition and blood anticoagulant

activity of cold and hot water extracts of different Codium species and those of other Codiacean species

under investigation are presented in Tables 2 and 3 respectively. Table 4 depicts the seasonal variation in chemical composition and blood anticoagulant activity of C. dwarkense.

Freeze-dried SPS of all Codium spp. were colorless to slightly yellowish in color. However, all cold water extracts were fluffy and smooth and hot water extracts were fibrous. SPS of Avrainvillea, Halimeda and Udotea indica species were yellow and powdery, and those of U. flabellum were colorless and fluffy in nature. Sulphated polysaccharides of all the Codiacean plants were easily soluble in water and sparingly soluble in dimethyl sulfoxide, however, the hot water extracts took longer than cold water extract to get dissolved. They were soluble in 0 to 10 N sulphuric acid, nitric acid and hydrochloric acid, 0 to 25 % ethyl alcohol, 0 to 10 % methanol, propanol and acetone and 0 to 5 N aqueous sodium hydroxide solution and insoluble in benzene, chloroform, ether and cyclohexane. Yields were calculated on the basis of dry weight of the sample. Yields, total sugar, sulphate, uronic acid and protein contents of cold water extracts of all Codium spp. mentioned were in the range of 1.2 to 7 % and hot water extracts range 2.4 to 5.0 % (Table 2). In all calcareous algae e.g., Avrainvillea, Halimeda and Udotea yields were very poor (Table 3). In all the extracts, ash content was directly proportional to the sulphate content. Ash

Table 1 ⎯ Collection data of Codiacean algal speciesa

Species Place (lat / long) Period of collection

Habitat

Codium dwarkense Okha (22°28'N; 69°05'E) December 1994- April 1995

Lower intertidal zone

C. coronatum Manapadu (8°45’N; 78°12'E) February 1997 Subtidal (10 m) C. decorticatum Tiruchendur (8°45'N; 78°12'E) February 1997 Subtidal (10 m) C. iyengarii Okha (22°28'N; 69°05'E) February 1996 Intertidal zone C. indicum Okha (22°28'N; 69°05′E) February 1996 Intertidal zone C. geppi Porbandar

(21°38′N; 69°37'E) February 1996 Intertidal zone

C. tenue Mandapam (09°16'N; 79°12′E)

March 1997 Lower intertidal zone

C. tomentosum Mandapam (09°16'N; 79°12'E)

March 1996 Lower intertidal zone

Avrainvillea erecta Mandapam (09°16'N; 79°12'E)

February 1997 Subtidal (10 m)

Udotea flabellum Mandapam (09°16'N; 79°12'E) February 1997 Subtidal (10 m) Udotea indica Okha (22°28'N; 69°05′E) March 1997 Intertidal zone Halimeda gracilis Mandapam

(09°16'N; 79°12'E) February 1997 Subtidal (10 m)

Halimeda tuna Diu (20°54'N; 70°22'E) March 1996 Intertidal zone

aVoucher specimen of each species has been deposited with CSMCRI Herbarium, Bhavnagar

Shanmugam et al.: Screening of Codiacean algae

35

contents of cold and hot water extract of Codium spp. were ca. 25 % and 15 % respectively. The total sugar content was higher in all cold water extracts of Codium species which was between 29 to 34 % but in hot water extract it ranged from 18 to 24 %. In other Codiacean plants total sugar content was higher in hot water extracts than in the cold water ones. Sulphate content was higher in all cold water extracts than that of hot water extracts of species of genera Codium and Avrainvillea. In Halimeda spp. hot water extracts contain more sulphate than the cold water extracts. But in Udotea spp. no clear trend in sulphate content could be observed. Uronic acid content in Codium spp. was 1.5 to 3.0 % and 2.7 to 6.0 % in cold and hot water extracts respectively. In Udotea spp. it was higher in cold water extract than in hot water extract, and in A. erecta and Halimeda spp. the difference in the uronic acid in these extracts was marginal. In A. erecta, Udotea spp. and Codium spp. cold water extracts contained lower protein than the hot water extracts.

Blood anticoagulant activity showed that all cold water extracts of Codium sp. exhibited higher CT ratio than those of hot water extracts. However, the highest activity was observed in the cold water extracts of C. tomentosum, C. dwarkense and C. tenue. In Udotea spp., hot water extracts showed relatively higher CT ratio whereas both cold and hot water extracts of A. erecta and Halimeda spp. exhibited marginal anticoagulant activity. Neutral sugar composition of the most active cold water extracts of C. dwarkense and C. tomentosum were composed of arabinose and galactose, whereas hot water extract of those species were a mixture of arabinose, galactose, mannose, glucose and rhamnose. Hot water extract of Udotea flabellum was a mixture of arabinose, galactose, mannose, xylose, rhamnose, ribose and glucose, of which galactose was pre-dominant. In Halimeda tuna and Avrainvillea spp. arabinose, galactose, mannose, xylose, glucose and rhamnose were detected in varying quantities (Table 3). On the basis of these data and biogenetic consi-deration, it is presumed that the carbohydrate profiles of Codium iyengarii, C. geppei, C. decorticatum, C. coronatum, C. indicum and C. tenue resembled those of C. dwarkense and C. tomentosum. Halimeda gracilis did not contain rhamnose.

The yields of cold water extracts of Codium species collected in different months ranged from 1.53 to 2.88 % and hot water extracts were 2.40 to 4.68 %

Indian J. Mar. Sci., Vol. 31, No. 1, March 2002

36

(Table 4). The yields of cold and hot water extract were higher for the samples collected in February and April respectively. Ash contents of cold (ca. 22 %) and hot water (ca. 14 %) extracts remained unchanged in all the respective samples. There was little difference in chemical composition e.g. total sugar, sulphate, protein and uronic acid contents of cold and hot water extracts of algal materials collected during different months in a season. However, all cold water extracts had higher sugar and sulphate contents and lower protein and uronic acid contents than those of their respective hot water extracts. Analysis of neutral sugar revealed that cold and hot water extracts contained different types of sugar. Cold water extract was characterised mainly by arabinose, galactose and trace amount of mannose whereas hot water extract contained mainly mannose and galactose with minor quantity of arabinose and trace amount of glucose. It is also interesting to note that xylose is present only in cold water extract of December collection i.e. in the younger plants, rhamnose being detected only in hot water extract of April collection i.e. in the matured plants. Cold water extracts showed relatively higher blood anticoagulant activity, the one of April collection exhibiting the highest. Analysis of neutral sugar composition of the most active cold water extract of April collection revealed the presence of only arabinose and galactose. Other cold water extracts were characterised by the predominance of

arabinose and galactose, and varying quantities of xylose or mannose (Table 4).

Discussion Cold water extracts of all Codium species contain

higher sugar and sulphate contents and lower protein and uronic acid contents. Also, cold water extracts exhibited higher blood anticoagulant activity than that of hot water extract. In Udotea spp. hot water extracts showed relatively higher blood anticoagulant activity which contained higher sugar and sulphate contents and lower protein and uronic acid contents. Hence it can be inferred that higher blood anticoagulant activity is associated with relatively higher contents of sugar and sulphate and lower protein and uronic acid contents. Both cold and hot water extracts of A. erecta and Halimeda spp. exhibited marginal blood anticoagulant activity. Similar pattern was reported10 for brown algal SPS.

Cold water extract of C. tomentosum, C. dwarkense and C. tenue containing comparatively lower uronic acid and higher sulphate showed stronger blood anticoagulant activity. Cold water extracts of some of the species like C. geppei and C. coronatum containing less sulphate and more uronic acid showed relatively poor blood anticoagulant activity. Therefore, it appears likely that higher blood anticoagulant activity is associated with higher sulphate content. Uehara et al.6 have reported that cold water extract of Codium latum

Table 3 ⎯ Chemical composition and blood anticoagulant activity of SPS of other Codiacean algae

A. erecta H. tuna H. gracilis U. indica U. flabellum a b a b a b a b a b

Yield (%) 0.06 0.12 0.15 0.30 0.21 0.33 0.43 0.90 0.65 0.65 Total sugar (%) 11.20 16.20 10.08 35.30 14.00 27.68 18.77 41.46 21.8 62.70 Protein (%) 7.60 14.94 22.00 13.03 18.89 17.46 8.96 12.50 2.30 9.00 Uronic acid (%) 5.60 7.68 5.88 5.17 7.15 6.79 2.37 1.43 1.83 1.41 Sulphate (%) 32.10 27.90 7.64 19.50 13.53 14.39 12.51 12.62 28.16 16.49 Arabinosec ND ⎯ 9.88 ⎯ ND ND ND ND ⎯ 1.09 Galactosec ND 7.94 55.58 18.70 ND ND ND ND ⎯ 32.44 Mannosec ND 8.10 15.40 ⎯ ND ND ND ND ⎯ 4.35 Xylosec ND 7.56 12.71 ⎯ ND ND ND ND ⎯ 2.33 Glucosec ND 66.32 6.43 81.30 ND ND ND ND 100 51.66 Rhamnosec ND 9.68 ⎯ ⎯ ND ND ND ND ⎯ 3.88 Ribosec ND ⎯ ⎯ ⎯ ND ND ND ND ⎯ 4.20 CT ratiod 1.17 1.11 1.17 1.23 1.00 1.50 2.45 2.72 1.11 2.05

a = cold water extracts; b = hot water extracts c = percent composition: calculated from gas chromatogram (GC⎯MS) taking the total area of all the peaks as 100% d = concentration 2 mg/ml in prothrombin time test; CT ratio of heparin (140.3 IU/mg) at 500μg/ml = 6.6 (cf. ref. 7, 9) ND = not done

Shanmugam et al.: Screening of Codiacean algae

37

from Japanese waters exhibited potent blood anticoagulant activity, whereas from Monostroma nitidum, a green alga, active fraction was obtained only by hot water extraction5. This seems to be due to the difference in sugar and sulphate contents of the SPS.

Sulphated polysaccharides obtained from Codiacean algal species, particularly plants of genus Codium, exhibited promising blood anticoagulant activity. SPS isolated from different Codiacean algae had different chemical composition and blood anticoagulant activity of the SPS were comparable, but differed in degree from one genus to another. As the cold water extracts of C. dwarkense and C. tomentosum showed the highest blood anticoagulant activity, they were selected for further studies to understand the molecular species present in those extracts which are responsible for blood anticoagulant activity. As reported earlier, it was found that highest blood anticoagulant activity was associated with arabinose7,9.

The SPS of matured plants (April collection) with higher arabinose content were more active than those of the younger plants. This is in confirmity with our earlier reports describing the fractions of cold water extract of C. dwarkense containing 100% arabinose exhibiting the highest blood anticoagulant activity7,9. Therefore, it appears likely that C. dwarkense synthesise more arabinose as it gains maturity.

Acknowledgement The authors are grateful to Dr. P. K. Ghosh,

Director, for helpful suggestions in preparing this manuscript. Sincere thanks are accorded to Prof. V. Krishnamurthy, Dr. V. D. Chauhan and Dr. H. V. Joshi for identification of algae. Thanks are also due to Mr A. Sathaku and Mr Seeni Ahmed, MARS, Mandapam, for collection of marine algae. Two authors ⎯ MS and ASK are grateful to the Department of Ocean Development, New Delhi for financial assistance under a National Programme (DOD/9-DS/1/96-2/10; dated 16/12/1996).

References 1 Chargaff E, Bancroft, F W & Stanely-Brown M, Studies

on the chemistry of blood coagulation II. On the inhibition of blood clotting by substance of high molecular weight, J Biol Chem, 115 (1936) 155-161.

2 Ofosu F A & Barrowcliffe, Mechanisms of action of low molecular weight heparin and heparinoids, Bailliére’s Clinical Haematology, 3 (1990) 505-529.

3 Deacon-Smith R A, Lee-Potter J P & Rogers D J, Anticoagulant activity in extracts of British marine algae, Bot Mar, 28 (1985) 333-338.

Indian J. Mar. Sci., Vol. 31, No. 1, March 2002

38

4 Rogers D J, Jurd K M, Blunden G, Paoleti S & Zanetti F, Anticoagulant activity of a proteoglycan in extracts of Codium fragile ssp. atlanticum, J Appl Phycol, 2 (1990) 357-361.

5 Maeda M, Uehara T, Harada N, Sekiguchi M & Hiraoka A, Heparinoid-active sulphated polysaccharides from Monostroma nitidum and their distribution in the Chlorophyta, Phytochemistry, 30 (1991) 3611-3614.

6 Uehara T, Takeshita M & Maeda M, Studies on anticoagulant-active arabinan sulfates from the green alga Codium latum, Carbohydr Res, 235 (1992) 309-311.

7 Siddhanta A K, Shanmugam M, Mody K H, Goswami A M & Ramavat B K, Sulphated polysaccharides of Codium dwarkense Boergs. from the west coast of India:

Chemical composition and blood anticoagulant activity, Int J Biol Macromol, 26 (1999) 151-154.

8 Siddhanta A K & Shanmugam M, Metabolites of tropical marine algae of the family Codiaceae (Chlorophyta): Chemistry and bioactivity, J Indian Chem Soc, 76 (1999) 323-334.

9 Shanmugam M, Mody K H & Siddhanta A K, Blood anticoagulant sulphated polysaccharides of the marine green algae Codium dwarkense (Boergs.) and C. tomentosum (Huds.) Stackh., Indian J Exp Biol, 39 (2001) 365-370.

10 Nishino T, Ura H & Nagumo T, The relationship between the sulphate content and the antithrombin activity of an α(1→2)-fucoidan purified from a commercial fucoidan fraction, Bot Mar, 38 (1995) 187-193.