Isolation and characterization of anticoagulant compound from marine mollusc Donax faba (Gmelin, 1791) from Thazhanguda, Southeast Coast of India

Glycosaminoglycans (GAGs) are linear polysaccharides found in the extracellular matrix and biological fluids of animals where they interact with hundreds of proteins and perform a variety of critical roles. There are five classes of animal GAGs: heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronan (HA). Many biological functions can be monitored directly by their impact on GAG quantity. In the present study, glycosaminoglycans were isolated from marine bivalve Donax faba . The amount of crude GAG was estimated as 12 gm/kg and of tissue in D. faba . After purification using gel chromatography, the yield was found to be 0.83 mg/kg. The bivalve showed the anticoagulant activity of the crude and purified samples 58 and 114 USP units/mg correspondingly in D. faba . The structural characterization of anticoagulant GAG was analyzed by Fourier transform infrared spectroscopy. Among the marine bivalve, D. faba purified showed more anticoagulant activity than that of crude sample. The results of this study suggest that the GAG from D. faba could be an alternative source of heparin.


INTRODUCTION
Glycosaminoglycans (GAGs) are long, linear, disaccharide repeats of hexosamine and highly sulphated galactose or hexuronic acids, and are usually found covalently attached to a protein 'core' to form proteoglycans (Kjellen and Lindahl, 1991;Silbert et al., 1997).GAGs are an extremely heterogeneous group of molecules that can be divided into several different general classes, such as haparan sulphate (HS) (and its model analogue, heparin), dermatan sulphate (DS) and chondroitin sulphate (CS), depending on the composition of the sugar backbone and the degree of sugar modification.Proteoglycans/GAGs are expressed by all nucleated cells and several bacterial pathogens such as Bordetella pertussis (Menozzi et al., 1994), Mycobacterium sp (Menozzi et al., 1996), and Listeria monocytogenes (Alvarez-Domingvez et al., 1997), encode surface proteins that recognize GAG, that is, GAG-binding adhesions (Rostand, 1997).The GAGs, HS and heparin have been implicated as participants in a variety of physiological processes including cell-cell recognition, blood coagulation, infection, and cell growth and differentiation.Both are polysaccharides that consist of repeating disaccharide units composed of uronic acid and an amino sugar.HS, which is sulfated lesser than heparin, is nearly ubiquities in the animal kingdom and is often a cell surface marker.On the other hand, heparin is found only in Chordata, Mollusca and Arthropoda (Nader et al., 1999) and over 80% of its glucosamine residues are N-sulfated (Gallagher and Walker, 1985).Heparin has been used for anticoagulant therapy for many years.Its anticoagulant effect appears to be mediated mainly through antithrombin III (AT III), which is a plasma protein and the main coagulation inhibitor in the blood.
Antithrobin III or heparin cofactor, as it is also called, inhibits thrombin and the activated forms of the coagulation factors IX, X, XI and XII.In the absence of heparin, the inhibition reactions are slow, but the addition of heparin strongly accelerates them.Several models have been proposed to explain the effect of heparin on the inhibition of thrombin by ATIII.Heparin is known to bind the ATIII; one widely accepted model assumes that heaprins forms a complex with AT III and transforms it into a more rapidly acting inhibitor (Holmer et al., 1979).Currently, commercial heparin preparations are obtained from mammalian sources, either from porcine, bovine intestine or bovine lung.Non-animal sources of heparin for pharmaceutical use are currently not available.However, the occurrence of the heparin is not restricted to mammals.Several heparin and heparin like polymers have been described in invertebrate animals such as crustaceans (Hoving and Linker, 1982), mollusks (Cavalcante et al., 1985) and ascidians (Cavalcante et al., 2000) also.It is real fact that the importance of marine organisms as a source of new substance is growing with marine species comprising approximately a half of the total global biodiversity, the sea offers an enormous resources for novel compounds, and it has been classified as the largest remaining reservoir of natural molecules to be evaluated for drug activity.A very different kind of substances have been obtained from marine organisms among other reasons because they are living in a very exigent, competitive and aggressive surrounding very different in many aspects from the terrestrial environment, a situation that demands the production of quite specific and potent active molecules (Aneiros and Garateix, 2004).
Molluscs contribute significantly to the total marine fish catch of the world.Marine bivalves are abundant in coastal and estuarine waters of India.The bivalve's fishery is constituted mainly by clams, mussels and oysters; molluscan fishery is not well-organized along the Indian coast.Molluscs are exploited in large quantities by traditional methods and sold live in the market for human consumption.The economically important species of marine bivalves are green mussel (Perna viridis), estuarine oyster (Crssostera madarasensis) and clam (Meretrix casta, M. meretrix, Donex sp.Phapia malabarica, Villorito cyprinoids) (Chattergi et al., 2002).Among the mollusks, some have pronounced pharmaceutical activities or other properties useful in the biomedical area.It is surprising that some of these pharmacological activities are attributed to the presence of polysaccharides, particularly those that are sulfated (Arumugam and Shanmugam, 2004).Hence, the attempt has been made to isolate, characterize and anticoagulant activity of the GAG from the marine bivalve Donax faba using chromatography.The GAGs Periyasamy et al. 5969 were purified by gel chromatography using sephadox G-100 column.The structure analysis of crude and purified GAGs was determined through Fourier Transform-Infra Red spectrum.

Isolation of glycosaminoglycans
The molluscs D. faba (Class-bivalvia; Family-Donacidae) was collected from the Thazhanguda coastal waters (Latitude, 11° 45' 0N; Longitude, 79° 45' 0E) Cuddalore, Southeast coast of India.Shells were opened and whole tissues were taken.They were blended in 0.4 M sodium sulfate solution (Na 2 SO 4 ; 3.5 1/kg of the tissue) and kept at 55°C for 1 h 30 min.The pH was adjusted to 11.5 by adding 10% sodium hydroxide (NaOH) solution.Aluminium sulfate (Al 2 (SO 4 ) 2 ) crystals (80 mg/kg tissues) were added to this solution, and the suspension was heated to 95°C for 1 h.Cetyl pyridinum chloride (CPC) solution (3 g/100 ml of 0.8 M Nacl) was used to precipitate the crude white heparin complex.The precipitate was redissolved in 150 ml of sodium chloride solution (2.0 M) and was incubated at 30°C for 30 min.The precipitate was washed with ethanol and methanol through centrifugation and vaccum dried.

Purification of GAGs -Gel chromatography
GAGs were purified on a 5 × 90-cm column of sephadex G-100 (Sigma).The elution rate was approximately 60 ml/h and 15-ml fractions were collected.The active fractions were pooled and extensively dialyzed against distilled water and freeze dried (Laurent, 1978).

Anticoagulant activity
The anticoagulant activities of crude and purified GAGs samples were determined by comparing with the concentration necessary to prevent the clotting of sheep plasma using USP (United State Pharmacopoeia) method.

Fourier transform-infra red spectrum analysis (FTIR)
FT-IR spectroscopy of crude and purified GAG sample of D. faba relied on a Bio-Rad FT-IR-40 model, USA.The sample (10 mg) was mixed with 100 mg of dried KBr and compressed to prepare a salt disc (10 mm diameter) for reading the spectrum further.

Estimation of GAGs
The amount of crude GAGs was estimated as 12 g/kg of tissue in D. faba.After purification using gel chromatography, the GAGs yield was found to be 0.83 mg/kg (Table 1).

Anticoagulant activity
By United States pharmacopoeia method, the anticoagulant activity of the D. faba crude and purified sample was reported to be 58 and 114 USP units/mg (Table 1 and Figure 1).

DISCUSSION
Heparin and heparin like compounds, which are present in some invertebrate molluscs, showed high anticoagu-lant activity and share most of the structural properties with mammalian heparins.Similarly, heparin has been prepared from a number of different species including humans (Linhardt et al., 1992), clams (Shuhei et al., 2011), shrimp (Muzaffer Demir et al., 2001) and seaweeds (Mahanama De Zoysa et al., 2008).Heparin and heparin like compounds, which are present in some invertebrate molluscs, showed high anticoagulant activity and share most of the structural properties with mammalian heparins.Homogenization of a 1 kg portion of whole D. faba in acetone and subsequent extraction in acetone /petroleum ether resulted in 250 g of defatted tissue.The heparin obtained from defatted tissue described in the present study has all of the features as that of heparin.Similarly, heparin has been prepared from a number of different species including (Linhardt et al., 1992), clams (Pejler et al., 1987;Dietrich et al., 1989) and seaweeds (Wladimir et al., 2000).Extraction of the defatted soft body tissue of the giant African snail and subsequent purification of its GAGs showed that this tissue contained a large amount of GAG and free from impurities (Kim et al., 1996).(1996).However, the cephalopods such as Sepia aculeate and S. brevimana, and Loligo duvauceli and Doryteuthis sibogae showed higher net yield of the heparin like sulfated polysaccharides 21.7, 24.0, 16.5 and 8.4 gm/kg, respectively (Mahalakshmi, 2003;Barwin vino, 2003).Arumugam et al. (2008) had quantified the heparin yield as 2.27 and 2.2 g/kg from Tridacna maxima and Perna viridis, respectively.Vijayabaskar et al. (2008) reported that the isolated glycosamnioglycans (GAG) and purified from both bivalves were estimated as 5.4, 4.1 and 1.4, 1.1 gm/kg wet tissue in K. opima and D. cuneatus.Vidhyanandhini (2010) reported that the amount of crude GAGs was estimated as 9.85 gm/kg and after purification by using amberlite and barium acetate, the yield was found to be 33 and 148 mg/kg of K. opima.Saravanan and Shanmugam (2010) reported the amount of crude and purified GAG was estimated as 17.2 g/kg and 48 mg/kg of tissue in A. pleuronectus.The result of the present study clearly shows that the yield of heparin and heparin-like glycoasaminoglycans is higher and lower than those of previously reports.Hence, it could be concluded that the molluscs might be used as potent sources for the extraction of heparin and heparin -like glycosaminoglycans.
The heparin isolated from marine clams and mussels has identical structural features and anticoagulant activity of mammalian polysaccharide (Pejler et al., 1987).Heparin with high anticoagulant activity was isolated from the marine molluscs, Anomalocardia brasiliana, Donax striatus and Tivela mactroides (Dietrich et al., 1985), which showed similar activity like mammalian heparin but differ in molecular weight; the molluscan heparin have a higher molecular weight and high anticoagulant activity (Dietrich et al., 1989).The biological activity of heparins in invertebrates remains enigmatic.The classes of Crustacea and Mollusca do not posses any blood coagulation system similar to that of mammals and other vertebrates and thus the presence of compounds that all specifically upon the proteins of the blood coagulation system is indeed remarkable (Dietrich et al., 1999).The anticoagulant activity of heparin differs from species to species due to their iteration with enzymes and inhibition of the coagulation system (Mulloy et al., 2000).The anticoagulant activity of the crude and purified sample of GAG from the whole body tissue of D. faba was reported as 58 and 114 USP units/mg.This variation might be due to the presence of non-anticoagulant substance in the samples since the activity of heparin depends upon the amount of impurity carried over in the isolated products.Burson et al. (1956) showed the activity ranging from 130 to 150 USP units/mg for extracted products of Spisula solidissima and Cyprina islandica.Arumugam and Shanmugam (2004) reported the anticoagulant activity crude and fractionated sample of GAG from T. attenuate -37 and 78 USP units/mg in T. attenuate.The anticoagulant activity of crude and purified sample of GAG from A. pleuronectus were 15.38 and 83.99 USP units/mg (Suganthi, 2007).
The crude and purified sample of E. berryi were 415 and 483.1 USP units/mg (Shanmugam et al., 2008).Vijayabaskar et al. (2008) reported that the extraction of GAGs from K. opima and D. cuneateus showed anticoagulant activity of 160 and 154 USP units/mg, and after the partial purification through DEAE cellulose column chromatography, the yields of anticoagulant activity were found to be 180 and 175 USP units/mg (Vidhyanandhini, 2010).The anticoagulant activity of crude and purified samples were estimated at 22.52, 20.00 and 18.60 USP units/mg for amberlite; 86.32, 83.06, and 92.43 USP units/mg for barium acetate and 80.36, 75.92, 89.68 USP units/mg for K. opima.From the aforementioned, it could be understood that whole body tissue of D. faba is also comparatively a good potential source of anticoagulant compounds.The molluscan GAGs were found to be structurally similar to the standard heparin as assessed by the FT-IR spectra.In the present study, the anticoagulant GAGs from whole body tissue of D. faba crude and purified sample showed a major peaks at 3437.15, 1639.49,1251.8,1197.79,1128.36 and 1078.21cm -1 which is said to be for the GAGs groups (Saravanan and Shanmugam, 2010).
For FT-IR spectrum of purified sample, the sulfate band stated from 1139.92 cm -1 and extended down to 995.12 cm -1 .The acetyl amino group was represented by a band at 1474.78 cm -1 and the carboxylic group at 1552.66 cm -1 .This was also well supported by the study of Rivera et al. (2002) who also claimed that the characterization of traces of contaminants in crude heparin by conventional physico-chemical techniques such as size-exclusion or ion-exchange chromatography is relatively difficult.The peak pattern between the standard heparin and the purified sample were at 3433.29, 1639.49cm -1 and 3437.15,1639.49cm -1 as indicating the presence of GAGs group in the samples analyzed.The FT-IR spectral analysis of the anticoagulant GAGs from D. faba showed more or less same number of peaks, lying within the same range of values of the commercial heparin used as a standard.The results in this finding show that bivalve, D. faba tissue had GAGs with high quantity of anticoagulant compounds.Thus, the result of the present investigation provides information about the isolation, purification and characterization of the heparin and heparin like glycosaminoglycans (heparin and heparin sulfate) compound from their chemical characteristic features.Further, it will pave the way for future researchers to take up research in this line to characterize fully the heparin and heparin like GAGs of molluscan source using chromatography different fraction, NMR, MS and other advanced techniques.
Besides the aforementioned, it could also have a very good anticoagulant activity as the anticoagulant compound from this species proves the possibility of its utilization as an additional potent source for the extraction of such anticoagulant compound since the anticoagulant activity of the presents isolated heparin and heparin like GAGs from D. faba was even more than that of many heparins obtained from commercial sources.

Table 1 .
The yield of glycosaminoglycans and their anticoagulant activity from D. faba.