|Year : 2013 | Volume
| Issue : 1 | Page : 39-45
Anticoagulant and antioxidant activity of sulfated chitosan from the shell of donacid clam Donax scortum (Linnaeus, 1758)
Namasivayam Subhapradha1, Shankar Suman1, Pasiyappazham Ramasamy1, Ramachandran Saravanan2, Vairamani Shanmugam1, Alagiri Srinivasan3, Annaian Shanmugam1
1 Center for Advanced Studies in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, India
2 Department of Pharmacology, Chettinad Hospital and Research Institute, Kelambakkam, India
3 Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India
|Date of Submission||10-Aug-2012|
|Date of Acceptance||18-Sep-2012|
|Date of Web Publication||6-Feb-2013|
CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai - 608 502
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: Sulfated chitosan was prepared from the shell of donacid clam Donax scortum by following demineralization, deproteinization, deacetylation and sulfating with chlorosulfonic acid. Results: FT-IR spectrum proved the structure of sulfated chitosan. The yield, sulfate content and molecular weight was found 80.30%, 14.2% and 18.65 × 10 4 Da. Their anticoagulant activity was determined for human plasma with respect to the activated partial thromboplastin time (APTT) and prothrombin time (PT) and it was 6.45 and 1.73 IU, respectively. The antioxidant activity of sulfated chitosan was evaluated as radical scavengers against superoxide anion (71.46% at 0.5 mg/ ml) and hydroxyl radicals (64.73% at 3.2 mg/ml). The antioxidant activity of sulfated chitosan was evaluated as radical scavengers against superoxide anion and hydroxyl radicals of 71.46% at 0.5 mg/ml and 64.73% at 3.2 mg/ml. The chelating ability and reducing power of sulfated chitosan was 43.24 at 10 mg/ml and 0.578 at 0.75 mg/ml. IC 50 values of scavenging abilities on superoxide anion and hydroxyl radicals were 0.398 and 0.527 mg/ml whereas those of chelating ability and reducing power were 5.01 and 0.728 mg/ml. Conclusion: These results indicated that sulfated chitosan from D. scortum had potent anticoagulant and antioxidant activities.
Keywords: Anticoagulant activity, antioxidant activity, Donax scortum, fourier transform - infra red spectroscopy, sulfated chitosan
|How to cite this article:|
Subhapradha N, Suman S, Ramasamy P, Saravanan R, Shanmugam V, Srinivasan A, Shanmugam A. Anticoagulant and antioxidant activity of sulfated chitosan from the shell of donacid clam Donax scortum (Linnaeus, 1758). Int J Nutr Pharmacol Neurol Dis 2013;3:39-45
|How to cite this URL:|
Subhapradha N, Suman S, Ramasamy P, Saravanan R, Shanmugam V, Srinivasan A, Shanmugam A. Anticoagulant and antioxidant activity of sulfated chitosan from the shell of donacid clam Donax scortum (Linnaeus, 1758). Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2020 Jan 25];3:39-45. Available from: http://www.ijnpnd.com/text.asp?2013/3/1/39/106990
| Introduction|| |
Chitosan is an abundant natural biopolymer obtained from the exoskeletons of crustaceans and arthropods, which is a non-toxic copolymer consisting of β-(1, 4)-2-acetamido -2-deoxy-D-glucose and β-(1,4)-2-amino-2-deoxy-D-glucose units.  As a natural renewable resource, chitosan has a number of unique properties such as biocompatibility, biodegradability, non-toxicity and antimicrobial activity, which have attracted much scientific and industrial interest.  Although, chitosan is soluble in aqueous dilute acids below pH 6.5, it is insoluble in water and most organic solvents. The poor solubility of chitosan is a major limiting factor to its utilization. Therefore, special attention has been paid to its chemical modification to obtain water soluble derivatives over a wide pH range. The chemical modification of the amino and hydroxyl group can generate products for pharmaceutical applications, for example sulfated chitosan possess a wide range of biological activities.  Thus, sulfated chitosan as the nearest structural analogue of the natural blood anticoagulant heparin, demonstrate anticoagulant, anti-sclerotic and antiviral activities. , Reactive oxygen species (ROS) and reactive nitrogen species (RNS), commonly called free radicals, are produced by the human body in the course of normal metabolism, by a variety of mechanisms. Under normal conditions free radicals can be beneficial to the body, being used for a number of physiological functions. Although there is always the potential for damage, this is kept in check by an array of intricately connected antioxidant defense and repair systems.  For the maintenance of tight homeostasis and to prevent stress due to free radical oxidation, the dietary antioxidant and/or herbal formulations proved their usefulness.  In recent years, there has been increasing interest in finding natural antioxidants, since they can protect the human body from free radicals and retard the progress of many chronic diseases. Je reported the antioxidant activity of nine hetero-chitooligosaccharides based on scavenging potency on the DPPH radical and found that hetero-chitooligosaccharides have an antioxidant activity, which is dependent on their degree of deacetylation and molecular weight.  Sun reported the antioxidant activity of chitosan oligomers and its scavenging ability against superoxide anion and hydroxyl radicals by flow injection chemiluminescence technology.  Vongchan reported the anticoagulant activities of chitosan polysulfate obtained from the synthesis in mild and semi-heterogeneous conditions.  Vikhoreva studied the anticoagulant activity of low molecular weight sulfated chitosan from the marine crab.  To the best of our knowledge, no study has been carried out for the preparation of sulfated chitosan from the shell of donacid clam Donax scortum. In the present paper, sulfated chitosan has been prepared by sulfation of chitosan from the shell of D. scortum using chlorosulfonic acid and Dimethylformamide. The structure of sulfated chitosan has been determined and the effect of coagulation assayed through APTT and PT. Antioxidant property of sulfated chitosan was studied as scavengers of superoxide anion, hydroxyl radical, reducing power and its chelating ability on metal ions.
| Materials and Methods|| |
Chemicals and reagents
D. scortum were collected from Porto Novo coastal region (11.49°N, 79.76°E). The shells were removed from the animal, washed and air-dried. Nitro blue tetrazolium (NBT), phenazine methosulfate (PMS), hydrogen peroxide (H 2 O 2 ), thiobarbituric acid (TBA), ethylene diamine tetra acetate (EDTA), ferrozine, nicotinamide adenine dinucleotide-reduced (NADH), trichloroacetic acid (TCA) and deoxyribose were purchased from Sigma Chemical Co. Milli-Q water was used to prepare reagents for antioxidant tests.
Preparation of sulfated chitosan
Chitin was extracted from the crushed shell of D. scortum by demineralization and deproteinization, and then the chitin was converted to chitosan through deacetylation using 40% NaOH by following Takiguchi procedure. , Sulfated chitosan was prepared based on the method of Xing.  Fifty ml of DMF.SO 3 was added into a 500 mL three-necked bottomed flask containing 50 ml of chitosan solution in a mixture of DMF-formic acid with swirling to get gelatinous chitosan. Then the reaction was run at adequate temperature (40-60˚C) for 1-2.5 h, and 95% of ethanol (300 ml) was added to precipitate the product. The mixture of products was filtered through a Buchner funnel under reduced pressure. The precipitate was washed with ethanol, and then re-dissolved in distilled water. The pH was adjusted to 7-8 with 2 M NaOH. The solution was dialyzed against distilled water for 48 h using a 12000 Da MW cut off dialysis membrane. The product was then concentrated and lyophilized to give sulfated chitosan.
Characterization of sulfated chitosan
The yield percentage was calculated from chitosan on dry weight basis. The average molecular weight of sulfated chitosan was determined by viscometric method using the Ostwald viscometer and the Mark-Houwink equation: [η] = 10 -5 4.97 M 0.77 , (0.5 M NaCl, and 25°C).  Sulfate content of sulfated chitosan was determined by following the method of Tehro and Hartiala.  FT-IR spectroscopy of solid sample of chitosan and sulfated chitosan from D. scortum shell and standard chitosan were relied on an AVATAR 330 Spectrometer. Sample (10 μg) was mixed with 100 μg of dried potassium bromide (KBr) and compressed to prepare a salt discs (10 mm diameter) for reading the spectrum.
The assay was carried out using heparan sulfate as standard. Sulfated chitosan was dissolved in Milli-Q water at various concentrations. Normal human plasma (90 μl) was mixed with 10 μl of a solution of sulfated chitosan (0-2 mg) and heparin sulfate (0- 100 μg).
APTT (Activated partial thromboplastin time)
APTT was performed using a kit obtained from Instrumentation Laboratory (Lexington, USA). The plasma (100 μl) containing various concentrations of sulfated chitosan and heparin sulfate was incubated at 37°C for 1 min. Bovine cephalin (100 μl) was then added and incubated at 37°C. After 3 min of incubation, 100 μl of pre-warmed 0.25 M CaCl 2 solution was added to the mixture and the clotting time was measured and compared with the standard; the activity was expressed as IU/mg.
The method used to measure PT (Prothrombin time) was as indicated by the manufacturer (Lexington, USA). One hundred microliter of plasma containing various concentrations of sulfated chitosan was pre-warmed for 5 min before adding of 200 μl of thromboplastin reagent. The clotting time was recorded simultaneously.
In vitro antioxidant assays
Superoxide radical scavenging assay
The superoxide scavenging ability of sulfated chitosan was assessed by the method of Nishikimi.  The reaction mixture, containing sulfated chitosan (0.05- 0.4 mg/ml), PMS (30 mM), NADH (338 mM) and NBT (72 mM) in phosphate buffer (0.1 M pH 7.4) was incubated at room temperature for 5 min and the absorbance was read at 560 nm against a blank.
Hydroxyl radicals scavenging assay
The reaction mixture containing sulfated chitosan (0.1- 3.2 mg/ml), was incubated with deoxyribose (3.75 mM), H 2 O 2 (1 mM), FeCl 3 (100 mM), EDTA (100 mM) and ascorbic acid (100 mM) in potassium phosphate buffer (20 mM, pH 7.4) for 60 min at 37˚C Halliwell.  The reaction was terminated by adding 1 ml of TBA (1%, w/v) and 1 ml of TCA (2%, w/v) and then heating the tubes in a boiling water bath for 15 min. The contents were cooled and the absorbance of the mixture was measured at 535 nm against the reagent blank. The decreased absorbance of the reaction mixture indicated decreased oxidation of deoxyribose.
Measurement of reducing power
The reducing power of sulfated chitosan was quantified by the method described by Yen and Chen  with minor modifications. One ml of reaction mixture containing different concentrations of sulfated chitosan in phosphate buffer (0.2 M, pH 6.6) was incubated with potassium ferricyanide (1%, w/v) at 50˚C for 20 min. The reaction was terminated by adding TCA solution (10%, w/v) and the mixture was centrifuged at 3000 rpm for 10 min. The supernatant was mixed with distilled water and ferric chloride (0.1%, w/v) solution and the absorbance was measured at 700 nm. The increased absorbance of the reaction mixture indicated increased reducing power.
Metal ion chelating assay
The ferrous ion-chelating potential of sulfated chitosan was investigated according to the method of Decker and Welch,  wherein the Fe 2+ -chelating ability of sulfated chitosan was monitored by measuring the ferrous iron-ferrozine complex at 562 nm. The reaction mixture containing sulfated chitosan of different concentrations, FeCl 2 (2 mM) and ferrozine (5 mM) was adjusted to a total volume of 0.8 ml with water, shaken well and incubated for 10 min at room temperature. The absorbance of the mixture was measured at 562 nm against blank. EDTA was used as a positive control.
All data are expressed as means ± SD. Data were analyzed by analysis of variance (P < 0.05) and the means separated by Duncan's multiple range tests. The results were processed using Ms-Excel and SPSS version 16.
| Results|| |
Yield, molecular weight, sulfate content, clotting assay and FT-IR spectral analysis
The yield, sulfate content and molecular weight of sulfated chitosan of D. scortum was found to be 84.03%, 14.2%, and 18.65 × 10 4 Da, respectively [Table 1]. The FT-IR spectrum of the standard chitosan [Figure 1] reported 12 major peaks lying between 673.97 cm -1 and 3445.59 cm -1 ; whereas the FT-IR spectrum of the chitosan sample from the shell of D. scortum [Figure 2] showed 12 peaks between 709.18 cm -1 and 3431.29 cm -1 . The absorption peaks at 668.90 cm -1 and 1134.36 cm -1 indicates the sulfo group [Figure 3].
|Table 1: Yield, molecular weight, sulfate content and APTT and PT activity of sulfated chitosan of D. scortum|
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The sulfated chitosan showed 6.45 IU/mg and 1.73 IU/mg of activity for APTT and PT, respectively [Table 1].
In vitro antioxidant assays
The inhibitory effect of sulfated chitosan on superoxide radicals was marked and concentration related [Figure 4]. The scavenging effect of superoxide radical (20.99-71.46%) was significant at all the concentrations (0.05-0.5 mg/ml) of sulfated chitosan tested whereas the BHA showed 28.49-88.41% of scavenging activity. The effect of sulfated chitosan on oxidative damage induced by hydroxyl radical at different concentrations (0.1-3.2 mg/ml) was found between 9.68% and 64.73%. The BHA exhibited the scavenging effect on hydroxyl radical was 84.36% at 3.2 mg/ml [Figure 5]. At different concentrations of 0.15-0.75 mg/ml, the reducing power of sulfated chitosan was found to range from 0.019-0.578%; whereas the reducing power of BHA was in the range of 0.966 - 1.893% [Figure 6]. The ferrous ion chelating effect of sulfated chitosan was found between 23.89% and 43.24% at different concentrations (0.1-10 mg/ml). The EDTA showed the chelating effect of 88.64% at 10 mg/ml [Figure 7]. The IC 50 value of sulfated chitosan against superoxide and hydroxyl radicals was 0.398 and 0.527 mg/ml whereas those of chelating ability and reducing power were 5.01 and 0.728 mg/ml.
| Discussion|| |
Characterization of sulfated chitosan
Yield, molecular weight, sulfate content and FT-IR spectral analysis
The yield, sulfate content and molecular weight of sulfated chitosan of D. scortum was found to be 84.03%, 14.2%, and 18.65 × 10 4 Da, respectively. The influence of molecular weight and substitution degree of sulfated polysaccharides plays a crucial role in biological activities such as anticoagulant, antioxidant and antimicrobial activities. Structural changes of chitosan and sulfated chitosan from D. scortum were confirmed by FT-IR spectroscopy and it was compared with the commercial chitosan from crab shell. The FT-IR spectrum of the standard chitosan reported 12 major peaks lying between 673.97/cm and 3445.59/cm; whereas the FT-IR spectrum of the chitosan sample from the shell of D. scortum recorded 12 peaks between 709.18/cm and 3431.29/cm. The FT-IR spectra of the standard chitosan showed the peaks at 3413.70/cm, 2923.27/cm, 2854.12/cm, 1741.22/cm, 1645.05/cm, 1456.53 cm - 1 , 1101.37/cm and 637.97/cm, corresponds to the D. scortum shell extracted chitosan showing nearly the absorbance band at 3431.29 cm - 1 , 2923.60/cm, 2849.32/cm, 1789.69/cm, 1633.44 cm - 1 , 1475.50/cm, 1125.37/cm, and 608.22/cm. The region between 3000/cm and 3500/cm shows the stretching of OH groups. This band is broad because of the hydrogen bonds. The OH band overlaps the stretching band of NH. Another significant change is observed in the region from 1000 cm -1 to 1200cm -1 . In this region chitosan presents a broad band centered it 1084.93/cm associated with the stretching of C=O. The characteristic absorption peaks at 668.90/cm and 1134.36/cm, which are due to sulfo groups, are reported to be found in the sulfated chitosan spectrum. When used the same methodology, in the FT-IR spectrum of sulfated chitosan from Sepioteuthis lessoniana also indicates the bands at 1254.79 and 1161.64/cm corresponding to asymmetric stretching vibrations of SO 3 . 
Heparin and chitosan sulfate are anticoagulants of direct action and prevent from activation of blood coagulation both in vivo and in vitro. Sulfated chitosan derivatives, as well as non-fractionated heparin, accelerate thrombin inactivation, forming an equimolar complex with antithrombin III was earlier demonstrated by Drozd and co-workers. , In the present study, sulfated chitosan had an effect on the APTT assay that was being expected because sulfate groups are necessary to provide anticoagulant effects and anticoagulant activities of polysaccharides; these are not only dependent on the sulfate content but also on the position of the sulfate groups.
In vitro antioxidant assays
Superoxide radical scavenging assay
Superoxide radical is known to be very harmful to cellular components as a precursor of more reactive oxidative species, such as single oxygen and hydroxyl radicals. The scavenging effect of superoxide radical was increasing significantly at all the tested concentration. The superoxide radical scavenging activity of sulfated chitosan was found significant at all the concentrations. When the concentration of sulfated chitosan was 0.398 mg/ml in the final solution, a superoxide anion inhibiting efficacy of approximately 50% was achieved, that is, the 50% inhibition concentration was 0.398 mg/ml. In the present study, sulfated chitosan effectively scavenged superoxide radicals in a concentration dependent manner. Further, superoxides are also known to indirectly initiate lipid peroxidation as a result of H 2 O 2 formation, creating precursors of hydroxyl radicals.  These results showed that the sulfated chitosan from
D. scortum has strong scavenging activity of the superoxide radical and clearly suggests that the antioxidant activity of sulfated chitosan was also related to its ability to scavenge superoxide radical.
Hydroxyl radical scavenging assay
Hydroxyl radicals, generated by the reaction of iron-EDTA complex with H 2 O 2 in the presence of ascorbic acid attack deoxyribose to form products that, upon heating with 2-thiobarbituric acid under acid conditions, yield a pink tint. Added hydroxyl scavengers compete with deoxyribose for the resulted hydroxyl radicals and diminish tint formation. The above mentioned model was used to measure inhibitory activities of sulfated chitosan on hydroxyl radicals. IC 50 of sulfated chitosan was 0.527 mg/ml. Earlier numerous workers have employed this system to assess the biological activity of various natural plant derived biomolecules.  Molecules that can inhibit deoxyribose degradation are those that can chelate iron ions and render them inactive or poorly active in a Fenton reaction. 
Measurement of reducing power
The reducing power of sulfated chitosan correlated well with increasing concentrations. The reducing power of high molecular weight and high sulfate content chitosan was 0.17 at 0.75 mg/ml.  IC 50 of sulfated chitosan from D. scortum was found to be 0.728 mg/ml. The reducing properties are generally associated with the presence of reductones,  which is been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom. 
Metal ion chelating assay
The ferrous ion-chelating effect of sulfated chitosan was concentration related and its IC 50 was 5.01 mg/ml. Presently, the chelating effect of sulfated chitosan was low, compared to EDTA at different concentrations. The chelating abilities of N-alkylated disaccharide chitosan derivatives were lesser than that observed with EDTA.  Transition metal ions can initiate lipid peroxidation and start a chain reaction, which leads to the deterioration of flavor and taste in food.  Since the ferrous ions are the most effective pro-oxidants in the food system  , the high ferrous-ion chelating abilities of sulfated chitosan would be beneficial if they were formulated into foods.
| Conclusion|| |
This study demonstrates the structural elucidation of sulfated chitosan from D. scortum through FT-IR and it has good anticoagulant activity due to the presence of sulfate group. Moreover, it possess various extents of antioxidant properties including scavenging activities for superoxide anion radicals, hydroxyl radicals, reducing power and ferrous-ion chelating activity. Based on the results obtained, sulfated chitosan may be used as a source of natural antioxidant, as a possible food supplement or ingredient in the nutraceutical or pharmaceutical industries.
Authors are thankful to the Director and Dean, CAS in Marine Biology, Faculty of marine sciences and Annamalai University for providing the necessary facilities. Two of the authors (NS and PR) are also thankful to the Centre for Marine Living Resources and Ecology (CMLRE), Ministry of Earth Sciences, Cochin for the financial assistance.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
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