Evaluation of antioxidant activities and chemical analysis of sulfated chitosan from Sepia prashadi

doi:10.1016/j.ijbiomac.2017.03.012

International Journal of Biological Macromolecules

Volume 99, June 2017, Pages 519-529

https://doi.org/10.1016/j.ijbiomac.2017.03.012 Get rights and content

Abstract

The chitin and chitosan of S. prashadi was prepared through demineralization, deproteinzation, deacetylation process and sulfation were carried by chlorosulfonic acid in N,N-dimethylformamide. The sulfate content in chitosan was found to be 18.9%. The carbon, hydrogen and nitrogen composition of the sulfated chitosan were recorded 39.09%, 6.95% and 6.58% respectively. The structural analysis was done by using FT-IR and NMR spectroscopy technique. The DSC curves of sulfated chitosan showed a large endothermic peak resolved with T_o value of 54.57 °C and T_P value of 97.46 °C. The morphology of sulfated chitin and sulfated chitosan were studied by SEM. The Further in vitro antioxidant activity of sulfated chitosan was screened by scavenging activity of superoxide radical assay, hydroxyl radical scavenging assay, metal-ion chelating effect and reducing power. Its anticoagulant activity was tested for human plasma with respect to Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT). Results prove that sulfated chitosan has potent antioxidant and anticoagulant activity.

Introduction

Approximately 70% of the landed value of shellfish is rejected as offal. This abundant waste material has either to be discarded at considerable cost or else be converted to value-added products. Concerns for environmental health and safety have compelled researchers/seafood processors to enhance value of seafood processing wastes and this has led to the production of several useful biochemicals and nutrients such as chitin, pigments, seafood peptones etc. from this resource material [1]. Chitin, a naturally abundant homopolymer, consists of β-(1 → 4) linked 2-acetamido-2-deoxy-d-glucopyranose units. A large quantity of chitin is manufactured from the exoskeleton of crustacean sources (shrimp, crab, lobster and crayfish), and from the shells of molluscs [2].

Chitin is regarded as a suitable functional material because it exhibits excellent properties such as biocompatibility, biodegradability, non-toxicity and adsorption properties [3]. However; this bio-functional polymer exhibits a limitation in processibility due to its low solubility in most of the organic solvents. Chitosan, (1-4)-2-amino-2-deoxy- β-d-glucan is found in all arthropods, in some other invertebrates (eg. squid and cuttlefish) and in some microorganisms and is readily prepared from chitin by N-deacetylation with alkali. Chitosan showed some biological activities such as immunological activity and antibacterial activity [4], [5] apart from its wide ranging applications in many areas, including the wastewater treatment, food, agriculture, cosmetic/personal care, biotechnological and pharmaceutical industries [6]. Since chitosan itself is insoluble in water at neutral or high pH region, the application of chitosan is quite limited. Therefore, chemical modification of chitosan to provide water- soluble materials is of prime interest to generate novel biomaterials.

Sulfation reactions of multi-functional polysaccharides are inevitably followed by the appearance of structural heterogeneity in a polymer chain. When chitosan is sulfated, a structural variety of products is obtained, which may be related to the various reactions of the three functional groups of the parent polymer, leading to different degrees of completion in the individual groups. On one hand this gives rise to uncertainty, but on the other hand, some structure that emerge from the random distribution of modified groups along the chain can reveal new features of biological functions like anticoagulant and antioxidant activity etc [7].

In general, the natural antioxidants mainly constitute a broad range of compounds including phenolic compounds, nitrogen compounds and caretenoids [8]. In the search of new antioxidants, exploration of aquatic habitats has led to the discovery that marine plants and invertebrates also contain antioxidants. Chitosan and its derivatives have attracted most attention because of their obvious antioxidant and anticoagulant activity. Moreover, the influence of molecular weight and substitution degree of sulfated polysaccharides on their biological activity is considered in majority of works involving the antioxidant or antiviral properties of these substances [9]. So, in the present attempt has been made to prepare sulfated chitosan from cuttlebone of S. prashadi and to investigate the in vitro antioxidant and anticoagulant activity.

Section snippets

Collection and preparation of cuttlebone powder

The cuttlefish, S. prashadi were collected from Thengaithittu landing centre of Puducherry region (Lat.11° 54′ 44′′ N; Long. 79° 49′ 13′′ E). The cuttlefishes were dissected and cuttlebones were taken out. They were washed, dried and were pulverized with pestle and mortar into fine powder and used for further analysis.

Extraction of chitin from cuttlebone of S. prashadi

Chitin was extracted from the cuttlebone of S. prashadi by following the method of Takiguchi [10]. 20 gm of squid pen powder was dematerialized with 300 ml of 2N HCl for 24 h with

Yield of chitin, chitosan and sulfated chitosan

The percentage yield of chitin and chitosan from the cuttlebone of S. prashadi was found to be 29% and 15% respectively. The percentage yield of sulfated chitosan was found to be 92.5%.

Sulfated content and CHN analysis

The sulfated chitosan reported 18.9% of sulfate content; whereas carbon, hydrogen and nitrogen contributed 39.09%, 6.95% and 6.58% respectively.

Molecular weight of sulfated chitosan

The molecular weight of sulfated chitosan was determined through viscometric analysis using Mark-Houwink equation. The molecular weight of cuttlefish S. prashadi sulfated

Discussion

Chitin has been extracted from many sources and its yield was found as low as 13%–90%: Krill (Euphasia superba) – 70%–90% Anderson et al. [17] prawn shells – 80% and shellfish waste – 14–27% Ashford et al. [1] and crab – 13–26% No and Meryers [18]. The cuttlebone of S. officinalis was found to contain 20% of chitin [19] whereas, in general, the squid/octopus reported 3–20% of chitin [20]. The yield of chitosan from the pen of D. singhalensis was found to contain 34.68% [21]. In the present

Acknowledgements

Authors are thankful to the Director and Dean, Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences and Annamalai University for providing necessary facilities. The authors 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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Research paperEvaluation of antioxidant activities and chemical analysis of sulfated chitosan from Sepia prashadi

Abstract

Introduction

Section snippets

Collection and preparation of cuttlebone powder

Extraction of chitin from cuttlebone of S. prashadi

Yield of chitin, chitosan and sulfated chitosan

Sulfated content and CHN analysis

Molecular weight of sulfated chitosan

Discussion

Acknowledgements

Int. J. Biol.

Carbohydr. Polym.

Anal. Biochem.

Anal. Biochem.

Polymer

Carbohydr. Polym.

Bioorg. Med. Chem. Lett.

Carbohydr. Polym.

Carbohydr. Polym.

Cell. Biol. Int.

Int. J. Biol. Macromol.

Food Chem.

Food Chem. Toxicol.

Bioorg. Med. Chem.

Thromb. Res.

Pract. Prevent. Med.

Mat. Sci. Eng.

Sci. Asia

J. Agric. Food Chem.

Japn. J. Cancer Res.

Gihodou Shupan Kabushki Kasisha. Japan

Biochem. Biophys. Res. Commmun.

Research paper
Evaluation of antioxidant activities and chemical analysis of sulfated chitosan from Sepia prashadi