International Journal of Biological Macromolecules 51 (2012) 826–830 Contents lists available at SciVerse ScienceDirect International Journal of Biological Macromolecules jo u rn al hom epa ge: www.elsevier.com/locate/ijbiomac Short communication Separation and scavenging superoxide radical activity of chitooligomers with degree of polymerization 6–16 Kecheng Li a,b , Ronge Xing a , Song Liu a,∗ , Yukun Qin a,b , Bing Li a,b , Xueqin Wang a,b , Pengcheng Li a,∗ a Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China b Graduate University of the Chinese Academy of Sciences, Beijing 100049, China a r t i c l e i n f o Article history: Received 30 June 2012 Received in revised form 17 July 2012 Accepted 29 July 2012 Available online 3 August 2012 Keywords: Fully deacetylated chitooligomers Degree of polymerization 6–16 Separation Scavenging superoxide radical activity a b s t r a c t The separation of chitooligomers (COS) with well-defined degree of polymerization (DP) is of interest to further study their bioactivity. However, there has been no report on separation of chitooligomers with DP > 6 and the activity of these oligomers is unknown. This paper focuses on separating COS with DP > 6 and five fractions were separated from the prepared fully deacetylated chitooligomers mixture by CM Sepharose Fast Flow column and analyzed by HPLC, which mainly contained glucosamine oligomers with DP6–7 (41.31%, 50.22%), DP7–8 (22.47%, 70.13%), DP9–10 (53.06%, 27.99%), DP10–12 (18.45%, 49.36%, 22.31%), and DP > 12, respectively. The superoxide radical scavenging activity of each fraction was inves- tigated. The oligomers with DP ranging from 10 to 12 exhibited higher scavenging activity than other fractions and in combination with the DP distribution of fractions, it was further concluded that the chitooligomers with DP11 was likely to be optimal for scavenging superoxide radical activity. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Chitooligomers is a partially hydrolyzed product of chitosan, mainly consisting of -1,4-linked d-glucosamine and partially of -1,4-linked N-acetyl-d-glucosamine. During the past decades, chi- tooligomers was known to possess various biological activities, including immunostimulating effect, antitumor, radical scaveng- ing, and antimicrobial actions [1]. The bioactivity of chitooligomers is significantly related to their degree of polymerization (DP) and degree of acetylation (DA). Previous studies on bioactivities of chi- tooligomers were mostly performed using mixtures with different molecular weights and various degree of acetylation. It is difficult to know which chitooligomer moleculars are playing a leading role in the biological assay. In order to further understand the biological action of chitooligomers, studies on chitooligomers with narrow or single DP have attracted interest. Ngo et al. reported that chitin oligosaccharides with molecular weight (Mw) 229.21–593.12 Da could effectively inhibit oxidative stress in live cells [2]. Chen et al. found that chitobiose and chi- totriose were more potent than glucosamine hydrochloride and three reference compounds (aminoguanidine, pyridoxamine and a water-soluble -tocopherol analogue) in scavenging hydroxyl rad- ical and both had the ability to exert a protective action against ∗ Corresponding authors. Tel.: +86 532 82898707; fax: +86 532 82968951. E-mail addresses: sliu@qdio.ac.cn (S. Liu), pcli@qdio.ac.cn, likecheng777@163.com (P. Li). CCl 4 -induced acute hepatoxicity [3,4]. Antitumor activity of chito- hexase has been reported and aroused researchers’ attention [5]. In addition, immunoregulatory function of two chitooligomers with narrow DP (tetra- to pentamers and hexamer) was studied [6]. However, to the best of our knowledge, there has been no report about the bioactivity of chitooligomers with narrow DP higher than 6, because the separation of these oligomers was extremely diffi- cult due to the decreasing percentage of difference between each individual oligomer. In the present study, we prepared a fully deacetylated chitooligomers with DP6–16 by acid hydrolysis of a highly deacety- lated chitosan and ethanol precipitation. Five narrow molecular weight fractions with DP > 6 were obtained for the first time by ion- exchange column chromatography. Subsequently, we investigated the antioxidant activity of fractions against superoxide radical. The effect of DP on superoxide radical scavenging activity was discussed in order to find the optimal DP. 2. Experiments 2.1. Materials Chitosan with degree of deacetylation of 82% and molecular weight of 658 kDa was obtained from Qingdao Baicheng Bio- chemical Corp. (China). Five standard chitooligomers, glucosamine dimer (≥98%), glucosamine trimer (≥96%), glucosamine tetramer (≥97%), glucosamine pentamer (≥90%), and glucosamine hexamer (≥80%), were purchased from Dalian Glycobio Co., Ltd. (Dalian, 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.07.031