Preparation of chitooligosaccharide acetate salts with narrow molecular size distribution and the antioxidative activity

Highlights

• COS acetate salts with narrow M_w distribution are prepared in kilogram scale.

• The anti-ROS activity is shown in cells, especially that with M_w 11.5 kDa.

• COS conc. over 3 mg/mL is efficient for exerting antioxidative activity.

• Better antioxidative activity than the commercial COS is obtained.

Abstract

Wide molecular weight distribution leads to uncertainties about the activity of chitooligosaccharide (COS). In the study, COS acetate salts with narrower size distribution were prepared in kilogram scale. The excellent water solubility and non-crystalline polysaccharide structure of COS products were observed. Furthermore, the antioxidative test results showed that the concentration of COS acetate salts exceeding 3 mg/mL was efficient for inhibiting free radicals and protecting human umbilical vein endothelial cells (HUVECs) against oxidative stress. Additionally, molecular weight of COS acetate played an important role in exerting antioxidative activity. COS acetate salt with M_w (weight average molecular weight) 41.2 or 4.22 kDa showed better radical scavenging activity and reducing power. Moreover, the cellular assay results suggested that COS acetate salts could significantly suppressed the reduction in cell viability under severe oxidative stress, especially that with M_w 11.5 kDa. In conclusion, COS acetate salts with narrow M_w distribution can be prepared in large scale and used as natural antioxidant, with the potential for preventing cardiovascular diseases.

Introduction

Oxidative stress, caused by excessive production of reactive oxygen species (ROS), induces damage to both cell biomolecules and membrane, which is linked to the pathogenesis of numerous degenerative diseases like cardiovascular diseases [1], [2]. Many factors can activate our body to produce elevated level of ROS, such as mental stress, pollution, ultraviolet irradiation, cigarette smoke and alcohol [3], [4]. Antioxidant is believed to be beneficial for human health via protecting or repairing cells from oxidative stress. Not only that, antioxidant also plays an important role in the extension of food shelf life by inhibiting the oxidative spoilage of nutritional ingredients. Consequently, there is a great demand for antioxidant in healthcare and food fields. Due to the potential health risks of synthetic antioxidants, natural antioxidant with low toxicity and high efficiency stimulates researchers’ enormous interests [5].

Chitosan is a natural linear polysaccharide, composed of glucosamine (GlcN) and N-acetyl-glucosamine (GlcNAc) units linked by β-(1, 4) glycosidic bonds, with numerous advantages including abundance, renewability, biodegradability, nontoxicity and good biocompatibility [6], [7]. Chitooligosaccharide (COS), partially hydrolyzed products of chitosan, is an attractive bioactive material because of improved solubility, low viscosity, as well as multiple biological activities [8]. Antioxidative activity is a prominent and fascinating property of COS. However, the term “COS” represents a large group of structurally different chemical entities with various degree of polymerization (DP) and degree of deacetylation (DD) [8], [9]. Molecular weight, in linear correlation with DP under a certain DD, is one of the most important factors affecting both solubility and bioactivity. Therefore, as a potential natural antioxidant, it is highly significant to reveal the relationship between molecular weight and antioxidative activity. Unfortunately, the previous studies present conflicting results on the issue. Some reports suggest that COS with low molecular weight was implicated in high antioxidative activity [10], [11], [12]. Chen et al. found that chitobiose showed markedly superiorly scavenging superoxide radical activity compared to COS with higher molecular weight [13]. But some available evidences point to the opposite conclusion [14], [15]. Furthermore, it was speculated that there may exist an optimum molecular weight of COS in terms of activity [16], [17]. In the previous reports, the concept of average molecular weight was usually underlined without consideration of molecular weight distribution, more importantly, the antioxidative activity of COS was widely studied based on cell-free chemical assay. As a result, it is hard to find out COS with high antioxidative activity for further application. Gradually, it is recognized that the molecular weight distribution of chitosan should be taken into account in the investigation of structure-property correlativity [18]. Much effort has been devoted to obtaining COS with narrow molecular weight distribution. Chromatography technology has been apply to isolate COS with DP less than 6, but not efficient for COS with larger molecular size [16]. In addition, organic solvent precipitation method is frequently adopted to narrow the molecular size of COS mixture [19]. But it causes several problems in practical application, such as low efficiency, solvent residual, and the recovery of a large amount of organic solvent. Furthermore, ultrafiltration treatment has been attempted to separate COS according to the molecular weight. Unfortunately, neither the molecular weight nor the distribution was investigated [20], [21]. As a result, the narrow and defined molecular size distribution is important for both study and application.

In this study, several green technologies, including oxidative pretreatment, enzymatic hydrolysis, membrane treatment and spray drying, were firstly combined for obtaining various molecular weight COS acetate salts with narrow molecular weight distribution. These COS products were well characterized via water-solubility test, GFC, FT-IR and XRD to investigate the physicochemical properties. Moreover, the antioxidative activity of COS acetate salts with different molecular weight was evaluated with strictly chemical methods and cellular assays. This work provides a practical reference for preparing and applying COS acetate salt with antioxidative activity in large scale.