Elsevier

Carbohydrate Polymers

Volume 79, Issue 1, 5 January 2010, Pages 137-144
Carbohydrate Polymers

Preparation of a series of chitooligomers and their effect on hepatocytes

https://doi.org/10.1016/j.carbpol.2009.07.039Get rights and content

Abstract

A series of chitooligomers with molecular weight ranging from 1.7  3.8 × 103 were prepared by degradation of a high molecular weight chitosan with hydrogen peroxide and selective precipitation in ethanol solutions. The prepared chitooligomers were characterized by gel permeation chromatography, elemental analysis, Fourier transform infrared spectra, 13C nuclear magnetic resonance spectroscopy and X-ray diffraction analysis. Cell culture experiments suggested that the effect of the chitooligomers on the proliferation of L02 hepatocytes was dependent on culture time, namely, at the initial stage of culture there was an inhibitory effect on proliferation of the cells; however, the cultures recovered in cell proliferation and exhibited promotion effect in following days. In the case of chitosan monomer (GlcN), high concentration of GlcN (1 mg/ml) produced a significant suppression in proliferation of L02 cells relative to control, with decreases of 35.2%, 60% and 72.9% on days 1, 3 and 5, respectively. In addition, there was no significant effect of the chitooligomers on the functions of albumin secretion and urea synthesis of the hepatocytes.

Introduction

Chitooligomers, a class of chitosans with degree of polymerization <20, are known to have some special biological activities such as antibacterial activity (Jeon, Park, & Kim, 2001), antitumor and immune enhancing effects (Jeon and Kim, 2002, Qin et al., 2002, Tokoro et al., 1988). These functions depend not only on their chemical structure but also on the molecular size. In recent years, studies of the effect of chitooligomers on various cells, e.g. polymorphonuclear cells (Usami, Okamoto, Takayama, Shigemasa, & Minami, 1998), macrophages (Okamoto et al., 2003), fibroblasts (Mori et al., 1997), endothelium (Okamoto et al., 2002), osteoblasts (Ohara et al., 2004) and red blood cells (Fernandes et al., 2008), have attracted more and more interest. The study of behaves of polymorphonuclear cells (PMNs), macrophages, endothelium and fibroblasts treated with chitooligomers have been related with the mechanisms of induction of acceleration of wound healing by chitin and chitosan. It has been demonstrated chitooligomers induced chemotactic migration of PMNs and stimulated the induction of interleukin-8 of fibroblast (Mori et al., 1997, Usami et al., 1998). However, to our best knowledge, there is no report on the effect of chitooligomers on hepatocytes.

Chitooligomers can be obtained by enzymatic depolymerization of chitosan (Qin, Wang, Peng, Hu, & Li, 2008) or by chemical depolymerization with acids such as hydrochloric acid (Belamie, Domard, & Giraud-Guille, 1997), nitric acid (Tommeraas, Varum, Christensen, & Smidrod, 2001) and sulfuric acid (Nagasawa, Tohira, Inoue, & Tanoura, 1971). In general, enzymatic methods have advantages over chemical reaction, since enzymes operate under milder conditions and are highly specific; however, their commercial use is limited due to cost and limited availability (Li et al., 2005). In addition, chitooligomers can also be obtained by oxidative depolymerization with oxidants, such as ozone (Yue, He, Yao, & Wei, 2009), sodium nitrite (Allan & Peyron, 1995) and hydrogen peroxide (Chang et al., 2001, Qin et al., 2002, Tian et al., 2004, Wang et al., 2005). Hydrogen peroxide has long been used in the treatment of chitosan because it is easy to handle, easily available and environmentally friendly. In particular, for cell culture applications, the method does not cause impurities in products. However, it was reported the reaction occurred in a random pattern, resulting in a broad distribution of molecular weight of the chitooligomers (Qin et al., 2002, Qin et al., 2002).

The goal of this work was to prepare a series of chitooligomers with different of molecular weights, and to explore preliminarily the effect of prepared chitooligomers on the hepatocytes. Here, we describe a quick and simple method for the production of a homogeneous series of chitooligomers, varying in molecular weight from 1.7 to 3.8 × 103, with low polydispersity. The prepared method involves a simple chemical process and only selective precipitations. Finally, cell proliferation, morphology and functions such as albumin secretion and urea synthesis were employed to determine the effect of the chitooligomers on the hepatocytes.

Section snippets

Materials

Original chitosan (CS0) with a degree of deacetylation (DD) of 91% was purchased from Zhejiang Aoxing Biotechnology Co., Ltd. (Zhejiang, China). Its viscosity average molecular weight is about 300,000. Chitosan monomer (d-glucosamine hydrochloride), penicillin and streptomycin were supplied by Solarbio (Beijing, China). RPMI-1640 was obtained from Gibco (USA). Fetal bovine serum (FBS) was purchased from HyClone (USA). Urea Nitrogen Kit and Albumin Detection Kit were from Nanjing Jiancheng

Molecular weight, polydispersity and DD of the prepared chitooligomers

The preparation of chitooligomers by degradation reaction with hydrogen peroxide has long been studied. In acid solution, chitosan was dissolved and the degradation reaction was homogenous. During the degradation reaction in our experiment, the solution viscosity dropped sharply at the beginning of the reaction (0  10 min), then decreased slowly. After reaction 2 h, there was almost no water-insoluble chitosan precipitation in the reaction solution when the pH value of the solution was adjusted to

Discussion

Chitooligomers are known to have some special biological activities. The effect of chitooligomers on various cell types, e.g. osteoblasts, macrophages, fibroblasts, endothelium and red blood cells, has been widely studied in recent years. It has been shown that chitooligomers exhibited effects on cell activity which were dependent on different targeted cells and concentration and molecular weight of chitooligomer. Ngo, Kim, and Kim (2008) examined the effects of the chitosan oligosaccharides

Conclusions

The present research showed that oxidative degradation of chitosan with hydrogen peroxide combined with a selective ethanol precipitation appears to be a quick and simple method to obtain chitooligomers with low polydispersity. The prepared chitooligomers were found to have effects on the proliferation of L02 hepatocytes which were dependent on culture time. In the case of chitosan monomer (GlcN), high concentration of GlcN produced a significant suppression in proliferation of L02 cells

Acknowledgements

This work is supported by the National Science Foundation of China (Project No. 30870614). The authors would like to thank Ms. Xiaoyan Wang, researcher at the Analytical & Testing Center of Sichuan University, for valuable suggestion and specimen characterization.

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