Preparation Of Chitooligosaccharides And Their Effects In Vivo

Chitosan, the deacetylated derivatives of chitin, is one of the nontoxic and biodegradable carbohydrate polymer, and has received much attention as a functional biopolymer for diverse Pharmaceuticals. Chitosan undergo enzymatic hydrolysis to give the chitooligosaccharides with great water-solubility. chitooligosaccharides are known to have biological activities such as antitumour activity, immuno-enhancing effects, enhancing protective effects against infection with certain pathogens in mice, antifungal activity. There is a defect that these chitooligosaccharides products easily turn browning in the process of preparation, thermal drying and preservation. These undesirable effects may be minimized by freeze-drying and proper storage conditions in laboratory scale. The large-scale production of chitooligosaccharides, to be economically feasible and technically acceptable, need to be studied.The recent progress on the preparation of chitooligosaccharides and their physiological activity was reveived in this paper. The modern analysis methods including high performance liquid chromatoghaphy(HPLC), Fourier transform infrared(FT-IR), magenetic resonance spectroscopy(¹H-NMR, ¹³C-NMR), elemental analysis, thermogravimetnc analysis(TG) and differential thermal analysis(DTA) were used to characterized the structure of product.The following are the main contents and conclusions:1. Chitosan was hydrolyzed to give chitooligosaccharides by alpha-amylase. The chitooligosaccharides was achieved from chitosan which was depolymerized by 3% alpha-amylase for 6h. The enzymatic hydrolysis could not change the structure of chitosan, but result in the decrease of thermo-stability.2. Chitooligosaccharides turn browning easily during the process of preservation. The factors influencing the browning of chitooligomers were investigated. The results indicated that the browning was attributed to the structure change of chitooligosaccharides. The water-solubility and moisture-adsorption of chitooligosaccharides decreased with the increase of browning. The time, temperature, pH, moisture, oxygen and reductant all had effect on the browning of chitooligomers. The optimal preservation condition for chitooligomers should be at low temperature and humidity, at pH below 4 or above 10, and in absence of oxygen.3. The chitosan was degraded by the cheap, efficient and commercially available enzymes in hydrochloric acid solution. The effect of molar ratio of added HCl to -NH₂ of chitosan on the degradation was investigated, and the favorable ratio was between 0.50 and 0.95. The chitooligomer hydrochlorides were directly prepared by the simple method. The chitooligomer hydrochlorides were characterized by thermogravimetric analysis, differential thermal analysis and Fourier transform infrared. These results show that the obtained chitooligomer hydrochlorides were much more stable than the chitooligomer acetates. This preparation procedure of chitooligomer hydrochlorides was simple and cheap, and helpful to preservation of chitooligomers.4. Chitosan, a cationic polysaccharide, has been widely used in biomedical fields. Although numerous studies have focused on its applications as controlled release or bioactive reagents, the effect of molecular weight and water-solubility of chitosan on its intestinal absorption, body distribution and bioactive properties still deserved to be investigated. As a preliminary study, we investigated the molecular weight -dependent intestinal absorption and body distribution patterns after oral administration of water-soluble chitosan (WSC, Mw 3.91×10⁴) and three fractions of chitosans with different molecular weight Mw (HCS 7.60×10⁵, MCS 3.60×10⁴ and LCS 5.30×10³). The results indicated that chtiosans could be absorbed, and the absorption and body distribution was significantly influenced by its M_W and water-solubility. As the M_W decreased, the absorption increased, high molecular weight chitosan (7.60×10⁵) was rarely absorbed by intestinal. The concentration of LCS in blood was 3 times and 7 times higher than MCS and HCS respectively at 0.5h. Four fractions of chitosans were distributed to all tested tissues such as liver, kidney, spleen, thymus, heart and lung. As Mw increased, the concentration in liver increased at 0.5h after administration. In consideration of different molecular weight chitosans in different body distribution manner, chitosan may be employed as material for drug delivery or controlled released reagents in biomedical applications.5. In order to investigate the effect of chitosan on the trace element level by oral administration, the Kunming strain mice was used as experiment object (10 mice in each group) and orally administrated for 90 days at dose of 1.50g/(kg.bw.day). The results indicated that the levels of Cu, Fe and Zn in liver and spleen were significantly raised, by oral administration middle molecular weight chitosan (Mw 3.60×10⁴) for long time. That was not significantly influenced by oral administration low molecular weight chitosan (Mw 5.30×10³), high molecular weight chitosan (Mw 7.60×10⁵) and water-solubility chitosan(Mw 3.91×10⁴) for long-term, respectively.