Determination Of Intestinal Absorption Rates Of Chito-oligosaccharide With Different Degree Of Polymerization

COS, known as Chito-oligosaccharide, is a kind of oligosaccharide linked by 2-10 glucosamine in the pattern ofβ-1, 4 glycosidic. Usually, it can be obtained from Chitin, which abundant in marine animals, through deacetylation and biological degraded engineering by using glycosidase. COS is the only prolific alkaline aminoglycan among all the natural sugar. It not only possess the features of water-soluble, safe non-toxic, easily absorbed and so on, but also have variety of biological activities such as enhancing the immunity of cellular and humoral, suppression the growth of tumor cell, regulation of blood lipids and increase the number of beneficial bacteria in intestinal canal.The biological activity of COS mainly includes following 2 aspects. Firstly, COS can play the role of Probiotics Bacteria as it can inhibit escherichia coli, staphylococcus aureus and other intestinal bacteria while increasing the number of Bifidobacterium and other Probiotics group when it enters into intestine. Secondly, COS can bring some positive functionalities after it has been absorbed into the bloodstream and reached the appropriate target organ in intestinal canal such as enhancing body's immunity, balancing the proportion of sugar in blood and inhibiting the growth of the tumor cells.Objectives: As far as we known from the literature, most of the research around the functionality of COS are focused on the culture of cell in vitro and the test and evaluation of impact efficiency through animal experiment. However, there is few report around the actually accurate absorbed status of COS when it enters into the digestive tract; the actual amount and proportion of COS being absorbed into the bloodstream and reached the target organ to reduce the amount of sugar, regulate immunity and play the anti-cancer effect; the actual amount and proportion of COS that play a role of regulating bacteria flora in intestinal so as to adjust immunity and inhibit tumor cell growth. The purpose of this study lies in following parts. Firstly, track the path of COS to see how it reaches the digestive tract. Secondly, make a comparison research to see if there are differences on the amount and proportion of COS being absorbed into the blood and intestine when use relatively large and small molecular weight of COS. Thirdly, study to identify the differences and relationships between the molecular weight of COS and the amount being absorbed into the blood and intestine. Finally, this study is trying to illustrate the main routes of COS through experiment, which could be used as the basis for further study on the functionality mechanism of COS.Methods: Firstly, we generated different polymerization degree of COS from snail enzymes through hydrolyze method. And then, we need to separate them according to their degree of polymerization through polyacrylamide gel chromatography method (Bio-Gel P-4). COS with polymerization degree within the range of 1-3 and 8-11 need to be collected and labeled by FITC, which is short for fluorescein isothiocyanate. The dissociative FITC should be removed from COS through the polyacrylamide gel column (Bio-Gel P-2/P-4) method. Two groups of 24 fasting hours mice will be lavaged by FITC labeled large and low molecular weight COS respectively. 1 hour latter, their blood serum and intestine should be taken and used as sample for detecting the intensity of fluorescence by using the fluorospectrophotometer after a series of treatment. The result could be used to determine whether COS is being absorbed into the bloodstream and the proportion between those being absorbed into blood and directly discharged from the intestinal tract, which in turn verifies if there is interrelationship between polymerization degree and the proportion of COS being absorbed into blood. Finally, all intestinal and blood serum sample need to pass polyacrylamide gel chromatography again (Bio-Gel P-4) to check and verify FITC labeled COS does not degrade in digestive tract.Results: 1. By adjusting the hydrolysis time, snail enzyme can be hydrolyzed into COS with different degree of polymerization, which could be eluted into COS with specific range of polymerization degree by using Bio-Gel P-4 polyacrylamide gel elution chromatography column.2. FITC can be used to label COS. The dissociative FITC can be eluted through Bio-Gel P-2, P-4 polyacrylamide gel elution chromatography column method. 3. After fed mice with FITC labeled large and small molecular weight COS fed mice, we can detect the intensity of fluorescence from both blood serum and intestinal object. The average ratios between them are 5.68:1 and 9.84:1 separately. 4. Based on the experiment result on the evaluation of intestinal juice and blood through the enteric material polyacrylamide gel column, there is significant peak around the position of FITC-COS elution volume, which confirmed that FITC labeled COS does not degrade in the digestive tract.Conclusion: FITC labeled COS can be smoothly absorbed while not being degraded in intestinal canal. The ratio of COS being absorbed into blood and intestine are 5.68:1 and 9.84:1 while we use large and small molecular weight COS. We can draw the conclusion that the absorption ratio of COS in intestinal canal will increase when the molecular weight decreases.