Anti-obesity effect of Chitosan-glycyrrhizin conjugate

Abstract

In this article, we first used FT-IR and H-NMR to confirm whether glycyrrhizin was properly oxidized by sodium periodate and generate aldehyde groups or whether glycyrrhizin is conjugated to glycol chitosan. Then, we used spectrophotometer to measure surface charge, because we thought that conjugating glycyrrhizin to amine group of glycol chitosan could affect the surface charge of glycol chitosan. As a result, negative surface charge was shifted to positive charge. This is thought to because pKa of glycol chitosan was changed due to conjugation of glycol chitosan and intra-/inter- formation between glycol chitosan. Also, by using 1-octanol we measure hydrophobicity of glycyrrhizin, glycol chitosan and Chito-GL. As mentioned earlier, conjugating glycyrrhizin to amine group of glycol chitosan has changed the surface charge of glycol chitosan. As a result, amine group decrease could make glycol chitosan more hydrophobic. Therefore, we dissolved Chito-GL, glycol chitosan and glycyrrhizin into distilled water and vortexed with same amount of 1-octanol. Consequently, conjugating glycyrrhizin made glycol chitosan more hydrophobic, which means that Chito-GL could enter the cell by passive diffusion rather than active transport which is used to transport glycol chitosan at low concentration. To confirm the difference on cellular uptake on 3T3-L1 cells, we used confocal microscope. Although glycol chitosan was more taken inside the cell in low concentration, at high concentration Chito-GL was more taken inside the cell whether it was differentiated or not differentiated 3T3-L1 cells. Before treating Chito-GL to the cells, we performed cell cytotoxicity test. Through this data we confirmed that from 0.7mg/ml Chito-GL shows cell cytotoxicity. As a result, we used 0.5mg/ml concentration of Chito-GL, because it was the highest concentration that did not show any cell cytotoxicity. In addition, we performed oil red o staining to confirm anti-obesity effect of Chito-GL. Chito-GL did not show anti-obesity effect at low concentration, however when it is treated at 500ug/ml it showed highest anti-obesity effect. Finally, we injected Chito-GL, glycol chitosan, glycyrrhizin to C57BL/6 mouse for 4 weeks daily. Consequently, Chito-GL showed 11.22% body weight decrease compare to high fat diet fed mouse. In case of glycyrrhizin, it showed high body weight gain and high level of blood glucose level. This is mainly due to low concentration of glycyrrhizin and degeneration of pancreas islet. Although glycol chitosan did not show statistic difference between normal diet fed mouse, it showed high blood glucose level. To sum up, When Chito-GL is intraperitoneally injected, it could suppress body weight gain. Additionally, it could prevent the pancreas islet from being degenerated from oxidative stress which is caused by obesity. Therefore, Chito-GL is efficient drug for treating obesity