|dc.description.abstract||New excipients to improve the oral absorption of drugs are developing significantly. Polymers are used to enhance drug absorption through the gastrointestinal tract. Chitosan is a biocompatible polymer that is capable of opening tight junctions in membranes and therefore widely used as an absorption enhancer. This polymer is, however, insoluble under normal physiological conditions and a wide range of more soluble chitosan derivatives was developed for the delivery of compounds in the more alkaline environment of the intestines. Furthermore, the ability of chitosan to act as a functional excipient is advantageous in terms of antioxidant- and antimicrobial activity. Chitosan, in combination with melittin, a cationic peptide component of bee venom, has been shown to have synergistic absorption effects in vitro. Therefore the aim and objectives of this study was to characterise chitosan derivatives and to evaluate their antioxidant- and antimicrobial activity and determine the cytotoxic effects. In vitro evaluation was performed on human hepatocellular liver carcinoma cell line (HepG2 cells) and human epithelial colorectal adenocarcinoma cell line (Caco-2 cells) and antimicrobial activity was determined on four bacterial strains.
The antioxidant activity of four different chitosan derivatives namely, trimethyl chitosan (TMC), triethyl chitosan (TEC), dicarboxymethyl chitosan (DCMC) and chitosan oligomers (TEO) was determined using the 1, 1- Diphenyl- 2- picrylhydrazyl (DPPH) assay. Additionally, the antimicrobial and in vitro cytotoxicity was determined of the four derivatives, melittin and a combination thereof. By using the disc and well diffusion assays and also the minimum inhibitory concentration (MIC) assay, the antimicrobial activity was determined. The 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT), lactate dehydrogenase (LDH) assays and flow cytometry were employed to determine the cytotoxicity.
It was determined that TEC displayed antioxidant activity with 25.37 ± 4.00%, 43.98 ± 6.67% and 47.66 ± 4.13% at 10 mg/ml, 25 mg/ml and 50 mg/ml, respectively, whereas the rest of the derivatives indicated as reactive oxygen species (ROS). Only TMC and melittin indicated antimicrobial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) at a concentration of 1.56 mg/ml for TMC and 0.0625 mg/ml and 0.03125 mg/ml for melittin respectively. TMC also displayed the most cytotoxicity in HepG2 cells with 27.35 ± 1.48%, 24.56 ± 1.19%, 19.66 ± 2.05% and 17.70 ± 1.54% at 5 mg/ml, 10 mg/ml, 25 mg/ml and 50 mg/ml, respectively and in Caco-2 cells 33.90 ± 3.40%, 32.43 ± 3.17%, 34.95 ± 4.34% and 26.82 ± 3.32% at 5 mg/ml, 10 mg/ml, 25 mg/ml and 50 mg/ml respectively. Other than TMC, DCMC indicated decreased cell viability at high concentrations with 27.08 ± 3.15% at 50 mg/ml in HepG2 cells and 34.45 ± 9.46% at 50 mg/ml in Caco-2 cells. Flow cytometry also indicated that the mechanism by which TMC decrease cell viability in HepG2 cells is through apoptosis and in Caco-2 cell necrosis.
It was concluded that TEC has antioxidant activity and TMC, melittin and a combination thereof has antimicrobial activity against selected bacterial strains, which might be an important contribution in the healing of wounds with skin infections. TMC indicated the most cytotoxicity in both cell lines and from previous results, it can be said that the molecular weight (MW) and degree of quaternisation (DQ) influences the functional properties and effect on cell viability of chitosan derivatives.||en_US