Purification and Characterization of Antimicrobial Peptides with Insulinotropic Activity from the Skin Secretion of Bornean Frogs

AMERA A M, MOHAMEED (2025) Purification and Characterization of Antimicrobial Peptides with Insulinotropic Activity from the Skin Secretion of Bornean Frogs. Masters thesis, Faculty of Medicine and Health Sciences (FMHS).

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Abstract

The antimicrobial and insulinotropic properties of skin secretions from two Bornean frog species in the Ranidae family—Pulchrana baramica and Hylarana erythraea—and Limnonectus paramacrodon from the Dicroglossidae family were investigated. A comprehensive approach utilizing various techniques was employed to study the characteristics of antimicrobial peptides (AMPs) isolated from these frogs. Initially, crude skin secretions were partially purified using Sep-Pak C-18 cartridges, followed by further purification through AKTA HPLC. SDS-PAGE analysis demonstrated that the AKTA HPLC method produced clearer, more distinct bands compared to Sep-Pak, confirming its superior effectiveness. In disc diffusion tests, the peptides exhibited significant inhibition zones against Escherichia coli and Staphylococcus aureus, indicating strong antibacterial activity. Specifically, P. baramica showed inhibition zones of 11.66±3.44 and 11.33±0.9 mm at 1000 µg/ml and 500 µg/ml concentrations, respectively, against E. coli, and 5.6±0.6 mm against S. aureus at 1000 µg/ml. Meanwhile, H. erythraea displayed inhibition zones of 7.33±0.5 and 4.3±0.6 mm at 1000 µg/ml and 500 µg/ml concentrations, respectively, against E. coli, and 4.6±0.6 mm against S. aureus. No antimicrobial activity was observed for L. paramacrodon. Minimum inhibitory concentration (MIC) tests revealed species-specific values, with AMPs from P. baramica demonstrating higher potency than those from H. erythraea (MIC: 250 µg/ml). Regarding cytotoxicity, AMPs purified from both Ranid frogs caused over 50% lysis of red blood cells, indicating potential impacts on mammalian erythrocyte membrane integrity, whereas L. paramacrodon peptides displayed lower toxicity with less than 50% cell lysis. Furthermore, the insulinotropic effects of Bornean frog AMPs on HIT-T15 cells were examined. These AMPs were found to enhance insulin secretion from pancreatic cells, with statistical analysis indicating a significant effect (p < 0.05) for P. baramica. In contrast, the effects of H. erythraea and L. paramacrodon were not significant (p > 0.05). These findings suggest that multifunctional peptides play a crucial role in protecting Bornean frogs against invading pathogens and hold potential as antidiabetic agents for managing glucose levels and diabetes.

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