Bioassay-guided purification of α-glucosidase inhibitor fatty acids from Padina tetrastromatica

Anti-diabetic potential of a brown alga, Padina tetrastromatica , from the coastline of the Persian Gulf, using bioassay-guided purification approach yielded the most active α-glucosidase inhibitor agents as fatty acids. Initially, the yeast’s α-glucosidase inhibition of the MeOH and 80% MeOH extracts were evaluated via a colorimetric assay. The liquid-liquid fractionation of 80% MeOH extract, as the most potent α-glucosidase inhibitor, resulted in four fractions, n -hexane, ethyl acetate, n -butanol and water. Hexane and ethyl acetate fractions were selected for further studies, with the IC 50 values of 38.0 ± 0.3 µg/mL and 53.7 ± 2.6 µg/mL, respectively. Followed by investigating α-glucosidase inhibition of the sub-fractions from the hexane fraction, using flash column chromatography, F18-21 selected as the most potent enzyme inhibitor. After further purifications of F18-21 by semi preparative HPLC, the mentioned fraction and two purified compounds, 8-octadecenoic acid ( 8 ) and all- cis -5,8,11,14-eicosatetraenoic acid ( 10) were subjected to GC-MS analysis for the identification. In addition, 1D and 2D NMR evaluations were performed for characterisation of 8-octadecenoic acid. Furthermore, three fatty acids, all- cis -8,11,14,17-eicosatetraenoic acid ( 6 ), cis -9,12-octadecadienoic acid ( 7 ), and all- cis -5,8,11,14,17-eicosapentaenoic acid ( 11 ), were isolated from the ethyl acetate fraction and identified, by HPLC and GC-MS techniques, respectively. Finally, α-glucosidase inhibition percent of the purified fatty acids were evaluated in two concentrations in the microplates, which showed their great potential for further investigations as anti-diabetic agents, in comparison with acarbose, as the positive control. Furthermore, molecular docking analysis and MD simulation were applied to investigate the structure activity of the purified compounds.