Isolation and characterization of pancreatic lipase inhibitors from endophytic fungi

Abstract

The present study was oriented to evaluate the potential of endophytic fungi to produce pancreatic lipase inhibitors. The cell free culture filtrates of 190 endophytic fungal isolates were screened for their pancreatic lipase inhibitory activity. The potential fungal culture filtrates were then partially purified using liquid‐liquid fractionation. Crude aqueous residue of #6AMLWLS inhibited porcine pancreatic lipase with an IC50 of 2.12 μg/ml, which showed better potential than the positive control Orlistat (IC50 2.73 μg/ml). The bioactive fungus (#6AMLWLS) was identified as Fusarium incarnatum both by morphological as well as molecular studies. Further TLC fractionation and silica gel column chromatography separated the crude residue into two pure bands (B1 and B2). The pure band B1 competitively inhibited pancreatic lipase with an inhibition coefficient (Ki) of 7.1 μM. It was a white amorphous solid, highly polar having melting point greater than 300°C. In phytochemically testing B1 showed positive blue coloration with ninhydrin, suggesting it to be an amino acid. 1HNMR, 13CNMR spectroscopy suggested the bioactive compound to be a four carbon amino acid derivative and peaks in FTIR spectroscopy confirmed the respective functional groups. Band 1 exhibited ESI-MS ions at m/z 422 confirming four amino acids to be linked by peptide bonds. The compound was chemically named as 11-amino-2,5,8-triethoxy-4,7,10-trioxo-12-oxa-3,6,9- triazatetradecanoic acid and subsequently called as Fustat. A literature search revealed that this compound did not match with any reported lipase inhibitors. Fustat showed 60% inhibition of lipid accumulation in 3T3-L adipocytes exhibiting no cytotoxicity against 3T3-L adipocytes. The present study establishes endophytic fungi as an important resource for molecules, which could be used as inhibitors of pancreatic lipase, a major target for controlling diet induced obesity. Fustat exhibits similar potential as orlistat for its further evaluation using animal model for pharmaceutical drug development.