Bioactivity-guided Isolation of the Antidiabetic Compound Fucosterol from the Leaves of Coccinia grandis (L.)

Abstract

Background: Coccinia grandis (L.) Voigt (Family: Curcurbitaceae) leaves are known to be a promising source for the isolation of antidiabetic compounds. Previous studies confirmed that the ethyl acetate extract of C. grandis leaves exerted potent α-amylase, α-glucosidase, and DPP IV enzymes inhibitory activities in-vitro. Objectives: To isolate and elucidate the structure/s of antidiabetic compound/s from the ethyl acetate leaf extract of C. grandis following the bioactivity-guided principle. Methods: The ethyl acetate leaf extract of C. grandis was fractionated by vacuum liquid chromatography (VLC). The resultant sub-fraction with the highest antidiabetic activity was further fractionated subsequently in a stepwise manner using flash chromatography and thin layer chromatography until a pure compound was isolated. Antidiabetic activity was determined in resulting fractions/sub-fractions and in the isolated compound using α-amylase, α glucosidase, and DPP-IV enzyme inhibitory assays. The structure of the isolated compound was elucidated using 1H and 13C Nuclear Magnetic Resonance (NMR) data and confirmed using FT IR spectroscopy. Physical appearance and melting point of the compound were noted. Results: The ethyl acetate extract of C. grandis yielded four VLC sub-fractions; EA1, EA2, EA3, and EA4. The EA4 was the most active sub-fraction which showed the highest inhibitory enzyme activities of α-amylase, α-glucosidase, and DPP-IV as IC50 7.13 (±0.36), 0.40 (±0.02) mg mL-1 , and 28 (±2) µg mL-1 , respectively. The subsequent isolation steps on EA4 yielded a pure pale-yellow compound with a melting point of 134-136⁰C. On the basis of the spectroscopic data, the pure compound was identified as fucosterol. α-Amylase, α-glucosidase, and DPP-IV inhibitory activities of fucosterol were determined to be IC50 0.77 (±0.01), 0.03 (±0.00) mg mL-1 , and 52.0 (±4.0) µg mL-1 , respectively. α-Glucosidase inhibitory activities of EA4 and fucosterol were significantly higher than that of acarbose (p<0.05). This is the first report on isolation of fucosterol from C. grandis leaves. Conclusions: The compound, fucosterol was isolated from the leaves of C. grandis for the first time and it exerted α-amylase, α-glucosidase, and DPP-IV inhibitory potential in-vitro, indicating its potency to act as an antidiabetic agent.