Chromatography separation, 1H NMR analysis and bioautography screening of methanol extract of Excoecaria agallocha L. from Bhitarkanika, Orissa, India

Abstract

ObjectiveTo separate the bioactive compounds from crude methanol extract of leaf of Excoecaria agallocha (E. agallocha), a mangrove plant from Bhitarkanika, Odisha, India. MethodsChromatographic separation was carried out by paper, thin layer chromatography (TLC) and column chromatographic techniques. Bioautography of the column fractionated extract and TLC chromatogram were evaluated in vitro for antibacterial and DPPH free radical scavenging activity. The HPLC and 1H NMR spectroscopy of the extract was also carried out to detect the presence of compounds and possible functional groups in the crude plant extract. Results: Evaluation of solvent system for chromatographic separation revealed that n-butanol: acetic acid: water in the ratio of 4:1:1 was the most appropriate for the separation of phenolic compounds in the crude extract. The antibacterial bioautography screening of the TLC fractionated extract of E. agallocha showed positive activity against Staphylococcus aureus (MTCC-1141) and Pseudomonas aeruginosa (MTCC 1034). Evaluation of antioxidant properties of TLC fraction of E. agallocha showed 23.36% scavenging activity using DPPH assay at 40 μL concentration. HPLC chromatogram of the plant extract produced seven peaks for the leaf and two peaks for the stem/bark with the highest peaks at 2.563 and 2.493 minutes of retention time, respectively. The 1H NMR spectroscopic analysis of the plant extract revealed the presence of acyclic aliphatic and α- mono substituted aliphatic group of compounds in the sample. ConclusionsThus, it is apparent from the present study that methanol crude extract of E. agallocha is rich in aliphatic group of compounds which can be purified and characterized for possible pharmaceutical utilization.