Isolation, Characterization of Neoandrographolide from Andrographis macrobotrys Nees and Evaluation of its effect on LPS induced TNF-α Activity

Introduction: Andrographis macrobotrys Nees is an important species of genus Andrographis with applications in traditional medicine. Neoandrographolide is one of the constituents in this plant. But there are no previous reports of isolation of neoandrographolide from A. macrobotrys. Current work is undertaken to concentrate on isolation, characterization, and evaluation of tumor necrosis factor-alpha (TNF-α) inhibition activity of neoandrographolide from A. macrobotrys. Materials and Methods: For the isolation process techniques like column chromatography, thin-layer chromatography (TLC), and preparative TLC were used. Characterization was done by ultra visible (UV)-visible spectroscopy, Fourier transform infrared (FTIR), proton nuclear magnetic resonance (1H NMR), carbon-13 (C13) nuclear magnetic resonance (13C NMR) analysis. 3-(4,5-dimethylthiaxo-2yl) 2, 5-diphenyl tetrazolium bromide (MTT) assay was done for the preliminary cytotoxicity test to standardize the sample concentration for the TNF-α inhibition study. The flowcytometric method was used to determine TNF-α inhibiting ability in a human monocytes cell line (THP-1). Results: Neoandrographolide was isolated from methanolic extract of A. macrobotrys which had a melting point of 174-175ºC. FTIR results had shown stretching for –OH, 3427.58 cm^-1,sp³-CH, lactone, and α, β unsaturated ester. NMR data confirmed 26 carbon structures. Cytotoxicity of isolated neoandrographolide was 22.59 μg/ml. Further lipopolysaccharide (LPS) induced TNF-α inhibition was highest in the case of isolated neoandrographolide in comparison with the crude extract of A. macrobotrys. Conclusion: A. macrobotrys can be used as a new source of neoandrographolide with anti-inflammatory abilities by inhibiting the TNF-α release in THP-1 cells.