Immuno-modulatory activities of crude extracts obtained from Echinodontium tinctorium. (a) At 1 mg/mL, crude extracts (80% ethanol, 50% methanol, water, 2% ammonium oxalate, and 5% NaOH) were assessed for their ability to induce TNF-α production in RAW264.7 macrophage cells as an indicator of immuno-stimulation. Lipopolysaccharide (LPS) was used as positive control, whereas media and water were used as negative controls. (b) Crude extracts (1 mg/mL) that were inactive for immuno-stimulation (80% ethanol, 50% methanol, and 5% NaOH) were tested for their ability to inhibit LPS-induced TNF-α production as an indicator of anti-inflammatory activity. Polymyxin B (PMB; an inhibitor of LPS) was used as a positive control, whereas media and water treated with LPS were used as negative controls. (c) Comparison of the anti-inflammatory activity of 5% NaOH extract from two different collections of E. tinctorium. Due to the large quantity of a new collection, the new NaOH extract was used for further purification studies and in vivo experiments. Data shown were pooled from three biological replicates (n = 3). Error bars are S.D., and one-way ANOVA was performed as statistical analysis. * denotes p < 0.05 as compared to the water control using Sidak’s multiple comparison test.