Effects of Clinacanthus nutans Extracts on Cytokine Secretion in PMA-Induced U937 Macrophage Cells

Background and objectives: Clinacanthus nutans (Burm f.) Lindau (C. nutans) is a wellknown traditional medicine in South East Asia and consists of abundant phytomedicinals properties. This study aimed to investigate the effects of C. nutans ethanol and aqueous extracts on interleukin4 (IL4) and interleukin13 (IL13) cytokines secretion in phorbol 12myristate 13acetate phorbol12myristate13acetate (PMA)induced U937 macrophages. Methods: Sequential ultrasonicassisted extraction was carried out using ethanol (ETOH) and water, by applying 1:10 ratio of leaves powder to the solvent volume. U937 cells were incubated with 25 nM PMA for 72 h to induce macrophage differentiation. The macrophage differentiation was assessed based on the cell morphological changes, cell viability and, CD14 and CD11b expression by using flow cytometry. The macrophages were incubated with both ETOH and aqueous extracts at 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg/mL concentration for 48 h. The viability of the extracttreated cells was assessed using PrestoBlue cell viability assay and the IL4 and IL13 secretions were assessed by using EnzymeLinked Immunosorbent Assay (ELISA). Results: PMA stimulation caused morphological changes of U937 cells from roundshaped, nonadherent to larger irregularshaped, adherent cells, and a reduction of cells viability to 87%. CD14 expression was downregulated from 7% to 4.5% upon PMA stimulation. CD11b expression was upregulated from 16% in untreated cells to 38% in PMAtreated cells. ELISA results showed that 1 mg/mL of ETOH and AQ extracts stimulated 1200 and 1800 pg/mL IL4 secretions, respectively. However, both extracts caused minimal IL13 secretion.  Conclusion: Clinacanthus nutans aqueous extracts stimulated IL4 production higher than ETOH extract in PMAinduced U937 macrophages.