METHODS: Raw 264.7 macrophages were used to asses G. cowa Roxb. immunomodulatory activity. The MTT assay was chosen to measure cell viability to evaluate the cytotoxic effect on cells. ELISA method was used to measure the concentration of Interleukin-6 (IL-6) and Tumor Necrosis Factor Alpha (TNF-α) secreted by cells after being treated with G. cowa Roxb. fraction. The neutral red uptake assay determined the effect of Garcinia cowa Roxb. on the phagocytic activity.
RESULTS: After Raw 264.7 macrophages were given the Hexan fraction (Hex) at concentrations of 12.5 and 25 μg/mL, there was a decrease in the concentration of IL-6, TNF-α, and the phagocytosis index of cells. Administration of the Ethyl Acetate fraction (EtOAc) at concentrations of 12.5 and 25 μg/mL on cells caused a decrease in IL-6 and TNF-α levels but did not affect the phagocytosis index. There was an increase in the level of TNF-α and the phagocytosis index after being given the Butanol fraction (BuOH) with concentrations of 12.5 and 25 μg/mL but there was a slight decrease in the level of IL-6.
CONCLUSIONS: Both Hex and EtOAc fractions could suppress immune responses through decreasing IL-6, TNF-α, and slightly decreased phagocytic activity. BuOH fraction could stimulate immunomodulatory activities through enhanced TNF-α levels and phagocytic index, but less potent in enhancing IL-6 production. The BuOH fraction could be developed as an immunostimulant.
AIM OF THE STUDY: This study explores the anti-inflammatory effect of TPTQ in silico, in vitro, and in vivo.
MATERIALS AND METHODS: In silico testing used the Gnina application, opened via Google Colab. The TPTQ structure was docked with the nuclear factor kappa B (NF-ĸB) protein (PDB: 2RAM). In vitro testing began with testing the cytotoxicity of TPTQ against Raw 264.7 cells, using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method. A phagocytic activity test was carried out using the neutral red uptake method, and interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) secretion tests were carried out using the enzyme-linked immunosorbent assay (ELISA) method. In vivo, tests were carried out on mice by determining cluster of differentiation 8+ (CD8+), natural killer cell (NK cell), and IL-6 parameters, using the ELISA method.
RESULTS: TPTQ has a lower binding energy than the native ligand and occupies the same active site as the native ligand. TPTQ decreased the phagocytosis index and secretion of IL-6 and TNF-α experimentally in vitro. TPTQ showed significant downregulation of CD8+ and slightly decreased NK cells and IL-6 secretion in vivo.
CONCLUSION: The potent inhibitory effect of TPTQ on the immune response suggests that TPTQ can be developed as an anti-inflammatory agent, especially in the treatment of Covid-19.