The challenges of the COVID-19 pandemic have highlighted an increasing clinical demand for safe
16 and effective treatment options against an overzealous immune defence response, also known as the “cytokine
17 storm”. Andrographolide is a naturally derived bioactive compound with promising anti-inflammatory activity
18 in many clinical studies. However, its cytokine-inhibiting activity, in direct comparison to commonly used
19 nonsteroidal anti-inflammatory drugs (NSAIDs), has not been extensively investigated in existing literature. The
20 anti-inflammatory activities of andrographolide and common NSAIDs, such as diclofenac, aspirin, paracetamol
21 and ibuprofen were measured on lipopolysaccharide (LPS) and interferon-γ induced RAW264.7 cells. The levels
22 of PGE2, nitric oxide (NO), TNF-α & LPS-induced release of pro-inflammatory cytokines on differentiated
23 human macrophage THP-1 cells were measured against increasing concentrations of andrographolide and
24 aforementioned NSAIDs. The associated mechanistic pathway was examined on NFκB using flow cytometry on
25 the human endothelial-leukocyte adhesion molecule (ELAM9) (E-selectin) transfected RAW264.7 cells with
26 green fluorescent protein (GFP). Andrographolide exhibited broad and potent anti-inflammatory and cytokine-
27 inhibiting activity in both cell lines by inhibiting the release of IL-6, TNF-α and IFN-γ, which are known to play
28 a key role in the etiology of cytokine storm and the pathogenesis of inflammation. In comparison, the tested
29 NSAIDs demonstrated weak or no activity against proinflammatory mediators except for PGE2, where the
30 activity of andrographolide (IC50 = 8.8 μM, 95% CI= 7.4 to 10.4 μM) was comparable to that of paracetamol (IC50
31 = 7.73 μM, 95% CI = 6.14 to 9.73 μM). The anti-inflammatory action of andrographolide was associated with its
32 potent downregulation of NFκB. The wide-spectrum anti-inflammatory activity of andrographolide
33 demonstrates its therapeutic potential against cytokine storms as an alternative to NSAIDs
