New natural pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and iNOS inhibitors identified from Penicillium polonicum through in vitro and in vivo studies

•Culture broth extract of Penicillium polonicum (endophytic fungi of P. nigrum fruit) was found to inhibit pro-inflammatory cytokines.•Three phenolics (1, 2 and 4) and one indole derivative (3) were isolated from the extract and explored as IL-1β, TNF-α, IL-6 and iNOS inhibitors.•The effect of pure compounds (1–4) and their combinations C1 (mixture of 1–4) and C2 (mixture of 1–4 & piperine) were studied through in vitro and in vivo methods.•Evaluation through mouse LPS-induced endotoxaemia and carrageenan-induced paw oedema models proved the anti-inflammation effect.•Compounds 1–4 and C1 attenuated inflammation by controlling the TNF-α, IL-6, IL-1β, iNOS, MPO and NO levels.•Compounds regulated the expression of pro-form and matured-form of IL-1β and NFκB. Overexpression of pro-inflammatory cytokines and iNOS have been found to be concomitant with several chronic inflammatory diseases and hence targeting their inhibition would be a useful therapy for inflammation. In view of this, study on discovery of natural pro-inflammatory cytokines inhibitory lead molecules from Penicillium polonicum, an endophytic fungus isolated from the fresh fruits of Piper nigrum was performed. When the culture broth extract of P. polonicum (EEPP) was subjected to LPS-induced cytokines expression (ELISA in RAW 264.7 cells), it exhibited inhibition of TNF-α, IL-6 and IL-1β and this encouraged us to do chemical investigation on EEPP to explore the bioactive components. Four compounds isolated and characterised as 3,5-di-tert-butyl-4-hydroxy-phenyl propionic acid (1), 2,4-di-tert-butyl phenol (2), indole 3-carboxylic acid (3) and tyrosol (4) were tested for their effect on the production of TNF-α, IL-1β and IL-6 in RAW 264.7 cells (ELISA). All the compounds exhibited a highly significant (P