ZCPG, a cysteine protease from Zingiber montanum rhizome exhibits enhanced anti-inflammatory and acetylcholinesterase inhibition potential.

A 48 kDa Zingiber montanum cysteine protease glycoprotein (ZCPG) purified previously was studied for anti-inflammatory and acetylcholinesterase inhibitory activity. The lipoxygenase inhibition by ZCPG was linear, with an IC50 value of 2.25 μM. MTT, LDH, and cell cycle analysis in THP-1 derived macrophages corroborate no significant cytotoxicity at a lower concentration. ZCPG inhibited the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in lipopolysaccharide-stimulated THP-1 macrophages. In contrast, an increase in the production of interleukin-10, an anti-inflammatory cytokine, was observed. A reverse-transcription polymerase chain reaction study further confirmed that ZCPG inhibited the expression of IL-1β, inducible nitric oxide synthase, and TNF-α by suppressing their mRNA transcription and expression in LPS stimulated THP-1 macrophages. Furthermore, the nature of acetylcholinesterase (AChE) inhibition by ZCPG is dose-dependent, competitive, and reversible. The AChE inhibitory activity was stable in a broad range of temperatures and pH. In vitro data were further validated by molecular interaction studies with a detailed inspection of the ZCPG probable binding modes in the active sites of AChE that provides the lead to deliver the structural determinants necessary for the activity towards AChE.