Ichnocarpus frutescens (L.) R. Br. root derived phyto-steroids defends inflammation and algesia by pulling down the pro-inflammatory and nociceptive pain mediators: An in-vitro and in-vivo appraisal

Abstract

Ichnocarpus frutescens, a climber plant, is distributed all over India. As its different parts are used as anti-inflammatory agent, so we re-investigated the roots to isolate compounds and evaluate its biological efficacy. Also, in-silico molecular docking was carried out to elucidate the structure activity relationship (SAR) of isolated compounds toward identifies the drug target enzyme with validation, which was further supported by anti-inflammatory in-vitro and in-vivo experimental models. The compounds have been undertaken mainly to investigate the anti-inflammatory and analgesic efficacy along with molecular docking investigation followed by anti-proteinase, anti-denaturation and cyclooxygenase (COX) inhibition studies. Inflammatory cytokines like TNF-α and IL-6 were assayed from lipopolysaccharides (LPS) and Concavallin (CON A) stimulated human PBMC derived macrophages by Enyme linked immune sorbent assay (ELISA) method. The purity index of the lead compound was determined by HPLC. The compounds were illustrated as 2-hydroxy tricosanoic acid (1), stigmasterol glucoside (2), stigmasterol (3), β-sitosterol (4) and β-sitosterol glucoside (5). The test molecules showed significant anti-denaturation, anti-proteinase and analgesic effect validated with docking study. Compounds exhibited anti-inflammatory and pain killing action due to dexamethasone like phytosterol property. Promising anti-denaturation and anti-proteinase activity offered by the compound 5, may hold its promise to fight against arthritis by rejuvenating the osteoblast cells and destroying the bone-resorpting complex of hydrated protein, bone minerals by secreting the acid and an enzyme collagenase along with pain management. The lead bioactive compound i.e. β-sitosterol glucoside (compound 5) demonstrated considerable anti-inflammatory activity showing more than 90% protection against the inflammatory cytokines at 50 µM dose. The anti-denaturation and COX-2 inhibition shown by the compound 5 was also noteworthy with the significant IC₅₀ (ranging from 0.25 to 2.56 µM) that also supporting its future promise for developing as anti-inflammatory agent. Since the most bio-active compound (5) elicit promising acute anti-inflammatory action and peripheral anti-nociceptive pain killing action with a significant ED₅₀ dose of 3.95 & 2.84 mg/kg i.p. respectively in the in-vivo animal model. It could suggest its potentiality as a good in-vivo bio available agent to be an emerging anti-inflammatory drug regimen scaffold in the future. It also establishes significant in-vitro and in-vivo result co-relation. Therefore, the compound 5 could be believed as a potent lead for designing anti-inflammatory, anti-arthritic drug or pain killer without showing any untoward effect.

Keywords: Anti-inflammatory; Cyclooxygenase-2; Cytokine; Ichnocarpus frutescens; Plant sterol; Proteinase.