Deciphering the anti-oxidant, anti-microbial and anti-inflammatory activity of Pilea umbrosa Blume and Indigofera linifolia (L.f.) Retz.by in vitro and in vivo assays

Abstract

Traditional medicines are preferred in developed and underdeveloped countries due to more efficacious potency and less toxic effects. Medicinal plants enriched in pharmaceutical components displayed promising antioxidant properties that reduce or delay in the onset of damaging effect caused by diseases, thereby gaining attention due to low cost, fewer side effects, and highly effective therapeutic performance in comparison to synthetic antioxidants that appears to be dangerous for human health. Current study aims to explore the phytochemical content, protective aptitude and therapeutic applications of Pilea umbrosa Blume and Indigofera linifolia (L.f.) Retz whole plant crude methanol extract and subsequent fractions: n-hexane, chloroform, ethyl acetate, n-butanol and residual aqueous fraction. Preliminary phytochemical analysis reveals the presence of phenols, flavonoids, alkaloids, anthraquinones, terpenoids, tri-terpenoids, β-cyanin, coumarins, protein, vitamin C, tannins, phlobatannins in all extracts of Pilea umbrosa and Indigofera linifolia plant. HPLC screening indicated the presence of significant amount of flavonoids namely rutin, catechin, myricetin, apigenin, gallic acid, and caffeic acid collectively in P. umbrosa (PUM, PUE, PUB, PUA) and I. linifolia extracts (ILM, ILE, ILB, ILA). GC/MS analysis displayed the existance of 23 compounds in PUM and 10 compounds in ILM extract which includes fatty acids, sugars, phenols, esters, and terpenoids in large amount that could play a major role as therapeutic applications. In vitro antioxidant activity using wide range of assays including DPPH, iron chelating assay, hydroxal radical, nitric oxide radical, β-carotene inhibition, total antioxidant activity and total reducing power assay reveal that butanol extract of P. umbrosa and ethyl acetate extract of I. linifolia extract indicated maximum antioxidant potential having lowest IC50 values, which may be parallel to the existence of higher phenolic and flavonoid content in admirable amount, reflecting their ability to donate hydroxyl moieties to neutralize radicals. Additionally, the strong antimicrobial potential against clinical, gram positive as well as gram negative MDR’s strains may prove promising antibacterial potential of P. umbrosa and I. linifolia extracts, thereby it can play potent role as a targeted approach in the treatment of bacterial diseases. In context of anti proliferative and cytotoxic potential of P. umbrosa and I. linifolia extracts against HCCLM3 and MDA-MB-231 cell lines, it was clearly observed that PUM and ILM extract play a promising anticancer role against liver and breast cancer cells in a dose xii and time dependent manner. PUM and ILM extract induced cell death, arrested cell cycle at subG1 phase. PUM and ILM extract also inhibited angiogenesis by targeting invasive and migratory ability of the cancer cells assessed through transwell and invasion matrigel system. These potent changes could be mediated through modulation of Bcl-xL, and Bcl-2, Cyclin D1 and caspases level. Analysis also reveal that PUM and ILM extract inhibited STAT3, P-Jak1, P-Jak2 and p-Src in MDA-MB 231 cell line, whereas induced the expression of JNK, MAPK and ERK signaling pathways in HCCLM3 cell line, which further provided a strong rationale to pursue the use of natural cytotoxic compound in anticancer therapy. Experimental investigation in in vivo modals reveals strong anti-inflammatory and analgesic potential of butanol and methanol extract of P. umbrosa, and ethyl acetate extract of I. linifolia against acute inflammation in carrageenan-induced rat’s modal and analgesic activity using Eddy’s hot plate method. In vivo studies pertaining to the hepatoprotective potential of PUM and ILM extract against CCl4-intoxicated rats indicated the restoring, repairing and regenerative ability of plant extracts in injured rats. The study confirmed the hepatoprotective ability of plant extract at the molecular level through modulating UPR pathway by normalizing ER stress markers, inflammatory mediators and antioxidant enzymes in a degenerated liver. The healing mechanism of plant extract attributed to the presence of the rich phytochemical content