Presence of pyrroloquinazoline alkaloid in Adhatoda vasica attenuates inflammatory response through the downregulation of proinflammatory mediators in LPS stimulated RAW 264.7 macrophages

Methods: This study focuses on the mode of action of ALK-F (alkaloid fraction) isolated from Adhatoda vasica in attenuating the nitric oxide production which was estimated by Griess assay, tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6) expression was analyzed by ELISA and expression of COX-2 and iNOS by RT-PCR and western blotting in LPS stimulated RAW 264.7 macrophages. PGE2 is one of the key PGs which is secreted by macrophages that regulates their activity in an autocrine manner.5 Cyclooxygenases (COXs) are responsible for the conversion of arachidonic acid to PGs.6 COX-1 is basically expressed in most cell types and it is liable for regulating normal physiological functions but COX-2 is inducible in cell types like macrophages, fibroblasts, and endothelial cells to play a focal role in inflammation.7 Many commercially available steroids and non-steroidal anti-inflammatory drugs have a boundless influence in the cure for inflammatory diseases, but due to their indeterminate side effects there arises a need for lead targets with the minimal adverse effect.8,9 Folklore medicinal plants with traditional medicinal values provide enormous health care benefits with new therapies and fewer side effects. The World Health Organization (WHO) estimated that 80% of the world population depends on these customary herbal plants for their health essentials due to their abundance and varied bioactivities compared to other commercial available non-steroidal anti-inflammatory drugs.10 Active leads from plant-derived alkaloids and phenolic compounds (flavonoids and tannins) were found to possess antioxidant and anti-inflammatory activities both in vitro and in vivo.11 Adhatoda vasica Nees is an evergreen shrub belonging to the family Acanthaceae, found in many regions of India and throughout the world, with expanded use in traditional medicine. The solution was once again separated into two layers and the upper aqueous layer was evaporated in a rotary evaporator and given for high performance liquid chromatography (HPLC) for identification of alkaloids with vasicine as a standard.17 Drugs and chemicals Classical media (Dulbecco's modified essential medium), dimethyl sulphoxide (DMSO), fetal bovine serum (FBS), L-nitro-arginine methyl ester (L-NAME) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), RIPA Buffer (10X), sulphuric acid, chloroform liquid ammonium hydroxide, and methanol was procured from HiMedia Laboratories (Mumbai, Maharas