Prophylactic efficacy of bioactive compounds identified from GC-MS analysis of Balarista formulation on adjuvant induced arthritic rats by inhibiting COX-2 inhibitor

Balarista, a classical ayurvedic formulation, is traditionally claimed for the treatment of rheumatoid arthritis (RA). The drawback behind this fermented product remains lack of its proper documentation and validation. In the present investigation, therefore, an in-house Balarista (IBF) formulation was prepared by using standard raw materials as mentioned in API (Ayurvedic Pharmacopoeia of India). As the preclinical scientific data regarding its molecular mechanism of action were not evaluated, so the present work was undertaken to investigate its anti-arthritic potential using Complete Freund's adjuvant (CFA) induced arthritic rat model and was compared with the marketed (AVS, BD, DB, RN) formulations. Rats were immunized by injecting CFA of 0.1 ml into the sub-plantar surface of right posterior paw. Animals were treated with formulation (2.31 ml/kg) and indomethacin (1 mg/kg) for 28 days and were sacrificed on 29ᵗʰ day. The hematological and biochemical parameters were studied. The histology and x-ray analysis were performed on proximal interphalangeal joints of the experimental rats. The molecular docking of the constituents identified from GC-MS analysis of IBF formulation was carried out with COX-2 receptor to find out the interaction using celecoxib as standard. The quantification of vasicine and total withanolides was performed by HPLC analysis for their standardization. A significant decrease in paw diameter, arthritic index and histological score was noticed in formulation treated groups. The decrease in body weight and alteration in hematological and biochemical parameters were also significantly checked by the IBF and marketed formulation in contrast to CFA treated groups. Remarkable reduction of TNF-α, IL-1β, and IL-6 were noticed in all the formulation treated rats. The histological study revealed decrease in synovial hyperplasia, pannus formation, and cartilage and bone erosion. The X-ray analysis showed decrease in soft tissue swelling, bone resorption and osteophyte formation upon the treatment with formulations. Based on docking score the components, 8-Azaspiro[4.5]decane-7,9-dione (-7.5); (3aS,7aR)-5,6,7a-trimethyl-4,7-dihydro-3aH-2-benzofuran-1,3-dione (-7.5); 2,6-Ditert-butylphenol (-7.2) exhibited significant binding affinity. The prophylactic activity of the formulation could be due to the synergistic effects produced by the phyto-constituents identified by GC-MS analysis. Moreover, the anti-arthritic activity of the IBF was attributed by the predominance of withaferin A.