RESUMEN
Intractable inflammation plays a key role in the progression of autoimmune diseases such as rheumatoid arthritis. Oedema and angiogenesis are the hall marks of chronic inflammation. The current study was aimed to investigate the pharmacological effects of the methanolic extract of Viola odorata (Vo.Me) on inflammation induced oedema and angiogenesis, and to identify the active principles and explore the molecular mechanisms thereof. Various models of inflammation were utilized in rats, including carrageenan- and histamine-induced acute oedema, as well as chronic models of Complete Freund's Adjuvant (CFA)-induced arthritis and cotton pellet-induced granuloma. Anti-angiogenic activity was evaluated by CAM assay followed by quantification of phytoconstituents through HPLC. Effect of Vo.Me treatment on the expression of various mediators (PGE-2 and NO) and genes (IL-1ß, TNF-α, NF-κB, and COX-2) were explored by qPCR and ELISA assays. HPLC analysis showed the presence of quercetin, chlorogenic acid, gallic acid, benzoic acid, m-coumaric acid, p-coumaric acid, synergic acid, caffeic acid, vanillic acid, sinapic acid, and cinnamic acid in Vo.Me. Significant dose-dependent inhibition of rats' paw oedema was observed in the Vo.Me administered groups (p < 0.05) in both acute and chronic inflammatory models. Moreover, at a dosage of 500 mg/kg, Vo.Me exhibited a comparable anti-inflammatory effect to indomethacin (p > 0.05). Additionally, Vo.Me demonstrated a remarkable anti-granulomatous activity. Histopathological findings demonstrated amelioration of inflammation in animal paws which were treated with Vo.Me and indomethacin. CAM assay also displayed significant inhibitory effect of Vo.Me on the blood vasculature growth. Vo.Me treatment also caused relatively less gastric irritation and hepatic damage as compared to indomethacin. At a molecular level, the down-regulation of NF-κB signalling leading to the decreased activation of pro-inflammatory mediators (such as IL-1ß, TNF-α, and COX-2) and their downstream molecules including prostaglandin E-2 (PGE-2) and nitric oxide (NO), is suggested to be responsible for these diverse anti-inflammatory effects. These findings confirmed the promising anti-inflammatory and anti-angiogenic activities of Vo.Me, which warrant bench-to-bedside translational studies to assess its safety and suitability for clinical usage.
RESUMEN
Uncontrolled inflammation plays a central role in the pathogenesis of various diseases. Currently available anti-inflammatory agents on prolonged use may lead to ulcers or thrombus formation. The present study was designed to evaluate the anti-inflammatory, anti-arthritic and anti-angiogenic potentials of methanol extract of Viola betonicifolia using battery of in vivo models. Methanol extract of Viola betonicifolia (Vb.Me) was prepared through maceration. High performance liquid chromatography (HPLC) and gas chromatography mass spectrometery (GC-MS) were performed to identify bioactive compounds present in Vb.Me. In vivo safety profile of Vb.Me was evaluated following OECD 425 acute toxicity guidelines. Anti-inflammatory potential of Vb.Me at three different dose levels was evaluated in in vivo acute (carrageenan and, histamine-induced paw oedema), sub-chronic (cotton pellet-induced granuloma) and chronic (Complete Freund's adjuvant-induced arthritis) models. Blood and paws samples were collected to study effects of Vb.Me treatment on the expression of various pro- and anti-inflammatory genes (RT-PCR) and to study the histopathological changes at tissue levels. Effects of Vb.Me on neovasculature development were studied in ex-ovo chicken chorioallantoic membrane (CAM) assay. Quercetin and n-hexadecanoic were identified as one of the major bioactive molecules in HPLC and GC-MS analysis of Vb.Me. Toxicity data revealed that Vb.Me was safe for administration up to the dose of 2000 mg/kg. Findings of inflammatory models showed that Vb.Me produced time and dose-dependent effects. 500 mg/kg Vb.Me showed significantly (p < 0.05) better effects as compared with 125 and 250 mg/kg. 500 mg/kg Vb.Me also showed comparable anti-inflammatory effects with indomethacin in both acute and chronic models respectively. RT-PCR data exhibited significant (p < 0.05) down-regulation of IL-6, IL-1ß, NF-kß, TNF-α and COX-2 genes with simultaneous up-regulation of IL-4 and IL-10 genes in the blood samples of animals treated with 500 mg/kg of Vb.Me and 10 mg/kg of indomethacin respectively. CAM assay data revealed arrest of microvessel outgrowth in Vb.Me-treated eggs. Altogether, findings of the current study indicate that Vb.Me exerts in vivo anti-inflammatory and anti-angiogenic effects through regulation of expression of various pro- and anti-inflammatory genes. Synergist actions of various bioactive molecules in Vb.Me are proposed to be responsible for these attributes. However, further studies to standardize the extract and evaluation of its potential in various inflammation-induced diseases are warranted.