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Background: Blepharis maderaspatensis is an ethnomedicinal plant used by the Mavilan and Koraga tribes of Kerala state, India for the treatment of liver diseases. Thus, the present study aims to evaluate the liver protective activity of defatted ethanolic extract of B. maderaspatensis on lipopolysaccharide-induced acute liver inflammation and oxidative stress in Wistar rat model. Methods: Preliminary phytochemical evaluation and high-performance thin-layer chromatography fingerprint validation were performed. The total phenolic and flavonoid contents were measured using the Folin-Ciocalteu method and the aluminium chloride method, respectively. The acute oral toxicity study was conducted in Swiss albino mice in accordance with OECD 423 guideline. Effect of defatted ethanolic extract of the whole plant of B. maderaspatensis (BmE) on liver inflammation was evaluated in LPS-induced Wistar rat model. The rats were treated with BmE (100, 250 and 500 mg/kg body weight) once daily for 7 days prior to the LPS (single dose i.p., 10 mg/kg b.w.) challenge. Liver tissue biochemicals (ALT, AST and ALP), and antioxidant status (SOD, CAT, GSH, MDA, NO and MPO) in euthanised experimental rats were carried out using commercial kits/standard procedures. Haematology (RBC, WBC, Hb and PLT; 4 and 24 h), and histopathology analysis were also performed. Serum IL-1β, IL-6, TNF-α and PGE2 production were determined by ELISA. The expressions of COX-2, iNOS, IL-1β, IL-6, TNF-α and NF-κB were analysed by qRT-PCR. Phosphorylation of IκB-α was measured by Western blotting. LC-MS analysis of BmE was also performed. Results: The LD50 value of BmE was found to be >5000 mg/kg b.w. BmE500 showed significant protection from LPS-induced liver injury as evidenced by reduced serum enzyme levels (AST, ALT and ALP; p≤ 0.001) and markedly improved the liver antioxidant status (MDA, GSH, SOD and CAT; p≤ 0.001), when compared to the LPS alone treated group. Haematology (24 h, RBC, WBC, Hb and PLT; p≤ 0.001) and histopathology also supported the above results. BmE500 pre-treatment significantly suppressed the LPS-induced expression of iNOS (p≤ 0.001) and COX-2 (p≤ 0.001), and the subsequent release of NO (p≤ 0.001) and PGE2 (p≤ 0.001). Moreover, BmE500 inhibited the gene expression of pro-inflammatory cytokines, including IL-1β (p≤ 0.001), IL-6 (p≤ 0.001), and TNF-α (p≤ 0.01), which is supported by ELISA results. In addition, BmE500 markedly attenuated the activation of transcription factor NF-κB (p≤ 0.001) as well as phosphorylation of IκBα, as evidenced from the qRT-PCR and Western blot analysis, respectively. The silymarin (100 mg/kg b.w.) drug standard also showed significant improvement in all parameters analysed. Conclusion: Results of the present investigation suggest that BmE has a significant protective effect on LPS-induced acute liver inflammation via attenuating inflammatory reactions, evidenced by the inhibition of NF-κB signalling cascade, and also through its antioxidant effects. Thus, the pharmacological data generated provide experimental evidence that clearly justifies the use of B. maderaspatensis as a liver protective agent in tribal medicine. |
