In-depth hepatoprotective mechanistic study of Phyllanthus niruri: In vitro and in vivo studies and its chemical characterization

Abstract

Phyllanthus niruri L. is a widespread tropical plant which is used in Ayurvedic system for liver and kidney ailments. The present study aims at specifying the most active hepatoprotective extract of P. niruri and applying a bio-guided protocol to identify the active compounds responsible for this effect. P. niruri aerial parts were extracted separately with water, 50%, 70% and 80% ethanol. The cytoprotective activity of the extracts was evaluated against CCl4-induced hepatotoxicity in clone-9 and Hepg2 cells. Bioassay-guided fractionation of the aqueous extract (AE) was accomplished for the isolation of the active compounds. Antioxidant activity was assessed using DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging method and ferric reducing antioxidant power (FRAP). The in vivo hepatoprotective activity of AE was evaluated in CCl4-induced hepatotoxicity in rats at different doses after determination of its LD50. Pretreatment of clone-9 and Hepg2 with different concentrations of AE (1, 0.1, 0.01 mg/ml) had significantly reduced the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) against CCl4 injures, and restored the activity of the natural antioxidants; glutathione (GSH) and superoxide dismutase (SOD) towards normalization. Fractionation of AE gave four fractions (I-IV). Fractions I, II, and IV showed a significant in vitro hepatoprotective activity. Purification of I, II and IV yielded seven compounds; corilagin C1, isocorilagin C2, brevifolin C3, quercetin C4, kaempferol rhamnoside C5, gallic acid C6, and brevifolin carboxylic acid C7. Compounds C1, C2, C5, and C7 showed the highest (p< 0.001) hepatoprotective potency, while C3, C4, and C6 exhibited a moderate (p< 0.001) activity. The AE exhibited strong antioxidant DPPH (IC50 11.6 ± 2 μg/ml) and FRAP (79.352 ± 2.88 mM Ferrous equivalents) activity. In vivo administration of AE in rats (25, 50, 100 and 200 mg/kg) caused normalization of AST, ALT, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total cholesterol (TC), triglycyrides (TG), total bilirubin (TB), glucose, total proteins (TP), urea and creatinine levels which were elevated by CCl4. AE also decreased TNF-α, NF-KB, IL-6, IL-8, IL10 and COX-2 expression, and significantly antagonizes the effect of CCl4 on the antioxidant enzymes SOD, catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSP). The histopathological study also supported the hepatoprotective effect of AE. P. niruri isolates exhibited a potent hepatoprotective activity against CCl4-induced hepatotoxicity in clone-9 and Hepg2 cell lines through reduction of lipid peroxidation and maintaining glutathione in its reduced form. This is attributable to their phenolic nature and hence antioxidative potential.

Fig 1. Chemical structures of the compounds isolated from P. niruri.

Fig 2. Effect of P. niruri AE on rats liver histopathology after acute CCl₄ intoxication.

(A) control portal area and surrounding hepatocytes; (B) CCl₄; (C) CCl₄ + 25 mg AE; (D) CCl₄+ 50 mg AE; (E) CCl₄+ 100 mg AE; (F) CCl₄+200 mg AE. (x100), n = 6. (1): severe ballooning degeneration; (2): fatty changes; (3): necrosis; (4): inflammatory cells infiltration; (5): marked congestion.

Fig 3. Immunohistochemical effect of P. niruri AE (100 mg kg^-1day^-1) on COX-2 and iNOS expression.

Expression of COX-2 (Higher panel) and iNOS (lower pannel) by immunohistochemical staining (x100); (A): control; (B) CCl₄; (C) CCl₄+ 100 mg kg^-1 day^-1 P. niruri AE.

Fig 4. Effect of P. niruri AE (100 mg kg^-1day^-1) on COX-2 and iNOS.

(A) % of COX-2; (B) % of iNOS positive cells as measured by image analysis; (C) NO levels; a: significantly different from control group; b: significantly different from CCl₄ group.