The scientific and traditional data revealed that Phyllanthus niruri Linn. (Euphorbiaceae) (PN), Andrographis paniculata Nees. (Acanthaceae) (AP) and Piper longum Linn. (Piperaceae) (PL) have their antioxidant and hepatoprotective role. In the present invention, we have investigated the synergistic effect these herbs against alcohol and HFD (High Fat Diet) induce hepatotoxicity in rats. Further mechanistic study was conducted to establish the mechanism of the developed formulation with respect to Hemeoxygenase-1 (HO-1) which involve in the hepatoprotective effect. The hepatoprotective activity of three herbal extracts combinations Phyllanthus niruri (PN), Andrographis paniculata (AP) and Piper longum (PL) were first studied in in-vitro on HepG-2 cell lines by using ethanol (100mM) and then in-vivo activity was explored again with 40% Alcohol 2ml/100g and HFD induce hepatotoxicity in Wistar rats. Oxidative stress parameters, liver enzymes, Lipid profile and histopathological and mechanistic study were assessed in liver homogenate. The body weight and urine analysis was done on 7th, 14th, 21st day. The herbal combinations with different ratios (AP: PN: PL, 1:1:1, 2:2:1, 3:1:1) showed significant hepatoprotective effect using in vitro (HepG-2) and in vivo models. Further herbal combination with equal ratio group showed significant hepatoprotective effect as compare to other groups. The herbal combination group showed significant reduction in oxidative stress parameters and improved antioxidant and liver enzymes level.The present studies suggest that the combination of herbal drugs showed synergistic hepatoprotective effect by antioxidant and induction of HO-1 mechanism. The combination of these three herbal drugs provide base for their clinical implications.
