Hepatoprotective Effects Of Active Principles From Boschniakia Rossica In Animal Models

Liver is a multifunctional organ and plays key roles in many physiological processes, including detoxification, synthesis of plasma proteins, glucose homeostasis, as well as utilization and cycling of various nutrients. Loss of liver function is the consequence of various liver diseases and toxic damage, and is a major health risk factor. Acute hepatic injury is usually a self-limiting disease, but may cause severe liver injury leading to fulminant hepatic failure and eventually need for liver transplantation to prevent death and, with some causes, may progress to chronic hepatitis. Carbon tetrachloride (CCl4) and D-galactosamine (GalN) are the well-known hepatotoxins that are widely used to induce toxic liver injury in a range of laboratory animals. A low dose of lipopolysaccharide (LPS), an outer membrane component of gram-negative bacteria, in combination with GalN, a hepatotoxin, has been shown to induce an experimental liver injury that is similar to clinical acute hepatic failure (ALF).Herbs have attracted a great deal of interest as physiologically functional foods and as a source for the development of drugs. Herbal medicines derived from plant extracts are increasingly being utilized to treat a wide variety of clinical diseases, with relatively little knowledge on their modes of action. So far, there has been considerable interest in the role of complementary and alternative medicines for treatment of liver diseases. Boschniakia rossica Fedtsch. et Flerov (Orobanchaceae), a parasitic plant often parasitizing on the root of plants of the genus Alnus (Betulaceae), distributes widely in Changbai Mountain of China, Fuji Mountain of Japan, the northern mountains of North Korea, and the western North America. Although the crude extracts of Boschniakia rossica have been reported to display a variety of pharmacological activities, the tonic principle has not been well elucidated. So far, the chemical constituent research had led to the isolation and identification of three major groups of compounds, namely phenylpropanoids, iridoids and polysaccharides, and the antioxidative and hepatoprotective effects of these three constituents have been demonstrated in animal models in our previous work.To ascertain their effects of inhibiting hepatocyte apoptosis and suppressing inflammation during liver injury, we conducted in vivo investigation to reveal the hepatoprotective function of active principles from Boschniakia rossica. We applied animal models of GalN/LPS-induced feminant hepatic failure and CCl4-induced acute liver injury, and compared the effects of active principles from Boschniakia rossica on liver injury with that of silymarin, which is used clinically in Europe and Asia for the treatment of liver diseases.Bioactive constituents from Boschniakia rossica were administered intragastrically once daily for a period of7days. One hour after the final treatment, mice were treated intraperitoneally with a single dose of CCl4or GalN/LPS with or without pretreatment of active principles from Boschniakia rossica. Liver and blood samples were collected different hours after the hepatoxin injection.The results showed that:1. Active constituents, refered to as BRE, BRPS and BRP, were prepared from Boschniakia rossica using different methods. BRE was mostly consisted of iridoid glycosides (30.1%), phenylpropanoid glycosides (32.2%) and polysaccharides (8.9%). BRPS contained67.3%of total sugar and3.11%of protein, while BRP was a neutral polysaccharide fraction with the total carbohydrate content of94.7%(w/w).2. CCl4challenge not only elevated the serum marker enzyme activities and reduced albumin (ALB) level, but also increased liver oxidative stress, as evidenced by elevated lipid hydroperoxide (LOOH) and malondialdehyde (MDA) concentrations, combined with suppressed potential of hepatic antioxidative defense system including superoxide dismutase (SOD), glutathione peroxidase (GPx) activities and reduced glutathione (GSH) content. Furthermore, serum tumor necrosis factor-a (TNF-a), hepatic nitrite level, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2(COX-2) protein contents were elevated while cytochrome P4502E1(CYP2E1) expression and function were inhibited. Preadministration of BRE not only reversed the significant changes in serum toxicity markers, hepatic oxidative stress, xenobiotic metabolizing enzymes and proinflammatory mediators induced by CCI4, but restored liver CYP2E1level and function. Interestingly, the protein expression of heme oxygenase-1(HO-1) was further elevated by BRE treatment, which was markedly increased after CCl4challenge. 3. Pretreatment of mice with BRPS reversed these altered parameters of mice with liver injury induced by CCl4-Furthermore, caspase-3cleavage and activities, and DNA fragmentation of liver in mice treated with BRPS were decreased than the mice treated with CCl4alone. The hepatoprotective effect of BRPS was further demonstrated by histopathological examination of liver sections.4. Marked reduction of hepatic necrosis and serum marker enzymes in BRP pretreated mice when compared with GalN/LPS challenged mice. Circulatory levels of tumor necrosis factor-a and interleukin-6were lower in BRP pretreated mice than the mice challenged with GalN/LPS. Mice pretreated with BRP decreased the activation of caspases-3and caspase-8, and showed a reduced level of DNA fragmentation of liver cells. BRP also reduced hepatic lipid peroxidation, increased potential of hepatic antioxidative defense system, and reduced hepatic nitric oxide level which was elevated by GalN/LPS injection. Immunoblot analysis showed down-regulation of iNOS and COX-2proteins of liver tissues in BRP pretreated group when compared with GalN/LPS challenged group. Furthermore, treatment with GalN/LPS markedly increased toll-like receptor4(TLR4), nuclear level of nuclear factor-κB (NF-κB) p65, and phosphorylation of both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in liver tissues. However, these increases were attenuated by pretreatment with BRP.The results indicate that:1. BRE, consisted of iridoid glycosides, phenylpropanoid glycosides and polysaccharides, had a protective effect against CCl4-induced acute hepatic damage in rats. The hepatoprotective effect of Boschniakia rossica extract is likely due to its ability to restore the CYP2E1function, induce the HO-1expression and suppress the inflammatory responses, in combination with the ability to scavenge free radicals.2. Polysaccharides from Boschniakia rossica alleviate CCl4-and GalN/LPS-induced acute liver injuries by enhancing antioxidative defense system, suppressing inflammatory responses and reducing apoptotic signaling.Hence, we propose that Boschniakia rossica might be useful as a potential therapeutic medication for attenuating hepatic damages. This study provides evidence that Boschniakia rossica may be useful as a potential pharmacological therapy for the prevention of hepatic failure.