An in vitro and in vivo hepatotoxicity study of an aspalathin- rich green rooibos extract
Hepatotoxicity is an undesirable condition of the liver, often occurring due to the interaction of drugs. Although different methods have been employed in the diagnosis of drug-induced hepatotoxicity, none have accurately proven the hepatotoxic effect of drug-drug interactions or drug-herb interactions. Flavonoids of aspalathin-rich green Rooibos extract (GRT) present many therapeutic properties that reduce the risk of developing cardiovascular diseases. However, there is a gap in literature regarding the efficacy or safety of this particular extract on the liver. Furthermore, Rooibos has been implicated in the development of hepatotoxicity which might have been triggered by drug-Rooibos interaction. This study therefore purposes to establish whether consumption of an aspalathin-rich green Rooibos extract (GRT) has any toxic effects on the liver using both an in vitro human liver cell line (C3A) model, as well as a sub-chronic Sprague- Dawley rat model. Cultured C3A cells were treated with different concentrations of GRT ranging from untreated control, 0.1, 1, 10, 100 and 1000 μg/mL and incubated for 1 hour, 24 hours or 48 hours. Thereafter, cell viability was evaluated using the mitochondrial activity assay 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphnyltetrazolium bromide (MTT) and adenosine triphosphate (ATP) assay. The impact of the GRT extract on enzyme release and membrane integrity was assessed using the adenylate kinase enzyme (AK). For the in vivo study, sub-chronic Sprague Dawley rats were used to investigate the toxicity of different concentrations of GRT extract. The rats were randomly divided into five groups with ten rats per group. Three of these groups were treated daily with different concentrations of GRT extract [10, 100 and 300 mg/kg body weight (bw)] for a period of 90 days, while the other groups were an untreated control and a carbon tetrachloride (CCl4) treated group for induction of hepatotoxicity. Treatments were administered using cubes made of jelly and gelatin for the duration of the study. At 24 hours before termination, the positive control group was treated with 1 mL/kg CCl4 in 1:1 ratio dilution with olive oil to induce liver injury. The body weights, food and water intakes were measured weekly during the period of the treatment. At termination, blood samples were collected for clinical biochemical tests [alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), Urea, total protein, total bilirubin, albumin uric acid and creatinine] and liver tissue collected for histology and protein expression (NF-kB, Bax, Caspase 3 and CYP3A4). The in vitro results revealed that lower concentrations of GRT significantly sustained cell viability in C3A which was associated with the increase in cell proliferation after 1 hour, 24 hours and 48 hours incubation compared to the control. A hepatotoxicity effect was only observed when the cells were treated with higher concentration of GRT extract (1000 μg/mL). The results of the in vivo study showed that the GRT extract had no effect on the body weights, food intake and water intake. Increased levels of liver enzymes, AST at the highest dose (300 mg/kg) and ALT in the CCl4 treated group were demonstrated. Total protein was unaffected by the GRT while the serum albumin was slightly increased. Histopathological changes showed diffuse marked hepatocellular damage caused by CCl4. There were no histopathological changes observed with 10 and 100 mg/kg while the 300 mg/kg displayed micro-and macrovesicular changes. There was a concentration-dependent inhibition of NF-KB and no significant effect was observed on the expression of caspase 3, Bax and CYP3A4. Taken together, these results suggest that lower concentrations of GRT extract have a hepatoprotective effect in vivo. Supporting these findings, in vitro results showed that low concentrations of GRT extract exhibited a hepatoprotective effect on the human hepatocellular carcinoma cells. However, there should be vigilance in the consumption of Rooibos tea as increased consumption may impair hepatocytes. .
hepatotoxicity --Rooibos --cytotoxicity --hepatocyte --anti-oxidant --anti inflammation
Networked Digital Library of Theses and Dissertations