As a preliminary test prior to the evaluation of the anti-oxidative effects of the E. japonicum and C. coreana Uyeki extracts, we first examined the clinical signs in rats induced by the extracts, such as effects on mortality, body weight, and the liver. In toxicological studies, body and organ weights generally indicate the presence of potentially toxic chemicals (Kim et al., 2004; Lee et al., 2009). Treatment-related mortality and clinical signs were not observed in all rats tested during the study period (results not shown). In contrast to the gain in body weight observed in rats in the control group, the rats treated with 1,3-DCP alone showed the most significant decrease in body weight (Table 1). However, 2% and 5% E. japonicum (or C. coreana Uyeki) treatment after 1,3-DCP administration resulted in a slight decrease in the weight, which was not statistically significant. Therefore, these results suggested that E. japonicum or C. coreana Uyeki extracts may exert protective effects against the decrease in body weight induced by 1,3-DCP. However, a dose-dependent protective effect was not observed, as the extent of weight loss was similar at both treatment concentrations. In contrast to the change in body weight, no significant differences was observed in the relative weights of the rat liver (weight ratio of body to tissue) in all groups tested (results not shown).
The results of serum biochemical analyses are summarized in Table 2. The marked increases in serum AST and ALT levels are attributed to severe damage in hepatic tissue membranes, because they are located in the cytoplasm and are released from cells after autolytic breakdown or cellular necrosis (Khan et al., 2012). The rats treated with 1,3-DCP showed a significant increase in the catalytic activities of serum AST and ALT, which indicated the induction of liver damage in the animals. However, the treatment of rats with 2% and 5% E. japonicum extract resulted in a significant decrease in the activities of these enzymes. Moreover, treatment with C. coreana Uyeki extract caused a more drastic reduction in enzyme activities, in a dose-dependent manner, and the AST and ALT levels almost reached the normal range following treatment with 5% C. coreana Uyeki extract. These results clearly suggested that E. japonicum and C. coreana Uyeki extracts may protect the liver against 1,3-DCP-induced damage in rats. However, the dose-dependency of extract was not observed in the E. japonicum-treated group. Therefore, further studies should be performed to elucidate the relationship between the attenuation of liver damage and the extract concentration used for treatment. Moreover, the molecular mechanism of the protective effect of extract remains unclear.
SOD and CAT are antioxidant enzymes known to scavenge free radicals in cells, and their catalytic activities indicate the oxidative status such as lipid peroxidation in organs with extensive cellular damage (Jurczuk et al., 2004). SOD and CAT assay with the liver homogenates was performed for all groups tested, and the results are summarized in Table 3. The activities of SOD and CAT were significantly diminished by treatment with 1,3-DCP. Based on this result and the increased values of AST and ALT shown in Table 2, it could be anticipated that 1,3-DCP induces liver toxicity by cellular necrosis, which may be associated with the increased production of reactive oxygen species due to decreased SOD and CAT activities in the liver (Haratake et al., 1993). In contrast, the activity of SOD and CAT were significantly restored by treatment with 5% E. japonicum or 5% C. coreana Uyeki extracts. However, the recovery effects were not apparent, or were marginal, in groups treated with 2% extracts. Nevertheless, these results indicated that the extracts of E. japonicum and C. coreana Uyeki play an important role in the recovery and/or protective effect against a decrease in anti-oxidant enzyme activities in the rat liver. The results also suggested that the reduction in AST and ALT activities in the serum following treatment with the extracts may be related with the restored enzymatic levels of SOD and CAT. However, the molecular mechanisms underlying the extract-mediated changes in enzyme activities of SOD and CAT, as well as AST and ALT, could not be elucidated through the current experiments.
To obtain further insight into 1,3-DCP-induced liver damage and the possible protective effect of E. japonicum and C. coreana Uyeki extracts on hepatic injury, we investigated the histopathological changes in the liver sections. It has been known that 1,3-DCP induces histopathological changes in the rat liver, including zonal necrosis of the centrilobular space, destruction of the sinusoidal structure, and increase in eosinophilic cellular debris and inflammatory cell infiltration in the necrotic areas (Choi et al., 2009; Stott et al., 1997). In the present study, the necrosis of hepatocytes and infiltration of inflammatory cells were observed around the central vein region of the rats in 1,3-DCP-treated group (80 mg/kg; Fig. 1B) compared with the normal liver tissue of control group (Fig. 1A). These are the typical histopathological features of liver injury caused by 1,3-DCP. However, the degree of hepatocyte necrosis and inflammation was alleviated around the central vein region of experimental groups treated with 5% E. japonicum (Fig. 1C) and 5% C. coreana Uyeki extracts (Fig. 1D). Based on these results, we concluded that 1,3-DCP-induced hepatic lesions were reduced by treatment with both extracts. The appearance of the hepatocytes in the groups treated with the extracts were similar to that of the control group, which further support our conclusion.
It has been suggested that chlorogenic acid from E. japonicum is a candidate compound with protective function against oxidative stress-induced hepatotoxicity (Seo et al., 2016). Four marker compounds from C. coreana Uyeki have been identified: bergenin (17.5%, w/w); isosalipurposide (8.6%, w/w); quercitrin (1.6%, w/w); and quercetin (0.05%, w/w). Bergenin has a protective effect against d-galactosamine-induced hepatotoxicity in rats (Lim et al., 2001). Isosalipurposide, a chalcone compound, exerts a cytoprotective effect against oxidative injury through Nrf2 activation (Han et al., 2015). Quercitrin and quercetin protect the liver from acetaminophen-induced injury (Shanmugam et al., 2016; Xie et al., 2016). Quercitrin is a glycoside formed from quercetin, which is known to deplete the heme pool and induce carbon monoxide release, thereby limiting the expression and activity of CYP2E1, a well-known enzyme that induce oxidative stress (Tang et al., 2013; Leung and Nieto, 2013). These results, therefore, suggest that certain component(s) in these plant extracts may alleviate the 1,3-DCP-mediated hepatotoxicity of rats.