Therefore we have further investigated the

Therefore, we have further investigated the anti-oxidant activity of isogarcinol in vitro by measuring the scavenging of 2,2′-diphenyl-1-1picrylhydrazyl (DPPH) and 2,2′-azino-bia (3-ethylben-zothiazoline-6-sulphonicacid) (ABTS), and determining its reducing power and ability to prevent lipid peroxidation. We also assessed the cytotoxicity and genotoxicity of isogarcinol, and observed its effect on cell viability, ROS, lactate dehydrogenase (LDH) release, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity and glutathione (GSH) levels, as well as its cytoprotective effect on mitochondria-dependent apoptosis.
Materials and methods
Results
Discussion It is widely believed that free radicals are responsible, at least in part, for many degenerative diseases such as receptor tyrosine kinase dysfunction, cancer and heart diseases (Kozics et al., 2013). Antioxidants can prevent oxidative-related disorders and in some cases help in their treatment (Pisoschi & Pop, 2015). Nowadays, phytochemical or antioxidant therapy is regarded as a promising strategy to protect cells from oxidative damage and natural products of plant origin are also important sources of antioxidants (Miremadi, Ayyash, Sherkat, & Stojanovska, 2014). Isogarcinol is a benzophenone with a variety of biological properties, such as antioxidant, antimicrobial, antifungal, anti-inflammatory, and anti-HIV activities (Baggett, Mazzola, & Kennelly, 2005). It also has antiplasmodial activity against P. Falciparum (Marti et al., 2010), acetylcholinesterase and butyrylcholinesterase inhibitory activities, antiprotozoal activity against Leishmania donovani (Lenta et al., 2007) and antibacterial activities (Deachathai, Mahabusarakam, Phongpaichit, & Taylor, 2005). In the present work, we confirmed the radical-scavenging activity, reducing power and anti-lipid peroxidation capacity of isogarcinol, and showed that it has significant antioxidant activity (Fig. 1), which furthermore confirmed the results of Stark et al. (2015). In particular, the IC50 of isogarcinol in the DPPH assay was almost 2-fold and 3-fold lower than those of VC and BHT, respectively (Table 1). Ito et al. (2003) also found that the DPPH radical-scavenging activity of isogarcinol (IC50 = 13.3 ± 1.3 µM) was about 2-fold lower than that of VE (IC50 = 22.8 ± 0.2 µM). The reducing power and anti-lipid peroxidation assays revealed that isogarcinol had antioxidant activity, but the results of both assays were lower than those obtained for VC. A different chemical reaction mechanism, leading to a different ranking order of antioxidant activity for isogarcinol, BHT and VC, in this context, could provide an explanation for the obtained results. In addition, we observed, in FRAP experiments, that isogarcinol possesses some reducing power (data not shown). Hence, our results confirmed that isogarcinol has strong antioxidant activity in vitro, so we explored its biological activity further. High levels of dietary compounds can be toxic and mutagenic in cell culture systems (Alía et al., 2006). Cytotoxic effects of isogarcinol have been reported against human diploid embryonic lung MRC-5 cells (IC50 value of 3.5 µM) (Marti et al., 2010) and human leukemia H-L60 cells (Matsumoto et al., 2003). However, isogarcinol showed toxicity at more than 10 µM and did not have any cytotoxic or promotional effects on HepG2 cells at 1–10 µM in our experiments (Fig. 2A). We also assessed the genotoxic activity of isogarcinol, using the SCGE assay, which is a trustworthy and rapid method for the quantification of DNA damage. The images clearly indicated that BHT was genotoxic at 100 and 500 µM in HepG2 cells (Fig. 3: E–G); in contrast isogarcinol had negligible genotoxic effects at 50, 100 and 500 µM (Fig. 3: B–D). HepG2 cells are a well-established model for studying the antioxidant effects of dietary compounds (Wen et al., 2015, Kong et al., 2016). They retain many of the functions of normal liver cells (Knowles, Howe, & Aden, 1980) and their morphological features and cell shapes are also similar to those of liver parenchymal cells (Zhou, Jiang, Geng, Cao, & Zhong, 2009). In the present study, treatment of HepG2 cells with 500 µM H2O2 for 3 h resulted in about 50% death (Fig. 2B) and preincubation with isogarcinol attenuated this death. These results imply that isogarcinol protected the HepG2 cells from H2O2-induced damage. Accumulating evidence has revealed that H2O2 triggers oxidative damage by elevating intracellular ROS (Parthasarathi et al., 2015). In this study, we found that H2O2 (500 µM) elevated intracellular ROS, leading to a roughly three-fold increase in DCF fluorescence intensity, and pretreatment with isogarcinol (0.5–1.5 µM) for 30 min greatly reduced the production of ROS. This suggests that isogarcinol directly scavenges ROS and/or free radicals formed in response to H2O2.