Regarding their reported beneficial properties
Regarding their reported beneficial properties on the digestive tract, the aim of this study was to evaluate the effect of water extracts of three Hypericum sps., HP, HA, HU and some of the main phenolic compounds (Ch, R and Q) on DNA protection against oxidative and alkylating DNA damage induced by hydrogen peroxide (H2O2) and MMS, respectively in colon cells (HT29 cells). Effects on 93 3 excision repair and nucleotide excision repair pathways were also assessed in HT29 cells by in vitro repair assay using comet assay. This repair assay measures the excision repair activity of an extract prepared from HT29 cells pretreated with HP, HA, HU, Ch, R or Q by providing the cell extract with a DNA substrate from HeLa cells (agarose-embedded nucleoids) containing specific damage in this case induced by MMS. Increases in DNA damage prevention and/or repair activity by Hypericum extracts will indicate an anticarcinogenic potential of these plants.
Materials and methods
Discussion In this study, we demonstrated that Hypericum water extracts and some of the main constituents have antigenotoxic effects, protecting colon cells from oxidative and alkylating DNA damage and also inducing BER repair activity. Protection of DNA is a cancer preventive mechanism; both oxidative and alkylating damage to colonocytes are known effects of dietary carcinogens. Concerning the DNA protection experiments, two different DNA damaging agents, an oxidant (H2O2) and alkylating (MMS) agent, were used. Hydrogen peroxide is a ubiquitous molecule and is one of the main reactive oxygen species (ROS) that cause DNA damage in cells mainly due to its conversion into hydroxyl radicals through the Fenton reaction. Hydroxyl radicals are highly reactive with biomolecules such as DNA inducing DNA strand breaks (SBs) (Breen and Murphy, 1995). Methyl methanesulfonate induces mainly N7meG (aprox. 83%) and N3meA (aprox. 10% of the total base alkylation) that are usually repaired by BER pathway (Drablos et al., 2004). Conventional comet assay allows detection of SBs induced by genotoxic agent, while inclusion of an extra step – digestion of nucleoids with FPG or AlkD – allows evaluation of modified bases, including some oxidative and alkylating damage, respectively (Speit et al., 2004). Since, FPG can also recognize alkylation damage such as N7meG, it was used to detect DNA damage induced by MMS. The three species of Hypericum tested had ability to protect HT29 cells from oxidative DNA damage induced by H2O2. Quercetin and R also decreased DNA damage induced by this oxidant. The antigenotoxic effect observed could be due, in part, to the ability of the extracts/compounds to act as antioxidant by free radical scavenging and/or their ability to chelate metal ions. Rainha and collaborators (Rainha et al., 2012) showed that antiradical activity is higher in HP, followed by HA and HU. The higher antioxidant activity of Hypericum extracts may be attributed to the presence of some phenolic constituints, such as Q, R and Ch in the water extracts. Rutin is the major phenolic compound present in HP (38.0μg/mg extract), less abundant in HA (2.7μg/mg extract) and absent in HU. Quercetin is present in the three species in similar concentrations (e.g. 5.0μg/mg extract in HU). Chorogenic acid is more abundant in HA (34.2μg/mg extract), followed by HU (6.4μg/mg extract) and HP (4.9μg/mg extract) (Rainha et al., 2012). Quercetin is present in vegetables and fruits especially in glycoside forms, such as rutin (Alia et al., 2005). In addition to high free radical scavenging activity, Q and R are able to chelate metal ions (Ramos et al., 2008). Quercetin also increases the levels of intracellular antioxidants, such as glutathione content, and increases activity of phase 2 enzymes as well as inhibits cytochrome P450s (Alia et al., 2005, Moon et al., 2006). Several studies, including our own showed the protective effects of Q against oxidative DNA damage in HepG2 cells (Lima et al., 2006, Ramos et al., 2008). Chlorogenic acid is an ester of caffeic acid with quinic acid present in a large variety of plants. In vitro, this polyphenol showed a strong free radical scavenging activity and decreased levels of reactive oxygen species (Abraham et al., 2007). However, in the present study, Ch seems not to be the main responsible for protection against oxidative damage, since the decrease of H2O2-induced DNA damage induced by Ch in HT29 cells was not statistically significant.