Hepatoprotective activity and anti-melanogenic properites of phlorotannins isolated from Ecklonia stolonifera

Abstract

Ecklonia stolonifera OKAMURA, the representative brown algae of Ecklonia sp., contains a various kinds of phlorotannins. Phlorotannins, secondary metabolites of Ecklonia sp., are tannin derivatives which are composed of phloroglucinol-based phenolics. Phlorotannins are well-known with their diverse biological activities such as antioxidation, anticancer, anti-diabetic, anti-obesity, anti-inflammation, anti-melanogenesis, and cytoprotective activities. In this study, six kinds of phlorotannins having strong antioxidant activity were isolated from E. stolonifera through silica, gel, and reverse-phase column chromatographies based on antioxidant-guided separation. The objectives of this study are to investigate the hepatoprotective and anti-melanogenic properties of phlorotannins isolated from E. stolonifera against tacrine-treated HepG2 cells as well as CCl4-treated ICR mice and a-MSH-treated B16F10 melanoma cells, respectively. The organic solvent-soluble fractions of ethanolic extract of E. stolonifera, such as n-hexane, ethyl acetate (EtOAc), and n-butanol (n-BuOH), and H2O fractions were evaluated via 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and intracellular reactive oxygen species (ROS) generation in tacrine-treated HepG2 cells. Among the four fractions tested, the EtOAc soluble fraction showed the highest antioxidant activity. From the EtOAc fraction, six phlorotannins, 2-phloroeckol, eckol, phlorofucofuroeckol B, 6,6'-bieckol, dioxinodehydroeckol, and phlorofucofuroeckol A were isolated. The structures of the phlorotannins were determined on the basis of spectroscopic evidence, including 1D and 2D nuclear magnetic resonance. The isolated phlorotannins showed potential radical scavenging activities against DPPH and suppressed the intracellular ROS in tacrine-treated HepG2 cells. Among them, eckol and 2-phloroeckol showed hepatoprotective activity in tacrine-treated HepG2 cells, however, phlorofucofuroeckol B and 6,6'-bieckol did not show the activity, even though having high antioxidant activity. Both eckol and 2-phloroeckol inhibited the expression of Fas-mediated cell-death proteins, including tBid, caspase-3, and poly (ADP-ribose) polymerase and suppressed the release of cytochrome c from mitochondria to cytosol in a dose-dependent manner in tacrine-treated HepG2 cells. Based on the in vitro assay, in vivo experiment was conducted to clarify the antioxidant and hepatoprotective activities using the CCl4-treated ICR mice. CCl4-induced oxidative damage decreased the hepatic antioxidant enzymes including catalase, superoxide dismutase (SOD), and glutathione S-transferase (GST) resulting in increase of malondialdehyde (MDA) level and release of ALT and AST levels. The pro-inflammatory cytokines including tumor necrosis factor a (TNF-a) and interleukin-6 also increased in CCl4-treated ICR mice. Among the isolated phlorotannins, eckol pretreatment exhibited protective activity on CCl4-induced oxidative stress through reducing lipid peroxidation and recovering hepatic antioxidant system including catalase, SOD and GST, as well as inducing heme oxygenase-1 expression. It also suppressed the production of pro-inflammatory cytokines including TNF-a and IL-6 in CCl4-treated ICR mice. Hepatoprotective property of eckol is resulted from downregulation of CCl4-induced mitogen-activated protein kinases (MAPKs) and Akt signal pathway activation with an exception of p38MAPK pathway through its antioxidant activity. To identify further biological activities of phlorotannins, in vitro anti-melanogenic activity was evaluated using the a-MSH-treated B16F10 melanoma cells. Among the isolated phlorotannins, dioxinodehydroeckol and phlorofucofuroeckol A reduced cellular melanin contents in the a-MSH-treated B16 melanoma cells. The results showed that dioxinodehydroeckol inhibited melanin synthesis and tyrosinase activity in a dose-dependent manner without any cytotoxicity. Dioxinodehydroeckol suppressed the expression of melanogenic proteins including tyrosinase, tyrosinase related proteins (TRP), and microphthalmia-associated transcription factor (MITF) at both protein and mRNA level. Moreover, dioxinodehydroeckol stimulated Akt phosphorylation in a dose-dependent manner; however, it did not change ERK phosphorylation. Co-treatment of LY294002 and a-MSH synergistically increased cellular melanin contents and tyrosinase activity, which were reduced by dioxinodehydroeckol treatment. On the other hands, phlorofucofuroeckol A suppressed the production of tyrosinase and TRP-1 at protein and mRNA level, however, the compound inhibited the production of MITF, not mRNA in a-MSH-treated B16 cells. Contrary to the results of dioxinodehydroeckol, phlorofucofuroeckol A stimulated ERK phosphorylation in a dose-dependent manner; however, it did not change Akt phosphorylation. Pretreatment of PD98059 did not decreased the melanogenesis and MITF protein expression in a-MSH-treated B16 melanoma cells, indicating that the phosphorylation of ERK in mainly contribute on the degradation of MITF at post-translational level. Furthermore, phosphorylation of CREB, which is a transcription factor of MITF, showed no changes and the level of intracellular cAMP activating CREB was not altered by phlorofucofuroeckol A treatment. No change of MITF mRNA expression at 1 h and decrease of MITF protein expression at 6 h, indicating that phlorofucofuroeckol A may regulate MITF activation at posttranslational level. These findings demonstrate that the phlorotannins are likely the principal constituent for the antioxidant, hepatoprotective and anti-melanogenic activities in E. stolonifera extract and may be used as a source of therapeutics or nutraceuticals for antioxidant, hepatoprotective, or skin whitening effects.