Asperflavin, an Anti-Inflammatory Compound Produced by a Marine-Derived Fungus, Eurotium amstelodami.

In the present study, 16 marine-derived fungi were isolated from four types of marine materials including float, algae, animals and drift woods along with the coast of Jeju Island, Korea and evaluated for anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW 24.7 cells. The broth and mycelium extracts from the 16 fungi were prepared and the broth extract (BE) of Eurotium amstelodami (015-2) inhibited nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells without cytotoxicity. By further bioassay-guided isolation, three compounds including asperflavin, neoechinulin A and preechinulin were successfully isolated from the BE of E. amstelodami. It was revealed that asperflavin showed no cytotoxicity up to 200 µM and significantly inhibited LPS-induced NO and PGE2 production in a dose-dependent manner. In the western blot results, asperflavin suppressed only inducible NOS (iNOS), but COX-2 were slightly down-regulated. Asperflavin was also observed to inhibit the production of pro-inflammatory cytokines including TNF-α, IL-1ß, and IL-6. In conclusion, this study reports a potential use of asperflavin isolated from a marine fungus, E. amstelodami as an anti-inflammatory agent via suppression of iNOS and pro-inflammatory cytokines as well as no cytotoxicity.

Stoichiometric expression of mtHsp40 and mtHsp70 modulates mitochondrial morphology and cristae structure via Opa1L cleavage.

Deregulation of mitochondrial heat-shock protein 40 (mtHsp40) and dysfunction of mtHsp70 are associated with mitochondrial fragmentation, suggesting that mtHsp40 and mtHsp70 may play roles in modulating mitochondrial morphology. However, the mechanism of mitochondrial fragmentation induced by mtHsp40 deregulation and mtHsp70 dysfunction remains unclear. In addition, the functional link between mitochondrial morphology change upon deregulated mtHsp40/mtHsp70 and mitochondrial function has been unexplored. Our coimmunoprecipitation and protein aggregation analysis showed that both overexpression and depletion of mtHsp40 accumulated aggregated proteins in fragmented mitochondria. Moreover, mtHsp70 loss and expression of a mtHsp70 mutant lacking the client-binding domain caused mitochondrial fragmentation. Together the data suggest that the molecular ratio of mtHsp40 to mtHsp70 is important for their chaperone function and mitochondrial morphology. Whereas mitochondrial translocation of Drp1 was not altered, optic atrophy 1 (Opa1) short isoform accumulated in fragmented mitochondria, suggesting that mitochondrial fragmentation in this study results from aberration of mitochondrial inner membrane fusion. Finally, we found that fragmented mitochondria were defective in cristae development, OXPHOS, and ATP production. Taken together, our data suggest that impaired stoichiometry between mtHsp40 and mtHsp70 promotes Opa1L cleavage, leading to cristae opening, decreased OXPHOS, and triggering of mitochondrial fragmentation after reduction in their chaperone function.

Whitening Effect of Octaphlorethol A Isolated from Ishige foliacea in an In Vivo Zebrafish Model.

In a previous study, we isolated octaphlorethol A (OPA) from Ishige foliacea and evaluated its anti-melanogenesis activity in a murine melanoma cell line. However, the whitening effect and toxicity of OPA have not yet been examined in vivo. Therefore, in this study, we investigated the inhibitory effect of OPA on melanin synthesis and tyrosinase activity in an in vivo zebrafish model. More than 90% of subject embryos survived upon exposure to OPA concentrations below 25 micrometer, which was not significantly different from the finding in the control group. OPA markedly inhibited melanin synthesis and tyrosinase activity in a concentration-dependent manner.

Octaphlorethol A: a potent α-glucosidase inhibitor isolated from Ishige foliacea shows an anti-hyperglycemic effect in mice with streptozotocin-induced diabetes.

α-Glucosidase inhibitors are important agents for decreasing postprandial hyperglycemia. The current study examined the inhibitory effects of octaphlorethol A (OPA) isolated from Ishige foliacea, a brown alga, on α-glucosidase, and analyzed the inhibitor's binding modes using the crystal structure of α-glucosidase. The effects of OPA on postprandial blood glucose levels after meals were also investigated. The IC50 value of OPA against α-glucosidase was 0.11 mM, which is higher than that of the commercial inhibitor acarbose. For further insights, we predicted the 3D structure of α-glucosidase and used a docking algorithm to simulate binding between α-glucosidase and OPA. These molecular modeling studies were successful, and indicated that OPA interacts with Phe575, His600, Arg526, Met444, Asp542, Tyr605, Ser448, Asp203, Lys480, and Phe450. Furthermore, increases in postprandial blood glucose levels were significantly suppressed in the OPA-treated group compared with those in the streptozotocin-induced diabetic or normal mice. Additionally, the area under the curve was significantly reduced following OPA administration (907 versus 1034 mg h dL(-1)) in the diabetic mice, along with a delay in the absorption of dietary carbohydrates. Collectively, these results indicated that OPA is a potent inhibitor of α-glucosidase, and shows potential to be used as an anti-diabetic agent.

Anti-inflammatory activity of questinol isolated from marine-derived fungus Eurotium amstelodami in lipopolysaccharide-stimulated RAW 264.7 macrophages.

In the present study, an anthraquinone derivative, questinol was successfully isolated from the broth extract of the marine-derived fungus Eurotium amstelodami for the first time. The structure of questinol was determined based on the analysis of the MS and NMR spectral data as well as comparison of those data with the published data. Moreover, the anti-inflammatory effect of questinol in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was investigated. The results showed that questinol did not exhibit cytotoxicity in LPS-stimulated RAW 264.7 cells up to 200 µM. Questinol could significantly inhibit NO and PGE2 production at indicated concentrations. Questinol was also found to inhibit the production of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IL-6. Furthermore, the western blot analysis showed that questinol suppressed the expression level of iNOS in a dose-dependent manner. However, questinol could slightly inhibit the expression of COX-2 at the concentration of 200 µM. Therefore, our study suggests that questinol might be selected as a promising agent for the prevention and therapy of inflammatory disease.

Chromene suppresses the activation of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 cells.

Inflammation is complex process involving a variety of immune cells that defend the body from harmful stimuli. However, pro-inflammatory cytokines and inflammatory mediators can also exacerbate diseases such as cancer. The aim of this study was to identify a natural effective remedy for inflammation. We isolated a functional algal chromene compound from Sargassum siliquastrum, named sargachromanol D (SD). We evaluated the anti-inflammatory effect of SD on lipopolysaccharide (LPS)-exposed RAW 264.7 cells by measuring cell viability, cytotoxicity, and production of inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6. SD inhibited production of NO and PGE2 from LPS-induced cells by preventing the expression of inflammatory mediators such as iNOS and COX-2 in a dose-dependent manner. Concurrently, levels of the pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 were reduced with increasing concentrations of SD. In addition, SD inhibited the activation of NF-κB and mitogen-activated protein kinases (MAPKs) pathways in a concentration-dependent manner. These results indicate that SD inhibits LPS-stimulated inflammation by inhibition of the NF-κB and MAPKs pathways in macrophages.

Protective effect of fucoidan against AAPH-induced oxidative stress in zebrafish model.

Fucoidan, extracted from Ecklonia cava, has been extensively studied because of its wide biological activities. However, antioxidative activities have not been yet examined. Therefore we evaluated in vitro and in vivo studies on antioxidative activities of E. cava fucoidan (ECF). ECF exhibited more prominent effects in peroxyl radical scavenging activity, compared to the other scavenging activities. Thus, ECF was further evaluated for its protective ability against 2,2'-azobis dihydrochloride induced oxidative stress in Vero cells and ECF strongly reduced the AAPH-induced oxidative damage through scavenging intracellular reactive oxygen species. Furthermore, we evaluated protective effect of ECF against AAPH-induced oxidative stress in zebrafish model. ECF significantly reduced ROS generation, lipid peroxidation and cell death in zebrafish model. These findings indicate that ECF has antioxidant activities in vitro Vero cells and in vivo zebrafish model, even though ECF is not a polyphenol or flavonoid compound and does not contain benzene rings or conjugated structures.

Protective effect of dieckol isolated from Ecklonia cava against ethanol caused damage in vitro and in zebrafish model.

In the present study, the protective effects of phlorotannins isolated from Ecklonia cava against ethanol-induced cell damage and apoptosis were investigated both in vitro and in vivo. Three phlorotannin compounds, namely phloroglucinol, eckol and dieckol, were successively isolated and identified from the extract. Dieckol showed the strongest protective effect against ethanol-induced cell apoptosis in Chang liver cells, with the lowest cytotoxicity. It was observed that dieckol reduced cell apoptosis through activation of Bcl-xL and PARP, and down-regulation of Bax and caspase-3 in Western blot analyses. In the in vivo study, the protective effect of ethanol induced by dieckol was investigated in a zebrafish model. The dieckol treated group scavenged intracellural reactive oxygen species and prevented lipid peroxidation and ethanol induced cell death in the zebrafish embryo. In conclusion, dieckol isolated from E. cava might possess a potential protective effect against ethanol-induced liver diseases.

Octaphlorethol A isolated from Ishige foliacea inhibits α-MSH-stimulated induced melanogenesis via ERK pathway in B16F10 melanoma cells.

In this study, the potent skin-whitening effects of Octaphlorethol A (OPA) isolated from Ishige foliacea was investigated through inhibitory effect of melanin synthesis and tyrosinase activity in alpha-melanocyte stimulating hormone (α-MSH) induced B16F10 melanoma cells. OPA markedly inhibited melanin synthesis and tyrosinase activity in a concentration-dependent manner. We also found that OPA decreased microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2) protein expressions. Moreover, OPA reduces p38 MAPK protein levels and activates extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinases (JNKs) protein expressions in B16F10 cells. A specific ERK inhibitor PD98059 significantly blocks OPA-inhibited melanin synthesis and tyrosinase activity, whereas a p38MAP and JNK inhibitor had no effect. These findings provide evidence demonstrating that the anti-melanogenic effect of OPA is mediated through the activation of ERK signal pathway in B16F10 cells. These results indicate that OPA has the potential to be used as a melanogenesis inhibitor in the food and cosmetics industry.

Octaphlorethol A, a novel phenolic compound isolated from Ishige foliacea, protects against streptozotocin-induced pancreatic ß cell damage by reducing oxidative stress and apoptosis.

Pancreatic ß cells are extremely sensitive to oxidative stress, which probably has an important role in ß cell damage in diabetes. The protective effect of octaphlorethol A (OPA), a novel phenolic compound isolated from Ishige foliacea, against streptozotocin (STZ)-induced pancreatic ß cell damage was investigated using a rat insulinoma cell line (RINm5F pancreatic ß cells). Pretreatment with OPA decreased the death of STZ-treated pancreatic ß cells at concentrations of 12.5 µg/ml or 50 µg/ml, and reduced the generation of thiobarbituric acid reactive substances and intracellular reactive oxygen species in a dose-dependent manner in STZ-treated pancreatic ß cells. In addition, the OPA pretreatment increased the activities of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase in STZ-treated pancreatic ß cells. Moreover, OPA treatment elevated the level of insulin, which was reduced by STZ treatment, and protected pancreatic ß cells against damage under STZ-treated conditions. These effects were mediated by suppressing apoptosis and were associated with increased anti-apoptotic Bcl-xL expression and reduced pro-apoptotic Bax and cleaved caspase-3 expression. These findings indicate that OPA may be useful as a potential pharmaceutical agent to protect against pancreatic ß cell damage caused by oxidative stress associated with diabetes.

The promoting effect of Ishige sinicola on hair growth.

This study was conducted to evaluate the promoting effect of Ishige sinicola, an alga native to Jeju Island, Korea, on hair growth. When vibrissa follicles were cultured in the presence of I. sinicola extract for 21 days, I. sinicola extract increased hair-fiber length. After topical application of I. sinicola extract onto the back of C57BL/6 mice, anagen progression of the hair shaft was induced. The I. sinicola extract significantly inhibited the activity of 5α-reductase. Treatment of immortalized vibrissa dermal papilla cells (DPCs) with I. sinicola extract resulted in increase of cell proliferation, which was accompanied by the increase of phospho-GSK3ß level, ß-catenin, Cyclin E and CDK2, whereas p27kip1 was down-regulated. In particular, octaphlorethol A, an isolated component from the I. sinicola extract, inhibited the activity of 5α-reductase and increased the proliferation of DPCs. These results suggest that I. sinicola extract and octaphlorethol A, a principal of I. sinicola, have the potential to treat alopecia via the proliferation of DPCs followed by the activation of ß-catenin pathway, and the 5α-reductase inhibition.

Protective effect of a marine polyphenol, dieckol against carbon tetrachloride-induced acute liver damage in mouse.

In this study, the hepatoprotective effect of dieckol on carbon tetrachloride (CCl4) induced hepatic damages in ICR mice liver was investigated. Mice were randomly divided into 4 groups such as saline treated (negative control), CCl4 treated (positive control), CCl4+dieckol (5mg/kg mouse) and CCl4+dieckol (25mg/kg mouse), respectively. The body weights and survival rates of mice, followed by dieckol treatments were significantly increased compared to the positive control. The level of GOT, GPT and MDA in the serum of the dieckol treated groups were reduced dose dependently than the control, significantly. The antioxidant enzymes including CAT, and GSH-px levels were increased significantly compared to the positive control. However, no significant differences were observed on hepatic histophathological analysis in dieckol treated groups dose dependently. Down-regulation of Bax and up-regulation of Bcl-xl protein expressions were observed in liver tissues of the dieckol administered groups. These results suggested that, dieckol can be developed as a therapeutic agent for liver disease by oxidative stress.

Protective effect of marine algae phlorotannins against AAPH-induced oxidative stress in zebrafish embryo.

In this study the protective effect of phlorotannins, including phloroglucinol, eckol, dieckol, eckstolonol and triphloroethol A, isolated from brown algae Ecklonia cava was investigated against AAPH-induced oxidative stress toxicity in zebrafish embryos. Zebrafish embryos were exposed to AAPH and compared with other groups that were co-exposed with phlorotannins until 2-days post-fertilisation. All phlorotannins scavenged intracellular ROS and prevented lipid peroxidation and reduced AAPH-induced cell death in zebrafish embryos. Negative changes in morphological phenomena, such as pericardial oedema, yolk sac oedema, and growth retardation in zebrafish embryos exposed to AAPH were not observed in groups exposed to phlorotannins. These results clearly indicate that phlorotannins possess prominent antioxidant activity against AAPH-mediated toxicity and might be potential therapeutic agents for treating or preventing several diseases implicated with oxidative stress. This study provides a useful tool for examining the protective effect of antioxidants against AAPH-induced oxidative stress in an alternative in vivo model.

Inhibition of tumor growth in vitro and in vivo by fucoxanthin against melanoma B16F10 cells.

The present study was designed to evaluate the molecular mechanisms of fucoxanthin against melanoma cell lines (B16F10 cells). Fucoxanthin reduced the proliferation of B16F10 cells in a dose-dependent manner accompanied by the induction of cell cycle arrest during the G(0)/G(1) phase and apoptosis. Fucoxanthin-induced G(0)/G(1) arrest was associated with a marked decrease in the protein expressions of phosphorylated-Rb (retinoblastoma protein), cyclin D (1 and 2) and cyclin-dependent kinase (CDK) 4 and up-regulation of the protein levels of p15(INK4B) and p27(Kip1). Fucoxanthin-induced apoptosis was accompanied with the down-regulation of the protein levels of Bcl-xL, an inhibitor of apoptosis proteins (IAPs), resulting in a sequential activation of caspase-9, caspase-3, and PARP. Furthermore, the anti-tumor effect of fucoxanthin was assessed in vivo in Balb/c mice. Intraperitoneal administration of fucoxanthin significantly inhibited the growth of tumor mass in B16F10 cells implanted mice.

Dieckol isolated from brown seaweed Ecklonia cava attenuates type ІІ diabetes in db/db mouse model.

In the present study, the attenuation of type ІІ diabetes by dieckol, a phlorotannin derivative isolated from brown seaweed, Ecklonia cava was investigated in C57BL/KsJ-db/db, a type ІІ diabetes mouse model. Dieckol was administered intraperitoneal injection at doses of 10 and 20 mg/kg body weight diabetes mice for 14 days. The blood glucose level, serum insulin level and body weight were significantly reduced in the dieckol administered group, compared to that of the saline administered group. Furthermore, reduced thiobarbituric acid reactive substraces (TBARS), as well as increased activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-px) in liver tissues were observed in the dieckol administered group. In addition, increased levels of the phosphorylation of AMPK and Akt were observed in the muscle tissues of the dieckol administered group in a Western blotting analysis. According to the findings of this study, it could be suggested that, dieckol can be developed as a therapeutic agent for type ІІ diabetes.

The JNk/NFkappaB pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from Ecklonia cava.

BACKGROUND: The proven immunomodulatory and immune system activating properties of Ecklonia cava (E. cava) have been attributed to its plentiful polysaccharide content. Therefore, we investigated whether the sulfated polysaccharide (SP) of E. cava specifically activates the protein kinases (MAPKs) and nuclear factor-kappaB (NFkappaB) to incite immune responses. METHODS: To assess immune responsiveness, lymphocytes were isolated from spleens of ICR mice and cultured with SP and its inhibitors. Assays included 3H-thymidine incorporation, flow cytometry, real time polymerase chain reaction (rtPCR), enzyme linked immunosorbent assay (ELISA), intracellular cytokine assay. Western blot, and electrophoretic mobility shift assay (EMSA). RESULTS: SP dose-dependently increased the proliferation of lymphocytes without cytotoxicity. In particular, SP markedly enhanced the proliferation and differentiation of CD3+ mature T cells and CD45R/B220+ pan B cells. Additionally, SP increased the expression and/or production of IL-2, IgG(1a), and IgG(2b) compared to that in untreated cells. The subsequent application of JNK (SP600125), NFkappaB (PDTC), and serine protease (TPCK) inhibitors significantly inhibited the proliferation and IL-2 production of SP-treated lymphocytes as well as the phosphorylation of JNK and IkappaB, the activation of nuclear NFkappaB p65, and binding of NFkappaB p65 DNA. Moreover, co-application of both JNK and NFkappaB inhibitors completely blocked the proliferation of lymphocytes even in the presence of SP. CONCLUSION: These results suggest that SP induced T and B cell responses via both JNK and NFkappaB pathways. GENERAL SIGNIFICANCE: The effect of SP on splenic lymphocyte activation was assayed here for the first time and indicated the underlying functional mechanism.

Dieckol isolated from Ecklonia cava protects against high-glucose induced damage to rat insulinoma cells by reducing oxidative stress and apoptosis.

Pancreatic ß cells are very sensitive to oxidative stress and this might play an important role in ß cell death with diabetes. The protective effect of dieckol, one of the phlorotannin polyphenol compounds purified from Ecklonia cava (E. cava), against high glucose-induced oxidative stress was investigated by using rat insulinoma cells. A high-glucose (30 mM) treatment induced the death of rat insulinoma cells, but dieckol, at a concentration 17.5 or 70 µM, significantly inhibited the high-glucose induced glucotoxicity. Treatment with dieckol also dose-dependently reduced thiobarbituric acid reactive substances (TBARS), the generation of intracellular reactive oxygen species (ROS), and the nitric oxide level increased by a high glucose concentration. In addition, the dieckol treatment increased the activities of antioxidative enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) in high glucose-pretreated rat insulinoma cells. Dieckol protected rat insulinoma cells damage under high glucose conditions. These effects were mediated by suppressing apoptosis and were associated with increased anti-apoptotic Bcl-2 expression, and reduced pro-apoptotic cleaved caspase-3 expression. These findings indicate that dieckol might be useful as a potential pharmaceutical agent to protect against the glucotoxicity caused by hyperglycemia-induced oxidative stress associated with diabetes.

Effect of Dieckol, a component of Ecklonia cava, on the promotion of hair growth.

This study was conducted to evaluate the effect of Ecklonia cava, a marine alga native to Jeju Island in Korea, on the promotion of hair growth. When vibrissa follicles were cultured in the presence of E. cava enzymatic extract (which contains more than 35% of dieckol) for 21 days, E. cava enzymatic extract increased hair-fiber length. In addition, after topical application of the 0.5% E. cava enzymatic extract onto the back of C57BL/6 mice, anagen progression of the hair-shaft was induced. The treatment with E. cava enzymatic extract resulted in the proliferation of immortalized vibrissa dermal papilla cells (DPC). Especially, dieckol, among the isolated compounds from the E. cava enzymatic extract, showed activity that increased the proliferation of DPC. When NIH3T3 fibroblasts were treated with the E. cava enzymatic extract and the isolated compounds from the E. cava enzymatic extract, the E. cava enzymatic extract increased the proliferation of NIH3T3 fibroblasts, but the isolated compounds such as eckol, dieckol, phloroglucinol and triphlorethol-A did not affect the proliferation of NIH3T3 fibroblasts. On the other hand, the E. cava enzymatic extract and dieckol significantly inhibited 5α-reductase activity. These results suggest that dieckol from E. cava can stimulate hair growth by the proliferation of DPC and/or the inhibition of 5α-reductase activity.

Quercitrin protects against ultraviolet B-induced cell death in vitro and in an in vivo zebrafish model.

Chronic exposure of skin to ultraviolet (UV) B radiation induces oxidative stress, which in turn, plays a crucial role in the induction of skin aging. The search for strategies to reverse skin aging is being constantly pursued. Here, the cytoprotective effect of quercitrin (QR) on UVB-induced cell injury in HaCaT human keratinocytes and in the zebrafish was investigated. Intracellular reactive oxygen species (ROS) generated by the exposure of HaCaT cells to UVB radiation were significantly decreased after treatment with QR, and significantly so with QR at 50 µM. As a result, QR reduced UVB-induced cell death and apoptosis in HaCaT cells. QR similarly reduced UVB-induced ROS generation and cell death in live zebrafish.