Ju-Young Ko

Purification and Determination of Two Novel Antioxidant Peptides from Flounder Fish (paralichthys olivaceus) Using Digestive Proteases

We investigated the effects of bioactive-peptides from hydrolysates of flounder fish muscle (FFM) on antioxidant activity. The hydrolysates were prepared by enzymatic reactions of FFM using eight commercial proteases such as papain, pepsin, trypsin, neutrase, alcalase, kojizyme, protamex, and α-chymotrypsin. The α-chymotrypsin hydrolysate showed the strongest antioxidant activity among the eight enzymatic hydrolysates. Further separation of the α-chymotrypsin hydrolysate was performed by ultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. Consequently, two novel peptides with high antioxidant activity were purified, and their amino acid sequences were determined (Val-Cys-Ser-Val [VCSV] and Cys-Ala-Ala-Pro [CAAP], respectively). The two peptides showed good scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical (IC(50) values, 111.32 and 26.89 μM, respectively) and high cytoprotective activities against 2,2-azobis-(2-amidino-propane) dihydrochloride (AAPH) without cytotoxicity and scavenged total reactive oxygen species in Vero cells. In particular, apoptotic bodies produced by AAPH dose-dependently decreased following treatment with the CAAP peptide. These results revealed firstly the two peptides with strong antioxidative effects from FFM.

Hepatoprotective effects of dieckol-rich phlorotannins from Ecklonia cava, a brown seaweed, against ethanol induced liver damage in BALB/c mice

Alcoholic liver disease, which is one of the most serious liver disorders, has been known to cause by ethanol intake. In the present study, in vivo hepatoprotective effects of dieckol-rich phlorotannins (DRP) from Ecklonia cava, a brown seaweed, on ethanol induced hepatic damage in BALB/c mice liver were investigated. After administration of 5 and 25mg/kg mouse of DRP and 4 g/kg mice ethanol, the body weights and survival rates were increased as compared to the control, which is ethanol-treated group without DRP. The glutamic oxaloacetic transaminase and glutamic pyruvic transaminase levels in the serum were lower than those of the control. DRP exhibited a reduction of the total cholesterol. The lower levels of SOD enzyme and a reduction of the formation of malondialdehyde were occurred in mice fed with 5 and 25mg/kg mouse of DRP. Finally the effect on improvement of fatty liver induced by ethanol was observed by taking out the liver immediately after dissecting the mouse. However, no significant difference was observed on hepatic histopathological changes. In conclusion, this study indicated that DRP could protect liver injury induced by ethanol in vivo. It suggested that DRP possesses the beneficial effect to human against ethanol-induced liver injury.

Neuroprotective effects of phlorotannins isolated from a brown alga, Ecklonia cava, against H2O2-induced oxidative stress in murine hippocampal HT22 cells.

Exposure of neurons to hydrogen peroxide (H(2)O(2)) results in oxidative stress and the activation of a cascade of intracellular toxic events resulting in oxidation, lipid peroxidation, and Ca(2+) elevation, ultimately resulting in cell death. In this study, we attempted to characterize the neuroprotective effects of phlorotannins isolated from Ecklonia cava, including phloroglucinol, eckol, triphloroethol A, eckstolonol, and dieckol, against H(2)O(2)-induced cell damage in murine hippocampus neuronal (HT22) cells. We measured the reactive oxygen species (ROS) and lipid peroxidation levels and evaluated the resultant cell death and alterations in Ca(2+)-concentrations. All phlorotannins were to scavenge intracellular ROS and repress ROS accumulation, thus preventing lipid peroxidation. Consquently, all phlorotannins reduced H(2)O(2)-induced cell death in HT22 cells. Moreover, phlorotannins inhibited H(2)O(2)-induced Ca(2+) release. This study provides a new useful strategy for preventing neuronal H(2)O(2)-induced oxidative stress.

Gallic acid isolated from Spirogyra sp. improves cardiovascular disease through a vasorelaxant and antihypertensive effect

In this study, we investigated the vasorelaxant and antihypertensive effects of gallic acid (GA), a polyphenol isolated from the green alga Spirogyra sp., to assess its suitability as a therapeutic for cardiovascular diseases (CVDs). We examined the effect of GA on endothelium-dependent vasorelaxation in human umbilical vein endothelial cells (HUVECs). GA increased nitric oxide (NO) levels by increasing phosphorylation of endothelial nitric oxide synthase (eNOS), and its effect on NO production was attenuated by pretreatment with the eNOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). We also investigated its antihypertensive effect by examining GA-mediated inhibition of angiotensin-I converting enzyme (ACE). GA inhibited ACE with a half-maximal inhibitory concentration (IC50) value of 37.38 ± 0.39 μg/ml. In silico simulations revealed that GA binds to the active site of ACE (PDB: 1O86) with a binding energy of -270.487 kcal/mol. Furthermore, GA clearly reduced blood pressure in spontaneously hypertensive rats (SHR) to an extent comparable to captopril. These results suggest that GA isolated from Spirogyra sp. exerts multiple therapeutic effects and has potential as a CVD treatment.

Preparative isolation and purification of phlorotannins from Ecklonia cava using centrifugal partition chromatography by one-step.

Various bioactive phlorotannins of Ecklonia cava (e.g., dieckol, eckol, 6,6-bieckol, phloroglucinol, phloroeckol, and phlorofucofuroeckol-A) are reported. However, their isolation and purification are not easy. Centrifugal partition chromatography (CPC) can be used to efficiently purify the various bioactive-compounds efficiently from E. cava. Phlorotannins are successfully isolated from the ethyl acetate (EtOAc) fraction of E. cava by CPC with a two-phase solvent system comprising n-hexane:EtOAc:methanol:water (2:7:3:7, v/v) solution. The dieckol (fraction I, 40.2mg), phlorofucofuroeckol-A (fraction III, 31.1mg), and fraction II (34.1mg) with 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol are isolated from the crude extract (500 mg) by a one-step CPC system. The purities of the isolated dieckol and phlorofucofuroeckol-A are ⩾90% according to high performance liquid chromatography (HPLC) and electrospray ionization multi stage tandem mass spectrometry analyses. The purified 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol are collected from fraction II by recycle-HPLC. Thus, the CPC system is useful for easy and simple isolation of phlorotannins from E. cava.

Preparative isolation of sargachromanol E from Sargassum siliquastrum by centrifugal partition chromatography and its anti-inflammatory activity

Centrifugal partition chromatography (CPC) can be used to isolate various bioactive compounds from natural materials by one-step. We confirmed antioxidative compounds existed in chloroform (CHCl3) fraction of Sargassum siliquastrum using online-HPLC. Fractions (A, B, C, D and E) were separated from the CHCl3 fraction by preparative CPC (n-hexane:ethyl acetate:methanol:water, 5:5:7:3, v/v). In this study, we proved that the isolated compounds exhibit anti-inflammatory activities using lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages. The fraction A which exhibited the strongest inhibitory effect on nitric oxide (NO) production level, was confirmed as sargachromanol E by LC-MS-ESI, (1)H NMR and (13)C NMR data. The sargachromanol E significantly reduced the inflammatory response in LPS induced macrophages, decreasing LPS-induced transcription factor of pro-inflammatory cyclooxygenase-2, NO synthase, phosphate P38, phosphate ERK1/2, LPS-stimulated tumor-necrosis factor alpha, interleukin-1 beta and prostaglandin E2 release. In conclusion, it was suggested that sargachromanol E inhibited inflammation in LPS induced RAW 264.7 cells via MAPK pathway.

Evaluation of the biological activities of marine bacteria collected from Jeju Island, Korea, and isolation of active compounds from their secondary metabolites

To explore marine microorganisms with medical potential, we isolated and identified marine bacteria from float s, marine algae, animals, and sponges collected from Jeju Island, Korea. We isolated and identified 21 different strains from the marine samples by 16S rRNA analysis, cultured them in marine broth, and extracted them with ethyl acetate (EtOAc) to collect secondary metabolite fractions. Next, we evaluated their anti-oxidative and anti-inflammatory effects. Among the 21 strains, the secondary metabolite fraction of Bacillus badius had both strong antioxidant and anti-inflammatory activity, and thus was selected for further experi ments. An antioxidant compound detected from the secondary metabolite fraction of B. badius was purified by preparative cen trifugal partition chromatography (n-hexane:EtOAc:methanol:water, 4:6:4:6, v/v), and identified as diolmycin A2. Additionally, diolmycin A2 strongly inhibited nitric oxide production. Thus, we successfully identified a significant bioactive compound from B. badius among the bacterial strains collected from Jeju Island.

Rapid preparation of functional polysaccharides from Pyropia yezoensis by microwave-assistant rapid enzyme digest system

This study describes a simple preparation of functional polysaccharides from Pyropia yezoensis using a microwave-assistant rapid enzyme digest system (MAREDS) with various carbohydrases, and evaluates their antioxidative effects. Polysaccharide hydrolysates were prepared using MAREDS under different hydrolytic conditions of the carbohydrases and microwave powers. Polysaccharides less than 10kDa (Low molecular weight polysaccharides, LMWP, ≤10kDa) were efficiently obtained using an ultrafiltration (molecular weight cut-off of 10kDa). MAREDS increases AMG activation via an increased degree of hydrolysis; the best AMG hydrolysate was prepared using a 10:1 ratio of substrate to enzyme for 2h in MAREDS with 400W. LMWP consisted of galactose (27.3%), glucose (64.5%), and mannose (8.3%) from the AMG hydrolysate had stronger antioxidant effects than the high molecular weight polysaccharides (>10kDa). We rapidly prepared functional LMWPs by using MAREDS with carbohydrases, and suggest that LMWP might be potentially a valuable algal polysaccharide antioxidant.

Identification and large isolation of an anti-inflammatory compound from an edible brown seaweed, Undariopsis peterseniana, and evaluation on its anti-inflammatory effect in in vitro and in vivo zebrafish

In this study, we isolated and purified secondary metabolites from an edible brown seaweed, Undariopsis peterseniana, and evaluated their anti-inflammatory effects in vitro and in vivo. Apo-9′-fucoxanthinone (Apo-9′), a main anti-inflammatory compound found in the chloroform fraction of U. peterseniana extracts, was first isolated by one-step centrifugal partition chromatography and identified by nuclear magnetic resonance. Apo-9′ significantly decreased nitric oxide (NO), prostaglandin E2, pro-inflammatory proteins (inducible NO synthase [iNOS] and cyclooxygenase [COX-2]), and pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6, and interleukin-1β) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells without exhibiting any cytotoxicity. In vivo, Apo-9′ protected LPS-stimulated zebrafish embryos against inflammatory stress and suppressed the expression of pro-inflammatory proteins such as COX-2 and iNOS. These findings suggest that U. peterseniana extract and Apo-9′ could be used as an anti-inflammatory agent in health care.

Evaluation on Anticancer Effect Against HL-60 Cells and Toxicity in vitro and in vivo of the Phenethyl Acetate Isolated from a Marine Bacterium Streptomyces griseus

We previously identified Streptomyces griseus as an anti-cancer agent (Kim et al., 2014). In this study, we isolated compounds from S. griseus and evaluated their anticancer effect and toxicity in vitro and in vivo. Preparative centrifugal partition chromatography (CPC) was used to obtain three compounds, cyclo(L-[4-hydroxyprolinyl]- L –leucine], cyclo(L-Phe-trans-4-hydroxy-L-Pro) and phenethyl acetate (PA). We chose PA, which had the highest anticancer activity, as a target compound for further experiments. PA induced the formation of apoptotic bodies, DNA fragmentation, DNA accumulation in G0/G1 phase, and reactive oxygen species (ROS) formation. Furthermore, PA treatment increased Bax/Bcl-xL expression, activated caspase-3, and cleaved poly-ADP-ribose polymerase (PARP) in HL-60 cells. Simultaneous evaluation in vitro and in vivo, revealed that PA exhibited no toxicity in Vero cells and zebrafish embryos. We revealed, for the first time, that PA generates ROS, and that this ROS accumulation induced the Bcl signaling pathway.