Ji-Hyeok Lee

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.

Dieckol, a phlorotannin isolated from a brown seaweed, Ecklonia cava, inhibits adipogenesis through AMP-activated protein kinase (AMPK) activation in 3T3-L1 preadipocytes.

In this study, we assessed the potential inhibitory effect of 5 species of brown seaweeds on adipogenesis the differentiation of 3T3-L1 preadipocytes into mature adipocytes by measuring Oil-Red O staining. The Ecklonia cava extract tested herein evidenced profound adipogenesis inhibitory effect, compared to that exhibited by the other four brown seaweed extracts. Thus, E. cava was selected for isolation of active compounds and finally the three polyphenol compounds of phlorotannins were obtained and their inhibitory effect on adipogenesis was observed. Among the phlorotannins, dieckol exhibited greatest potential adipogenesis inhibition and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding proteins (C/EBPα), sterol regulatory element-binding protein 1 (SREBP1) and fatty acid binding protein 4 (FABP4) in a dose-dependent manner. The specific mechanism mediating the effects of dieckol was confirmed by AMP-activated protein kinase (AMPK) activation. These results demonstrate inhibitory effect of dieckol compound on adipogenesis through the activation of the AMPK signal pathway.

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.

Antioxidant activity of polysaccharide purified from Acanthopanax koreanum Nakai stems in vitro and in vivo zebrafish model

In this study, the antioxidative effects of a purified polysaccharide isolated from the stems of Acanthopanax koreanum Nakai (ASP) on hydrogen peroxide-induced oxidative stress was investigated both in vitro and in vivo using a zebrafish model. A. koreanum Nakai stem was hydrolyzed using five carbohydrases and five proteases for the enzyme-assistant extraction. Of the enzyme-assistant extracts, the Protamex extract exhibited the highest yield and a potent scavenging activity against free radicals. Ethanol-added separation and anion exchange chromatography were conducted to identify the active polysaccharide. The purified polysaccharide significantly scavenged hydrogen peroxide and reduced hydrogen peroxide-induced cell death in Vero cells and in zebrafish. The results reveal that ASP is a useful antioxidant polysaccharide and might be available for relevant industrial applications.

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.

A marine algal polyphenol, dieckol, attenuates blood glucose levels by Akt pathway in alloxan induced hyperglycemia zebrafish model

Recently, zebrafish (Danio rerio) have been used as a powerful model for human disease. The anti-diabetes activity of the anti-diabetic compound, dieckol (DK), one of the marine algal polyphenols isolated from Ecklonia cava, was confirmed in the zebrafish model. The zebrafish were divided to four groups, the normal (alloxan-untreated), alloxan-induced diabetic zebrafish without (control) and with DK, as well as those treated with metformin, a commercial drug. The blood glucose levels of the DK group of the hyperglycemic zebrafish at 90 min were decreased more than 3.3 times compared with the control group. Furthermore, reduced glucose-6-phosphate and phosphoenolpyruvate carboxykinase were observed with the treatments of DK and metformin in the liver tissues, and there was increased phosphorylation of protein kinase B (Akt) in the muscle tissue in the zebrafish model. Akt activation was involved in mediating the effect of DK on glucose transport activation and insulin sensitivity. These results prove that DK exerts a strong anti-diabetic effect by improving blood glucose regulation, hepatic glucose metabolic regulation and Akt up-regulation in alloxan induced hyperglycemic zebrafish.

Protective effect of marine brown algal polyphenols against oxidative stressed zebrafish with high glucose

The zebrafish (Danio rerio) is one of the most widely used vertebrate models in research studies in molecular genetics, development biology, drug discovery and human disease. This study has confirmed an increase in the production of reactive oxygen species (ROS) and induction of cell death by high glucose treatment in zebrafish. We observed that exposure to phlorotannins, which include 6,6-bieckol, phloroeckol, dieckol and phlorofucofuroeckol isolated from an edible brown alga, Ecklonia cava, significantly inhibited high glucose induced ROS and cell death. Among the phlorotannins, DK (Dieckol) significantly reduced heart rates, ROS, nitric oxide (NO), lipid peroxidation generation and cell death in high glucose induced oxidative stress. Further, high glucose levels induced the over expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), whereas DK treatment reduced its over expression. These findings indicate that the zebrafish model is an efficient animal model that can be used to investigate hyperglycemia-stimulated oxidative stress. Therefore, this model can be used as an in vivo experiment to confirm the antioxidant properties of functional foods and nutraceuticals.

A sulfated polysaccharide of Ecklonia cava inhibits the growth of colon cancer cells by inducing apoptosis

We investigated anticancer effects of the crude polysaccharides (CPs) isolated from Ecklonia cava enzymatic extracts using AMG, Viscozyme, Protamex, and Alcalase enzyme against a colon cancer cell line, CT26 cells. Among them, the CP of Protamex extract (PCP) contained the highest fucose and sulfated group contents and showed the highest growth inhibitory effect against CT-26 cells. In addition, PCP dose-dependently increased the formation of apoptotic body and the percentage of Sub-G1 DNA contents. Also, PCP activated caspase 9 and PARP as regulating the expressions of Bax and Bcl-2. Moreover, PPP2, a fraction purified from PCP showed the highest growth inhibitory effect against CT 26 cells with the increased fucose and sulfated group contents. The results demonstrate that the isolated SP containing plentiful fucose and sulfated group contents has the anticancer effect on colon cancer cells via regulation of Bcl-2/Bax signal pathway.

The Growth, Innate Immunity and Protection against H 2 O 2 -Induced Oxidative Damage of a Chitosan-Coated Diet in the Olive Flounder Paralichthys olivaceus

We demonstrate enhanced growth, innate immunity and protection against hydrogen peroxide (H2O2)-induced protein oxidation and cellular DNA damage in olive flounder Paralichthys olivaceus fed a chitosan-coated moist pallet (MP) diet. A chitosanbased biopolymer coated MP as the experimental diet and a non-coated MP (control) was fed to olive flounder fish. Growth, including the average weight gain (g/fish), weight gain (%) and feed intake (g) of the fish group fed a chitosan-coated MP diet increased significantly. The survival rate was reported as 100% throughout the experimental period. Immunological parameters indicated higher mucus lysozyme activity and significantly higher fish skin mucus total protein content was observed in fish fed the chitosan-coated MP diet compared to the control. A blood plasma analysis revealed attenuation of cellular DNA and protein oxidative damage caused by H2O2-induced oxidative stress in the fish fed the chitosan-coated MP diet compared to the control group. Moreover, blood serum biochemical analysis revealed health-promoting effects, including significantly higher hemoglobin and total cholesterol levels in the fish fed the chitosan-coated MP diet compared to the control group. In conclusion, growth, in nate immunity and protection against oxidative stresses were improved by feeding of the chitosan-coated MP diet to olive flounder reared in aquaculture.