• Journal of Life Science
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• v.20 no.2
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• pp.260-268
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• 2010

The lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes in tissues from Channa argus were purified and characterized by biochemical, immunochemical and kinetic methods. The activity of LDH in skeletal muscle was the highest at 380.4 units and those in heart, eye and brain tissues were 13.4, 3,5 and 5.4 units, respectively. Citrate synthase (EC 4.1.3.7, CS) activity in heart tissue was the highest at 20.7 units. LDH/CS in skeletal muscle, heart, eye and brain tissues were 172.9, 0.6, 0.32 and 0.47. Protein concentration in skeletal muscle tissue was 14.7 mg/g and specific activities of LDH in skeletal muscle, heart, eye and brain tissues were 25.88, 0.79, 0.31 and 1.38 units/mg, respectively. Therefore, skeletal muscle tissue was anaerobic and heart tissue was aerobic. The LDH isozymes in tissues were identified by polyacrylamide gel electrophoresis, immunoprecipitation and Western blot with antiserum against $A_4$, $B_4$, and eye-specific $C_4$. LDH $A_4$, $A_3B$, $A_2B_2$. $AB_3$ and $B_4$ isozymes were detected in every tissue, $C_4$, $AC_3$, $A_2C_2$ and $A_3C$ were detected in eye tissue, and $A_3C$ was found in brain tissue. LDH $A_4$, $A_3B$, $A_2B_2$, $AB_3$, $B_4$, eye-specific $C_4$ isozymes were purified by affinity chromatography and Preparative PAGE Cells. The LDH $A_4$ isozyme was purified in the fraction from elution with $NAD^+$ containing buffer of affinity chromatography. Eye-specific $C_4$ isozyme was eluted right after $A_4$, after which $B_4$ isozyme was eluted with plain buffer. As a result, one part of molecular structures in $A_4$, $B_4$ and eye-specific $C_4$ were similar, but were different from each other in $B_4$ and $C_4$. Therefore the subunit A may be conservative in evolution, and the evolution of subunit B seems to be faster than that of subunit A. The activity of LDH $A_4$, $A_2B_2$, $B_4$, and eye-specific $C_4$ isozymes remained at 39.98, 21.28, 19.67 and 16.87% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The $Km^{PYR}$ values were 0.17, 0.27 and 0.133 mM in $A_4$, $B_4$ and eye-specific $C_4$ isozymes, respectively. The optimum pH of LDH $A_4$, $B_4$, eye-specific $C_4$, $A_2B_2$, $A_3B$, and $AB_3$ were pH 6.5, pH 8.5, pH 5.5, pH 6.0-6.5, pH 5.0 and pH 7.5. The $A_4$ and heterotetramer isozymes stabilized a broad range of pH. Especially, LDH activities in skeletal muscle tissue were high, resulting in a high degree of muscle activity.LDH metabolism in eye tissue seems to be converted faster from pyruvate to lactate by eye-specific $C_4$ isozyme as eye-specific $C_4$ have the highest affinity for pyruvate, and right after the conversion, oxidation of lactate was induced by $A_4$ isozyme. It was found that expression of Ldh-C, affinity to substrate and reaction time of $C_4$ isozyme were different according to the ecological environmental and feeding capturing patterns.

• Journal of Life Science
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• v.28 no.2
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• pp.247-256
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• 2018

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used drugs for lowering blood lipid levels and preventing cardiovascular diseases. HMG-CoA reductase is a key enzyme to control the biosynthesis of cholesterol. We have tested HMG-CoA reductase-inhibitory activity on the flavonoids of 98 species in vitro. The anti-hypercholesterolemic activities of flavonoids were studied using an HMG-CoA reductase assay equipped with a 96-well UV plate. This assay was based on the spectrophotometric measurement of the decrease in absorbance, which represents the oxidation of NADPH by the catalytic subunit of HMG-CoA reductase in the presence of the substrate HMG-CoA. Among the clinically available statins, pravastatin was used as a positive control. Among the tested compounds, kuraridin, morin and sophoraflavanone G showed strong inhibition activities. In particular, morin and sophoraflavanone G inhibited HMG-CoA reductase by 45.0% and 54.6% at a concentration of $10{\mu}g/ml$, and the $IC_{50}$ values were calculated to $13.31{\mu}g/ml$ and $7.26{\mu}g/ml$ respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.42 no.12
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• pp.1899-1907
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• 2013

We investigated the effects of unripe black raspberry water extract (UBR-W) and oxidation-LDL treatment on cholesterol levels. Experiments using an established human hepatocellular carcinoma cell line (HepG2) showed a time-dependent increase in expression of LDL receptor after UBR-W treatment. Expression of LDL receptor-related genes, such as SREBP1 and 2, increased upon UBR-W treatment. However, expression of HDL-related genes was unaffected by UBR-W. HMG-CoA reductase activity was reduced by UBR-W treatment, whereas HMG-CoA mRNA expression significantly increased. In addition, the ApoB/ApoA1 mRNA level, which is a predictor of cardiovascular risk, was reduced in a time-dependent manner by UBR-W treatment. Macrophage-like cells (RAW 264.7) showed increased expression of ox-LDL-related genes, such as CD36, scavenger receptor-A, adipophilin, and PPAR-gamma, upon ox-LDL treatment compared to untreated control cells, and quantitative lipid analysis indicated a dramatic increase in lipid accumulation. However, UBR-W treatment significantly reduced expression of ox-LDL-related genes and largely prevented lipid accumulation. The results indicate that UBR-W mediates a cholesterol-lowering effect via inhibition of cholesterol synthesis and induction of LDL uptake through SREBP.

• Journal of Microbiology and Biotechnology
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• v.30 no.10
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• pp.1458-1466
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• 2020

Oligomeric proanthocyanidins (OPCs), classified as condensed tannins, have significant antioxidation, anti-inflammation and anti-cancer effects. This study was performed to investigate the anti-inflammatory effects of OPCs and the mechanism underlying these effects in lipopolysaccharide (LPS)-stimulated bovine mammary epithelial cells (MAC-T). Real-time PCR and ELISA assays indicated that OPC treatment at 1, 3 and 5 ㎍/ml significantly reduced the mRNA and protein, respectively, of oxidant indicators cyclooxygenase-2 (COX-2) (p < 0.05) and inducible nitric oxide synthase (iNOS) (p < 0.01) as well as inflammation cytokines interleukin (IL)-6 (p < 0.01), IL-1β (p < 0.01) and tumor necrosis factor-α (TNF-α) (p < 0.05) in LPS-induced MAC-T cells. Moreover, OPCs downregulated LPS-induced phosphorylation of p65 and inhibitor of nuclear factor kappa B (NF-κB) (IκB) in the NF-κB signaling pathway (p < 0.01), and they inhibited p65 translocation from the cytoplasm to the nucleus as revealed by immunofluorescence test and western blot. Additionally, OPCs decreased phosphorylation of p38, extracellular signal regulated kinase and c-jun NH₂-terminal kinase in the MAPK signaling pathway (p < 0.01). In conclusion, the anti-inflammatory and antioxidant activities of OPCs involve NF-κB and MAPK signaling pathways, thus inhibiting expression of pro-inflammatory factors and oxidation indicators. These findings provide novel experimental evidence for the further practical application of OPCs in prevention and treatment of bovine mastitis.

• The Korean Journal of Pharmacology
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• v.23 no.2
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• pp.113-121
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• 1987

Local anesthetics were investigated for their effects on mitochondrial electron transport system, production of superoxide radical from submitochondrial particles and malondialdehyde production through lipid per oxidation. Local anesthetics had various effects on activities of enzymes in electron transport chain. The activities of NADH dehydrogenase, NADH oxidase and NADH-ubiquinone oxidoreductase were effectively inhibited by lidocaine, procaine and dibucaine but slightly influenced by cocaine. The activities of succinate dehydrogenase, succinate-cytochrome c oxidoreductase and succinate-ubiquinone oxidoreductase were inhibited by lidocaine and dibucaine, but the succinate oxidase activity was stimulated by local anesthetics. Both dihydroubiquinone-cytochrome c oxidoreductase and cytochrome c oxidase activities were inhibited by local anesthetics. In these reactions, the response of Complex I segment to local anesthetics was greater than other Complex segments. Local anesthetics inhibited both the superoxide production from submitochondrial particles supplemented with succinate or NADH and the enhanced production of superoxide radicals by antimycin. The malondialdehyde production by oxygen free radicals was inhibited by local anesthetics. These results suggest that the inhibition of superoxide and malondialdehyde production caused by local anesthetics may be brought by suppression of the electron transport in mitochondria at sites in or near complex I segment.

• Journal of Life Science
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• v.26 no.12
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• pp.1400-1408
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• 2016

The black currant (Ribes nigrum L.) is belong to the Grossulariaceae family, and has piquant berries, which can be eaten as raw or as processed foods, such as jams, jelly, juice and syrups. In this study, the fresh juice of black currant (FJBC) from Austria and its subsequent organic solvent fractions, such as hexane fraction, ethylacetate (EA) fraction, butanol fraction and water residue, were prepared and their in-vitro anti-oxidant, anti-coagulation and anti-platelet aggregation activities were evaluated. The FJBC and EA fraction, which has concentrated polyphenol and flavonoid, showed strong radical scavenging activities and reducing power. The $RC_{50}s$ of EA fraction against DPPH anion, ABTS cation, nitrite were 136.3, 66.2 and $115.5{\mu}g/ml$, respectively, those are 1/10, 1/16, and 1/7.7 of $RC_{50}s$ of vitamin C. In anti-coagulation assay, the FJBC, EA and butanol fraction showed significant inhibitory activities against thrombin, prothrombin and coagulation factors. Furthermore, the anti-platelet aggregation activities of EA and butanol fraction were the stronger than that of aspirin. The concentrations required for 50% platelet aggregation inhibition of aspirin, EA and butanol fraction were 0.395, 0.192 and 0.261 mg/ml, respectively. The EA and butanol fraction have no hemolysis activities up to 0.5 mg/ml against human red blood cells. The results suggest that the FJBC and its EA and butanol fraction have high potentials as novel anti-thrombosis agents. This report provides the first evidence of anti-thrombosis activity of black currant.

• Journal of Food Hygiene and Safety
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• v.33 no.5
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• pp.389-398
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• 2018

The purpose of this study was to evaluate anti-obesity activity of Cirsium setidens Nakai test material (CNTM) in 3T3-L1 adipocytes and obese C57BL/6J mice fed with a high-fat diet using various obesity-related in vitro experiments. During adipocyte differentiation, CNTM significantly inhibited lipid accumulation and ROS production compared to controls. To evaluate whether CNTM could exert glycerol release effects on mature 3T3-L1 adipocytes, we treated cells with various concentrations of CNTM for 1 h. Treatment of mature adipocytes with $160-320{\mu}g/mL$ of CNTM increased the release of glycerol, but not in a significant dose-dependent manner. Anti-adipogenic and anti-lipogenic effects of CNTM seemed to be mediated by the inhibition of $PPAR{\gamma}$ and $C/EBP{\alpha}$. Moreover, CNTM stimulated fatty acid oxidation in an AMPK-dependent manner. CNTM-treated groups of C57BL/6J mice showed reduced body weights and adipose tissue weight with improving serum lipid profiles and adiponectin protein expression in obese C57BL/6J mice fed with a high-fat diet. These results suggest that CNTM might have anti-obesity effect on adipogenesis and lipid metabolism in vitro and in vivo. This presents the possibility of developing a treatment for obesity using nontoxic natural resources.

• Journal of Life Science
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• v.23 no.1
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• pp.31-37
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• 2013

The present study aimed to investigate effects of ethanol extracts from Hydropsyche kozhantschikovi on cell and DNA damage caused by oxidative stress. In a radical scavenging assay, compared with ascorbic acid used as a control, the level of DPPH (1,1-diphenyl-2-picrylhydrazyl) and that of hydroxyl radicals in H. kozhantschikovi extracts were 60.0% and 43.7%, respectively. The ferrous iron chelating level was 37.5% compared to the chelating value of EDTA (ethylenediaminetetraacetic acid) as a positive control at the same concentration. To verify inhibitory effects of oxidative cell damage induced by reactive oxygen species (ROS), the relative level of lipid peroxidation and the expression level of the p21 protein were compared in extracts-treated and untreated groups. Lipid peroxidation was completely inhibited in the extracts-treated group compared with the radical-only treated group. The level of p21 protein expression was restored to 92.2% of p21 protein expression in the control sample. In addition, DNA cleavage inhibition in the H. kozhantschikovi extracts was 74.1% compared with that of the control group, suggesting that H. kozhantschikovi extracts repress DNA cleavage induced by ROS. Moreover, the phosphorylation ratio of the H2AX protein was 16.7% in the radical-treated group, indicating that the ethanol extracts inhibited 83.3% of DNA damage. Our findings suggest that ethanol extracts from H. kozhantschikovi are effective not only in repressing the oxidation of free radicals and highly toxic hydroxyl radicals, but also in decreasing cell and DNA damage caused by oxidative stress.

• Korean Journal of Food Science and Technology
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• v.34 no.2
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• pp.157-163
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• 2002

An investigation was carried out to study the characteristics of lipoxygenase in dioctyl sulfosuccinate (aerosol-OT, AOT)/isooctane revered micelles of microaqueous system containing infinitesimal water. ${\alpha}-Linoleic$ acid as a substrate could be analyzed by the colorimetric methodology using 5%(w/v) cupric acetate-pyridine solution and the activity of lipoxygenase was able to be assayed by the degree of ${\alpha}-linoleic$ acid consumption per minute. Optimal pH, temperature, and R-value ([water]/[AOT]) were determined as the value of 5.0, $25^{\circ}C$, and 10.0, respectively. Kinetic analysis of the enzyme reaction under the optimal conditions showed that the values of $K_m$ and $V_{max}$ were 0.31 mM of ${\alpha}-linoleic$ acid and $384.16{\mu}mol$ of ${\alpha}-linoleic$ acid decomposed/min, respectively. The results indicate the reaction to be lipoxygenase-catalyzed oxidation of ${\alpha}-linoleic$ acid in AOT/isooctane reversed micellar system. The inhibitory effect of natural antioxidants on lipoxygenase showed little inhibitory effect of L-ascrobic acid while ${\alpha}-tocopherol$ showed 72% of inhibitory effect.

• Korean Journal of Plant Resources
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• v.22 no.5
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• pp.425-430
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• 2009

The objective of this study was to investigate the antioxidative capacity of oregano extracts. Amount of oregano extract at which DPPH radical scavenging activity was inhibited by 50% was $21.8{\mu}g$ as compared to 100% by pyrogallol as a reference. Total antioxidant status was examined by total antioxidant capacity against potent free radical reactions. Total antioxidant capacities of oregano extract at the amounts of 7.5 and $15{\mu}g$ were 15.1 and 31.4 nmol Trolox equivalents, respectively. Oxygen radical absorbance capacities of oregano extract at the amounts of 0.2 and $0.4{\mu}g$ were 1.4 and 2.4 nmol gallic acid equivalents, respectively. Total phenolic contents of oregano extract at the amounts of 30 and $75{\mu}g$ were 40.5 and 83.9 nmol gallic acid equivalents, respectively. The inhibitory effect of oregano extract on lipid peroxidation was examined using rat liver mitochondria induced by $FeSO_4$/ascorbic acid. Oregano extracts at the amounts of 20 and $50{\mu}g$ decreased TBARS level by 20 and 64%, respectively. Thus strong antioxidant effects of oregano extract seem to be due to, at least in part, the prevention against free radicals-induced oxidation, followed by inhibition of lipid peroxidation.