• Korean Journal of Fisheries and Aquatic Sciences
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• v.42 no.6
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• pp.614-620
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• 2009

We report non-specific immune responses and its disease resistance against Edwardsiella tarda by alga mixture (HE; Hizikia:Ecklonia) in olive flounder for the first time. Five isonitrogenous (44% crude protein) and isocaloric (17.1 MJ $kg^{-1}$) diets were formulated to have 0%, 2%, 4%, 6% and 8% of the alga mixture. One of five experimental diets was fed triplicate groups of fish (30 fish/group) to apparent satiation in a flow through system. After a two week feeding, blood was sampled at 3, 6, 12, 24 h after the last feeding for a kinetic measurement of nitroblue tetrazolium (NBT) activity and healthy fish with similar sizes in each tank were selected and injected with 1 mL of E. tarda suspension ($1.0\times10^7$ CFU/mL) to evaluate the disease resistance of the fish. Dietary supplementation of alga mixtures resulted in significantly higher non-specific immune responses compared with the fish fed the control diet. The cumulative mortality was significantly lower in the fish groups fed alga mixture containing diets than control group in the challenge test with E. tarda. Therefore, the results in this study indicate that dietary supplementation of Hizikia and Ecklonia mixtures enhance the non-specific immune responses and a disease resistance of olive flounder.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.5
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• pp.369-378
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• 2000

This study was undertaken to investigate an involvement of nitroxergic innervation in gastric smooth muscle of rat. Isometric tension study, the measurement of single cell length, NADPH diaphorase stain of smooth muscle layers and neuronal nitric oxide synthase (nNOS) western blotting were performed. Sodium nitroprusside (SNP), a nitric oxide donor, relaxed the muscle strips precontracted by acetylcholine (ACh) in a concentration-dependent manner. Pretreatment of L-arginine decreased the contraction induced by electric field stimulation (EFS). Pretreatment of $N^G-nitro-L-arginine$ methyl ester (L-NAME), a NOS inhibitor, increased the EFS-induced contractions. LY 83583, a guanylate cyclase (GC) inhibitor, reversed the inhibitory actions of L-arginine on the muscle contractions. The effects of L-Arginine, L-NAME and LY 83583 on ACh-induced contractions were not significant. L-arginine reduced the EFS-induced contraction in circular muscle, whereas L-NAME enhanced the EFS-induced contraction in longitudinal strips. By EFS, the phasic contractions appeared approximately $20{\sim}25$ seconds later. L-NAME significantly shortened the delay time to about $2{\sim}3$ seconds. In single cell study, ACh contracted gastric smooth muscle cells, SNP relaxed the cells, and the latter also inhibited the ACh-induced contraction. LY 83583 enhanced the ACh-induced contraction and antagonized SNP-induced relaxation. NADPH diaphorase activity was assessed by a histochemistry, nitroblue tetrazolium (NTB) staining. Positive staining was observed in both circular and longitudinal muscle layers. L-arginine increased the staining, while L-NAME decreased the staining. Western blotting for nNOS proved the presence of nNOS in rat gastric smooth muscle. EFS and additional $Ca^{2+}$ increased nNOS protein expression. These results suggest that in rat stomach, both circular and longitudinal muscle layers are innervated with nitroxergic nerves which relax the gastric smooth muscle via NO-cGMP pathway.

• Korean Journal of Food Science and Technology
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• v.32 no.3
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• pp.706-712
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• 2000

The superoxide dismutase(SOD)-like activities for 26 kinds of herbs and spices and 10 kinds of instant curry products were determined by measuring their abilites to reduce nitroblue tetrazolium. All samples showed the SOD-like activities. Rosemary, cassia, tarragon, allspice, oregano, bay leaves, basil, marjoram, thyme and star anise had higher activities than $10^5\;unit/g$ and clove had highest activity of $232,143{\pm}19.989\;unit/g$. The SOD-like activities for 10 kinds of instant curry products were in the range of $400{\sim}700\;unit/g$ when measured after heat treatment at $100^{\circ}C$ for 10 min. The water extracts of spices, herbs and curries were obtained by heat treatments of $25^{\circ}C$ for 60 min or $100^{\circ}C$ for 10 min, and their nitrite scavenging activity was measured at different pH conditions(1.2, 4.2 or 6.0). The nitrite scavenging activities were higher at acidic pH. However, the effects were not different from two heat treatments. The water extracts from cassia, bay leaves, allspices, oregano, staranise, rosemary, clove and tarragan had high nitrite scavenging activity(>90%) when they were measured at pH 1.2, and those from clove was highest $(97.58{\pm}0.88%)$. The pure curry used as raw materials for instant curry products had the nitrite scavenging activity in the range of $50{\sim}60%$ at pH 1.2 and the activity was not changed during the aging period$(0{\sim}12weeks)$. The ten brands of instant curry products had the nitrite scavenging activities of $12{\sim}28%$ at pH 1.2

• Advances in Traditional Medicine
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• v.5 no.3
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• pp.216-230
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• 2005

Although the field of study in immune enhancing compounds is relatively new, natural products from plants represent a rich and promising source of novel molecules with immunomodulating properties, Microglial cells, the main immune effector cells of the brain, usually display a ramified morphology and low expression levels of immunologically relevant antigens such as MHC class I and class II. Since any compound which participates in activation of phagocytic cells contributes to the production of potentially toxic factors, the search for convenient in vitro test-systems and study of mechanisms of action of these agents are of great interest. Human blood polymorphonuclear (PMN) cells and primary microglial cells isolated from Sprague-Dawley rats were used as cellular screening tests for study of phagocytosis-stimulating action of immunomodulating agents. Numbers of phagocytic activity were evaluated by the phagocyte ingestion of yeast cells and NO-synthase activity, nitrite production, and nitroblue tetrazolium test were determined after phagocyte stimulation. It was possible to demonstrate that indexes of phagocytic activity can be used as quantitative indicators for measurement immunomodulating activity. As a positive control, Zymosan A-induced phagocytosis in both PMN cells and primary microglial cells was used. $IFN-{\gamma}$ (0.1 -1 U/ml) stimulated phagocytosis in PMN cells 1.2 times after 2 - 3 h incubation, although at higher concentrations (10 - 100 U/ml) it strongly inhibited phagocytosis. In a similar way, at higher concentrations, $IFN-{\gamma}$ (100 - 500 U/ml) suppressed phagocytosis in zymosan-A stimulated microglial cells. When Polypodium leucotomus, cambricum and vulgare extracts were tested alone, increased levels of phagocytosis were observed in PMN. In addition, microglial cells showed both increased phagocytosis and MHC class-II antigen expressions. Surprisingly, when PMN and microglia were treated with a combination of Polypodium and $IFN-{\gamma}$, phagocytosis was not inhibited. We did not find changes in NO-synthase activity and nitrite production in both microglia and PMN cells activated by different immunomodulating agents. These results indicate that primary microglial cell cultures as well as human PMN cells can provide reproducible quantitative results in screening phagocytic activity of different immunoactive compounds. Furthermore, both inhibitory or activation mechanisms might be studied using these in vitro experimental approaches.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.40 no.5
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• pp.300-307
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• 2007

This study investigated the effects of dietary supplementation with hot water extracts (HE) of selfheal (Prunella vulgaris) and Lactobacillus rhamnosus culture fluid (LF) on the growth and non-specific immune responses of juvenile olive flounder. A total of 270 fish ($5.07{\pm}0.01g$, average $weight{\pm}SD$) were divided randomly into nine groups, and three groups were fed one of three isonitrogenous (51% crude protein) and isocaloric (17.6 MJ/kg) diets. The diets contained no supplement, 50 mL hot water extract, or 50 mL L. rhamnosus fluid (designated as diets CON, HE and LF, respectively). During the 8-week feeding trial, the growth, feed utilization and survival of the fish were not significantly affected by the experimental diets. There were no significant differences in the hematocrit and hemoglobin of fish fed each of the experimental diets. However, the serum alanine aminotransferase and aspartate aminotransferase activity were lower with the dietary supplements containing HE and LF. The alanine aminotransferase activity of fish fed the HE diet was significantly lower than that of fish fed the control diet. The hematocrit, hemoglobin, and nitroblue tetrazolium (NBT) activity were higher 3 hr after feeding than 24 hr afterwards. The NBT activity of fish fed the HE and LF diets were significantly higher than that of fish fed the control diet 3 hr after feeding. The findings suggest that dietary supplementation with HE and LF could enhance the nonspecific immune responses of juvenile olive flounder.

• Journal of Aquaculture
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• v.16 no.4
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• pp.210-215
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• 2003

When fed on one of the six isonitrogenous (45%) and isolipic (8%) feed containing 5 or 10% Undaria, 2% wasabi leaf, 2% wasabi stem and 0.5% herb (Obosan) for a period of 8 weeks, 95-98% juvenile flounder survived. Growth, feed efficiency and condition factor of the flounder fed on diet containing 0.5% herb were significantly (p<0.05) higher than those fed on diet supplemented with 10% Undaria. There was no significant (p>0.05) differences in moisture, crude protein, lipid and ash of the flounder receiving the different diets. The flounder fed on diet supplemented with 10% Undaria had the highest moisture but the lowest lipid in liver. Hematological parameters such as red blood cell, hematocrit and hemoglobin and serum constituents such as glucose, total cholesterol and glutamic-oxaloacetic transaminase of the flounder fed on the diets varied but no specific trend became apparent. Lysozyme activity in the serum of the flounder fed on diet supplemented with 5% Undaria and the herb, as well as nitroblue tetrazolium (NBT) reduction of macrophage in the head kidney of the flounder fed on diet containing the herb and 2% wasabi stem were significantly (p<0.05) higher than those receiving control diet. Briefly, the herb supplementation promoted growth and that of Undaria and wasabi stem enhanced non-specific immune response.

• Food Science and Preservation
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• v.19 no.3
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• pp.455-461
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• 2012

Obesity, a strong risk factor for the development of chronic diseases, is characterized by an increase in the number and size of adipocytes differentiated from precursor cells, preadipocytes. Recent research suggests that increased reactive oxygen species (ROS) production in 3T3-L1 adipocyte facilitates adipocyte differentiation and fat accumulation. This study was to investigate whether reduced ROS production by Sargassum micracanthum extract (SME) could protect the development of obesity through inhibition of adipogenesis. 3T3-L1 preadipocytes were treated SME for up to 8 days following standard induction of differentiation. The extent of differentiation reflected by amount of lipid accumulation and ROS production was determined by Oil red O staining and nitroblue tetrazolium (NBT) assay. Treatment of SME significantly inhibited ROS production and adipocyte differentiation that is depend on down regulation of NADPH oxidase 4 (NOX4), a major ROS generator, and peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$) and CCAAT/enhancer-binding protein alpha ($C/EBP{\alpha}$), a key adipogenic transcription factor. These results indicate that SME can inhibit adipogenesis through a reduced ROS level that involves down-regulation of NOX4 expression or via modulation of adipogenic transcription factor.

• Journal of fish pathology
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• v.23 no.1
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• pp.69-83
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• 2010

This study was aimed to investigate the effects of injection of garlic, Allium sativum, extract and immersion in garlic juice on the nonspecific immunity and the resistance against the artificial infection of Streptococcus iniae and Edwardsiella tarda of the olive flounder, Paralichthys olivaceus. The nonspecific immune mechanisms were assessed in terms of lysozyme activity, nitroblue-tetrazolium (NBT) assay and superoxide dismutase (SOD) activity etc. Relative percent survival (RPS) was assessed by the challenge with S. iniae BS10 or E. tarda KE-1. Almost of the garlic extract injected groups showed the enhanced level of the tested nonspecific immune factors. In the challenge test with S. iniae and E. tarda, RPS of 5% garlic extract pre-injected group was much higher than that of any other tested groups, respectively. Almost of the garlic juice immersion tested groups exhibited strengthened nonspecific immune defence factors, lysozyme activity, the number of lymphocytes and neutrophils, NBT reduction and SOD activity in kidney. In the challenge with S. iniae and E. tarda, RPS in the 0.25 g/L of garlic juice immersed group was much higher than any other tested groups, respectively. The results suggest that the garlic extract and juice would be effective to enhance the nonspecific immunity and protective ability of olive flounder against fish disease such as S. iniae and E. tarda.

• Journal of Animal Science and Technology
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• v.52 no.4
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• pp.337-346
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• 2010

We report non-specific immune responses and disease resistance against Vibrio anguillarum, Streptococcus iniae and Edwardsiella tarda by dietary supplementation of fermented garlic powder (FGP) in olive flounder for the first time. Four isonitrogenous (45% crude protein) and isocaloric (17.1 MJ/kg) diets were formulated to have 0%, 0.5%, 1% and 2% of the FGP (G-0, G-0.5, G-1 and G-2). The experimental diets were fed to juvenile olive flounder averaging 23.4 g in triplicate groups (90 fish/group) in a flow-through system. After a five-week feeding trial, healthy fish with similar sizes from each tank were selected and injected with 1 ml of three bacteria each to evaluate disease resistance of the fish. During the 5-week feeding trial, the weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio, and survival of the fish were not significantly affected by the experimental diets. However, feed intake was significantly lower (P<0.05) in the fish fed the G-2 diet compared with the control group. Hemoglobin, myeloperoxidase activity, cholesterol and HDL-cholesterol were not different between the dietary groups. However, hematocrit, nitroblue tetrazolium (NBT) activity, and lysozyme activity were increased (P<0.05) with an increment of dietary FGP. Plasma triglyceride of the fish fed the G-0.5 diet was significantly lower than that of fish fed the control diet. The cumulative mortality was lower in the fish fed diets containing FGP compared with the control group in the challenge test except for the bacteria Edwardsiella tarda. The results in this study indicate that dietary supplementation of FGP can enhance the non-specific immune responses and disease resistance of olive flounder against V. anguillarum and S. iniae.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.1
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• pp.163-170
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• 2006

Nardostachyos Rhizoma (N. Rhizoma) belonging to the family Valerianaceae has been anti-arrhythmic effect, and sedation to the central nerve and a smooth muscle. We reported that the water extract of N. Rhizoma induced apoptotic cell death and differentiation in human promyelocytic leukemia (HL-60) cells. Cytotoxicity of N. Rhizoma was detected only in HL-60 cells (IC50 is about 200 ${\mu}g/ml$). The cytotoxic activity of N. Rhizoma in HL-60 cells was increased in a dose-dependent manner. We used several measures of apoptosis to determine whether these processes were involved in N. Rhizoma-induced apoptotic cell death. The high-dose (200 ${\mu}g/ml$) treatment of N. Rhizoma to HL-60 cells showed cell shrinkage, cell membrane blobbing, apoptotic bodies, and the fragmentation of DNA, suggesting that these cells underwent apoptosis. Treatment of HL-60 cells with N. Rhizoma time-dependently induced activation of caspase-3, caspase-8, and caspase-9 and proteolytic cleavage of poly(ADP-ribose) polymerase. Also, we investigated the effect of N. Rhizoma on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells was determined through expression of CD11b and CD14 surface antigens using flow cytometry and nitroblue tetrazolium (NBT) assay, and through analysis of cell cycle using propidium iodide assay, respectively. N. Rhizoma induced the differentiation of HL-60 at the low-dose (100 ${\mu}g/ml$) treatment, as shown by increased expression of differentiation surface antigen CD11b, but not CDl4 and increased reducing activity of NBT. When HL-60 cells were treated with N. Rhizoma at concentration of $50{\mu}g/ml\;and\;100{\mu}g/ml$, NBT-reducing activities induced approximately 1.5-fold and 20.0-fold as compared with the control. In contrast, HL-60 cells treated with the N. Rhizoma-ATRA combination showed markedly elevated levels of 26.3-fold at $50{\mu}g/ml$ N. Rhizoma-0.1 ${\mu}M$ ATRA combination and 27.5-fold at 50 ${\mu}g/ml$ N. Rhizoma-0.2 ${\mu}M$ ATRA combination than when treated with N. Rhizoma alone or ATRA alone. It may be that N. Rhizoma plays important roles in synergy with ATRA during differentiation of HL-60 cells. DNA flow-cytometry indicated that N. Rhizoma markedly induced a G1 phase arrest of HL-60 cells. N. Rhizoma-treated HL-60 cells increased the cell population in G1 phase from 32.71% to 42.26%, whereas cell population in G2/M and S phases decreased from 23.61% to 10.33% and from 37.78% to 33.98%, respectively. We examined the change in the $p21^{WAF1/Cip1}\;and\;p27^{Kip1}$ proteins, which are the CKIs related with the G1 phase arrest. The expression of the CDK inhibitor $p27^{Kip1},\;but\;not\;p21^{WAF1/Cip1}$ were markedly increased by N. Rhizoma. Taken together, these results demonstrated that N. Rhizoma induces apoptotic cell death through activation of caspase-3, and potently inhibits the proliferation of HL-60 cells via the G1 phase cell cycle arrest in association with $p27^{Kip1}$ and granulocytic differentiation induction .