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Genistein is a polyphenolic nonsteroidal isoflavonoid with estrogen-like activity has been shown to have anticancer, antioxidant, and anti-inflammatory activities. Fractalkine is a unique chemokine that functions as a chemoattractant and an adhesion molecule on endothelial cells activated by proinflammatory cytokines. In this study, we investigated the effects of genistein (5-25 muM) on fractalkine expression in human umbilical vein endothelial cells (HUVECs) and on its receptor, CX3CR1, in THP-1 cells in response to treatment with tumor necrosis factor (TNF)- alpha. TNF-alpha significantly induced fractalkine expression in endothelial cells. Genistein decreased TNF-alpha-induced fractalkine expression through suppression of Akt and p38 phosphorylation and NF-kappaB activities. Genistein also strongly suppressed TNF-alpha-induced expression of CX3CR1 in monocytes. Genistein suppressed TNF-alpha-stimulated adhesion of monocytes to HUVECs. Immunohistochemical analysis revealed that genistein suppressed the in vivo lipopolysaccharide (LPS)-induced arterial endothelial fractalkine expression in the heart, kidney, and small intestine. These results suggest that genistein may provide a new pharmacological approach for suppressing fractalkine/CX3CR1-mediated injury under vascular inflammatory conditions., (Copyright 2010 S. Karger AG, Basel.)

Exendin-4, a long-acting glucagon-like peptide-1-receptor agonist, is known to enhance beta-cell function, but the active mechanism by which it modulates beta-cell mass still remains unclear. We investigated what the long-term effects of exendin-4 (300 pmol/kg body weight per day) on beta-cell function and mass would be in 90% pancreatectomized (Px) Sprague Dawley rats; half of whom were intraperitoneally injected with streptozotocin (STZ, 20 mg/kg body weight) and half of whom were not. Exendin-4 improved glucose tolerance by elevating serum insulin levels in both STZ-treated and untreated Px rats. At hyperglycemic clamp, STZ attenuated both first and second phase insulin secretion in STZ- and saline-treated Px rats, but exendin-4 incompletely reversed the attenuation. Since STZ mostly removed the remaining beta-cells by increasing apoptosis after Px, their regeneration was initiated through neogenesis, which was determined by the number of beta-cells budding from pancreatic duct layers and small clusters. Exendin-4 enhanced beta-cell proliferation and neogenesis in STZ-treated and -untreated Px rats and reduced beta-cell apoptosis partly by attenuating the expression of endoplasmic reticulum stress-response genes such as X-box-binding protein-1, activating transcription factor (ATF)-4, ATF6, and C/EBP-homologous protein. In conclusion, exendin-4 improved glycemic control by potentiating beta-cell function and increasing beta-cell mass by increasing beta-cell neogenesis and proliferation and by decreasing apoptosis in diabetic rats.

3,3'-diindolylmethane (DIM) is the major in vivo product of the acid-catalyzed oligomerization of indole-3-carbinol present in cruciferous vegetables, and it has been shown to exhibit anticancer properties. In this study, we assessed the effects of DIM on the metastasis of 4T1 mouse mammary carcinoma cells. In vitro culture studies showed that DIM dose-dependently inhibited the migration, invasion, and adhesion of 4T1 cells at concentrations of 0-10 micromol/L without attendant changes in cell viability. In an in vivo lung metastasis model, 4T1 cells (2 x 10(5) cells/mouse) were injected into the tail veins of syngeneic female BALB/c mice. Beginning on the second day, the mice were subjected to gavage with 0-10 mg DIM/(kg body weight x d) for 13 d. Oral DIM administration resulted in a marked reduction in the number of pulmonary tumor nodules. DIM treatment significantly reduced the levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and vascular cell adhesion molecule (VCAM)-1 and increased TIMP-2 levels in the sera and lungs of mice injected with 4T1 cells. Additionally, DIM treatment reduced the serum concentrations of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)alpha. We have demonstrated that DIM profoundly inhibits the lung metastasis of 4T1 cells, which was accompanied by reduced levels of MMP, adhesion molecules, and proinflammatory cytokines. These results indicate that DIM has potential as an antimetastatic agent for the treatment of breast cancer.

This study investigated the apoptotic regulation by green tea catechin epigallcatechin-3-gallate (EGCG) on colon cancer cells in the presence of low-dose H(2)O(2) known to exert the activation of signal pathways leading to cell proliferation. In the presence of low-dose H(2)O(2), EGCG induced apoptosis and abolished the cell-proliferative effect exhibited by low-dose H(2)O(2). This reduction of growth was accompanied by an activation of AMP-activated kinase (AMPK), a decrease in cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) levels, and the induction of apoptotic markers such as p53 and poly(ADP-ribose) polymerase (PARP) cleavage. The low-dose H(2)O(2) stimulated COX-2 expression, and treating cells with synthetic AMPK activator AICAR (5-aminoimiazole-4-carboxamide-1-beta-d-ribofuranoside) resulted in greater suppression of COX-2 expression and PGE(2). By treating cells with high concentrations of the reactive oxygen species (ROS) scavenger NAC (N-acetyl-1-cysteine), the apoptotic effect of EGCG was abolished and led to suppression of AMPK and COX-2, indicating that the liberation of excessive ROS might be the upstream signal of the AMPK-COX-2 signaling pathway even in the presence of low-dose H(2)O(2).

Luteolin (3',4',5,7-tetrahydroxyflavone), a flavonoid, has been known to possess antimutagenic, antitumorigenic, antioxidant, and anti-inflammatory properties. In this study, we investigated the role of luteolin in the regulation of adipogenic differentiation in 3T3-L1 preadipocytes. Luteolin inhibited intracellular triglyceride accumulation in a dose-dependent manner without cytotoxicity. Western blot and reverse transcription-polymerase chain reaction analyses showed that this inhibition was accompanied by attenuated expression of the adipogenic transcription factors: peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein alpha. Luteolin inhibited the PPARgamma transactivation stimulated by rosiglitazone, a synthetic agonist, in COS-7 cells and inhibited rosiglitazone-induced adipogenic differentiation in 3T3-L1 cells. These data suggest that luteolin exerts antiadipogenic effects by suppressing adipogenic transcription factors and by inhibiting the transactivation of PPARgamma.

A new resveratrol dimer, (+)-vitisinol E (1) which demonstrated inhibitory activity on BACE-1 (beta-site APP-cleaving enzyme 1) in vitro, was isolated from the stembark extract of Vitis vinifera (Vitaceae) together with four known resveratrol oligomers, (+)-epsilon-viniferin (2), (+)-ampelopsin A (3), (+)-vitisin A (4) and (-)-vitisin B (5). The chemical structure of 1 was established by MR spectroscopic analyses, including HMBC. All isolated resveratrol derivatives (1-5) demonstrated significant inhibition on baculovirus-expressed BACE-1 in a dose-dependent manner, which was assessed with the FRET assay using Rh-EVNLDAEFK as a substrate in vitro.

Epigallocatechin-3-O-gallate (EGCG), the main catechin in green tea, has anti-oxidant, anti-atherosclerotic and anti-inflammatory properties. Fractalkine, a chemokine involved in inflammation and early atherosclerotic processes, acts as a chemoattractant as well as an adhesion molecule in endothelial cells activated by proinflammatory cytokines. In the present study, we investigated the effect of EGCG on fractalkine expression in TNF-alpha-induced human umbilical vein endothelial cells (HUVECs). EGCG decreased TNF-alpha-induced fractalkine mRNA and protein expression in HUVECs in a time-dependent manner. EGCG suppressed the TNF-alpha-induced phosphorylation and degradation of IkappaB-alpha, thereby decreasing the phosphorylation and nuclear translocation of the NF-kappaB p65 subunit in HUVECs. The DNA binding activity of the NF-kappaB p65 subunit was lower in EGCG-pretreated HUVECs than in those treated with TNF-alpha alone. Furthermore, EGCG inhibited monocyte adhesion to HUVECs stimulated by TNF-alpha. The silencing of fractalkine with an siRNA or treatment with a blocking antibody against fractalkine suppressed the TNF-alpha-induced increase in monocyte adhesion. These results demonstrate that EGCG prevents TNF-alpha-induced vascular endothelial fractalkine expression., (2009 S. Karger AG, Basel.)

Ginseng root is known to induce anti-diabetic activity, but the key components involved are unknown. We investigated which major ginsenosides in ginseng enhanced glucose homeostasis by in vitro studies. Rb1 and Rg1 reduced the triglyceride accumulation in 3T3-L1 adipocytes by activating PKA with increased intracellular cAMP. However, the insulin-stimulated glucose uptake was enhanced by Rb1 and Rg1 via activation of phosphatidylinositol-3 kinase. Rb1 and Rg1 promoted glucose-stimulated insulin secretion and cell viability in Min6 cells through PKA which augmented IRS2 expression to enhance insulin/IGF-1 signaling. These results suggest that Rb1 and Rg1 improved glucose homeostasis through the activation of a PKA like glucagon-like peptide-1 receptor agonist.

Lactobacillus paraplantarum KM (Lp), Weissella sp. 33 (Ws), and Enterococcus faecium 35 (Ef) were used in single (Lp, Ws, Ef) or mixed cultures (Lp+Ws, Lp+Ef, Ws+Ef) for soymilk fermentation (37 degrees C, 12 h). After 12 h, the cell numbers, pH, and TA of soymilk were 7.4x108 -6.0x109 CFU/ ml, 3.8-4.5, and 0.59-0.70%, respectively. Changes in the contents of glycitin and genistin in soymilk fermented with Ef were not significant (p<0.05). The contents of isoflavone glucosides in soymilk fermented with the other cultures decreased significantly with an increase of aglycone contents (p<0.05). It corresponded well with a sharp increase in beta- glucosidase activity during fermentation. About 92-100% of the daidzin and 98-100% of the genistin in soymilk were converted to corresponding aglycones by Lp, Ws, or Lp+Ef within 12 h.

Folate deficiency increases stroke risk. We examined whether folate deficiency affects platelet endothelial cell adhesion molecule-1 (PECAM-1), which is an immunoglobulin-associated cell adhesion molecule and mediates the final common pathway of neutrophil transendothelial migration, in blood vessels in the gerbil dentate gyrus after transient forebrain ischemia. Gerbils were exposed to a folic acid-deficient diet (FAD) for 3 months and then subjected to common carotid artery occlusion for 5 min. In the control diet (CD)- and FAD-treated sham-operated groups, weak PECAM-1 immunoreactivity was detected in the blood vessels located in the dentate gyrus. PECAM-1 immunoreactivity in both groups was increased by 4 days after ischemic insult. PECAM-1 immunoreactivity in the FAD-treated group was twice as high that in the CD-treated-sham-operated group 4 days after ischemic insult. Western blot analyses showed that the change patterns in PECAM-1 protein levels in the dentate gyrus in both groups after ischemic insult were similar to changes in PECAM-1 immunohistochemistry in the ischemic dentate gyrus. Our results suggest that folate deficiency enhances PECAM-1 in the dentate gyrus induced by transient ischemia.

Recent studies have revealed that beta-cell dysfunction is an important factor in developing type 2 diabetes. beta-cell dysfunction is related to impairment of the insulin/IGF-1 signaling cascade through insulin receptor substrate-2 (IRS2). The induction of IRS2 in beta-cells plays an important role in potentiating beta-cell function and mass. In this study, we investigated whether herbs used for treating diabetes in Chinese medicine-Galla rhois, Rehmanniae radix, Machilus bark, Ginseng radix, Polygonatum radix, and Scutellariae radix-improved IRS2 induction in rat islets, glucose-stimulated insulin secretion and beta-cell survival. R. radix, Ginseng radix and S. radix significantly enhanced glucose-stimulated insulin secretion compared to the control, i.e., by 49, 67 and 58%, respectively. These herbs induced the expression of IRS2, pancreas duodenum homeobox-1 (PDX-1), and glucokinase. The increased level of glucokinase could explain the enhancement of glucose-stimulated insulin secretion with these extracts. Increased PDX-1 expression was associated with beta-cell proliferation, which was consistent with the cell viability assay. In conclusion, R. radix, Ginseng radix and S. radix had an insulinotropic action similar to that of exendin-4.

This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice.

Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H(2)O(2) in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H(2)O(2). Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H(2)O(2)-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders.

The effect of Schisandra fructus extract (SFE) on doxorubicin (Dox)-induced cardiotoxicity was investigated in H9c2 cardiomyocytes. Dox, which is an antineoplastic drug known to induce cardiomyopathy possibly through production of reactive oxygen species, induced significant cytotoxicity, intracellular reactive oxygen species (ROS), and lipid peroxidation. SFE treatment significantly increased cell survival up to 25%, inhibited intracellular ROS production in a time- and dose-dependent manner, and inhibited lipid peroxidation induced by Dox. In addition, SFE treatment induced expression of cellular glutathione S-transferases (GSTs), which function in the detoxification of xenobiotics, and endogenous toxicants including lipid peoxides. Analyses of 31,100 genes using Affymetrix cDNA microarrays showed that SFE treatment up-regulated expression of genes involved in glutathione metabolism and detoxification [GST theta 1, mu 1, and alpha type 2, heme oxygenase 1 (HO-1), and microsomal epoxide hydrolase (mEH)] and energy metabolism [carnitine palmitoyltransferase-1 (CPT-1), transaldolase, and transketolase]. These data indicated that SFE might increase the resistance to cardiac cell injury by Dox, at least partly, together with altering gene expression, especially induction of phase II detoxification enzymes.

Bioactivity-guided fractionation of Saururus chinensis (Saururaceae) using a lymphoproliferation assay led us to isolate 5 lignans (compounds 1 - 5). Compounds 1 - 5 were identified as sauchinone, (-)-saucerneol, saucerneol C, manassantin A, and manassantin B, respectively, by spectroscopic analyses. The immunosuppressive activities of the active compounds were evaluated using lymphoproliferation, mixed leukocyte response, and Th1/Th2 cytokine assays. The relative potency was in the order: manassantin A, B > (-)-saucerneol > saucerneol C > sauchinone.

Bacillus subtilis CH3-5 was isolated from cheonggukjang prepared according to traditional methods. CH3-5 secreted at least four different fibrinolytic proteases (63, 47, 29, and 20 kDa) into the culture medium. A fibrinolytic enzyme gene, aprE2, encoding a 29 kDa enzyme was cloned from the genomic DNA of CH3-5, and the DNA sequence determined. aprE2 was overexpressed in heterologous B. subtilis strains deficient in extracellular proteases using a E. coli-Bacillus shuttle vector. A 29 kDa AprE2 band was observed and AprE2 seemed to exhibit higher activities towards fibrin rather than casein.

Over 500 microorganisms isolated from Korean traditional foods, Maeju (source of soybean paste) and Nuruk (Korean koji), were screened to obtain an isomaltulose-producing microorganism. It was identified as Enterobacter sp. FMB-1 by 16S rRNA sequencing and the API 20E system. It had a greater than 90% conversion of sucrose (as 4 g/l) to isomaltulose in 2 days. Small amounts of trehalulose, glucose, and fructose were produced as byproducts, implying that this strain could be possibly employed in the production of isomaltulose in industry.

Plantainoside D (PD), was isolated from the leaves of Picrorhiza scrophulariiflora (Scrophulariaceae). The anti-oxidative activity of PD was evaluated based on scavenging effects on hydroxyl radicals and superoxide anion radicals. Adriamycin (ADR) is a potent anti-tumor drug known to cause severe cardiotoxicity. Although ADR generates free radicals, the role of free radicals in the development of cardiac toxicity has not been understood. This study was undertaken to investigate the protective effect of PD against ADR-induced apoptosis. In vitro, ADR caused dose-dependent toxicity in H9c2 cardiac muscle cells. Pre-treatment of the cardiac muscle cells with PD significantly reduced ADR-induced apoptosis of cardiac muscle cells. PD inhibited the ROS produced by ADR in the cardiac muscle cells. As well, PD increased GSH(glutathione), compared with ADR. In response to ADR, NF-kappaB was activated in H9c2 cells. However the treatment of PD reduced the activation of NF-kappaB. We also observed that the NF-kappaB inhibitor, PDTC, inhibited the cytotoxic effect on ADR-induced apoptosis in cardiac muscle cells. In parallel, IkappaBalpha-dominant negative plasmid-overexpression abrogated ADR-induced apoptosis in H9c2 cardiac muscle cells. In conclusion, these results suggest that Plantaionoside D can inhibit ADR-induced apoptosis in H9C2 cardiac muscle cells via inhibition of ROS generation and NF-kappaB activation. The pure compound PD can be a potential candidate agent which protects cardiotoxicity in ADR-exposed patients.

In this study, we examined the protective effects of Caesalpinia sappan L. and its major component, brazilin, against tert-butylhydroperoxide (t-BHP)-induced cell death in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. We found that the extract of C. sappan L. and brazilin induced antioxidant response element (ARE)-luciferase activity and heme oxygenase-1 (HO-1) expression in a concentration-dependent manner. The inductive effect of brazilin was more potent than the extract of C. sappan L. and the expression of HO-1 reached a peak at 12 h after brazilin treatment. The extract and brazilin protected the cells against t-BHP-induced cell death. Their protective effects were abrogated by zinc protoporphyrin IX (ZnPP IX), a HO inhibitor. These results demonstrate that the extract of C. sappan L. and brazilin induce the expression of HO-1 and the enzyme diminishes t-BHP-induced cell death in HEI-OC1 cells.

3,3'-Diindolylmethane (DIM) is the major in vivo product of acid-catalyzed oligomerization of indole-3-carbinol, which is a promising anticancer agent present in cruciferous vegetables and has itself been reported to have anticarcinogenic properties. This study examined DIM-mediated regulation of apoptosis in the HCT116 (wild-type p53) and HT-29 (mutant p53) human colon cancer cell lines. DIM (0-30 micromol/L) substantially decreased the number of viable cells and induced apoptosis of HCT116 and HT-29 cells in a concentration-dependent manner. Western-blot analyses of total cell lysates revealed that DIM increased the activation of caspase-3, -7, -8, and -9 and enhanced poly(ADP-ribose) polymerase cleavage in both HCT116 and HT-29 cells. In addition, DIM increased the translocation of cytochrome c and Smac/Diablo from the mitochondria to the cytoplasm. In concert with the caspase-8 activation by DIM, increased levels of Fas and truncated Bid were observed. DIM did not affect the protein levels of p53, Bcl-2, Bax, or Fas ligand (FasL) in HCT116 cells. In HT-29 cells, however, DIM decreased Bcl-2 levels, although the protein levels of Bax or FasL were not affected. The caspase-8 inhibitor Z-IETD-FMK attenuated the DIM-induced apoptosis, indicating that increased activation of this enzyme contributed to the increase in p53-independent apoptosis that was observed in colon cancer cells. We have demonstrated that DIM induces apoptosis in colon cancer cells, providing insights into the mechanisms underlying its antitumorigenic activities.

Ulmus davidiana Planch (Ulmaceae) extract (UD) has long been known to have anti-inflammatory and anticancer activities. UD has been also known to have protective effects on damaged tissue, inflammation and bone among other functions. Effects of UD on inflammatory and immune responses and its mechanisms in collagen-induced inflammation (CII) rat were studied. Hind paw volumes of rats were measured by volume meter; lymphocyte proliferation, interleukin (IL)-1, IL-2, tumor necrosis factor (TNF)-α level was determined by 3-(4,5-2dimethylthiazal-2yl)2,5-diphenyltetrazoliumbromide assay. Antibodies to collagen type II (BC-II) were determined by enzyme-linked immunosorbent assay. There was a marked secondary inflammatory response in CII model, which accompanied with the decrease of body weight and the weight of immune organs simultaneously. The administration of UD (20, 80, 150mg/kg, intragastrically×10 days) inhibited the inflammatory response and restored body weight and the weight of immune organs of CII rats. Lymphocyte proliferation and IL-2 production of CII rats increases, together with IL-1 and TNF-α in peritoneal macrophages and synoviocytes. The administration of UD (20, 80, 150mg/kg, 10 days) reduced above changes significantly. UD had no effect on the concentration of antibodies to BC-II. From the results, it was concluded that UD possesses anti-inflammatory and immunoregulatory activities and has a therapeutic effect on CII rats., (Copyright © 2006 Elsevier B.V. All rights reserved.)

The effect of bee venom acupuncture (BVA) (api-toxin) on the development of type II collagen (CII)-induced arthritis (CIA) in rats has been studied. We have compared the levels of activity of a comprehensive range of cytoplasmic, lysosomal and matrix protease types, together with the levels of free radical-induced protein damage (determined as protein carbonyl derivative) in synovial fluid from CIA-treated, BVA-treated and normal rats. Many protease types showed significantly increased activity in CIA compared with normal rats. BVA (5 and 10 microl/100g) significantly reduced these enzyme activities by some 80% each, but levels of plasma proteases activity (including those enzyme types putatively involved in the immune response, such as dipeptidyl aminopeptidase IV and proline endopeptidase) in CIA, BVA (5 microl/100g)-treated and normal plasma samples were not significantly different. The level of free radical induced damage to synovial fluid proteins was approximately three-fold higher in CIA compared with normal rats. However, BVA (5 microl/100g) significantly decreased the level of reactive oxygen free radical species (ROS) induced oxidative damage to synovial fluid proteins. It was concluded that activation of proteolytic enzymes and free radicals are likely to be of equal potential importance as protein damaging agents in the pathogenesis of rheumatoid arthritis (RA), and the development of novel therapeutic strategies for the latter disorder should include both protease inhibitory and free radical scavenging elements. In addition, the protease inhibitory element should be designed to inhibit the action of a broad range of enzymatic mechanistic types (cysteine, serine, metallo proteinases and peptidases). In conclusion, BVA is considered to be an effective RA modulator, inhibiting protease activities and removing ROS.

Ondamtanggamibang (ODG) has been used as a remedy to treat psychological anxiety and depression in Oriental medicine. In this study, we found the protective effects of ODG against oxidative stress by induction of the antioxidative enzyme, heme oxygenase (HO)-1 in neuronal PC12 cells. Pretreatment with ODG extract protected neuronal cells from damage induced by H(2)O(2) and 6-hydroxydopamine (6-OHDA), but simultaneous treatment with ODG extract did not. ODG also inhibited the intracellular reactive oxygen species elevation by H(2)O(2) and 6-OHDA. ODG stimulation strongly induced the expression of HO-1 in PC12 cells. The protective effect of ODG extract on oxidative stress-induced damage was suppressed by HO inhibitor, zinc protoporphyrin IX (ZnPP-IX). Taken together, these data suggest that ODG treatment has potential protective effects in neuronal cells under oxidative stress.

The vascular relaxant effect of the rootbark extract of Paeonia moutan was evaluated in isolated rat thoracic aorta. The methanolic extract of the rootbark showed a vasorelaxant activity in rat aortic preparations precontracted with 0.3 microM phenylephrine (IC50 value: 16.8 microg/mL). The activity-guided fractionation of the extract led to the isolation of five active principles such as paeoniflorin (1), paeonidanin (2), methylpaeoniflorin (4), tetragalloylglucose (5) and pentagalloylglucose (6), and these active ingredients potently relaxed phenylephrine-induced contraction of rat aortic preparations in a concentration-dependent manner (IC50 values: 19.4, 7.9, 10.1, 5.1 and 3.6 microM, respectively). These results suggest that pinane glycosides and galloylglucoses might be the components responsible for the vasorelaxant properties of the rootbark extract of P. moutan, and their vasorelaxant effects may be mediated through increases in the release of nitric oxide from endothelial cells.

Aim: To observe neuroprotective effects of raw and roasted licorice against hypoxia and ischemic damage., Methods: When elucidating the protective effects of raw and roasted licorice, we analyzed the lactate dehydrogenase (LDH) release using PC12 cells after hypoxia in an in vitro study and after transient forebrain ischemia in an in vivo study on Mongolian gerbils., Results: Raw and roasted licorice significantly reduced LDH release from PC12 cells exposed to an hypoxic chamber for 1 h. In the roasted licorice-treated group, the decrease of LDH release was more pronounced compared to that of the raw licorice-treated group. In roasted licorice-treated animals, approximately 66%-71% of CA1 pyramidal cells in the ischemic hippocampus were stained with cresyl violet compared to the control group. However, in the raw licorice-treated animals, no significant neuroprotection against ischemic damage was shown. In addition, ischemic animals in roasted licorice-treated group maintained the Cu, Zn-superoxide dismutase (SOD1) activity and protein levels compared to the control group, while in raw licorice-treated group SOD1 activity and protein levels were reduced significantly. High pressure liquid chromatography analysis showed that non-polar compounds containing glycyrrhizin-degraded products, such as glycyrrhetinic acid (GA) and glycyrrhetinic acid monoglucuronide (GM), were increased in roasted licorice., Conclusion: Roasted licorice had neuroprotective effects against ischemic damage by maintaining the SOD1 levels. In addition, the difference in protective ability between raw and roasted licorice may be associated with non-polar compounds, such as GA and GM.

Adriamycin is a potent antitumor drug that is known to cause severe cardiotoxicity. This study examined the protective effect of calceolarioside on adriamycin-induced cardiomyocyte toxicity. Calceolarioside significantly inhibited the adriamycin induced cell death and caspase-3 activation, which may be explained by the increase in Bcl-2 expression and the inhibition of Bax expression. Calceolarioside increased the expression of the antioxidant molecules and decreased the level of intracellular reactive oxygen species. Catalase, glutathione, N-acetylcysteine, Mannitol and Mn-TBAP (manganese (III) tetrakis-(4-benzoic acid) porphyrin) significantly inhibited the H9c2 cell death induced by adriamycin. Calceolarioside significantly inhibited H9c2 cell death, and was more effective than that observed with the other antioxidants, including probucol, ascorbic acid, and alpha-tocopherol. Overall, these results suggest that calceolarioside can inhibit adriamycin-induced apoptosis in H9c2 cardiomyocyte by inhibiting the generation of reactive oxygen species. Calceolarioside may be a potential candidate agent that inhibits cardiomyocyte-toxicity in adriamycin-exposed patients.

Ondamtanggagambang (ODG) has been used as a prescription for psychological anxiety and depression in Korean medicine. In this study, we found that ODG have protective effects against oxidative stress in cardiomyocytes. Pretreatment with ODG extract prevented H2O2 and ZnCl2-induced cell damage in H9c2 cardiomyocytes, whereas simultaneous treatment of ODG extract did not. The protective effect of ODG extract on oxidative stress-induced damage was suppressed significantly by heme oxygenase (HO) inhibitors, zinc protoporphyrin-IX (ZnPP-IX, p < 0.01) and tin protoporphyrin-IX (SnPP-IX, p < 0.01) in H9c2 cells. ODG stimulation of cells strongly induced the expression of HO-1 protein. Taken together, it is suggested that ODG-induced expression of HO-1 may have a beneficial role in cardiomyocytes under oxidative stress., (Copyright 2006 John Wiley & Sons, Ltd.)

We investigated anti-diabetic candidates and their mechanisms from the fractions of Chungkookjang (CKJ), a traditional fermented unsalted soybean, by investigating insulin signaling, peroxisome proliferator-activated receptor (PPAR)-gamma activity and glucose-stimulated insulin secretion, in vitro. Cooked soybeans (CSB) and CKJ, fermented predominantly with Bacillus subtilis, were extracted by 70% EtOH followed by an XAD-4 column chromatography with a serial mixture of solvents comprised of MeOH and water. During fermentation, the contents of isoflavonoid aglycones were elevated, and the fractions enriched with aglycones enhanced insulin-stimulated glucose uptake in 3T3-L1 adipocytes. This increase in glucose uptake resulted from stimulating a translocation of the glucose transporter (GLUT)-4 into the plasma membrane through the phosphorylation of insulin receptor substrate (IRS)-1 and Akt. Especially, daidzein enriched fractions elevated insulin-stimulated glucose uptake by acting as PPAR-gamma agonist up to levels exhibited when 10 nM insulin is administered. Fractions containing small peptides with low polarity in CKJ slightly increased glucose-stimulated insulin secretion. The data suggest that an increase in isoflavonoid aglycones in CKJ, in comparison to CSB, enhances glucose utilization via activating insulin signaling and stimulates PPAR-gamma activity in adipocytes. In addition, CKJ contains small peptides improving glucose-stimulated insulin secretion in insulinoma cells. Overall, CKJ is superior to CSB in anti-diabetic action.

This study investigates a proposed design of a peptide sequence that is based on a bioactive conformation of statins that act as the competitive inhibitors of HMG-CoA for HMGR. To bridge these heterogeneous organic compounds, a conformational aspect relating to an analysis of the flexibility of the peptide molecules and their occupied volumes was applied to the peptide design. The design criterion was formulated in terms of a proximity parameter (Pr), reflecting the probability of an active peptide conformation to approximate the statin. Through a structure-functional analysis of previously synthesized peptides and statin molecules, nine peptides were selected for the peptide library. Comparing the calculated proximity parameters, four peptides (IAVE, YAVE, IVAE, and YVAE) from the library were selected and synthesized. In vitro assays elucidated the inhibition properties for HMGR that are exhibited by these peptides. Among all peptides, YVAE showed the highest ability to inhibit HMGR. A kinetic analysis revealed that this peptide is a competitive inhibitor of HMG-CoA with an equilibrium constant of inhibitor binding (K(i)) of 15.2 +/- 1.4 microM. The calculated coefficient correlation (R) between log (IC(50)) and the inverse value of proximity parameter (1/Pr) was found to be 0.99, indicating a high degree of correlation and efficacy of the given approach in the peptide sequence design., ((c) 2006 Wiley Periodicals, Inc.)

Fisetin, a natural flavonol present in edible vegetables, fruits, and wine, was reported to exert anticarcinogenic effects. The objective of the current study was to examine the effect of fisetin on the cell cycle progression of the human colon cancer cell line HT-29. HT-29 cells were cultured in serum-free medium with 0, 20, 40, or 60 micromol/L fisetin. Fisetin dose dependently inhibited both cell growth and DNA synthesis (P < 0.05), with a 79 +/- 1% decrease in cell number observed 72 h after the addition of 60 micromol/L fisetin. Perturbed cell cycle progression from the G(1) to S phase was observed at 8 h with 60 micromol/L fisetin treatment, whereas a G(2)/M phase arrest was observed after 24 h (P < 0.05). The phosphorylation state of the retinoblastoma proteins shifted from hyperphosphorylated to hypophosphorylated in cells treated with 40 micromol/L fisetin. (P < 0.05). Fisetin decreased the activities of cyclin-dependent kinases (CDK)2 and CDK4; these effects were likely attributable to decreases in the levels of cyclin E and D1 and an increase in p21(CIP1/WAF1) levels (P < 0.05). However, fisetin also inhibited CDk4 activity in a cell-free system (P < 0.05), indicating that it may directly inhibit CDk4 activity. The protein levels of cell division cycles (CDC)2 and CDC25C and the activity of CDC2 were also decreased in fisetin-treated cells (P < 0.05). These results indicate that inhibition of cell cycle progression in HT-29 cells after treatment with fisetin can be explained, at least in part, by modification of CDK activities.

Aim: To examine whether trans-10,cis-12 CLA (t10c12) or cis-9,trans-11 CLA (c9t11) inhibits heregulin (HRG)-beta-stimulated cell growth and HRG-beta-ErbB3 signaling in HT-29 cells., Methods: We cultured HT-29 cells in the absence or presence of the CLA isomers and/or the ErbB3 ligand HRG-beta. MTT assay, [3H]thymidine incorporation, Annexin V staining, RT-PCR, Western blotting, immunoprecipitation, and in vitro kinase assay were performed., Results: HRG-beta increased cell growth, but did not prevent t10c12-induced growth inhibition. T10c12 inhibited DNA synthesis and induced apoptosis of HT-29 cells, whereas c9t11 had no effect. T10c12 decreased the levels of ErbB1, ErbB2, and ErbB3 proteins and transcripts in a dose-dependent manner, whereas c9t11 had no effect. Immunoprecipitation/Western blot studies revealed that t10c12 inhibited HRG-beta-stimulated phosphorylation of ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) to ErbB3, ErbB3-associated PI3K activities, and phosphorylation of Akt. However, c9t11 had no effect on phospho Akt levels. Neither t10c12 nor c9t11 had any effect on HRG-beta-induced phosphorylation of ERK-1/2., Conclusion: These results indicate that the inhibition of HT-29 cell growth by t10c12 may be induced via its modulation of ErbB3 signaling leading to inhibition of Akt activation.

Thermostable Pseudomonas fluorescens SIK W1 lipase (PFL), which is responsible for the spoilage of milk, was overexpressed as inclusion bodies in Escherichia coli. Renaturation of solubilized PFL was achieved by using size-exclusion protein refolding chromatography. The renatured enzyme was purified homogeneously using a combination of gel filtration and ion-exchange FPLC. Its specific activity was found to be enhanced in the presence of Ca2+. Secondary structural changes induced by Ca2+ were monitored by circular dichroism, which demonstrated that the activity increase of PFL in the presence of Ca2+ is strongly correlated with significant increases in alpha-helix and beta-sheet content. In the presence of Ca2+, the PFL structure was found resistant to denaturation by guanidine hydrochloride and to enzyme activity loss due to cosolvents like DMSO and trifluoroethanol, suggesting that Ca2+ plays an important role in inducing conformational changes and consequently in maintaining enzyme structural stability.

Garcinia extract was used as a potential anti-obesity agent. In this study, we found that Garcinia extract inhibits the cytoplasmic lipid accumulation as well as adipogenic differentiation of preadipocytes. The mechanisms that regulate the inhibition of insulin-induced differentiation by Garcinia extracts include the inhibition of expression of the early adipogenic transcription factor, CCAAT element binding protein (C/EBP)alpha that regulate adipogenesis. These results suggest that the specific targets of Garcinia extract on differentiation process of 3T3-L1 cells could be, at least, early adipogenic differentiation factor.

The binding properties of S- and R-ofloxacin to poly[d(A-T)(2)], poly[d(G-C)(2)] and poly[d(I-C)(2)] were studied by circular dichroism (CD) and various fluorescence techniques. The spectral properties of R-ofloxacin did not change when it was mixed with poly[d(A-T)(2)] and poly[d(I-C)(2)], indicating that R-enantiomer does not interact with these polynucleotides. On the other hand, when S-ofloxacin was mixed with any polynucleotide, or R-enantiomer with poly[d(G-C)(2)], characteristic changes in CD and fluorescence were observed. Therefore, it is clear that enantiomers of ofloxacin selectively recognize B-form DNA. The overall spectral properties of the ofloxacin-polynucleotide complex are similar to those of the norfloxacin-polynucleotide complex [Eur. J. Biochem. 267 (2000) 6018], suggesting that this quinolone also binds in the minor groove of DNA and therefore it may be partially inserted between DNA bases or interact with purine bases.