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In spite of their potential as biologically active compounds, the high molecular mass and viscous natures of fucoidans have hampered their applications especially as a therapeutic agent. Herein the fucoidan-degrading enzyme activities were partially purified from the cultured cells of Sphingomonas paucimobilis PF-1 mainly by ammonium sulfate precipitation. This enzyme preparation degraded fucoidans from the Korean Undaria pinnatifida sporophyll into several low-molecular weight fuco-oligosaccharides (LMFOs) with less than 3,749 Da. The FTIR spectra of intact fucoidan and mixture of LMFOs (1,389-3,749 Da) showed no significant structural difference except for about 10% reduced level of sulfate esters in LMFOs. The LMFOs have exerted strong anticoagulating activities at which the activated partial thromboplastin time (APTT) and thrombin time (TT) were significantly prolonged, although 3 approximately 20 times weaker activities were observed than those of intact fucoidan. In addition, unlike intact fucoidan, LMFOs did not affect significantly to the prothrombin time (PT). These results suggest that the partially purified fucoidan-degrading enzyme preparation is valuable for the production of fuco-oligosaccharides having anticoagulating activities, and that the molecular weight and/or sulfate content of the fucoidan from the Korean Undaria pinnatifida sporophyll could be important factors for its anticoagulating activity.

Four dammarane glycosides, namely ginsenosides Rk1 (1), Rg5 (2), 20(S)-Rg3 (3), and 20(R)-Rg3 (4), isolated from a new processed ginseng, were evaluated for their inhibitory activity against platelet aggregation induced by adenosine diphosphate (ADP), collagen, arachidonic acid (AA) and U46619 (thromboxane A2 mimetic agent). Ginsenoside Rk1 and Rg5 inhibited AA-induced platelet aggregation in a dose dependent manner. Their activity against AA-induced platelet aggregation were found to be 8-22 fold higher than that of a known antiplatelet drug acetylsalicylic acid (ASA). They also inhibited U46619-induced platelet aggregation. Ginsenoside 20(S)-Rg3 and 20(R)-Rg3 showed mild inhibitory activity against AA and U46619-induced aggregation.

Lichen species were collected from King George Island (Antarctica) and were screened for their immunomodulatory effect. Among the lichens tested, the methanol extract (CR-ME) of Caloplaca regalis showed the highest nitric oxide (NO) production in murine peritoneal macrophages. Therefore, this study further examined the ability of C. regalis to induce secretory and cellular responses in macrophages. Macrophages were treated with various concentrations of CR-ME for 18 h. The CR-ME treatment induced tumoricidal activity and increased the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide by macrophages. However, CR-ME had a little effect on the levels of reactive oxygen species, interleukin-1 and IFN-gamma in CR-ME-treated macrophages. The CR-ME-induced tumoricidal activity was partially abrogated by a NO inhibitor and the anti-TNF-alpha antibody. Thus, the tumoricidal effect of CR-ME appeared to be mainly mediated by NO and TNF-alpha production from macrophages. Treating the macrophages with a p38 mitogen-activated protein kinase (MAPK) inhibitor partially blocked the tumoricidal activation induced by CR-ME, whereas inhibitors of the other kinases did not have an inhibitory effect. These results suggest that CR-ME induces the tumoricidal activity via the p38 MAPK-dependent pathway. Furthermore, electrophoretic mobility shift assay analyses revealed that the CR-ME treatment induced the activation of the NF-kappaB transcription factor. Overall, these results indicate that the tumoricidal activity induced by CR-ME is mainly due to TNF-alpha and NO production, and the activation of macrophage by CR-ME is mediated probably via the p38 MAPK and NF-kappaB pathway. Our results may also provide some leads in the development of new immunomodulating drugs.

Introduction: In acute respiratory distress syndrome (ARDS), adequate positive end-expiratory pressure (PEEP) may decrease ventilator-induced lung injury by minimising overinflation and cyclic recruitment-derecruitment of the lung. We evaluated whether setting the PEEP using decremental PEEP titration after an alveolar recruitment manoeuvre (ARM) affects the clinical outcome in patients with ARDS., Methods: Fifty-seven patients with early ARDS were randomly assigned to a group given decremental PEEP titration following ARM or a table-based PEEP (control) group. PEEP and inspired fraction of oxygen (FiO2) in the control group were set according to the table-based combinations of FiO2 and PEEP of the ARDS network, by which we aimed to achieve a PEEP level compatible with an oxygenation target. In the decremental PEEP titration group, the oxygen saturation and static compliance were monitored as the patients performed the ARM along with the extended sigh method, which is designed to gradually apply and withdraw a high distending pressure over a prolonged period, and the decremental titration of PEEP., Results: The baseline characteristics did not differ significantly between the control and decremental PEEP titration groups. Initial oxygenation improved more in the decremental PEEP titration group than in the control group. However, dynamic compliance, tidal volume and PEEP were similar in the two groups during the first week. The duration of use of paralysing or sedative agents, mechanical ventilation, stay in the intensive care unit and mortality at 28 days did not differ significantly between the decremental PEEP titration and control groups., Conclusions: The daily decremental PEEP titration after ARM showed only initial oxygenation improvement compared with the table-based PEEP method. Respiratory mechanics and patient outcomes did not differ between the decremental PEEP titration and control groups., Trial Registration: ClinicalTrials.gov identifier: ISRCTN79027921.

A methanol extract of Sophora japonica was subjected to anti-platelet activity guided fractionation affording the isolation of four flavonoids and six flavonoid-glycosides: biochanin A (1), irisolidone (2), genistein (3), sissotrin (4), sophorabioside (5), genistin (6), tectoridin (7), apigenin (8), quercitrin (9), and rutin (10). The structure of each compound was determined by a variety of spectroscopic methods. Among the compounds, 1, 3, and 7 showed approximately 2.5-6.5 fold greater inhibitory effects on arachidonic acid (AA) and U46619 induced platelet aggregation (IC50: 19.9 and 99.8 microM; 20.3 and 53.8 microM; 25.9 and 123.4 microM, respectively) than acetylsalicylic acid (ASA, IC50: 63.0 and 350.0 microM). Compound 2 was an approximately 22-40 fold stronger inhibitor than ASA on AA and U46619 induced aggregation (IC50: 1.6 and 15.6 microM, respectively).

This study examined the effects of red ginseng acidic polysaccharide (RGAP) on macrophage-mediated cytotoxicity towards murine melanoma B16 cells. RGAP alone had no effect on killing of tumor cells. RGAP treatment increased the production of interleukin-1 (IL-1), IL-6, and nitric oxide (NO) by macrophages, whereas tumor necrosis factor (TNF-alpha) and reactive oxygen species (ROS) production were not changed by RGAP. However, treatment of macrophages with a combination of RGAP and recombinant interferon-gamma (rIFN-gamma) enhanced killing of tumor cells. In addition, the combination treatment showed marked cooperative induction of IL-1, IL-6, TNF-alpha, and NO production. Electrophoretic mobility shift assay analysis revealed that treatment of macrophages with RGAP plus rIFN-gamma induced the activation of the nuclear factor-kappa B (NF-kappaB) transcription factor. In agreement with this, the combination treatment resulted in increased NF-kappaB-p65 expression. The present results demonstrate synergistic effects on macrophage function of RGAP in combination with rIFN-gamma, and suggest that NF-kappaB plays an important role in mediating these effects. These data also support the development of clinical studies of this combination.

The magnesium (Mg) and manganese (Mn) were evaluated for its effectiveness as an immunomodulator in rats. The treatments were as follows: Group 1, AIN-93M diet (0.05% Mg, 0.001% Mn); Group 2, high-dose Mg (0.1% Mg, 0.001% Mn); and Group 3, high dose Mn (0.05% Mg, 0.01% Mn) (n-12/group). After 12 weeks of supplementation, rats were sacrificed to assess the effect on a range of innate responses (tumoricidal activity, oxidative burst and nitric oxide) and the mitogen-stimulated lymphoproliferative response. Immune function was significantly affected in both the high dose Mg and the Mn group. Lymphocyte proliferative responses and NK cell activity were measured in pooled spleen from each group. The mitogen response of lymphocytes to LPS in the spleen was significantly reduced in high dose Mg-treated groups, whereas the response to ConA was not affected in both high dose minerals-treated groups. The reactive oxygen species level of macrophages was decreased in both groups. These effects were more pronounced in high dose Mg-treated group. Nitric oxide production was also decreased in high dose minerals-treated group. In addition, tumoricidal activities of splenic NK cell and peritoneal macrophage in mineral exposed rats were significantly increased. Moreover, percent death of macrophage was reduced in two groups receiving high dose mineral supplements. Taken together, the present data suggest that high dose trace min erals exert a differential effect on the function of immune cells.

An aryltetralin lactone, (-)-(1S,2R,3R)-1-(3',4'-methylenedioxyphenyl)-3-(hydroxymethyl)-6,7-dimethoxy-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid lactone, was isolated from the methanol extract of the aerial parts of Bupleurum falcatum and named bupleurumin. The structure of the compound was determined on the basis of chemical and spectroscopic methods, including two-dimensional NMR spectrometry (e.g., 1H- and 13C-NMR, homo- and heteronuclear COSY, HMBC). Bupleurumin showed an 8-fold potent inhibitory effect (IC50: 47.5 uM) compared to that of acetylsalicylic acid (ASA, IC50 : 420 uM) on collagen induced platelet aggregation and comparable effects as ASA on arachidonic acid-induced platelet aggregation.

Pharmacological modulation of heme oxygenase (HO) gene expression may have significant therapeutic potential in oxidant-induced disorders, such as ischemia reperfusion (I/R) injury. Higenamine is known to reduce ischemic damages by unknown mechanism(s). The protective effect of higenamine on myocardial I/R-induced injury was investigated. Ligation of rat left anterior descending coronary artery for 30 min under anesthesia was done and followed by 24 h reperfusion before sacrifice. I/R-induced myocardial damages were associated with mitochondria-dependent apoptosis as evidenced by the increase of cytochrome c release and caspase-3 activity. Administration of higenamine (bolus, i.p) 1 h prior to I/R-injury significantly decreased the release of cytochrome c, caspase-3 activity, and Bax expression but up-regulated the expression of Bcl-2, HO-1, and HO enzyme activity in the left ventricles, which were inhibited by ZnPP IX, an enzyme inhibitor of HO-1. In addition, DNA-strand break-, immunohistochemical-analysis, and TUNEL staining also supported the anti-apoptotic effect of higenamine in I/R-injury. Most importantly, administration of ZnPP IX inhibited the beneficial effect of higenamine. Taken together, it is concluded that HO-1 plays a core role for the protective action of higenamine in I/R-induced myocardial injury.

It has been suggested that nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) may act as a mediator of cytokine-induced effects on bone turn-over. NO is also recognized as an important factor in bone remodeling, i.e., participating in osteoblast apoptosis in an arthritic joint. The components of Agastache rugosa are known to have many pharmacological activities. In the present study, we investigated the effects of Agastache rugosa leaf extract (ELAR) on NO production and the iNOS expression in ROS 17/2.8 cells activated by a mixture of inflammatory cytokines including TNF-alpha and IL-1beta. A preincubation with ELAR significantly and concentration-dependently reduced the expression of iNOS protein in ROS 17/2.8 cells activated with the cytokine mixture. Consequently, the NO production was also significantly reduced by ELAR with an IC50 of 0.75 mg/mL. The inhibitory mechanism of iNOS induction by ELAR prevented the activation and translocation of NF-kappaB (p65) to the nucleus from the cytosol fraction. Furthermore, ELAR concentration-dependently reduced the cellular toxicity induced by sodium nitroprusside, an NO-donor. These results suggest that ELAR may be beneficial in NO-mediated inflammatory conditions such as osteoporosis.

The effect of YS 51, a synthetic 1-(beta-naphtylmethyl)6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline alkaloid, on the expression of manganese-superoxide dismutase (Mn-SOD), an antioxidant enzyme, was examined in sheep pulmonary artery endothelial cells (SPAEC) and a human cervical carcinoma cell line (Hela). YS 51 alone or in combination with cytokines enhanced the expression of Mn-SOD mRNA in SPAEC and Hela cells. YS 51 also showed synergistic effects on the induction of Mn-SOD mRNA with phorbol-12-myristate-13-acetate (TPA) and/or tumor necrosis factor-alpha (TNF-alpha). In Hela cells, the induction of Mn-SOD mRNA by YS 51 was in a time- and dose-dependent manner and the expression of Mn-SOD mRNA was increased to a maximum of 4-fold in 9 h. Enhancement of Mn-SOD mRNA by YS 51 was completely abolished by actinomycin D but not cycloheximide, suggesting that the induction of Mn-SOD mRNA byYS 51 is independent of new protein synthesis. Pretreatment of curcumin, an inhibitor of c-jun N-terminal kinase (JNK), dose-dependently suppressed the induction of Mn-SOD mRNA by YS 51, but not by 2'-amino-3'-methoxyflavone (PD98059) and 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazol (SB203580), inhibitors of mitogen-activated protein kinase. Also, YS 51 induced the phosphorylation activity of JNK in a time-dependent manner without affecting the phosphorylation activity of the extracellular signal-regulated kinase 1 (ERK1) and p38 MAP kinase. These results implicated that the JNK pathway appears to play a crucial role in mediating the YS 51-induced Mn-SOD gene expression, and that up-regulation of Mn-SOD would contribute to the anti-inflammatory actions mediated by YS 51., (Copyright 2004 S. Karger AG, Basel)

Nine phenolic (1-9) and two furan type (10, 11) compounds, were isolated from the methanolic extract of the tuber of Gastrodia elata Blume (Orchidaceae) in the course of continuing search for platelet anti-aggregating plant components. Compound 1 was identified as 4,4'-dihydroxybenzyl sulfone, a novel compound for the best of our knowledge. Compound 10, 5-hydroxymethyl-2-furancarboxaldehyde, was isolated for the first time from this plant. Compound 1 (IC50; 83 microM) was about four times more inhibitory to U46619 induced aggregation than ASA (IC50; 340 microM). Compound 9, 4,4'-dihydroxy-dibenzylether, (IC50; 5 microM, 3 microM and 33 microM, respectively) was 10-80 fold more potent than ASA (IC50; 420 microM, 53 microM and 340 microM respectively) to collagen, epinephrine and U46619 induced aggregation, although it is less active than ASA to AA induced aggregation.

Five pentacyclic triterpenoids, oleanolic acid (1), hederagenin (2), ursolic acid (3), tormentic acid (4) and myrianthic acid (5), were isolated from the methanol extract of the leaves of Campsis grandiflora, and structures of the compounds were established by the spectroscopic methods. Compounds 2, 3, 4, and 5 were isolated for the first time from the genus Campsis. All of the compounds (IC50: 45.3, 32.8, 82.6, 42.9 and 46.2 microM respectively) were as equivalently inhibitive as acetylsalicylic acid (IC50: 57.0 microM) on epinephrine induced platelet aggregation.

Disseminated intravascular coagulation (DIC) is a pathological syndrome, which occurs following the uncontrolled widespread activation of blood coagulation, resulting in the intravascular formation of fibrin, which may lead to thrombotic occlusion of small and midsize vessels. The effects of 1-(alpha-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (YS-49, CAS 132836-42-1) and 1-(beta-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetra-hydroisoquinoline (YS-51, CAS 213179-96-5) on the experimental DIC induced by lipopolysaccharide (LPS) in rats, were investigated. The oral administration of YS-49 and YS-51 (10 or 50 mg/kg) attenuated the dramatic increase of serum fibrinogen/fibrin degradation product (FDP) level, the decrease of plasma fibrinogen concentration and the number of platelets in blood and the prolongation of prothrombin time (PT) and activated partial thromboplastin time (aPTT) induced by LPS. The liver and kidney function parameters, aspartate amino-transferase (AST) and blood urea nitrogen (BUN), were also improved with YS-49 and YS-51. The above results suggest that YS-49 and YS-51 have therapeutic potential for DIC and/or accompanying multiple organ failure.

Five coumarins, isoimperatorin (1), pabulenol (2), isooxypeucedanin (3), oxypeucedanin hydrate (4) and osthol (5) were isolated from the MeOH extract of Angelica genuflexa in the course of searching for anti-platelet and anti-coagulant components from plants. Pabulenol (2) was isolated from A. genuflexa for the first time. The five compounds isolated from A. genuflexa, together with decursinol angelate (6), decursin (7) and nodakenin (8) from A. gigas were evaluated for their effects on platelet aggregation and blood coagulation. Compounds 2, 5, 6 and 7 were observed to be either equally effective or 2-4 times more inhibitory than ASA in both arachidonic acid and U46619 (TXA2 mimetic) induced platelet aggregations.

In the course of our work on anti-platelet constituents from plants, five phenolic compounds, magnolol, honokiol, obovatol, methyl caffeate, and syringin, were isolated from the methanol extracts of the barks and fruits of Magnolia obovata. The compounds were identified based on the spectroscopic data. Methyl caffeate was isolated for the first time from the genus Magnolia and it showed 3 approximately 4-folds higher potency than ASA. The activities of obovatol and honokiol were comparable to ASA. Magnolol and syringin showed only very mild inhibitory effects to all the stimulators.

In the present study, the effects of 1-(beta-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (YS 51), a positional isomer of 1-(alpha-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (YS 49), on nitric oxide production and inducible nitric oxide synthase (iNOS) mRNA expression were investigated in RAW 264.7 cells, mouse monocyte macrophage, exposed to lipopolysaccharide (LPS) plus interferon (IFN)-gamma. In addition, the effects of YS 51 on vascular reactivity in vitro and ex vivo, iNOS protein expression (rat lung) and survival rate (mice), were also investigated in LPS-treated rodents. Treatment with YS 51 reduced not only nitric oxide production (IC(50), 23.5 microM), but also expression of iNOS mRNA in RAW 264.7 cells in a concentration-dependent manner. Incubation of rat endothelium-denuded thoracic aorta with LPS (300 ng/ml) for 8 h in vitro resulted in suppression of vasoconstrictor effects to phenylephrine, which was restored by coincubation with YS 51. Treatment with YS 51 before (30 min) injection of LPS resulted in significant reduction of the expression of iNOS protein in rat lung tissue and restored the vascular contractility to 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha) (U46619), ex vivo. The plasma concentration of nitrite/nitrate (NOx) level was significantly (p < 0.01) reduced by YS 51 (10 and 20 mg/kg, i.p) in LPS-treated (10 mg/kg, i.p) rats. Furthermore, YS 51 significantly increased the survival rate in LPS-injected mice. In RAW 264.7 cells, YS 51 inhibited the formation of nuclear factor-kappaB-DNA complex and iNOS promoter activity in a concentration-dependent manner, indicating that iNOS gene expression was modified transcriptionally by YS 51. These data strongly suggest that YS 51, a positional isomer of YS 49, might be beneficial in septic shock and/or endotoxin-induced inflammatory disorders.