A new lignan from Balanophora abbreviata and inhibition of lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression

Six lignans including a new lignan (1), beta-sitosterol glucopyranoside and phenylpropanoids were isolated from the whole plants of Balanophora abbreviata Bl. (Balanophoraceae). Their structures were determined by NMR, MS analysis and other spectroscopic methods. Lignans (1, 2 and 4) showed potent inhibitory activities on the lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells.     

Structure-activity relationships of 2-aminothiazole derivatives as inducible nitric oxide synthase inhibitor

Nitric oxide synthase (NOS) has been divided into two major sub-enzymes, i.e. inducible NOS (iNOS) and constitutive NOS (cNOS). Although nitric oxide (NO) plays an important role as host defense mediator, excessive production of NO by iNOS has been involved in the pathology of many inflammatory diseases. Recently, we reported that the 2-imino-1,3-oxazolidine (1a) weakly inhibits iNOS and that introduction of an alkyl moiety on the oxazolidine ring of 1a enhances the inhibitory activity and selectivity for iNOS. In our search for better iNOS inhibitors, we focused our efforts on the 2-aminothiazole scaffold 3 as it possesses a ring similar to that of 1a. In this study, we evaluated the inhibitory activity of a series of 2-aminothiazole derivatives against both iNOS and neuronal NOS (nNOS). Our results show that introduction of appropriately-sized substituents at the 4- and 5-position of the 2-aminothiazole ring improves the inhibitory activity and selectivity for iNOS. We also found that the selectivity of 5a [5-(1-methyl)ethyl-4-methylthiazol-2-ylamine] and 5b [5-(1,1-dimethyl)ethyl-4-methylthiazol-2-ylamine] for iNOS was similar to that of oxazolidine derivative 1b (4-methyl-5-propyl-2-imino-1,3-oxazolidine) and much higher than that of L-NAME. However, we could not enhance the inhibitory activity against iNOS by introducing an alkyl substituent into the 2-aminothiazole ring as we could in the case of oxazolidine one. On the other hand, introduction of bulky or hydrophilic substituent at any position of the 2-aminothiazole ring remarkably decreased or even abolished the inhibitory activity against NOS.     

Anti-Phototoxicity Effect of Phenolic Compounds from Acetone Extract of Entada phaseoloides Leaves via Activation of COX-2 and iNOS in Human Epidermal Keratinocytes

The extract from Entada phaseoloides was employed as active ingredients of natural origin into cosmetic products, while the components analysis was barely reported. Using LC-DAD-MS/qTOF analysis, eleven compounds (1–11) were proposed or identified from acetone extract of E. phaseoloides leaves (AE). Among them, six phenolic compounds, protocatechuic acid (2), 4-hydroxybenzoic acid (3), luteolin-7-O-β-d-glucoside (5), cirsimaritin (6), dihydrokaempferol (9), and apigenin (10), were isolated by various chromatographic techniques. Protocatechuic acid (2), epicatechin (4), and kaempferol (11) at a concentration 100 μM increased the HaCaT cells viability of the UVB-irradiated cell without any cytotoxicity effect and reduced the expression of COX-2 and iNOS inflammation gene. Moreover, compounds 2 and 4 could have potent effects on cell migration during wound closure. These results suggest that compounds 2, 4, and 11 from AE have anti-photoaging properties and could be employed in pharmaceutical and cosmeceutical products.     

Absolute structures of some naturally occurring isopropenyldihydrobenzofurans,remirol, remiridiol,angenomalin, and isoangenomalin

The absolute structures of some naturally occurring chiral 2-isopropenyl-2,3-dihydrobenzofurans, (+)-remirol (1a), (+)-remiridiol (1b), (+)-angenomalin (2), and (+)-isoangenomalin (3), were studied by respective chiral synthesis. Kinetic resolutions of racemic 2-isopropenyl-2,3-dihydrobenzofurans, 2-isopropenyl-4,6-dimethoxy-2,3-dihydrobenzofuran (4), 4-hydroxy-2-isopropenyl-2,3-dihydrobenzofuran-5-carbaldehyde (8), and 2-isopropenyl-6-(MOM)oxy-2,3-dihydrobenzofuran-5-carbaldehyde (11c), by Sharpless dihydroxylation using (DHQ)(2)AQN or (DHQD)(2)AQN gave the corresponding chiral 2-isopropenyl-2,3-dihydrobenzofurans. Chiral (S)-(+)-4 (99% ee, using (DHQD)(2)AQN) was converted to natural remirol (S)-(+)-1a and then to natural remiridiol (S)-(+)-1b. (S)-(+)-8 (97% ee, using (DHQD)(2)AQN) was converted to natural angenomalin (S)-(+)-2. (R)-(-)-11c (>99% ee, using (DHQ)(2)AQN), was converted to natural isoangenomalin (R)-(+)-3. Thus, the absolute structures of natural remirol (+)-1a and remiridiol (+)-1b and angenomalin (+)-2 were determined to be S, and the structure of natural isoangenomalin (+)-3 was R.     

Induction of hepatic inducible nitric oxide synthase by cholesterol in vivo and in vitro

Cholesterol-rich diet impairs endothelial NO synthase (eNOS) and enhances inducible NOS (iNOS) expression. In this study, we investigated effects of cholesterol on iNOS expression in high-fat-fed rat models, HepG2 and RAW264.7 cells. The high-fat diet increased the plasma total cholesterol level 6-7 fold and low-density lipoprotein cholesterol level (LDL-C) approximately 70 fold and slightly increased the level of lipid peroxidation as determined by thiobarbituric acid-reactive substance assay. The high-fat diet also increased plasma nitric oxide (NO) concentrations up to 5 fold, and induced iNOS mRNA expression in liver. The contractile responses of the endothelium-denuded thoracic aortic rings to phenylephrine were significantly damaged in high-fat-fed rats when assessed by organ chamber study. Treatment with estrogen for 4 days failed to reduce iNOS expressions as well as aortic contractility, although it improved lipid profiles. In cultured HepG2 or murine macrophage RAW264.7 cells, 3 days treatment with either 25-hydroxycholesterol or 7-ketocholesterol induced iNOS mRNA expression, as determined by RT-PCR. Our data suggested that the chronic exposure of hepatocytes and macrophage cells to high concentration of cholesterol or oxysterols may induce iNOS expression and subsequent synthesis of NO, which may be important in the pathogenesis of atherosclerosis.     

Chlorinated briarane-type diterpenoids from the gorgonian coral Ellisella robusta (Ellisellidae)

Four chlorinated metabolites featuring briarane carbon skeletons have been isolated from the gorgonian coral Ellisella robusta, which was collected off the coast of southern Taiwan: two new natural products, robustolides D (1) and E (2), and two known metabolites, robustolides F (3) and G (4). The structures of metabolites 1–4 were determined by spectroscopic methods, using 1D and 2D NMR in particular. The structures and absolute stereochemistry of robustolides D (1), F (3), and G (4) were directly established by X-ray diffraction analysis. Robustolide D (1) is the first metabolite of briarane-related natural products found to possess two halogen atoms.     

Stereochemical determination of tetrahydropyran-substituted xanthones from fungus Chaetomium murorum

Chaetoxanthone D (1), a new tetrahydropyran-substituted xanthone originated from polyketide pathway, together with the four known natural products chaetoxanthone C (2), alternariol methyl ether (3), alternariol (4) and 2,5-dimethyl-7-hydroxychromone (5) was isolated from a strain of Chaetomium murorum. The structures of these compounds were elucidated based on extensive spectroscopic analyses. The absolute configurations of 1 and 2 were determined by using quantum chemical electronic circular dichroism (ECD) calculations.     

Klysimplexins I-T, eunicellin-based diterpenoids from the cultured soft coral Klyxum simplex

New eunicellin-base diterpenoids, klysimplexins I-T (1-12), were isolated from a cultured soft coral Klyxum simplex. Their structures were elucidated by spectroscopic methods, particularly in 1D and 2D NMR experiments. The absolute stereochemistry of 4 was determined by Mosher's method. Compounds 9 and 12 have been shown to exhibit cytotoxicity toward a limited panel of cancer cell lines. Compounds 2-6, 10 and 11 were found to display significant in vitro anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells by inhibiting the expression of the iNOS protein. Compounds 10 and 11 also could effectively reduce the level of COX-2 protein.     

New abietane-type diterpenes from the heartwood of Picea morrisonicola

New abietane-type diterpenes, 15-acetoxy-7-oxodehydroabietic acid (1), picealactones A (2), B (3), and C (4), together with the known 7-oxodehydroabietic acid (5) were isolated and identified from the heartwood of Picea morrisonicola. The structures of 1-4 were determined on the basis of spectral data explanation. Compounds 2-4 possessed a rare 5-dehydro-18, 6-olide functionality. Compounds 1 and 2 were first isolated from natural source.     

In vitro anti-inflammatory activities of naucleoffieine H as a natural alkaloid from Nauclea officinalis Pierrc ex Pitard,through inhibition of the iNOS pathway in LPS-activated RAW 264.7 macrophages

Abstract

Naucleoffieine H, a natural indole alkaloid, was isolated and identified from Nauclea officinalis Pierrc ex Pitard which is a traditional Chinese medicine used for the treatment of various diseases, such as cold, fever, bronchitis, pneumonia, acute tonsillitis, etc. In the present study, the effect of naucleoffieine H on the anti-inflammatory activities was investigated in LPS-induced RAW 264.7 macrophages. The results showed that naucleoffieine H significantly inhibited the release of nitric oxide (the level of nitrite as a stable biomarker of NO production) and tumor necrosis factor-α (TNF-α). Interesting, naucleoffieine H down-regulated the overexpression of inflammatory protein induced nitric oxide synthase (iNOS), but no effect on the expression cyclooxygenase-2 (COX-2) protein. In addition, this bioactive alkaloid suppressed enzymatic activity of iNOS activated by LPS. The above results indicated that naucleoffieine H suppress NO and TNF-α overproduction via block the iNOS pathway in LPS-induced RAW 264.7 macrophages.     

Total synthesis and structural elucidation of ent-micropyrone and (+)-ascosalipyrone

The total synthesis of 6-[(1S,3S)-1,3-dimethyl-2-oxopentyl]-4-hydroxy-3,5-dimethyl-2H-pyran-2-one (22), the enantiomer of the natural product micropyrone (1), was achieved in 9 linear steps (10% overall yield), from Evans auxiliary (R)-12 with key coupling of the dianion of dione 17 and aldehyde 11. Formation of the pyrone ring and subsequent oxidation at C7 was achieved without epimerization of the sensitive position α to both the pyrone ring and the carbonyl. The same sequence using the alternate dione 24 achieved the total synthesis of 6-[(1S,3S)-1,3-dimethyl-2-oxopentyl]-4-hydroxy-3-methyl-2H-pyran-2-one (28), the (+)-enantiomer of the natural product, ascosalipyrone (2). In both cases diastereomeric aldehydes 11 and 16 were taken through the synthetic sequence to give two possible diastereomers of the natural products. Comparison of the (1)H and (13)C NMR data for the synthetic isomers with that reported for the natural products determined their relative stereochemistry. Comparison of the optical rotation obtained for 22 established it to be the enantiomer of micropyrone.     

Induction of nitric oxide synthase (NOS) by soluble glucocorticoid induced tumor necrosis factor receptor (sGITR) is modulated by IFN-gamma in murine macrophage

Earlier study showed that glucocorticoid induced tumor necrosis factor receptor (GITR), a new TNFR family, activated murine macrophages to express inducible nitric oxide synthase (iNOS) and to generate nitric oxide (NO). A possible involvement of pro-inflammatory cytokines on NO production by GITR was investigated in vitro systems and signaling molecules contributing to sGITR-induced iNOS production are determined in Raw 264.7 cells, a murine macrophage cell line. The result showed that the synergy was afforded by the combination of GITR with IFN-g in a dose-dependent manner but IFN-gamma alone was not able to induce NOS. No effects were observed with TNF-alpha, IL-1beta, or IL-6 co-treated with GITR. To determine signaling molecules contributing to sGITR-induced iNOS production, a specific inhibitor for signal pathway proteins tested showed that PDTC (NF-kappaB) and genistein (tyrosine kinase) inhibited NOS induction significantly, while sodium orthovanadate (tyrosine phosphatase) potentiated NOS expression. These results suggest that activations of NF-kappaB were involved in induction of iNOS by GITR and IFN-gamma priming caused earlier and stronger NF-kappaB activation.     

Soluble factor from tumor cells induces heme oxygenase-1 by a nitric oxide-independent mechanism in murine peritoneal macrophages

Tumor target-derived soluble secretary factor has been known to influence macrophage activation to induce nitric oxide (NO) production. Since heme oxygenase-1 (HO-1) is induced by a variety of conditions associated with oxidative stress, we questioned whether soluble factor from tumor cells induces HO-1 through NO-dependent mechanism in macrophages. We designated this factor as a tumor-derived macrophage-activating factor (TMAF), because of its ability to activate macrophages to induce iNOS. Although TMAF alone showed modest activity, TMAF in combination with IFN-gamma significantly induced iNOS expression and NO synthesis. Simultaneously, TMAF induced HO-1 and this induction was slightly augmented by IFN-gamma. Surprisingly, however, induction of HO-1 by TMAF was not inhibited by the treatment with the highly selective iNOS inhibitor, 1400 W, indicating that TMAF induces the HO-1 enzyme by a NO-independent mechanism. While rIFN-gamma alone induced iNOS, it had no effect on HO-1 induction by itself. Collectively, the current study reveals that soluble factor from tumor target cells induces HO-1 enzyme in macrophages. However, overall biological significance of this phenomenon remains to be determined.     

New benzoyl glucosides and cytotoxic pterosin sesquiterpenes from Pteris ensiformis Burm

Three new compounds: 2R,3R-pterosin L 3-O-beta-D-glucopyranoside (1), beta-D-xylopyranosyl(1-->2)-7-O-benzoyl-beta-D-glucopyranoside (2) and 4-O-benzoyl-beta-D-xylo-pyranosyl(1-->2)-7-O-benzoyl-beta-D-glucopyranoside (3), together with nine known compounds, were isolated from the ethyl acetate extract of Pteris ensiformis. 5-[2-Hydroxyethylidene]-2(5H)-furanone (4), which had been synthesized, was isolated from natural sources for the first time. The structures of all isolated compounds were determined on the basis of mass and spectroscopic evidence. Compound 1 and pterosin B (5) show cytotoxicity against HL 60 cells (human leukemia) with the IC(50) values of 3.7 and 8.7 microg/mL, respectively.     

Aurones and Flavonols from Coreopsis lanceolata L. Flowers and Their Anti-Oxidant,Pro-Inflammatory Inhibition Effects,and Recovery Effects on Alloxan-Induced Pancreatic Islets in Zebrafish

(1) Background: Many flavonoids have been reported to exhibit pharmacological activity; a preparatory study confirmed that Coreopsis lanceolata flowers (CLFs) contained high flavonoid structure content; (2) Methods: CLFs were extracted in aqueous methanol (MeOH:H₂O = 4:1) and fractionated into acetic ester (EtOAc), normal butanol (n-BuOH), and H₂O fractions. Repeated column chromatographies for two fractions led to the isolation of two aurones and two flavonols; (3) Results: Four flavonoids were identified based on a variety of spectroscopic data analyses to be leptosidin (1), leptosin (2), isoquercetin (3), and astragalin (4), respectively. This is the first report for isolation of 2–4 from CLFs. High-performance liquid chromatography (HPLC) analysis determined the content levels of compounds 1–4 in the MeOH extract to be 2.8 ± 0.3 mg/g (1), 17.9 ± 0.9 mg/g (2), 3.0 ± 0.2 mg/g (3), and 10.9 ± 0.9 mg/g (4), respectively. All isolated compounds showed radical scavenging activities and recovery activities in Caco-2, RAW264.7, PC-12, and HepG2 cells against reactive oxygen species. MeOH extract, EtOAc fraction, and 1–3 suppressed NO formation in LPS-stimulated RAW 264.7 cells and decreased iNOS and COX-2 expression. Furthermore, all compounds recovered the pancreatic islets damaged by alloxan treatment in zebrafish; (4) Conclusions: The outcome proposes 1–4 to serve as components of CLFs in standardizing anti-oxidant, pro-inflammatory inhibition, and potential anti-diabetic agents.     

In vitro anti-inflammatory activity of 4-arylcoumarins from endophytic Streptomyces aureofaciens CMUAc130 in murine macrophage RAW 264.7 cells

This research was undertaken to find the in vitro inflammatory action of 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin produced by Streptomyces aureofaciens CMUAc130. We investigated the effects of 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin not only on the formation of nitric oxide (NO), PGE(2), TNF-alpha, IL-6 and IL-1beta, but also on inducible NO synthase and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells. The data obtained were consistent with the modulation of iNOS enzyme expression. A similar fashion was also observed when LPS-induced PGE(2) release and COX-2 expression were tested. The significant inhibitory effects were shown in concentration-dependent manners. In addition, 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin also mildly but significantly reduced the formation of TNF-alpha. These findings support the application of 5,7-dimethoxy-4-p-methoxylphenylcoumarin and 5,7-dimethoxy-4-phenylcoumarin as anti-inflammatory agents.     

Effect of sildenafil citrate on interleukin-1beta-induced nitric oxide synthesis and iNOS expression in SW982 cells

The purpose of this study was to identify the effect of sildenafil citrate on IL-1β-induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1β stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1β-induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1β treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1β-induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1β-induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.     

In vivo study of the inflammatory modulating effects of low-level laser therapy on iNOS expression using bioluminescence imaging

This study was designed to demonstrate that bioluminescence imaging (BLI) can be used as a new tool to evaluate the effects of low-level laser therapy (LLLT) during in vivo inflammatory process. Here, the efficacy of LLLT in modulating inducible nitric oxide synthase (iNOS) expression using different therapeutic wavelengths was determined using transgenic animals with the luciferase gene under control of the iNOS gene expression. Thirty transgenic mice, FVB/N-Tg(iNOS-luc)Xen, were allocated randomly to one of four experimental groups treated with different wavelengths (lambda = 635, 785, 808 and 905 nm) or a control group (nontreated). Inflammation was induced by intra-articular injection of zymosan A in both knee joints. Laser treatment (25 mW cm(-2), 200 s, 5 J cm(-2)) was applied to the knees 15 min after inflammation induction. Measurements of iNOS expression were performed at various times (0, 3, 5, 7, 9 and 24 h) by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The results showed a significant increase in BLI signal after irradiation with 635 nm laser when compared to the nonirradiated animals and the other LLLT-treated groups, indicating wavelength dependence of LLLT effects on iNOS expression during the inflammatory process, and thus demonstrating an action spectrum of iNOS gene expression following LLLT in vivo that can be detected by BLI. Histological analysis was also performed and demonstrated the presence of fewer inflammatory cells in the synovial joints of mice irradiated with 635 nm compared with nonirradiated knee joints.