Structures and bioactivities of monomeric and dimeric carvotacetones from Sphaeranthus africanus.

Four previously undescribed carvotacetones including one monomeric (1) and three dimeric (8, 9, 10) derivatives, together with six known compounds were isolated from the n-hexane extract of the aerial parts of Sphaeranthus africanus L. The structures of the previously undescribed compounds were elucidated as 3-angeloyloxy-5-isobutanoyloxy-7-hydroxycarvotacetone (1), 7,7'-oxybis{3-angeloyloxy-5-[(2R*,3R*)-2,3-dihydroxy-2-methylbutanoyloxy]carvotacetone} (8), (2″S*,3″R*)-7-{3-angeloyloxy-5-[(2R*,3R*)-2,3-dihydroxy-2-methylbuta-noyloxy]carvotaceton-7-yloxy}-3-angeloyloxy-5-(2,3-dihydroxy-2-methylbutanoyloxy)carvo-tacetone (9), and 7,7'-oxybis{3-angeloyloxy-5-[(2S*,3R*)-2,3-dihydroxy-2-methylbutanoyl-oxy]carvotacetone} (10). The three dimeric derivatives (8-10) showed potent anti-proliferative activity against human cancer cell lines (CCRF-CEM, MDA-MB-231, U-251, HCT-116) with IC50 values ranging from 0.2 to 2.0 µM. Caspases 3 and 7 were found to be activated by all compounds, indicating apoptosis induction activity. Monomers exhibited a specific inhibition of NO production in BV2 and RAW 264.7 cells with IC50 values ranging from 4.2 to 6.8 µM which were 2-3.5-fold lower than IC50 values causing cytotoxicity. In addition, the carvotacetones reduced NF-κB1 (p105) mRNA expression at concentrations of 10 and 2.5 µM. Altogether, the results indicate that carvotacetones may be interesting lead structures for the development of anti-cancer and anti-inflammatory drugs.

Formation of 5-Aminomethyl-2,3-dihydropyridine-4(1H)-ones from 4-Amino-tetrahydropyridinylidene Salts.

Various 4-aminotetrahydropyridinylidene salts were treated with aldehydes in an alkaline medium. Their conversion to 5-substituted ß-hydroxyketones in a one-step reaction succeeded only with an aliphatic aldehyde. Instead, aromatic aldehydes gave 5-substituted ß-aminoketones or a single δ-diketone. The new compounds were characterized using spectroscopic methods and a single crystal structure analysis. Some of them showed anticancer and antibacterial properties.

Characterization of potentially health-promoting constituents in sea fennel (Crithmum maritimum) cultivated in the Conero Natural Park (Marche region, Central Italy).

CONTEXT: Sea fennel (Crithmum maritimum L. [Apiaceae]) is an aromatic herb rich in bioactive molecules, such as polyphenols, with potential positive effects on human health. OBJECTIVE: This study aimed at the characterization of sea fennel secondary metabolites, focusing on the phenolic fraction. MATERIALS AND METHODS: Samples of whole sprouts, sole leaves and sole stems were subjected to accelerated solvent extraction with methanol, and the resulting extracts were analyzed by high­performance thin­layer chromatography, high-performance liquid chromatography, and liquid chromatography coupled with diode array detection and high-resolution mass spectrometry (LC-DAD-HRMS). RESULTS: HPTLC and HPLC analyses of sea fennel extracts showed similar chromatographic profiles among the tested samples, and the prevalence of chlorogenic acid within the phenolic fraction was verified. Ten hydroxycinnamic acids, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, 11 flavonoid glycosides, e.g., rutin, hyperoside, isoquercitrin, two triterpene saponins and two hydroxylated fatty acids, were detected and annotated via liquid chromatography coupled with diode array detection and high-resolution mass spectrometry. DISCUSSION AND CONCLUSIONS: The use of accelerated solvent extraction and LC-DAD-HRMS for the characterization of sea fennel secondary metabolites allowed the annotation of seven compounds newly detected in sea fennel, including triterpene saponins and hydroxylated fatty acids.

Glycolipid-enriched fraction of Osmanthus fragrans inhibits LPS-induced expression of inflammatory genes, COX-2, E-selectin, and Interleukin-8.

ETHNOPHARMACOLOGICAL RELEVANCE: Osmanthus fragrans Lour. is a small ornamental tree native to the Southeastern parts of China. It is mainly cultivated because of its characteristic fragrance, and used in the food and perfume industry. Besides, its flowers are used in traditional Chinese medicine to treat a variety of diseases including those related to inflammation. AIM OF THE STUDY: The aim of the study was to investigate in more detail the anti-inflammatory properties of O. fragrans flowers, and to characterize their active principles and mechanisms of action. MATERIALS AND METHODS: O. fragrans flowers were successively extracted with n-hexane, dichloromethane and methanol. The extracts were further fractionated by chromatographic separation. COX-2 mRNA expression in PMA-differentiated, LPS-stimulated THP-1 cells was used as lead assay for activity-guided fractionation. The most potent fraction was chemically analyzed by LC-HRMS. The pharmacological activity was also evaluated in other inflammation-related in-vitro models, such as analysis of IL-8 secretion and E-selectin expression in HUVECtert cells and selective inhibition of COX-isoenzymes. RESULTS: n-Hexane and dichloromethane extracts of O. fragrans flowers significantly inhibited COX-2 (PTGS2) mRNA expression. Additionally, both extracts inhibited COX-2 enzyme activity, whereas COX-1 enzyme activity was affected to a significantly lower extent. Fractionation of the extracts led to a highly active, glycolipid-containing fraction. In total, 10 glycolipids were tentatively annotated by LC-HRMS. This fraction also inhibited LPS-induced COX-2 mRNA expression, IL-8 secretion and E-selectin expression. The effects were limited to LPS-induced inflammation and not observed when inflammatory genes were induced by TNF-α, IL-1ß or FSL-1. Since all these inducers of inflammation act via different receptors, it is likely that the fraction interferes with the binding of LPS to the TLR4-receptor, which mediates pro-inflammatory effects of LPS. CONCLUSION: Taken together, the results demonstrate the anti-inflammatory potential of O. fragrans flower extracts in general, and of the glycolipid-enriched fraction in particular. The effects of glycolipid-enriched fraction are potentially mediated via the inhibition of the TLR4 receptor complex.

Author Correction: S-nitrosoglutathione inhibits adipogenesis in 3T3-L1 preadipocytes by S-nitrosation of CCAAT/enhancer-binding protein ß.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

C13 Megastigmane Derivatives From Epipremnum pinnatum: ß-Damascenone Inhibits the Expression of Pro-Inflammatory Cytokines and Leukocyte Adhesion Molecules as Well as NF-κB Signaling.

In order to identify active constituents and to gain some information regarding their mode of action, extracts from leaves of Epipremnum pinnatum were tested for their ability to inhibit inflammatory gene expression in endothelial- and monocyte-like cells (HUVECtert and THP-1, respectively). Bioactivity-guided fractionation using expression of PTGS2 (COX-2) mRNA as a readout resulted in the isolation of two C13 megastigmane glycosides, gusanlungionoside C (1) and citroside A (3), and the phenylalcohol glycoside phenylmethyl-2-O-(6-O-rhamnosyl)-ß-D-galactopyranoside (2). Further analysis identified six additional megastigmane glycosides and the aglycones ß-damascenone (10), megastigmatrienone (11), 3-hydroxy-ß-damascenone (12), and 3-oxo-7,8-dihydro-α-ionol (13). Pharmacological analysis demonstrated that 10 inhibits LPS-stimulated induction of mRNAs encoding for proinflammatory cytokines and leukocyte adhesion molecules, such as TNF-α, IL-1ß, IL-8, COX-2, E-selectin, ICAM-1, and VCAM-1 in HUVECtert and THP-1 cells. 10 inhibited induction of inflammatory genes in HUVECtert and THP-1 cells treated with different agonists, such as TNF-α, IL-1ß, and LPS. In addition to mRNA, also the upregulation of inflammatory proteins was inhibited by 10 as demonstrated by immune assays for cell surface E-selectin and secreted TNF-α. Finally, using a luciferase reporter construct, it was shown, that 10 inhibits NF-κB-dependent transcription. Therefore, we hypothesize that inhibition of NF-κB by ß-damascenone (10) may represent one of the mechanisms underlying the in vitro anti-inflammatory activity of Epipremnum pinnatum extracts.

S-nitrosoglutathione inhibits adipogenesis in 3T3-L1 preadipocytes by S-nitrosation of CCAAT/enhancer-binding protein ß.

Murine 3T3-L1 adipocytes share many similarities with primary fat cells and represent a reliable in vitro model of adipogenesis. The aim of this study was to probe the effect of S-nitrosoglutathione (GSNO) on adipocyte differentiation. Adipogenesis was induced with a mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine in the absence and presence of increasing GSNO concentrations. Biochemical analysis after 7 days of differentiation showed a prominent anti-adipogenic effect of GSNO which was evident as reduced cellular triglycerides and total protein content as well as decreased mRNA and protein expression of late transcription factors (e.g. peroxisome proliferator activated receptor γ) and markers of terminal differentiation (e.g. leptin). By contrast, the nitrosothiol did not affect mRNA and protein expression of CCAAT/enhancer-binding protein ß (C/EBPß), which represents a pivotal early transcription factor of the adipogenic cascade. Differentiation was also inhibited by the NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate. Biotin switch experiments showed significantly increased S-nitrosation of C/EBPß variants indicating that posttranslational S-nitrosative modification of this transcription factor accounts for the observed anti-adipogenic effect of NO. Our results suggest that S-nitrosation might represent an important physiological regulatory mechanism of fat cell maturation.

Anti-inflammatory and antiproliferative compounds from Sphaeranthus africanus.

BACKGROUND: Sphaeranthus africanus has been used in traditional Vietnamese medicine to treat sore throat, and to relieve pain and swelling. However, the anti-inflammatory activity of this plant had not yet been investigated. Previously, we isolated five carvotacetones (1-5) from this plant that displayed cytotoxicity against several cancer cell lines. PURPOSE: The objective of this study was to isolate further constituents from S. africanus and to investigate the anti-inflammatory activity of all constituents. Furthermore, the anti-proliferative activity of the newly isolated compounds was evaluated. STUDY DESIGN AND METHODS: Compounds were isolated from the upper parts of S. africanus by chromatographic methods. Structures were determined using spectroscopic techniques, like NMR and MS. All nine compounds isolated from S. africanus were evaluated for inhibitory activity against COX-1 and COX-2 isoenzymes in-vitro, COX-2 mRNA expression and influence on NO production. The anti-proliferative activities of newly isolated compounds (6-9) were evaluated by XTT viability assay with four cancer cell lines, namely CCRF-CEM, MDA-MB-231, HCT-116, and U-251 cells. RESULTS: Two diastereomeric carvotacetones (3-angeloyloxy-5-[2″S,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (6) and 3-angeloyloxy-5-[2″R,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (7), asperglaucide (8) and chrysoplenol D (9) were isolated from S. africanus. COX-1 and COX-2 assays of compounds 1-9 revealed that compounds 1 and 2 possess potent and selective COX-2 inhibitory activity with IC50 values of 3.6 and 0.5 µM, respectively. COX-2 gene expression assay showed that some carvotacetones exhibited inhibitory effects on COX-2 gene expression in THP-1 macrophages. Compound 4 is the most active compound inhibiting the synthesis of COX-2 by 55% at 2.06 µM. In the iNOS assay, all seven carvotacetones inhibited NO production in BV2 and RAW cell lines with IC50 values ranging from 0.2 to 2.9 µM. Compound 4 showed potent inhibitory activity with IC50 values of 0.2 µM in both BV2 and RAW cell lines. Molecular docking studies revealed the binding orientations of 1 and 2 in the active sites of COX-2. XTT assay of the newly isolated compounds revealed that the two isomeric carvotacetones (6-7) exhibited considerable anti-proliferative activity against four cancer cell lines (CCRF-CEM, MDA-MB-231, HCT-116, U-251) with IC50 values ranging from 1.23 to 8 µM. CONCLUSION: For the first-time, the diastereomeric carvotacetones (6-7) were isolated as separate compounds, and their anti-proliferative activity was determined. Selective COX-2 inhibitory, COX-2 mRNA expression and NO production inhibitory activities by some of the major constituents of S. africanus supports the traditional medical application of this plant for the treatment of inflammation-related disorders.