Efficient identification of flavones, flavanones and their glycosides in routine analysis via off-line combination of sensitive NMR and HPLC experiments.

We present a standardized, straightforward and efficient approach applicable in routine analysis of flavonoids combining sensitive NMR and HPLC experiments. The determination of the relative configuration of sugar moieties usually requires the acquisition of 13C NMR shift values. We use a combination of HPLC and sensitive NMR experiments (1D-proton, 2D-HSQC) for the unique identification of known flavones, flavanones, flavonols and their glycosides. Owing to their broad range of polarity, we developed HPLC and UHPLC methods (H2O/MeOH/MeCN/HCOOH) which we applied and validated by analyzing 46 common flavones and flavanones and exemplified for four plant extracts. A searchable data base is provided with full data comprising complete proton and carbon resonance assignments, expansions of HSQC-spectra, HPLC parameters (retention time, relative retention factor), UV/Vis and mass spectral data of all compounds, which enables a rapid identification and routine analysis of flavones and flavanones from plant extracts and other products in nutrition and food chemistry.

Network pharmacology of cancer: From understanding of complex interactomes to the design of multi-target specific therapeutics from nature.

Despite massive investments in drug research and development, the significant decline in the number of new drugs approved or translated to clinical use raises the question, whether single targeted drug discovery is the right approach. To combat complex systemic diseases that harbour robust biological networks such as cancer, single target intervention is proved to be ineffective. In such cases, network pharmacology approaches are highly useful, because they differ from conventional drug discovery by addressing the ability of drugs to target numerous proteins or networks involved in a disease. Pleiotropic natural products are one of the promising strategies due to their multi-targeting and due to lower side effects. In this review, we discuss the application of network pharmacology for cancer drug discovery. We provide an overview of the current state of knowledge on network pharmacology, focus on different technical approaches and implications for cancer therapy (e.g. polypharmacology and synthetic lethality), and illustrate the therapeutic potential with selected examples green tea polyphenolics, Eleutherococcus senticosus, Rhodiola rosea, and Schisandra chinensis). Finally, we present future perspectives on their plausible applications for diagnosis and therapy of cancer.

Natural product agonists of peroxisome proliferator-activated receptor gamma (PPARγ): a review.

Agonists of the nuclear receptor PPARγ are therapeutically used to combat hyperglycaemia associated with the metabolic syndrome and type 2 diabetes. In spite of being effective in normalization of blood glucose levels, the currently used PPARγ agonists from the thiazolidinedione type have serious side effects, making the discovery of novel ligands highly relevant. Natural products have proven historically to be a promising pool of structures for drug discovery, and a significant research effort has recently been undertaken to explore the PPARγ-activating potential of a wide range of natural products originating from traditionally used medicinal plants or dietary sources. The majority of identified compounds are selective PPARγ modulators (SPPARMs), transactivating the expression of PPARγ-dependent reporter genes as partial agonists. Those natural PPARγ ligands have different binding modes to the receptor in comparison to the full thiazolidinedione agonists, and on some occasions activate in addition PPARα (e.g. genistein, biochanin A, sargaquinoic acid, sargahydroquinoic acid, resveratrol, amorphastilbol) or the PPARγ-dimer partner retinoid X receptor (RXR; e.g. the neolignans magnolol and honokiol). A number of in vivo studies suggest that some of the natural product activators of PPARγ (e.g. honokiol, amorfrutin 1, amorfrutin B, amorphastilbol) improve metabolic parameters in diabetic animal models, partly with reduced side effects in comparison to full thiazolidinedione agonists. The bioactivity pattern as well as the dietary use of several of the identified active compounds and plant extracts warrants future research regarding their therapeutic potential and the possibility to modulate PPARγ activation by dietary interventions or food supplements.

Polyacetylenes from Radix et Rhizoma Notopterygii incisi with an inhibitory effect on nitric oxide production in vitro.

Notopterygium roots (Qiang Huo) have been used in traditional Chinese medicine for treating colds, inflammatory diseases like rheumatoid arthritis, and as an analgesic. The anti-inflammatory activity of the roots of Notopterygium incisum has been evaluated by testing the inhibitory activity on nitric oxide production by inducible nitric oxide synthase. The apparent authenticity of the sample was checked by DNA sequence comparison. Using activity-guided isolation, different compounds were isolated and structurally characterized by means of NMR and mass spectroscopy. Eight polyacetylenes could be identified and were tested on their inhibitory activity on nitric oxide production in RAW 264.7 mouse macrophages using the Griess assay. Different 3-hydroxy allyl polyacetylenes exhibited significant activity (IC50: 8-acetoxyfalcarinol, 20.1 µM; falcarindiol, 9.2 µM; 9-epoxyfalcarindiol, 8.8 µM; and crithmumdiol, 23.6 µM).

Polyacetylenes from Notopterygium incisum--new selective partial agonists of peroxisome proliferator-activated receptor-gamma.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism and therefore an important pharmacological target to combat metabolic diseases. Since the currently used full PPARγ agonists display serious side effects, identification of novel ligands, particularly partial agonists, is highly relevant. Searching for new active compounds, we investigated extracts of the underground parts of Notopterygium incisum, a medicinal plant used in traditional Chinese medicine, and observed significant PPARγ activation using a PPARγ-driven luciferase reporter model. Activity-guided fractionation of the dichloromethane extract led to the isolation of six polyacetylenes, which displayed properties of selective partial PPARγ agonists in the luciferase reporter model. Since PPARγ activation by this class of compounds has so far not been reported, we have chosen the prototypical polyacetylene falcarindiol for further investigation. The effect of falcarindiol (10 µM) in the luciferase reporter model was blocked upon co-treatment with the PPARγ antagonist T0070907 (1 µM). Falcarindiol bound to the purified human PPARγ receptor with a Ki of 3.07 µM. In silico docking studies suggested a binding mode within the ligand binding site, where hydrogen bonds to Cys285 and Glu295 are predicted to be formed in addition to extensive hydrophobic interactions. Furthermore, falcarindiol further induced 3T3-L1 preadipocyte differentiation and enhanced the insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes confirming effectiveness in cell models with endogenous PPARγ expression. In conclusion, we identified falcarindiol-type polyacetylenes as a novel class of natural partial PPARγ agonists, having potential to be further explored as pharmaceutical leads or dietary supplements.

Naphthoquinones from Onosma paniculata induce cell-cycle arrest and apoptosis in melanoma Cells.

Activity-guided fractionation of a petroleum ether-soluble extract of the roots of Onosma paniculata, which has been shown to affect the cell cycle and to induce apoptosis in melanoma cells, led to the isolation of several shikonin derivatives, namely, ß-hydroxyisovalerylshikonin (1), acetylshikonin (2), dimethylacrylshikonin (3), and a mixture of α-methylbutyrylshikonin and isovalerylshikonin (4+5). All compounds exhibited strong cytotoxicity against eight cancer cell lines and MRC-5 lung fibroblasts, with 3 found to possess the most potent cytotoxicity toward four melanoma cell lines (SBcl2, WM35, WM9, and WM164). Furthermore, 3 and the mixture of 4+5 were found to interfere with cell-cycle progression in these cell lines and led to an increasing number of cells in the subG1 region as well as to caspase-3/7 activation, indicating apoptotic cell death.

Cytotoxic furanogermacranolides from the flowers of Helianthus angustifolius.

The dichloromethane extract of the flowers of Helianthus angustifolius L. (Asteraceae, Heliantheae) was investigated in vitro for its cytotoxic activity using human cancer cell lines: CCRF-CEM leukemia, MDA-MB-231 breast cancer, U251 glioblastoma, HCT 116 colon cancer cells, and the human lung fibroblast cell line MRC-5. Cytotoxicity-guided fractionation led to the isolation of four related heliangolide-type sesquiterpene lactones. The structures were elucidated by means of NMR spectroscopy and high-resolution mass spectrometry. Of the investigated compounds, 8-methacrylyl-4,15-iso-atriplicolide (1) showed the highest activity against all tested cancer cell lines with IC50 values ranging from 0.26 ± 0.01 µM for CCRF-CEM cells to 4.22 ± 0.26 µM for MRC-5 cells.

Synthesis of N-substituted 2-[(1E)-alkenyl]-4-(1H)-quinolone derivatives as antimycobacterial agents against non-tubercular mycobacteria.

In an effort to improve biological activities and to examine antimycobacterial-lipophilicity relationships of 2-[(1E)-alkenyl)]-4-(1H)-quinolones, we have synthesized a series of 30 quinolones by introducing several alkyl groups, an alkenyl and an alkynyl group at N-1. All synthetic compounds were first tested in vitro against Mycobacterium smegmatis and the most active compounds (MIC values ∼3.0-7.0 µM) were further examined against three other rapidly growing strains of mycobacteria using a microtiter broth dilution assay. The Clog P values of the synthetic compounds were calculated to provide an estimate of their lipophilicity. Compounds 18e, 19a and 19b displayed the most potent inhibitory effect against M. smegmatis mc(2)155 with an MIC value of ∼1.5 µM, which was twenty fold and thirteen fold more potent than isoniazid and ethambutol, respectively. On the other hand, compounds 17e, 18e and 19a were most active against Mycobacterium fortuitum and Mycobacterium phlei with an MIC value of ∼3.0 µM. In the human diploid embryonic lung cell line MRC-5 cytotoxicity assay, the derivatives showed moderate to strong cytotoxic activity. Although the antimycobacterial activity of our synthetic compounds could not be correlated with the calculated log P values, an increase in lipophilicity enhances the antimycobacterial activity and C13-C15 total chain length at positions 1 and 2 is required to achieve optimal inhibitory effect against the test strains.

Inhibition of inducible nitric oxide synthase by bis(helenalinyl)glutarate in RAW264.7 macrophages.

Nitric oxide (NO) plays a role in various physiological and pathophysiological conditions such as immunoregulatory and inflammatory processes. Hence, NO and its generating enzyme, inducible nitric oxide synthase (iNOS) may not only be of diagnostic and prognostic value, but may also serve as targets for novel therapeutic options. In the present investigation, we have screened a phytochemical library by correlating the IC(50) values for 531 natural products of 60 cell lines with the microarray-based mRNA expression of 95 genes known to be involved in NO metabolism and signaling with the aim to identify candidate compounds as inhibitors for NO metabolism and signaling. We identified bis(helenalinyl)glutarate (BHG) as putative candidate compound. Indeed, BHG inhibited NO production (IC(50) value: 0.90+/-0.04microM) and down-regulated iNOS protein expression (IC(50) value: 1.12+/-0.16microM) of RAW264.7 mouse macrophages in the presence of lipopolysaccharide and interferon-gamma. Performing XTT cytotoxicity assays, we found that BHG inhibited cell growth in a dose-dependent manner with an IC(50) value of 5.6microM. To gain insight into molecular pathways involved in NO inhibition and cytotoxicity, we performed microarray experiments which were exemplarily validated by real-time RT-PCR. A total of 227 genes (67 up- and 160 down-regulated) were obtained, which exhibited significant differences in mRNA regulation between BHG-treated and untreated RAW264.7 macrophages. Sixteen of 227 genes are known to be involved in NO-signaling. Pathway analyses revealed that further five and four down-regulated genes belong to the glucocorticoid receptor and interleukin-1 and interleukin-10 pathways, respectively. An interference of these two pathways and NO is known for inflammation and auto-immune diseases. The therapeutic potential of this compound has to be explored in the future.