Silymarin Ameliorates Diabetes-Induced Proangiogenic Response in Brain Endothelial Cells through a GSK-3 β

Diabetes mellitus (DM) is a major risk factor for cardiovascular disease. Additionally, it was found to induce a dysfunctional angiogenic response in the brain that was attributed to oxidative stress. Milk thistle seed extract (silymarin) has potent antioxidant properties, though its potential use in ameliorating diabetes-induced aberrant brain angiogenesis is unknown. Glycogen synthase kinase-3β is a regulator of angiogenesis that is upregulated by diabetes. Its involvement in diabetes-induced angiogenesis is unknown. To evaluate the potential of silymarin to ameliorate diabetes-induced aberrant angiogenesis, human brain endothelial cells (HBEC-5i) were treated with 50 μg/mL advanced glycation end (AGE) products in the presence or absence of silymarin (50, 100 μM). The angiogenic potential of HBEC-5i was evaluated in terms of migration and in vitro tube formation capacities. The involvement of GSK-3β was also evaluated. AGE significantly increased the migration and tube formation rates of HBEC-5i by about onefold (p=0.0001). Silymarin reduced AGE-induced migration in a dose-dependent manner where 50 μM reduced migration by about 50%, whereas the 100 μM completely inhibited AGE-induced migration. Similarly, silymarin 50 μg/mL blunted AGE-induced tube formation (p=0.001). This effect was mediated through a GSK-3β-dependent inhibition of VEGF release. In conclusion, silymarin inhibits AGE-induced aberrant angiogenesis in a GSK-3β-mediated inhibition of VEGF release

Scaffold Diversity of Fungal Metabolites

Many drug discovery projects rely on commercial compounds to discover active leads. However, current commercial libraries, with mostly synthetic compounds, access a small fraction of the possible chemical diversity. Natural products, in contrast, possess a vast structural diversity and have proven to be an outstanding source of new drugs. Several chemoinformatic analyses of natural products have demonstrated their diversity and structural complexity. However, to our knowledge, the scaffold content and structural diversity of fungal secondary metabolites have never been studied. Herein, the scaffold diversity of 223 fungal metabolites was measured and compared to the diversity of approved drugs and commercial libraries for HTS containing natural, synthetic, and semi-synthetic compounds. In addition, the global diversity of the fungal isolates was assessed and compared to other reference data sets using Consensus Diversity Plots, a chemoinformatic tool recently developed. It was concluded that fungal secondary metabolites are cyclic systems with few ramifications and more diverse than the commercial libraries with natural products and semi-synthetic compounds. The fungal metabolites data set was one of the most structurally diverse, containing a large proportion of different and unique scaffolds not found in the other compound data sets including ChEMBL. Therefore, fungal metabolites offer a rich source of molecules suited for identifying diverse candidates for drug discovery

Simultaneous determination of warfarin and 7-hydroxywarfarin in rat plasma by HPLC-FLD

In this study, high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been used for the first time, for direct determination of warfarin and its major metabolite, 7-hydroxywarfarin, in rat plasma. The simple and sensitive method was developed using Fortis® reversed-phase diphenyl column (150 × 4.6 mm, 3 μm) and a mobile phase composed of phosphate buffer (25 mmol L–1)-methanol-acetonitrile (70:20:10, V/V/V), adjusted to pH 7.4, at a flow rate of 0.8 mL min–1. The diphenyl chemistry of the stationary phase provided a unique selectivity for separating the structurally related aromatic analytes, warfarin and 7-hydroxywarfarin, allowing their successful quantification in the complex plasma matrix. The method was linear over the range 0.01–25 μg mL–1, for warfarin and 7-hydroxywarfarin, and was found to be accurate, precise and selective in accordance with US FDA guidance for bioanalytical method validation. The method was sensitive enough to quantify 0.01 μg mL–1 of warfarin and 7-hydroxywarfarin (LLOQ) using only 100 µL of plasma. The applicability of this method was demonstrated by analyzing samples obtained from rats after oral administration of a single warfarin dose, and studying warfarin and 7-hydroxywarfarin pharmacokinetics

Discovering new structural diversity from unexplored fungi

Discovery of anticancer drugs with high efficacy coupled with action at novel target sites is necessary to combat cancer. As part of a multidisciplinary project to identify anticancer leads from diverse natural product resources, our group has been studying fungi from different ecological habitats, including filamentous Ascomycota from terrestrial, freshwater, and symbiotic fungi (fungal endophytes), as a source of novel scaffolds for drug design and development. During the course of my research work, 56 bioactive compounds have been isolated and identified, with 30 of the isolated leads representing new chemical entities. Our lab relies on bioactivity-directed fractionation methodology for the isolation and purification of cytotoxic lead compounds from fungi, in which the bioassay results guide the purification processes. However, one of the inefficient outputs of utilizing this methodology is the re-isolation of previously known compounds, particularly mycotoxins. It is hypothesized that discovery of cytotoxic bioactive compounds with novel structures will be expedited by development and application of a dereplication methodology that has the capability to identify known compounds at the level of the crude extract. A dereplication methodology has been developed and implemented successfully for the identification of fungal secondary metabolites in crude culture extracts using a UPLC-PDA-HRMS-MS/MS method. Finally, the chemical diversity of the isolated compounds was analyzed through principal component analysis

UPLC-HRESI-MS and GC-MS analysis of the leaves of Nicotiana glauca

The alkaloid-rich fraction obtained by fractionation of the crude methanolic extract of the leaves of wild Tobacco tree Nicotiana glauca Graham (Solanaceae) was analyzed using UPLC-MS and GC-MS. Anabasine, a piperidine alkaloid, was identified as the major constituent with approximately 60 % (m/m) of the alkaloid-rich fraction. In addition to anabasine, six secondary metabolites were identified using high-resolution UPLC-MS. Anabasine was quantified in the leaves to be 1 mg g–1 dry plant material. The GC-MS analysis revealed five compounds with anabasine as the major component, while nicotine was not detected. Moreover, GC-MS was used for the analysis of the volatile oil that was obtained by hydrodistillation from the leaves of N. glauca. The volatile plant oil was found to be rich in oxygenated sesquiterpenes (e.g., β-bisabolol) and carboxylic acids and esters (e.g., ethyl linoleate and hexadecanoic acid), whereas anabasine was not detected

Graviola Inhibits Hypoxia-Induced NADPH Oxidase Activity in Prostate Cancer Cells Reducing Their Proliferation and Clonogenicity

Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1–5?µg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47phox). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1a levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity

Cytotoxic Homoisoflavonoids from the Bulbs of Bellevalia flexuosa

Four new homoisoflavonoids, 7-O-methyl-8-demethoxy-3'-hydroxy-3,9-dihydropunctatin (4), 6-hydroxy-8-demethoxy-4'-O-methyl-3,9-dihydropunctatin (8), 7,4'-O-dimethyl-8-demethoxy-3,3'-dihydroxy-3,9-dihydropunctatin (13), and 7-O-methyl-3-hyroxy-3,9-dihydropunctatin (14) were identified from a chloroform extract of the bulbs of Bellevalia flexuosa, along with 13 known analogues. The structures were determined by analysis of HRMS and NMR data, while ECD spectroscopy enabled the assignment of the absolute configurations of the new compounds 4, 8, 13 and 16. The cytotoxic activities of the isolated compounds (1–17) were evaluated using a panel of human cancer cell lines. Compounds 2 and 7 were the most potent against the MDA-MB-435 (melanoma) cancer cell line with IC50 values of 1.6 and 2.0?µM, respectively, and were essentially equipotent against the OVCAR3 (ovarian) cancer cell line with IC50 values of 9.5 and 10.8?µM, respectively. However, compound 7, with an IC50 value of 3.6?µM, was the most potent against the MDA-MB-231 (breast) cancer cell line

Greensporone C, a Freshwater Fungal Secondary Metabolite Induces Mitochondrial-Mediated Apoptotic Cell Death in Leukemic Cell Lines

Therapeutic agents used in the treatment of cancer are known to develop resistance against cancer cells. Hence, there is a continuing need to investigate novel agents for the treatment and management of cancer. Antitumor activity of greensporone C (GC), a new resorcylic acid lactone isolated from an organic extract of a culture of a Halenospora sp. freshwater fungus, was subjected for screening against a panel of leukemic cell lines (K562, U937, and AR320). In all the three cell lines, cell proliferation was inhibited in dose-dependent fashion. GC further arrested the cells in SubG0 phase in dose-dependent manner. Annexin V/PI dual staining data confirmed apoptotic death of treated K562 and U937 leukemic cells. Treatment with GC suppressed constitutively phosphorylated AKT and downregulated expression of inhibitor of apoptotic proteins XIAP, cIAP-1, and cIAP-2. In summation to this, GC-treated leukemic cells upregulated protein expression of pro-apoptotic proteins, Bax with concomitant decrease in expression of anti-apoptotic proteins including Bcl-2 and Bcl-xL. Upregulation of Bax was associated with cytochrome c release which was confirmed from the collapse of mitochondrial membrane. Released cytochrome c further activated caspase cascade which in turn initiated apoptosis process. Anticancer activity of this isolated fungal compound GC was potentiated via stimulating production of reactive oxygen species (ROS) along with depletion of reduced glutathione (GSH) levels in K562 and U937 leukemic cells. Pretreatment of these cells with N-acetyl cysteine prevented GC-induced depletion of reduced GSH level and mitochondrial-caspase-induced apoptosis. Altogether, our data show that GC modulates the apoptotic response of human leukemic cells and raises the possibility of its use as a novel therapeutic strategy for hematological malignancies

In Situ Analysis of Asimina triloba (Paw Paw) Plant Tissues for Acetogenins via the Droplet-Liquid Microjunction-Surface Sampling Probe Coupled to UHPLC-PDA-HRMS/MS

Asimina triloba, commonly known as paw paw, is one of approximately 2100 species in the Annonaceae family, scores of which are known to biosynthesize bioactive secondary metabolites, termed Annonaceous acetogenins. Even with over 400 acetogenins identified, a high-throughput screening protocol for these compounds does not exist. Advances in direct ambient ionization mass spectrometry have opened the door to many metabolite profiling methodologies, but for acetogenins, this is often complicated by the abundance of isomers that are present. A droplet-liquid microjunction-surface sampling probe coupled to UPLC-PDA-HRMS/MS system was employed to detect acetogenins in situ from A. triloba. The seeds, fruit pulp, twigs, leaves, and flowers of A. triloba were all examined for acetogenins. Additionally, lithium was infused post-column to increase the sensitivity of the fragments, thus allowing for characterization of the structural classes, and mass defect filtering was used to mine the data for the various acetogenin analogues. This surface sampling system allowed for the rapid identification and differentiation of Annonaceous acetogenins directly from the various organs of A. triloba, including the never before studied flowers

Mass Spectrometry Imaging of Secondary Metabolites Directly on Fungal Cultures

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient ionization technique that enables imaging experiments directly on fungal cultures. Much information can be gained by examining an organism directly from culture, rather than through an extraction process, as the regional and temporal distribution of bioactive compounds can give a better understanding of interactions in the environment. However, this technique has been underutilized towards the direct analysis of fungal cultures. A major challenge of fungal culture analysis with DESI-MS is the requirement of a firm, flat surface for effective ionization. The media upon which fungi grow can be easily deformed by the pressures from the solvent spray and gas, and the fungal topography is naturally uneven, often containing mycelium and spores that move freely. Furthermore, DESI-MS imaging can only analyse the surface of a sample, thus internal compounds remain undetected. This project first sought to overcome these issues, and then, apply the newly developed methodology to explore the chemical interactions between two distinct fungi. To test the methodology, a fungus that produces antifungal metabolites was grown against a mycotoxin producing fungus, both of the phylum Ascomycota. By comparing the spatial and temporal distribution of secondary metabolites between both isolated cultures and co-cultures, the chemical exchanges that took place were visualized