Synthesis and anti-proliferative activities of new derivatives of embelin

Embelin (1), a benzoquinone isolated from Embelia ribes is known to possess variety of biological activities. Despite of several promising biological activities, preclinical efforts on embelin were hampered because of its poor aqueous solubility. In order to address the solubility issue, herein, we have synthesized a series of Mannich products of embelin by treating it with various secondary amines. The synthesized compounds were screened for antiproliferative and antimicrobial activities. In cytotoxicity screening, the benzyl-piperidine linked derivative 8m was found to possess better antiproliferative activity compared to parent natural product embelin against a panel of cell lines including HCT-116, MCF-7, MIAPaCa-2 and PC-3 with IC₅₀ values of 30, 41, 34 and 36 μM, respectively. The mechanistic study of compound 8m revealed that it exhibits cytotoxicity via induction of apoptosis and mitochondrial membrane potential loss. Further, the compounds were tested for antimicrobial activity where dimethylamino- 8a and piperidine linked derivative 8b displayed antibacterial activity against Staphylococcus aureus with MIC values of 8 and 16 μg/mL, respectively. Mannich derivatives did now show improved aqueous solubility, however their hydrochloride salts 8a·HCl, 8b·HCl and 8m·HCl showed significantly improved aqueous solubility without affecting biological activities of parent Mannich derivatives.     

Development of glial cells cultured from prenatally alcohol treated rat brain: Effect of supplementation of the maternal alcohol diet with a grape extract

The aim of this work was to investigate the effect of supplementation of a maternal alcohol diet with a grape extract on glial cell development. Glial cells were cultured during 4 weeks from cortical brain cells of the new born offspring in DMEM medium supplemented with fetal calf serum. Enzymatic markers of nerve cell development were measured (enolase isoenzymes and glutamine synthetase). Since alcohol consumption produces free radicals the antioxidant system superoxide dismutase was also investigated. Compared to the decrease found in only alcohol treated animals, all parameters except neuron-specific enolase were antagonized and even stimulated after grape extract supplementation. The effect was more important after only 1 month than 3 months of treatment. Also in the total brain an alcohol antagonizing effect and a glutamine synthetase activation were found. Our data demonstrate that addition of a grape extract to the maternal alcohol diet may partially or completely overcome the alcohol induced retardation of glial cell development.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

Determination of phenolic compounds and diterpenes in roots of Salvia miltiorrhiza and Salvia przewalskii by two LC–MS tools: Multi-stage and high resolution tandem mass spectrometry with assessment of antioxidant capacity

Comparative phytochemical analyses of hydroalcoholic (50% EtOH) extracts from roots of S. miltiorrhiza (SM) and S. przewalskii (SP) were performed using two complementary LC–MS systems: the first system HPLC-DAD-MSⁿ an ion trap mass spectrometer and the second system consisted high resolution MS/MS Orbitrap mass spectrometer. The individual compounds were identified using a previously published approach via comparison of the exact molecular masses, mass spectra and retention times to those of standard compounds, online available databases and literature data. Moreover, the determination of antioxidative activities of extracts by DPPH and FRAP methods was carried out. Analysis allowed to identify 39 chemical compounds in extracts from both species. Extract from root of SP differs from SM in the presence of several metabolites such as: przewalskinic acid and their derivatives, przewaquinone C, przewaquinonate A, glycosides of rosmarinic acid, methyltanshinonate, whereas tanshinones, salvianolic acids and lithospermic acids occurred in both species. Moreover, it was shown that hydroalcoholic extract from roots of SM exerted stronger antioxidant properties in a FRAP test (max. 323.92 μM Fe²⁺/L) and in DPPH test (max. 78.64 nM TE) in comparison with SP extract.     

Dormancy break of celery (Apium graveolens L.) seeds by plant derived smoke extract

Seed dormancy of a highly-dormant cultivar of celery (Apium graveolens L.) was broken by combinations of plant-derived smoke extract or N⁶-benzyladenine (BA) and gibberellins A_4/7 (GA_4/7) in the dark at temperatures between 18 and 26°C. A less dormant cultivar which responded to GA_4/7 alone showed no additional response to smoke extract or BA. Neither smoke extract nor BA affected either cultivar in the dark in the absence of GA_4/7. The partial dormancy-breaking effect of short exposures to red-light was also enhanced by smoke extracts in this highly-dormant cultivar. The results suggest that smoke extracts act in a similar way to cytokinins, by enhancing gibberellin activity in the celery seed system.Abbreviations BA N⁶-benzyladenine - GA_4/7 A₄ and A₇ gibberellin mixture     

Variation in economically and ecologically important traits in onion plant organs during reproductive development

The spatial distribution of organosulphur compounds throughout the onion (Allium cepa L.) plant body during reproduction is of ecological and horticultural interest. These secondary metabolites are associated with both pest resistance and many of the vegetable's culinary and medicinal properties, including the ability to inhibit platelet aggregation. Inhibition of platelet aggregation can be of benefit to human cardiovascular health. Organosulphur compound concentrations are associated with elemental sulphur, pungency, soluble solids and effect on human platelet aggregation. These parameters were evaluated in extracts collected separately from bulb scales, leaf blades, scapes and umbels biweekly throughout the reproductive phase of the life cycle of the onion. Significant variation in pungency, platelet inhibition, total sulphur content and soluble solids existed among samples of organs and within organs over time during reproductive growth. Furthermore, some extracts from leaf, scape and bulb induced rather than inhibited platelet aggregation.     

Effect of high pressure homogenization and papain on the preparation of autolysed yeast extract

The effect of high pressure homogenization (600 and 1000 bar) prior to autolysis of a commercial pressed baker's yeast was examined. High pressure homogenization released a maximum of 30% of the solids and 34% of the total nitrogen (TN). After autolysis of the whole homogenized slurry, high yields of solids and TN (up to 81 and 85%, respectively) were obtained. Autolysis of non-homogenized controls yielded much lower yield values (30 and 39%, respectively), whereas autolysis in the presence of papain but without prior disruption gave intermediate values (50 and 61%, respectively). The various treatments led to changes in the extract composition: standard autolysates had the highest total nitrogen and true protein weight contents and the lowest carbohydrate content, whereas this trend was reversed when cells were first disrupted before autolysis. In contrast to controls obtained by standard autolysis without or with papain, centrifuged autolysates from pre-homogenized fractions were not clear. Treatment with a combination of a flocculation and a weighting agent clarified the extracts but resulted in a loss of solids (approximately 20%), including nitrogen and carbohydrates.     

Potential effects and relevant lead compounds of Vigna mungo (L.) Hepper seeds against bacterial infection,helminthiasis, thrombosis and neuropharmacological disorders

Multidrug-resistant bacterial infections, helminthiasis, thrombosis, anxiety and insomnia are some of the major global health concerns. Vigna mungo (L.) Hepper (VM) has been used traditionally to treat microbial infection, helminthic disorder, schizophrenia, memory loss, and blood circulatory problem. This research aims to discover antibacterial, anthelmintic, thrombolytic and neuropharmacological effects of the methanol extract of Vigna mungo seeds (MESVM), and also in-silico prediction of relevant lead compounds by molecular docking and ADME/T analysis. The crude extracts and subsequent fractions of MESVM were investigated for antibacterial activity by disc diffusion method, anthelmintic activity by paralysis and death test on earthworms, and thrombolytic activity by in vitro blood clot dissolution test. Open-field test and elevated plus maze test were performed for evaluating anxiolytic activity of the extracts. Using molecular docking, ligand poses of selected VM seeds’ phytoconstituents were predicted targeting tubulin, GlcN-6-P synthase, and human tissue plasminogen activator proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. In the antibacterial activity test, the MESVM at 10000 μg/mL concentration created highest and significant (P < 0.001) zone of inhibition against Staphylococcus aureus (15.42 mm) and Escherichia coli (12 mm) compared with tetracycline. The MESVM exhibited remarkable anthelmintic activity at 50 mg/mL concentration with 35.4 min paralysis time, 75.2 min death time and were closer to the durations of standard drug albendazole. No test extract showed anxiolytic activity. In thrombolytic activity test, all concentrations of MESVM produced clot lytic activity with high significance (P < 0.001) in comparison with the blank. In docking, 2′-hydroxygenistein, cyclokievitone hydrate, and aureol displayed maximum affinity to the target proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. This research revealed that the MESVM demonstrated potential anthelmintic, antibacterial and thrombolytic effects that confirmed the folkloric uses of VM and the found relevant lead compounds might be further optimized in future drug development.     

6-Shogaol induces apoptosis in acute lymphoblastic leukaemia cells by targeting p53 signalling pathway and generation of reactive oxygen species

Combination therapies, using medicinal herbs, are broadly recommended to attenuate the chemotherapy adverse effects. Based on our previous findings considering the anti-leukaemic effects of ginger extract on acute lymphoblastic leukaemia (ALL) cells, the present study was aimed to investigate the anti-cancer role of this pharmaceutical plant on ALL mice models. Moreover, we worked towards identifying the most anti-leukaemic derivative of ginger and the mechanism through which it may exert its cytotoxic impact. In vivo experiments were performed using five groups of six C57BL/6 nude mice, and the anti-leukaemic activity of ginger extract alone or in combination with methotrexate (MTX) was examined. Results showed increased survival rate and reduced damages in mice brain and liver tissues. Subsequently, MTT assay demonstrated synergistic growth inhibitory effect of 6-shogaol (6Sh) and MTX on ALL cell lines and patients primary cells. Eventually, the molecular anti-neoplastic mechanism of 6Sh was evaluated using Bioinformatics. Flow cytometry illustrated 6Sh-mediated apoptosis in Nalm-6 cells confirmed by Western blotting and RT-PCR assays. Further analyses exhibited the generation of reactive oxygen species (ROS) through 6Sh. The current study revealed the in vivo novel anti-leukaemic role of ginger extract, promoted by MTX. Moreover, 6-shogaol was introduced as the major player of ginger cytotoxicity through inducing p53 activity and ROS generation.     

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1 hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.