9 research outputs found
Bioactive secondary metabolites from marine and under explored habitats
No full textThis thesis presents results obtained from the investigation of secondary metabolites through screening of marine organisms, marine-derived microbes, and microbes form under-explored habitats. The first part includes the isolation of eight cytotoxic diterpene derivatives of which four were new from the organic extract of the sponge Spongionella sp. obtained from the U.S. National Cancer Institute’s Open Repository Program, the isolation of three new antibacterial dibenzofuran derivatives and a known butyrolactone from ascomycete Super1F1-09 isolated from the Indo-Pacific sponge Acanthella cavernosa. An attempt to synthesize these compounds was conducted. This part also includes the isolation of five known pyrroloiminoquinone alkaloids, from the Fijian sponge Zyzzya sp., which showed potent antiprotozoal activity. The second part comprises the use of OSMAC approach for the isolation of four new ansamycin-type polyketides, three new macrolactones and one known siderophore from Streptomyces strain C34 isolated from Atacama Desert, Chile. These compounds showed good antibacterial activity with one of the ansamycins showed pronounced antibacterial activity against a panel of clinical isolates of methicillin-sensitive as well as methicillin-resistant S. aureus (MRSA). This part also contains the use of microbial co-culture for the induction of secondary metabolites. It comprises the isolation of ten antiprotozoal fungal metabolites, of which one was new, from Aspergillus fumigatus when co-cultured with the novel strain Streptomyces C2 isolated from Atacama Desert. In conclusion, natural products from diverse sources proved to be the major resource of drug discovery. This thesis describes the isolation and structural characterisation of 35 compounds, 15 of which were new. Extremophiles proved to be a good source for new secondary metabolites.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Evaluation of derived compounds from sponges against induced oxidative stress in cortical neurons
No full textIntroduction
Marine sponges are a huge source of different and active biochemical structures (Sagar et al., 2010;Abbas et al., 2011;Mollica et al., 2012). Many of these secondary metabolites are created with defensive purposes (Sagar et al., 2010;Mollica et al., 2012) and liberated into the water, so its action occurs at very low concentrations and therefore they are usually very potent and active compounds (Rane et al., 2014). The family of makaluvamines (MKs) is one of these active groups of secondary metabolites. They are iminoquinone alkaloids isolated from sponges of the genera Zyzzya (Zhang et al., 2012) that have been described as anticancer (Wang et al., 2009), antimalarial (Davis et al., 2012), topoisomerase II inhibitors (Shinkre et al., 2007) and antioxidant compounds (Utkina, 2013). Since previously antioxidant research was done in silica, in this work, we delve into the activity of this compounds family testing five MKs (H, J, F, L and G) in a cellular in vitro model of oxidative stress, also used for the study of neurodegenerative diseases (Facecchia et al., 2011). This model consists on the treatment of primary cortical neurons with hydrogen peroxide, an oxidant inductor, and the co-treatment with the tested compounds to evaluate the neuroprotection and the antioxidant capacity thereof. The fact is that oxidative stress is associated to mitochondrial dysfunction and a common feature in neurodegenerative diseases. Thus, increasing antioxidant defenses activity, in neurodegeneration cellular models, can elicit cellular protection and therefore diminish the risk of neurodegenerative diseases (Bonda et al., 2010).
Methods
Makaluvamines information: The Marine Biodiscovery Centre (Department of Chemistry, University of Aberdeen) supplied the library of pure compounds isolated from Zyzzya sources. Makaluvamines H, J, F, L and G, we will refer to them by its letter.
Primary cortical neurons: were obtained from embryonic day 15−18 mice fetuses as described elsewhere (Vale et al., 2010).
Cytotoxicity assay: The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) assay was used to analyzed cell viability as previously described (Alonso et al., 2011a;b). Primary cortical neurons were grown in 96 well plates and exposed for 48h to different compound concentrations (0.01, 0.05, 0.1 and 1 µM) added to the culture medium. Saponin was used as cellular death control. After treatment, MTT was performed and absorbance was measured at 595 nm in a spectrophotometer plate reader.
Neuroprotection assays: Treatments were carried out in 96-well plate, as co-incubations of 200 µM H2O2 and the compounds H, L and G at 1 and 0.1 µM and J and F at 0.1 and 0.05 µM for 12h on 4-5 div neurons.
Mitochondrial function: mitochondrial function wastudied by MTT test following the method previously described.
Mitochondrial membrane potential (Ψm): Ψm was determined by the tetramethylrhodamine methyl ester (TMRM) assay (White et al., 2011). TMRM incubation was with 1 µM for 30 min and cells were solubilized with 50% DMSO/water. Fluorescence was measured at 535 nm excitation, 590 nm emission.
Reactive oxygen species (ROS) generation: ROS production was analyzed by a fluorescence test using 7′,2′-dichlorofluorescein diacetate (DCFH-DA), as previously described (Kim et al., 2002). Neurons were loaded with 20 µM DCF-DA for 30 min at 37 °C. Cells were washed and kept at room temperature for 30 min to allow a complete de-esterification. DCF fluorescence was measured at 475 nm and emission at 525 nm.
Glutathione (GSH) levels measurement: GSH levels were evaluated with a thiol tracker dye. Cells were loaded with 10 µM ThiolTracker™ Violet dye for 1 h at 37 °C. After incubation, fluorescence was read at 404 nm excitation and 526 nm emission.
Catalase (CAT) activity determination: CAT activity was measured with Amplex® Red Catalase Assay Kit, following the commercial protocol.
Statistical analysis: Statistical analysis was performed by Student's t-test. P values < 0.05 were considered statistically significant. Results, expressed in percentage of no-treated neurons control, are presented as means ± SEM of three or more experiments. Assays were carried out at least by triplicate.
Results and discussion
Neuronal viability is well related with mitochondrial activity, so MKs cytotoxicity was studied by MTT assay. Cortical neurons were exposed for 48h to MKs H, L and G at 0.01, 0.05, 0.1 and 1 µM. No signs of cytotoxicity were observed, even at the highest concentration tested and we follow the investigations with 1 and 0.1 µM. However, MKs J and F were cytotoxic at the highest concentration and therefore, 0.1 and 0.05 µM were chosen for the experiments.
In order to study the MKs antioxidant activity, compounds were tested in a cellular oxidative stress in vitro model previously set up [25], consisting of cortical neurons co-incubated with H2O2 (200 µM) and the MKs at the selected concentrations for 12h. Then, we evaluated the mitochondrial function, Ψm, ROS and GSH levels and CAT activity.
Firstly, the possible MKs protection against mitochondrial dysfunction caused by oxidative stress was tested. Mitochondrial function was analyzed by MTT, also correlated with neurons survival measurements (Varming et al., 1996). MKs, at the two chosen concentrations, were co-incubated with H2O2 (200 µM) for 12h, and viability assays were performed. Results demonstrated that the viability of neurons treated with the oxidant decreased a 31.6 ± 2.0% (p < 0.001), and MKs H, J and L protected neurons at this level. MK L protected cells reaching a basal percentage of viability, with a 90.5 ± 8.0% (p = 0.013) at 0.1 µM.
Ψm analyses contribute to comprehend the MKs activity against H2O2 insults. TRMR test reveals a diminution of 33.6 ± 4.3% (p < 0.001) in Ψm when neurons were treated with the oxidant inductor for 12h. The same MKs treatments were performed, and the TRMR was executed. The Ψm decrease produced by the oxidant agent, was only reduced by MK J at 0.1 µM (84.5 ± 1.5%, p = 0.009).
Oxidative stress is an imbalance between ROS generation and antioxidants levels (Gandhi and Abramov, 2012). Thus, in oxidative stress conditions ROS levels are augmented compared to basal conditions. H2O2 treatments in neurons elevated ROS production in a 20.0 ± 2.5% (p 2O2 as previously described and ROS levels were measured. A reduction of ROS levels regarding the oxidant treatment was observed in MKs H, J, F and G treatments.
In physiological conditions, low concentrations of H2O2 are transformed to water and molecular oxygen by GSH–peroxidase, with GSH as a proton donor. But when H2O2 amounts are high, they are instead eliminated by CAT. GSH is one of the antioxidant mitochondrial systems of protection against oxidative damage (Bains and Shaw, 1997). So to conclude the antioxidant research, MKs effects over GSH and CAT were evaluated. GSH is the main intracellular thiol in cells (Zampagni et al., 2012) and a thiol tracker was used to evaluate it. 12h H2O2 incubation produces a GSH level reduction of 25.8 ± 3.1% (p 2O2, as detailed above, and only MK J increased its levels to a 92.5 ± 9.4% (p = 0.048), achieving GSH basal amounts. Moreover the oxidation treatment decreases CAT activity in neurons in a 24.4 ± 5.5% (p < 0.01) however, the co-incubation with MKs increased CAT activity. MKs J, L and G treatments produced a significant elevation with a complete reestablishment of the activity.
Neurons consume an elevated percentage of total body oxygen and consequently they are one of the most vulnerable cell populations to oxidative stress, which plays an important role in neurodegenerative pathology . After MKs evaluation in neurons under oxidative stress condition, we conclude that all of them afford some protection against oxidation, which is consistent with the already published about MKs H, L and G (Utkina, 2013). Once again compound H was the less active in our cellular model and MKs L and G denoted some antioxidant protection. Above all the MKs tested, the no-previously tested MK J at 0.1 µM highlights with a complete neuroprotection, reducing oxidation consequences, such as mitochondrial dysfunction and ROS generation, and increasing antioxidant defenses by maintaining GSH basal levels and CAT activity. All these antioxidant effects might be explained for an activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response element (ARE) pathway, the main sensor and modulator of oxidative stress, that trigger the transcription of genes like superoxide dismutase 1, CAT, sulforedoxin, thioredoxin, peroxiredoxin and proteins responsible for the synthesis and metabolism of GSH. It has been reported that Nrf2-ARE pathway activation ameliorates the animal symptoms in research models for neurodegenerative diseases (Gan and Johnson, 2013) and numerous scientists of this area are focusing their experiments on the modulation of enzymatic regulatory components, that protect against oxidative stress, to emulate their restorative effects and consequently slow down the illness progression (Andersen, 2004).
The results presented in this work elucidate that makaluvamine J is a potent molecule for neuroprotection against oxidative stress. Nevertheless, the precise mechanism by which MK J activates the antioxidant cell defenses is still unknown. For that reason, further studies about the MK J activity over the Nrf2-ARE pathway and its possible implications in neurodegenerative disorders will be required
Isolation of phenolic constituents from <i>Rhododendron yunnanense</i> flowers as a potent cyclooxygenase-2 and vascular endothelial growth factor receptor-2 inhibitor:phytochemical and molecular simulation studies
Get PDFThe genus Rhododendron is a rich source of phenolic compounds that possess a wide range of biological activities. Phytochemical investigation of the methanolic extract of the flowers of Rhododendron yunnanense Franch. led to the isolation and characterization of 13 phenolic compounds isolated for the first time from this plant species.These compounds were identified as quercetin (1), quercitrin (2), avicularin (3), taxifolin-3-O-α-L-arabinoside(4), azalein (5), kaempferol-3-O-rhamnoside (6), kaempferol-4ʹ-methoxy-3-O-rhamnoside (7), kaempferol-3-β-Dglucopyranoside(8), catechin (9), epicatechin (10), catechin-3-O-gallate (11), 5-O-Z-p-coumaroylquinic acid methyl ester (12), and 5-O-caffoeylquinic acid methyl ester (13). The structures of compounds 1–13 were determined by 1D and 2D nuclear magnetic resonance and comparison with reported spectral data. A molecular simulation study was carried out on the binding mode of the isolated compounds as anti-inflammatory agents through cyclooxygenase 2 (COX-2) inhibition and as mediators of tumor angiogenesis through vascular endothelial growth factor inhibition. The docking results of the isolated compounds revealed promising binding affinities to the examined enzymes. Compound 2 showed predominant affinity for the two examined receptors [COX-2 (−19.4542 kcal/mol) and vascular endothelial growth factor receptor 2 (−17.6036 kcal/mol)]. The isolated compounds offered significantly active phytoconstituents for drug discovery and development.</p
Isolation of Pandangolide 1 from Cladosporium oxysporum, An Endophyte of the Terrestrial Plant Alyxia reinwardtii
Get PDFPandangolide 1 was isolated from the ethyl acetate extract of Cladosporium oxysporum cultures. The fungus was originally obtained from Alyxia reinwardtii. The structure of pandangolide 1 was elucidated on the basis of nuclear magnetic resonance (NMR) spectroscopy and accurate mass spectrometric data. This is the first report of the isolation of pandangolide 1 from endophytic C. oxysporum derived from a terrestrial host plan
Wound healing and antioxidant properties of <i>Launaea procumbens</i> supported by metabolomic profiling and molecular docking
Get PDFWounds adversely affect people’s quality of life and have psychological, social, and economic impacts. Herbal remedies of Launaea procumbens (LP) are used to treat wounds. In an excision wound model, topical application of LP significantly promoted wound closure (on day 14, LP-treated animals had the highest percentages of wound closure in comparison with the other groups, as the wound was entirely closed with a closure percentage of 100%, p < 0.05). Histological analysis revealed a considerable rise in the number of fibroblasts, the amount of collagen, and its cross-linking in LP-treated wounds. Gene expression patterns showed significant elevation of TGF-β levels (2.1-fold change after 7 days treatment and 2.7-fold change in 14 days treatment) and downregulation of the inflammatory TNF-α and IL-1β levels in LP-treated wounds. Regarding in vitro antioxidant activity, LP extract significantly diminished the formation of H2O2 radical (IC50 = 171.6 μg/mL) and scavenged the superoxide radical (IC50 of 286.7 µg/mL), indicating antioxidant potential in a dose-dependent manner. Dereplication of the secondary metabolites using LC-HRMS resulted in the annotation of 16 metabolites. The identified compounds were docked against important wound-healing targets, including vascular endothelial growth factor (VEGF), collagen α-1, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and transforming growth factor-β (TGF-β). Among dereplicated compounds, luteolin 8-C-glucoside (orientin) demonstrated binding potential to four investigated targets (VEGF, interleukin 1β, TNF-α, and collagen α-1). To conclude, Launaea procumbens extract could be regarded as a promising topical therapy to promote wound healing in excisional wounds, and luteolin 8-C-glucoside (orientin), one of its constituents, is a potential wound-healing drug lead
Identification and characterization of the actinomycin G gene cluster in Streptomyces iakyrus
No full textThe gene cluster directing actinomycin G biosynthesis in Streptomyces iakyrus has been identified and sequenced. It contains one actinomycin synthetase I (ACMS I) gene and two copies of ACMS II and III genes. Genetic analysis demonstrates a unique partnership between the putative hydroxylation and chlorination activities as both acmG8 and acmG9 genes need to be transcribed for the biosynthesis of actinomycin G2–3, respectively
Spongionella Secondary Metabolites Protect Mitochondrial Function in Cortical Neurons against Oxidative Stress
Get PDFThe marine habitat provides a large number of structurally-diverse bioactive compounds for drug development. Marine sponges have been studied over many years and are found to be a rich source of these bioactive chemicals. This study is focused on the evaluation of the activity of six diterpene derivatives isolated from Spongionella sp. on mitochondrial function using an oxidative in vitro stress model. The test compounds include the Gracilins (A, H, K, J and L) and tetrahydroaplysulphurin-1. Compounds were co-incubated with hydrogen peroxide for 12 hours to determine their protective capacities and their effect on markers of apoptosis and Nrf2/ARE pathways was evaluated. Results conclude that Gracilins preserve neurons against oxidative damage, and that in particular, tetrahydroaplysulphurin-1 shows a complete neuroprotective activity. Oxidative stress is linked to mitochondrial dysfunction and consequently to neurodegenerative disorders like Parkinson and Alzheimer diseases, Friedreich ataxia or Amyotrophic lateral sclerosis. This neuroprotection against oxidation conditions suggest that these metabolites could be interesting lead candidates in drug development for neurodegenerative diseases
Unveiling the antibacterial potential of Arctotis aurantiaca roots: Isolation, characterization, and computational studies
No full text337348Phytochemical investigation of the total ethanolic extract and various derived fractions of Arctotis aurantiaca roots resulted in the isolation of eight chemically diverse metabolites, including two fatty alcohols [n-octacosanol (1) and n-pentacosanol (2)], three sesquiterpene lactones [9β‑hydroxy-11 β,13-dihydrozaluzanin C (3), 11α, 13-dihydroglucozaluzanin C (5), and ixerin F (6)], one coumarin [6-Methoxy-7-hydroxycoumarin (scopoletin) (4)], one lignan [4,8-dimethoxyeudesmin (7)] and one phenolic acid derivative [3,5-dicaffeoylquinic acid methyl ester (8)] using different chromatographic techniques. The antibacterial activity of these isolated compounds was investigated against Escherichia coli ATCC 25,922 using well-diffusion assay technique. Among the tested compounds, compound (6) exhibited the strongest antibacterial activity with a MIC of 1.7 µg/mL, whereas compound (1) was the least potent with a MIC of 218.2 µg/mL compared to gentamicin (MIC= 2 µg/mL) as a positive control. Additionally, molecular docking and 140 ns molecular dynamics (MD) simulations were performed to evaluate the binding interactions of the isolated compounds with E. coli DNA gyrase A. The results indicated that ixerin F (6) showed strong binding affinity and stability over 140-nanosecond simulation, highlighting its potential as an antibacterial agent. This study underscores the integration of computational and experimental methods to develop novel antibacterial strategies against E. coli, presenting A. aurantiaca as a candidate antibacterial plant by targeting bacterial DNA gyrase, a key enzyme involved in regulating DNA topology and supercoiling, which indirectly impacts essential cellular processes such as replication and transcription.18
Isolation of Pandangolide 1 from Cladosporium oxysporum, An Endophyte of the Terrestrial Plant Alyxia reinwardtii
No full textPandangolide 1 was isolated from the ethyl acetate extract of Cladosporium oxysporum cultures. The fungus was originally obtained from Alyxia reinwardtii. The structure of pandangolide 1 was elucidated on the basis of nuclear magnetic resonance (NMR) spectroscopy and accurate mass spectrometric data. This is the first report of the isolation of pandangolide 1 from endophytic C. oxysporum derived from a terrestrial host plan
