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2.
Rev Environ Health ; 2022 Sep 22.
Article in English | MEDLINE | ID: mdl-36129168

ABSTRACT

The fifth generation of radiofrequency communication, 5G, is currently being rolled out worldwide. Since September 2017, the EU 5G Appeal has been sent six times to the EU, requesting a moratorium on the rollout of 5G. This article reviews the 5G Appeal and the EU's subsequent replies, including the extensive cover letter sent to the EU in September 2021, requesting stricter guidelines for exposures to radiofrequency radiation (RFR). The Appeal notes the EU's internal conflict between its approach to a wireless technology-led future, and the need to protect the health and safety of its citizens. It critiques the reliance of the EU on the current guidelines given by the International Commission on Non-Ionizing Radiation Protection (ICNIRP), that consider only heating and no other health relevant biological effects from RFR. To counteract the ICNIRP position, the 2021 cover letter briefly presented recent research from the EU's own expert groups, from a large collection of European and other international studies, and from previous reviews of the effects of RFR on humans and the environment. The 5G Appeal asserts that the majority of scientific evidence points to biological effects, many with the potential for harm, occurring below the ICNIRP public limits. Evidence to establish this position is drawn from studies showing changes to neurotransmitters and receptors, damage to cells, proteins, DNA, sperm, the immune system, and human health, including cancer. The 2021 Appeal goes on to warn that 5G signals are likely to additionally alter the behaviour of oxygen and water molecules at the quantum level, unfold proteins, damage skin, and cause harm to insects, birds, frogs, plants and animals. Altogether, this evidence establishes a high priority for the European Union towards (i) replacing the current flawed guidelines with protective thresholds, and (ii) placing a moratorium on 5G deployment so as to (iii) allow industry-independent scientists the time needed to propose new health-protective guidelines. This 2021 Appeal's relevance becomes even more pressing in the context of the EU plans to roll out the sixth generation of wireless technologies, 6G, further adding to the known risks of RFR technology for humans and the environment. This all leads to an important question: Do EU decision makers have the right to ignore EU´s own directives by prioritising economic gain over human and environmental health?

3.
Curr Biol ; 32(17): 3676-3689.e5, 2022 09 12.
Article in English | MEDLINE | ID: mdl-35863351

ABSTRACT

Much of our understanding of navigation comes from the study of individual species, often with specific tasks tailored to those species. Here, we provide a novel experimental and analytic framework integrating across humans, rats, and simulated reinforcement learning (RL) agents to interrogate the dynamics of behavior during spatial navigation. We developed a novel open-field navigation task ("Tartarus maze") requiring dynamic adaptation (shortcuts and detours) to frequently changing obstructions on the path to a hidden goal. Humans and rats were remarkably similar in their trajectories. Both species showed the greatest similarity to RL agents utilizing a "successor representation," which creates a predictive map. Humans also displayed trajectory features similar to model-based RL agents, which implemented an optimal tree-search planning procedure. Our results help refine models seeking to explain mammalian navigation in dynamic environments and highlight the utility of modeling the behavior of different species to uncover the shared mechanisms that support behavior.


Subject(s)
Spatial Navigation , Animals , Hippocampus , Humans , Learning , Mammals , Rats , Reinforcement, Psychology
4.
Neuron ; 110(3): 394-422, 2022 02 02.
Article in English | MEDLINE | ID: mdl-35032426

ABSTRACT

The mammalian hippocampal formation contains several distinct populations of neurons involved in representing self-position and orientation. These neurons, which include place, grid, head direction, and boundary cells, are thought to collectively instantiate cognitive maps supporting flexible navigation. However, to flexibly navigate, it is necessary to also maintain internal representations of goal locations, such that goal-directed routes can be planned and executed. Although it has remained unclear how the mammalian brain represents goal locations, multiple neural candidates have recently been uncovered during different phases of navigation. For example, during planning, sequential activation of spatial cells may enable simulation of future routes toward the goal. During travel, modulation of spatial cells by the prospective route, or by distance and direction to the goal, may allow maintenance of route and goal-location information, supporting navigation on an ongoing basis. As the goal is approached, an increased activation of spatial cells may enable the goal location to become distinctly represented within cognitive maps, aiding goal localization. Lastly, after arrival at the goal, sequential activation of spatial cells may represent the just-taken route, enabling route learning and evaluation. Here, we review and synthesize these and other evidence for goal coding in mammalian brains, relate the experimental findings to predictions from computational models, and discuss outstanding questions and future challenges.


Subject(s)
Goals , Spatial Navigation , Animals , Hippocampus/physiology , Learning , Mammals , Neurons/physiology , Prospective Studies , Spatial Navigation/physiology
5.
Curr Biol ; 31(6): 1221-1233.e9, 2021 03 22.
Article in English | MEDLINE | ID: mdl-33581073

ABSTRACT

Flexible navigation relies on a cognitive map of space, thought to be implemented by hippocampal place cells: neurons that exhibit location-specific firing. In connected environments, optimal navigation requires keeping track of one's location and of the available connections between subspaces. We examined whether the dorsal CA1 place cells of rats encode environmental connectivity in four geometrically identical boxes arranged in a square. Rats moved between boxes by pushing saloon-type doors that could be locked in one or both directions. Although rats demonstrated knowledge of environmental connectivity, their place cells did not respond to connectivity changes, nor did they represent doorways differently from other locations. Place cells coded location in a global reference frame, with a different map for each box and minimal repetitive fields despite the repetitive geometry. These results suggest that CA1 place cells provide a spatial map that does not explicitly include connectivity.


Subject(s)
Hippocampus/cytology , Place Cells , Space Perception , Action Potentials , Animals , Place Cells/cytology , Rats
6.
J Ethnopharmacol ; 242: 112061, 2019 Oct 05.
Article in English | MEDLINE | ID: mdl-31283956

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: Berries of Sorbus species have been used to treat type 2 diabetes in many regions in Europe. AIMS OF THE STUDY: To investigate the inhibitory activity of berry extract of Sorbus on the digestive enzymes α-amylase and α-glucosidase, two important targets for management of blood glucose for type 2 diabetics. Furthermore, to test the anti-hyperglycaemic potential of S. norvegica berry extract in vivo. MATERIALS AND METHODS: 70% acetone berry extracts of 16 Sorbus species were tested in vitro for inhibition of α-amylase and α-glucosidase. Single berry extracts were analysed by 1H-NMR spectroscopy and principal component analysis to evaluate the chemical profiles of the extracts. The anti-hyperglycaemic effect was evaluated in an oral starch tolerance test in STZ-treated C57BL/6 mice. RESULTS: The lowest IC50 values against α-amylase and α-glucosidase were obtained with the Sorbus species belonging to the subspecies Aria, which have simple leaves compared to pinnately compound leaves of the other Sorbus species. Species belonging to subspecies Aria grouped together and away from the other Sorbus species in the score plot, indicating a difference in chemistry. Both the carbohydrate- and polyphenol-fraction contributed to the enzyme inhibition. Extract of the most active species, S. norvegica, had anti-hyperglycaemic activity, at a level 36 times lower than clinically used acarbose, corresponding to a needed daily dose of 900 mg extract. CONCLUSIONS: Sorbus species of subspecies Aria have the potential to be used for management of type 2 diabetes.


Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Hypoglycemic Agents/therapeutic use , Plant Extracts/therapeutic use , Sorbus , Animals , Fruit , Hypoglycemic Agents/chemistry , Male , Metabolomics , Mice, Inbred C57BL , Plant Extracts/chemistry , Proton Magnetic Resonance Spectroscopy , alpha-Amylases/antagonists & inhibitors , alpha-Amylases/chemistry , alpha-Glucosidases/chemistry
7.
J Neurosci ; 39(25): 4847-4863, 2019 06 19.
Article in English | MEDLINE | ID: mdl-30926746

ABSTRACT

The glutamate transporter GLT-1 is highly expressed in astrocytes but also in neurons, primarily in axon terminals. We generated a conditional neuronal GLT-1 KO using synapsin 1-Cre (synGLT-1 KO) to elucidate the metabolic functions of GLT-1 expressed in neurons, here focusing on the cerebral cortex. Both synaptosomal uptake studies and electron microscopic immunocytochemistry demonstrated knockdown of GLT-1 in the cerebral cortex in the synGLT-1 KO mice. Aspartate content was significantly reduced in cerebral cortical extracts as well as synaptosomes from cerebral cortex of synGLT-1 KO compared with control littermates. 13C-Labeling of tricarboxylic acid cycle intermediates originating from metabolism of [U-13C]-glutamate was significantly reduced in synGLT-1 KO synaptosomes. The decreased aspartate content was due to diminished entry of glutamate into the tricarboxylic acid cycle. Pyruvate recycling, a pathway necessary for full glutamate oxidation, was also decreased. ATP production was significantly increased, despite unaltered oxygen consumption, in isolated mitochondria from the synGLT-1 KO. The density of mitochondria in axon terminals and perisynaptic astrocytes was increased in the synGLT-1 KO. Intramitochondrial cristae density of synGLT-1 KO mice was increased, suggesting increased mitochondrial efficiency, perhaps in compensation for reduced access to glutamate. SynGLT-1 KO synaptosomes exhibited an elevated oxygen consumption rate when stimulated with veratridine, despite a lower baseline oxygen consumption rate in the presence of glucose. GLT-1 expressed in neurons appears to be required to provide glutamate to synaptic mitochondria and is linked to neuronal energy metabolism and mitochondrial function.SIGNIFICANCE STATEMENT All synaptic transmitters need to be cleared from the extracellular space after release, and transporters are used to clear glutamate released from excitatory synapses. GLT-1 is the major glutamate transporter, and most GLT-1 is expressed in astrocytes. Only 5%-10% is expressed in neurons, primarily in axon terminals. The function of GLT-1 in axon terminals remains unknown. Here, we used a conditional KO approach to investigate the significance of the expression of GLT-1 in neurons. We found multiple abnormalities of mitochondrial function, suggesting impairment of glutamate utilization by synaptic mitochondria in the neuronal GLT-1 KO. These data suggest that GLT-1 expressed in axon terminals may be important in maintaining energy metabolism and biosynthetic activities mediated by presynaptic mitochondria.


Subject(s)
Excitatory Amino Acid Transporter 2/metabolism , Glutamic Acid/metabolism , Homeostasis/physiology , Mitochondria/metabolism , Neurons/metabolism , Synapses/metabolism , Animals , Aspartic Acid/metabolism , Cerebral Cortex/metabolism , Excitatory Amino Acid Transporter 2/genetics , Mice , Mice, Knockout , Mitochondria/genetics , Oxygen Consumption/physiology , Presynaptic Terminals/metabolism , Synapses/genetics , Synaptosomes/metabolism
8.
Plant Physiol ; 174(1): 56-72, 2017 May.
Article in English | MEDLINE | ID: mdl-28275147

ABSTRACT

The Mediterranean plant Thapsia garganica (dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase calcium pump in mammals and is of industrial importance as the active moiety of the anticancer drug mipsagargin, currently in clinical trials. Knowledge of thapsigargin in planta storage and biosynthesis has been limited. Here, we present the putative second step in thapsigargin biosynthesis, by showing that the cytochrome P450 TgCYP76AE2, transiently expressed in Nicotiana benthamiana, converts epikunzeaol into epidihydrocostunolide. Furthermore, we show that thapsigargin is likely to be stored in secretory ducts in the roots. Transcripts from TgTPS2 (epikunzeaol synthase) and TgCYP76AE2 in roots were found only in the epithelial cells lining these secretory ducts. This emphasizes the involvement of these cells in the biosynthesis of thapsigargin. This study paves the way for further studies of thapsigargin biosynthesis.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Plant Proteins/metabolism , Thapsia/metabolism , Thapsigargin/metabolism , Cytochrome P-450 Enzyme System/classification , Cytochrome P-450 Enzyme System/genetics , Gas Chromatography-Mass Spectrometry , Gene Expression Profiling/methods , Gene Expression Regulation, Plant , Models, Chemical , Molecular Structure , Phylogeny , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Roots/cytology , Plant Roots/genetics , Plant Roots/metabolism , Plants, Genetically Modified , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Thapsia/cytology , Thapsia/genetics , Thapsigargin/chemical synthesis , Nicotiana/genetics , Nicotiana/metabolism
9.
Molecules ; 22(3)2017 Mar 06.
Article in English | MEDLINE | ID: mdl-28272319

ABSTRACT

Radix Astragali is a component of several traditional medicines used for the treatment of type 2 diabetes in China. Radix Astragali is known to contain isoflavones, which inhibit α-glucosidase in the small intestines, and thus lowers the blood glucose levels. In this study, 21 samples obtained from different regions of China were extracted with ethyl acetate, then the IC50-values were determined, and the crude extracts were analyzed by 1H-NMR spectroscopy. A principal component analysis of the 1H-NMR spectra labeled with their IC50-values, that is, bioactivity-labeled 1H-NMR spectra, showed a clear correlation between spectral profiles and the α-glucosidase inhibitory activity. The loading plot and LC-HRMS/NMR of microfractions indicated that previously unknown long chain ferulates could be partly responsible for the observed antidiabetic activity of Radix Astragali. Subsequent preparative scale isolation revealed a compound not previously reported, linoleyl ferulate (1), showing α-glucosidase inhibitory activity (IC50 0.5 mM) at a level comparable to the previously studied isoflavones. A closely related analogue, hexadecyl ferulate (2), did not show significant inhibitory activity, and the double bonds in the alcohol part of 1 seem to be important structural features for the α-glucosidase inhibitory activity. This proof of concept study demonstrates that bioactivity-labeling of the 1H-NMR spectral data of crude extracts allows global and nonselective identification of individual constituents contributing to the crude extract's bioactivity.


Subject(s)
Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Glycoside Hydrolase Inhibitors/chemistry , Metabolomics , Principal Component Analysis , Proton Magnetic Resonance Spectroscopy , Astragalus propinquus , Chromatography, High Pressure Liquid , Enzyme Activation/drug effects , Glycoside Hydrolase Inhibitors/pharmacology , Inhibitory Concentration 50 , Metabolomics/methods , alpha-Glucosidases/chemistry
10.
Article in English | MEDLINE | ID: mdl-28326125

ABSTRACT

The present study was carried out to investigate the wound healing potentials of 17 medicinal plants historically used in Ghana for wound healing. Warm and cold water extracts were prepared from the 17 dried plant species and tested in vitro in the scratch assay with NIH 3T3 fibroblasts from mice. The wound healing scratch assay was used to evaluate the effect of the plants on cell proliferation and/or migration in vitro, as a test for potential wound healing properties. After 21 hours of incubation increased proliferation and/or migration of fibroblasts in the scratch assay was obtained for 5 out of the 17 plant species. HPLC separation of the most active plant extract, which was a warm water extract of Philenoptera cyanescens, revealed the wound healing activity to be attributed to rutin and a triglycoside of quercetin. The present study suggests that Allophylus spicatus, Philenoptera cyanescens, Melanthera scandens, Ocimum gratissimum, and Jasminum dichotomum have wound healing activity in vitro.

11.
Bioorg Med Chem Lett ; 27(2): 319-322, 2017 01 15.
Article in English | MEDLINE | ID: mdl-27908763

ABSTRACT

In drug discovery, lipophilicity is a key parameter for drug optimization. Lipophilicity determinations can be both work and time consuming, especially for non-UV active compounds. Herein, an improved and simple 1H NMR-based method is described to estimate the lipophilicity at physiological pH (logD7.4) in 1-octanol and D2O buffer. The method can be applied to both UV and non-UV active compounds. In addition, neither calibration curves nor internal/external standards are needed. We have demonstrated that logD7.4 can be accurately measured using 1H NMR for compounds within the logD7.4 interval between 0.7 and 3.3. The method was also compared to a previously described HPLC method.


Subject(s)
Pharmaceutical Preparations/chemistry , 1-Octanol/chemistry , Deuterium Oxide/chemistry , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Proton Magnetic Resonance Spectroscopy
12.
J Exp Biol ; 219(Pt 16): 2504-13, 2016 08 15.
Article in English | MEDLINE | ID: mdl-27307488

ABSTRACT

Drosophila, like most insects, are susceptible to low temperatures, and will succumb to temperatures above the freezing point of their hemolymph. For these insects, cold exposure causes a loss of extracellular ion and water homeostasis, leading to chill injury and eventually death. Chill-tolerant species are characterized by lower hemolymph [Na(+)] than chill-susceptible species and this lowered hemolymph [Na(+)] is suggested to improve ion and water homeostasis during cold exposure. It has therefore also been hypothesized that hemolymph Na(+) is replaced by other 'cryoprotective' osmolytes in cold-tolerant species. Here, we compared the hemolymph metabolite profiles of five drosophilid species with marked differences in chill tolerance. All species were examined under 'normal' thermal conditions (i.e. 20°C) and following cold exposure (4 h at 0°C). Under benign conditions, total hemolymph osmolality was similar among all species despite chill-tolerant species having lower hemolymph [Na(+)]. Using NMR spectroscopy, we found that chill-tolerant species instead have higher levels of sugars and free amino acids in their hemolymph, including classical 'cryoprotectants' such as trehalose and proline. In addition, we found that chill-tolerant species maintain a relatively stable hemolymph osmolality and metabolite profile when exposed to cold stress while sensitive species suffer from large increases in osmolality and massive changes in their metabolic profiles during a cold stress. We suggest that the larger contribution of classical cryoprotectants in chill-tolerant Drosophila plays a non-colligative role for cold tolerance that contributes to osmotic and ion homeostasis during cold exposure and, in addition, we discuss how these comparative differences may represent an evolutionary pathway toward more extreme cold tolerance of insects.


Subject(s)
Adaptation, Physiological , Cold Temperature , Drosophila/physiology , Hemolymph/metabolism , Metabolome , Aging/physiology , Animals , Ions , Metabolomics , Models, Biological , Osmolar Concentration , Principal Component Analysis , Proton Magnetic Resonance Spectroscopy , Species Specificity , Tropical Climate
13.
PLoS One ; 11(2): e0149148, 2016.
Article in English | MEDLINE | ID: mdl-26882071

ABSTRACT

Protein fibrillation is the pathological hallmark of several neurodegenerative diseases and also complicates the manufacturing and use of protein drugs. As a case study, the inhibitory activity of the natural compound aloin against insulin fibrillation was investigated. Based on Thioflavin T assays, high-performance liquid chromatography and transmission electron microscopy it was found that a degradation product of aloin, formed over weeks of storage, was able to significantly inhibit insulin fibrillation. The activity of the stored aloin was significantly reduced in the presence of small amounts of sodium azide or ascorbic acid, suggesting the active compound to be an oxidation product. A high-performance liquid chromatography method and a liquid chromatography-mass spectrometry method were developed to investigate the degradation products in the aged aloin solution. We found that the major compounds in the solution were aloin A and aloin B. In addition, 10-hydroxy aloin and elgonica dimers were detected in smaller amounts. The identified compounds were isolated and tested for activity by means of Thioflavin T assays, but no activity was observed. Thus, the actual fibrillation inhibitor is an as yet unidentified and potentially metastable degradation product of aloin. These results suggest that degradation products, and in particular oxidation products, are to be considered thoroughly when natural products are investigated for activity against protein fibrillation.


Subject(s)
Emodin/analogs & derivatives , Insulin/chemistry , Anthraquinones/chemistry , Benzothiazoles , Biological Products , Chemistry, Pharmaceutical , Chromatography, High Pressure Liquid , Emodin/chemistry , Excipients/chemistry , Fluorescent Dyes/chemistry , Glucosides/chemistry , Microscopy, Electron, Transmission , Oxidation-Reduction , Protein Stability , Proteolysis , Solutions , Spectrometry, Fluorescence , Thiazoles/chemistry
14.
Phytochemistry ; 119: 62-9, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26386983

ABSTRACT

Inhibition of the necrotizing hyaluronidase, phospholipase A2 and protease enzymes in four snake venoms by crude water and ethanol extracts of 88 plant species used against snakebites in traditional Chinese medicine was measured. High-resolution hyaluronidase inhibition profiles were constructed for the 22 plants showing highest hyaluronidase inhibition, and the results were used to guide subsequent structural analysis towards specific hyaluronidase inhibitors. Structural analysis was performed by high-performance liquid chromatography, high-resolution mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy, i.e., HPLC-HRMS-SPE-NMR. This allowed identification of four non-tannin inhibitors, i.e., lansiumamide B (6) from Clausena excavata Burm.f., myricetin 3-O-ß-D-glucopyranoside (7) from Androsace umbellata (Lour.) Merr., and vitexin (8) and 4',7-dihydroxy-5-methoxyflavone-8-C-ß-D-glucopyranoside (9) from Oxalis corniculata L. Absolute configuration of 2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide (1) was determined using the Mosher method, which revealed two enantiomers, i.e., (2S,3R)-2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide and (2R,3S)-2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide with a ratio of 7:3.


Subject(s)
Hyaluronoglucosaminidase/antagonists & inhibitors , Phospholipase A2 Inhibitors/isolation & purification , Phospholipase A2 Inhibitors/pharmacology , Snake Bites/drug therapy , Tannins/isolation & purification , Tannins/pharmacology , Apigenin/isolation & purification , Chromatography, High Pressure Liquid , Glycoside Hydrolase Inhibitors/chemistry , Hyaluronoglucosaminidase/metabolism , Medicine, Chinese Traditional , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Oxalidaceae/chemistry , Phospholipase A2 Inhibitors/chemistry , Plant Extracts/chemistry , Solid Phase Extraction , Tannins/chemistry
15.
Eur J Med Chem ; 102: 574-81, 2015 Sep 18.
Article in English | MEDLINE | ID: mdl-26318064

ABSTRACT

Although cyclic peptide structures are usually investigated as highly constrained scaffolds, cyclic antimicrobial peptides of natural origin often feature flexible residues. Hereby we report our findings concerning a structure-activity study conducted on a model sequence by replacing a glycine residue with a variety of flexible residues (i.e. ω-amino and α,ω-diamino acids). The resulting library has been tested for antimicrobial activity against a wide range of clinically relevant pathogens as well as for toxicity to red blood cells. Circular dichroism and molecular modelling have been used to study changes in conformation. Increments as high as 16-fold in antimicrobial activity (as effective as lipidation) and >2-fold in haemolytic EC50 values were observed. Interestingly, secondary structures can be stabilized by increasing, rather than decreasing, ring flexibility.


Subject(s)
Amino Acids/chemistry , Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Glycine/chemistry , Peptides, Cyclic/pharmacology , Surface-Active Agents/pharmacology , Anti-Bacterial Agents/chemistry , Antifungal Agents/chemistry , Bacteria/drug effects , Dose-Response Relationship, Drug , Fungi/drug effects , Hemolysis/drug effects , Microbial Sensitivity Tests , Molecular Structure , Peptides, Cyclic/chemistry , Structure-Activity Relationship , Surface-Active Agents/chemistry
16.
J Chromatogr A ; 1408: 125-32, 2015 Aug 21.
Article in English | MEDLINE | ID: mdl-26187760

ABSTRACT

In this work, development of a new microplate-based high-resolution profiling assay using recombinant human aldose reductase is presented. Used together with high-resolution radical scavenging and high-resolution α-glucosidase assays, it provided the first report of a triple aldose reductase/α-glucosidase/radical scavenging high-resolution inhibition profile - allowing proof of concept with Radix Scutellariae crude extract as a polypharmacological herbal drug. The triple bioactivity high-resolution profiles were used to pinpoint bioactive compounds, and subsequent structure elucidation was performed with hyphenated high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy. The only α-glucosidase inhibitor was baicalein, whereas main aldose reductase inhibitors in the crude extract were baicalein and skullcapflavone II, and main radical scavengers were ganhuangemin, viscidulin III, baicalin, oroxylin A 7-O-glucuronide, wogonoside, baicalein, wogonin, and skullcapflavone II.


Subject(s)
Aldehyde Reductase/metabolism , Free Radical Scavengers/analysis , Hypoglycemic Agents/analysis , Scutellaria baicalensis/chemistry , alpha-Glucosidases/metabolism , Chromatography, High Pressure Liquid , Glycoside Hydrolase Inhibitors/analysis , Humans , Magnetic Resonance Spectroscopy/methods , Plant Extracts/analysis , Recombinant Proteins/metabolism , Solid Phase Extraction
17.
J Pharm Biomed Anal ; 107: 333-40, 2015 Mar 25.
Article in English | MEDLINE | ID: mdl-25645337

ABSTRACT

Concentrated solutions containing 6-aminocaproic acid and the excipients citric acid and sorbitol have been studied at temperatures of 50°C, 60°C, 70°C and 80°C as well as at 20°C. It has previously been reported that the commonly employed citric acid is a reactive excipient, and it is therefore important to thoroughly investigate a possible reaction between 6-aminocaproic acid and citric acid. The current study revealed the formation of 3-hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid between 6-aminocaproic acid and citric acid by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR). Less than 0.03% of 6-aminocaproic acid was converted to 3-hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid after 30 days of storage at 80°C. Degradation products of 6-aminocaproic acid were also observed after storage at the applied temperatures, e.g., dimer, trimer and cyclized 6-aminocaproic acid, i.e., caprolactam. No reaction products between D-sorbitol and 6-aminocaproic acid could be observed. 3-Hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid, dimer and caprolactam were also observed after storage at 20°C for 3 months. The findings imply that an oral solution of 6-aminocaproic acid is relatively stable at 20°C at the pH values 4.00 and 5.00 as suggested in the USP for oral formulations. Compliance with the ICH guideline Q3B is expected.


Subject(s)
Aminocaproic Acid/chemistry , Citric Acid/chemistry , Excipients/chemistry , Pharmaceutical Solutions/chemistry , Administration, Oral , Chemistry, Pharmaceutical/methods , Drug Contamination , Magnetic Resonance Spectroscopy/methods , Mass Spectrometry/methods , Sorbitol/chemistry , Temperature
18.
J Nat Prod ; 78(2): 294-300, 2015 Feb 27.
Article in English | MEDLINE | ID: mdl-25679337

ABSTRACT

The crude methanol extract of Pueraria lobata was investigated by dual high-resolution α-glucosidase inhibition and radical scavenging profiling combined with hyphenated HPLC-HRMS-SPE-NMR. Direct analysis of the crude extract without preceding purification was facilitated by combining chromatograms from two analytical-scale HPLC separations of 120 and 600 µg on-column, respectively. High-resolution α-glucosidase and radical scavenging profiles were obtained after microfractionation of the eluate in 96-well microplates. This allowed full bioactivity profiling of individual peaks in the HPLC chromatogram of the crude methanol extract. Subsequent HPLC-HRMS-SPE-NMR analysis allowed identification of 21 known compounds in addition to two new compounds, i.e., 3'-methoxydaidzein 8-C-[α-D-apiofuranosyl-(1→6)]-ß-D-glucopyranoside and 6″-O-malonyl-3'-methoxydaidzin, as well as an unstable compound tentatively identified as 3'-de-O-methylpuerariafuran.


Subject(s)
Glucosides/isolation & purification , Glucosides/pharmacology , Glycoside Hydrolase Inhibitors/isolation & purification , Glycoside Hydrolase Inhibitors/pharmacology , Isoflavones/isolation & purification , Isoflavones/pharmacology , Pueraria/chemistry , alpha-Glucosidases/drug effects , Chromatography, High Pressure Liquid , Glucosides/chemistry , Glycoside Hydrolase Inhibitors/chemistry , Isoflavones/chemistry , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Plant Extracts/chemistry , Solid Phase Extraction
19.
Food Chem ; 161: 192-8, 2014 Oct 15.
Article in English | MEDLINE | ID: mdl-24837940

ABSTRACT

Bulbs and leaves of 35 Allium species and cultivars bought or collected in 2010-2012 were investigated with multivariate data analysis, high-resolution α-glucosidase inhibition assays and HPLC-HRMS-SPE-NMR with the aim of exploring the potential of Allium as a future functional food for management of type 2 diabetes. It was found that 30 out of 106 crude extracts showed more than 80% inhibition of the α-glucosidase enzyme at a concentration of 40mg/mL (dry sample) or 0.4g/mL (fresh sample). High-resolution α-glucosidase biochromatograms of these extracts allowed fast identification of three analytes with α-glucosidase inhibitory activity, and subsequent HPLC-HRMS-SPE-NMR experiments allowed identification of these as N-p-coumaroyloctopamine, N-p-coumaroyltyramine, and quercetin. The distribution of these three compounds was mapped for all samples by HPLC-ESI-HRMS. Unsupervised principal component analysis of samples from 2012 indicated that a major difference between fresh material and dried material is the increased amount of quercetin, a known α-glucosidase inhibitor.


Subject(s)
Allium/chemistry , Chromatography, High Pressure Liquid/methods , Magnetic Resonance Spectroscopy/methods , Glycoside Hydrolase Inhibitors , Multivariate Analysis , Principal Component Analysis
20.
Mar Drugs ; 12(4): 2228-44, 2014 Apr 10.
Article in English | MEDLINE | ID: mdl-24727393

ABSTRACT

Extracts of five halophytes from southern Portugal (Arthrocnemum macrostachyum, Mesembryanthemum edule, Juncus acutus, Plantago coronopus and Halimione portulacoides), were studied for antioxidant, anti-inflammatory and in vitro antitumor properties. The most active extracts towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were the methanol extracts of M. edule (IC50 = 0.1 mg/mL) and J. acutus (IC50 = 0.4 mg/mL), and the ether extracts of J. acutus (IC50 = 0.2 mg/mL) and A. macrostachyum (IC50 = 0.3 mg/mL). The highest radical scavenging activity (RSA) against the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical was obtained in the ether extract of J. acutus (IC50 = 0.4 mg/mL) and H. portulacoides (IC50 = 0.9 mg/mL). The maximum total phenolic content (TPC) was found in the methanol extract of M. edule (147 mg gallic acid equivalents (GAE)/g) and in the ether extract of J. acutus (94 mg GAE/g). Significant decreases in nitric oxide (NO) production were observed after incubation of macrophages with lipopolysaccharide (LPS) and the chloroform extract of H. portulacoides (IC50 = 109 µg/mL) and the hexane extract of P. coronopus (IC50 = 98.0 µg/mL). High in vitro cytotoxic activity and selectivity was obtained with the ether extract of J. acutus. Juncunol was identified as the active compound and for the first time was shown to display selective in vitro cytotoxicity towards various human cancer cells.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antioxidants/pharmacology , Plant Extracts/pharmacology , Salt-Tolerant Plants/chemistry , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/isolation & purification , Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Antioxidants/chemistry , Antioxidants/isolation & purification , Biphenyl Compounds/pharmacology , Cell Line , Cell Line, Tumor , Free Radical Scavengers/chemistry , Free Radical Scavengers/isolation & purification , Free Radical Scavengers/pharmacology , Humans , Inhibitory Concentration 50 , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Mice , Nitric Oxide/metabolism , Phenols/chemistry , Phenols/isolation & purification , Picrates/pharmacology , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Portugal
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