Screening for acetylcholinesterase inhibition, lipid peroxidation inhibition and antioxidant activity of medicinal plants from Morocco / Detección de inhibición de acetilcolinesterasa, inhibición de peroxidación de lípidos y actividad antioxidante de plantas medicinales de Marruecos

Acetylcholinesterase (AChE), hydrolyzes acetylcholine to choline and acetate, thereby terminating this neurotransmitter effect at cholinergic synapses. Therefore, AChE inhibition is used for counterbalance the cholinergic deficit in Alzheimer's disease (AD) patients. In the present work, in order to find new plant acetylcholinesterase inhibitors, the hydroalcoholic extracts from seventeen medicinal plant species were screened for their acetylcholinesterase inhibition activity, as well as total phenolic (TPC) and flavonoids contents (TFC) and antioxidant activity using ORAC (Oxygen Radical Absorbance Capacity) assay, and their ability to inhibit lipid peroxidation. The results revealed that Rumex acetosa, Taraxacum officinale and Hypericum perforatum extracts possessing the highest TPC and TFC, were the most effective in terms of ORAC antioxidant activity, and acetylcholinesterase inhibition, in addition to their ability to inhibit liposomes peroxidation, suggesting that those plant species may provide a substantial source of secondary metabolites, which act as natural antioxidants and acetylcholinesterase inhibitors, and may be beneficial in the treatment of AD.

La acetilcolinesterasa (AChE) hidroliza la acetilcolina se hidroliza en colina y acetato, terminando así este efecto neurotransmisor en las sinapsis colinérgicas. Por lo tanto, la inhibición de la AChE se utiliza para contrarrestar el déficit colinérgico en pacientes con enfermedad de Alzheimer (EA). En el presente trabajo, con el fin de encontrar nuevos inhibidores de la acetilcolinesterasa vegetal, se analizaron los extractos hidroalcohólicos de diecisiete especies de plantas medicinales para determinar su actividad inhibidora de la acetilcolinesterasa, así como el contenido total de fenólicos (TPC) y flavonoides (TFC) y la actividad antioxidante utilizando ORAC (Capacidad de absorbancia de radicales de oxígeno) y su capacidad para inhibir la peroxidación de lípidos. Los resultados revelaron que los extractos de Rumexacetosa, Taraxacum officinale e Hypericum perforatum que poseen los más altos TPC y TFC, fueron los más efectivos en términos de actividad antioxidante ORAC e inhibición de acetilcolinesterasa, además de su capacidad para inhibir la peroxidación de los liposomas, sugiriendo que esas especies de plantas puede proporcionar una fuente sustancial de metabolitos secundarios, que actúan como antioxidantes naturales e inhibidores de la acetilcolinesterasa, y puede ser beneficioso en el tratamiento de la EA.

Chemical and Biological Properties of Three Poorly Studied Species of Lycium Genus-Short Review.

The genus Lycium belongs to the Solanaceae family and comprises more than 90 species distributed by diverse continents. Lycium barbarum is by far the most studied and has been advertised as a "superfood" with healthy properties. In contrast, there are some Lycium species which have been poorly studied, although used by native populations. L. europaeum, L. intricatum and L. schweinfurthii, found particularly in the Mediterranean region, are examples of scarcely investigated species. The chemical composition and the biological properties of these species were reviewed. The biological properties of L. barbarum fruits are mainly attributed to polysaccharides, particularly complex glycoproteins with different compositions. Studies regarding these metabolites are practically absent in L. europaeum, L. intricatum and L. schweinfurthii. The metabolites isolated and identified belong mainly to polyphenols, fatty acids, polysaccharides, carotenoids, sterols, terpenoids, tocopherols, and alkaloids (L. europaeum); phenolic acids, lignans, flavonoids, polyketides, glycosides, terpenoids, tyramine derivatives among other few compounds (L. schweinfurthii), and esters of phenolic acids, glycosides, fatty acids, terpenoids/phytosterols, among other few compounds (L. intricatum). The biological properties (antioxidant, anti-inflammatory and cytotoxic against some cancer cell lines) found for these species were attributed to some metabolites belonging to those compound groups. Results of the study concluded that investigations concerning L. europaeum, L. intricatum and L. schweinfurthii are scarce, in contrast to L. barbarum.

Edible Coatings Enriched with Essential Oils on Apples Impair the Survival of Bacterial Pathogens through a Simulated Gastrointestinal System.

Edible coatings supplemented with essential oil components have been investigated to control spoilage microorganisms. In this study, the survival of Listeria monocytogenes and Salmonella enterica serovar Typhimurium on apples treated with edible coatings based on sodium alginate (2%) (ECs) and supplemented with essential oil components, namely eugenol (Eug) at 0.2% or in combination with 0.1% (v/v) of Eug and citral (Cit) at 0.15% was determined. Both bacterial pathogens were exposed on apples treated with ECs supplemented with Eug or Eug + Cit and challenged with gastrointestinal fluids and their survival was examined. Both pathogens were able to survive on the surface of 'Bravo de Esmolfe' apple. The use of ECs in fresh-cut fruits impaired the survival of both bacterial populations over 72 h at 4 °C. The exposure of the pathogens on apples with ECs supplemented with Eug and Cit and challenged with gastrointestinal fluids significantly reduced their survival. This study evidences that the use of alginate edible coating enriched with Eug or the combination of Eug and Cit can contribute to the safer consumption of minimally processed fruits.

Moroccan Propolis: A Natural Antioxidant, Antibacterial, and Antibiofilm against Staphylococcus aureus with No Induction of Resistance after Continuous Exposure.

This study was performed to evaluate the total phenols, flavonoids, and antioxidant activities of twenty-four propolis samples from different regions of Morocco. In addition, two samples were screened regarding the antibacterial effect against four Staphylococcus aureus strains. Gas chromatography coupled to mass spectra (GC-MS) analysis was done for propolis samples used in antibacterial tests. The minimum inhibitory and minimum bactericidal concentration (MIC, MBC) were determined. The potential to acquire the resistance after sequential exposure of bacterial strains and the impact of adaptation to propolis on virulence using the Galleria mellonella were evaluated. Additionally, the effects of propolis extract on the bacterial adherence ability and its ability to inhibit the quorum sensing activity were also examined. Among the twenty-four extracts studied, the samples from Sefrou, Outat el Haj, and the two samples marketed in Morocco were the best for scavenging DPPH, ABTS, NO, peroxyl, and superoxide radicals as well as in scavenging of hydrogen peroxide. A strong correlation was found between the amounts of phenols, flavonoids, and antioxidant activities. Propolis extract at the MIC value (0.36 mg/mL) significantly reduced (p < 0.001) the virulence potential of S. aureus ATCC 6538 and the MRSA strains without leading to the development of resistance in the sequence of continuous exposure. It was able to impair the bacterial biofilm formation. The results have revealed that sample 1 reduces violacein production in a concentration dependent manner, indicating inhibition of quorum sensing. This extract has as main group of secondary metabolites flavonoids (31.9%), diterpenes (21.5%), and phenolic acid esters (16.5%).

Antioxidant and anti-lipoxygenase activities of extracts from different parts of Lavatera cretica L. grown in Algarve (Portugal).

BACKGROUND: Lavatera cretica L. was used in folk medicine as anti-inflammatory among other applications. As inflammation is many times associated with oxidative processes, the aim of the present work was to evaluate the ability of aqueous extracts obtained from different parts of L. cretica to prevent oxidation or inflammation using several methods in vitro. MATERIALS AND METHODS: The capacity of samples for preventing lipid peroxidation, scavenging free radicals, chelating metal ions, reducing power, and inhibiting lipoxygenase activity was investigated. This last assay also permits to evaluate the anti-inflammatory activity. The quantification of total phenols was performed using Folin-Chiocalteu reagent. RESULTS: The highest concentrations of total polyphenols and flavonoids were found in the leaf extract (254.62 ± 6.50 mg gallic acid equivalent/gram; dry weight). Leaf and flower extracts were the most active for scavenging 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt free radicals [Inhibition concentration (IC 50 = 2.88 ± 0.54 and IC50 = 4.37 ± 0.54 ΅g/mL, respectively)], and leaf extract was also the best for scavenging hydroxyl radicals (IC50 = 0.81 ± 0.05 µg/mL). Bract plus sepal extract possessed the best capacity for preventing lipid peroxidation when lecithin liposome was the lipid substrate (IC50 = 0.19 ± 0.03 µg/mL) and scavenging superoxide anion radicals (IC50 = 1.13 ± 0.48 µg/mL). Leaf and flower extracts were the best lipoxygenase inhibitors (IC50 = 0.013 ± 0.0034 µg/mL in both extracts). CONCLUSIONS: L. cretica extracts were able to scavenge free radicals, inhibit lipid peroxidation and lipoxygenase activity. With these attributes, this plant can have an important role in the treatment of neurodegenerative disorders.

In depth analysis of the quenching of three fluorene-phenylene-based cationic conjugated polyelectrolytes by DNA and DNA bases.

The interaction of three cationic poly {9,9-bis[N,N-(trimethylammonium)hexyl]fluorene-co-1,4-phenylene} polymers with average chain lengths of ∼6, 12, and 100 repeat units (PFP-NR36(I),12(Br),100(Br)) with both double and single stranded, short and long, DNA and DNA bases have been studied by steady state and time-resolved fluorescence techniques. Fluorescence of PFP-NR3 polymers is quenched with high efficiency by DNA (both double and single stranded) and DNA bases. The resulting quenching plots are sigmoidal and are not accurately described by using a Stern-Volmer quenching mechanism. Here, the quenching mechanism is well modeled in terms of an equilibrium in which a PFP-NR3/DNA aggregate complex is formed which brings polymer chains into close enough proximity to allow interchain excitation energy migration and quenching at aggregate or DNA base traps. Such an analysis gives equilibrium constants of 8.4 × 10(6) (±1.2 × 10(6)) M(-1) for short-dsDNA and 8.6 × 10(6) (±1.7 × 10(6)) M(-1) for short-ssDNA with PFP-NR36(I).

Effect of aggregation on the photophysical properties of three fluorene-phenylene-based cationic conjugated polyelectrolytes.

The effect of aggregation on the photophysical properties of three cationic poly{9,9-bis[N,N-(trimethylammonium)hexyl] fluorene-co-l,4-phenylene} polymers with average chain lengths of ∼6, 12, and 100 repeat units (PFP-NR3(6(I),12(Br),100(Br))) has been studied by steady-state and time-resolved fluorescence techniques. Conjugated polyelectrolytes are known to aggregate in solution and for these PFP-NR3 polymers this causes a decrease in the fluorescence quantum yield. The use of acetonitrile as a cosolvent leads to the breakup of aggregates of PFP-NR3 in water; for PFP-NR3(6(I)), this results in an ∼10-fold increase in fluorescence quantum yield, a ca. 2-fold increase in the molar extinction coefficient at 380 nm, and an increase in the emission lifetime, as compared with polymer behavior in water. Fluorescence anisotropy also decreases with increasing aggregation, and this is attributed to increased fluorescence depolarization by interchain energy transfer in aggregate PFP-NR3 clusters. Förster resonance energy transfer along the polymer chain is expected to be very fast, with a calculated FRET rate constant of 7.3 × 10(12) s(-1) and a Förster distance of 2.83 nm (cf. the polymer repeat unit separation of 0.840 nm) for PFP-NR3(100(Br)). The complex polymer excited-state decay kinetics in aggregated PFP-NR3 systems have been successfully modeled in terms of intrachain energy transfer via migration and trapping at interchain aggregate trap sites, with model parameters in good agreement with data from picosecond time-resolved studies and the calculated theoretical Förster energy-transfer rates.

Inclusion of a single-tail amino acid-based amphiphile in a lipoplex formulation: effects on transfection efficiency and physicochemical properties.

Effects of the addition of a cationic amino acid-based synthetic amphiphile, arginine N-lauroyl amide dihydrochloride (ALA), to a lipid-based transfection formulation have been investigated. It is shown that the inclusion of ALA results in a substantial enhancement of the transfection capability of lipoplexes prepared with liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine and cholesterol, which themselves mediate highly efficient transfection. A possible explanation for the increased biological activity is that ALA adsorbed to the surface of the DNA-lipid complexes is involved in triggering internalization. However, in order to identify possible additional factors underlying the enhanced transfection efficiency, the physical properties of formulations with and without ALA were characterized using cryo-transmission electron microscopy, dynamic light scattering, and an ethidium bromide intercalation assay. ALA seems to have limited influence on the initial internal structure of the complexes and the protection of DNA, but its presence is found to decrease the average effective size of the dispersed particles; this change in size may be important in improving the biological activity. Furthermore, ALA can act to influence the transfection efficiency of the formulation by promoting the release of DNA following internalization in the transfected cells.

Size and morphology of assemblies formed by DNA and lysozyme in dilute aqueous mixtures.

Assemblies formed by a well-defined quality of DNA (4331 bp T7 DNA) and the small net-cationic protein lysozyme in dilute aqueous solutions have been characterized using cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS) as the main techniques. In a wide range of different DNA to lysozyme ratios in solutions of low ionic strength, dispersions of aggregates with the same general morphology and a practically constant hydrodynamic size are formed. The basic structure formed in the dispersions is that of rather flexible worm-like assemblies with a diameter of 10-20 nm, which are suggested to be made up by bundles of on the order of 10 DNA chains with an intervening matrix of lysozyme. With increased ionic strength, the worm-like appearance of the assemblies is lost and they adopt a less well-defined shape. The results suggest that the formation of the DNA-lysozyme aggregates is strongly influenced by cooperative assembly of the components and that, in addition to the electrostatic attraction between DNA and lysozyme, attractive interactions between the protein units are important in governing the behavior of the system.

Interactions between cationic lipid bilayers and model chromatin.

Complexes formed in mixtures of cationic liposomes of varying charge density and nucleosome core particles (NCPs) or nucleosome arrays have been characterized. Under most of the conditions studied, the lipids and NCPs or arrays formed lamellar structures similar to those obtained with the liposomes and pure DNA. Thus, the dissociation of DNA from the NCP or nucleosome array and the formation of a DNA-lipid complex is thermodynamically favored, which can likely be ascribed mainly to the gain in entropy on release of the small counterions. Only at very low liposome charge densities are there indications that the NCPs/arrays do not dissociate upon interaction with the lipid bilayers. The reported results can serve as a valuable reference point in investigations of biologically more relevant systems.

Phase behavior and coassembly of DNA and lysozyme in dilute aqueous mixtures: a model investigation of DNA-protein interactions.

Results from an experimental investigation of the phase behavior of an aqueous system of DNA from salmon testes and the protein lysozyme are presented. At very low concentrations of either or both of the macromolecular components, wormlike assemblies with a width of the order of 10 nm are formed. There are strong indications that direct interactions between the protein units are instrumental both in driving the phase separation and in controlling the morphology of the formed assemblies.

Cationic fluorene-based conjugated polyelectrolytes induce compaction and bridging in DNA.

The cationic conjugated polymer (CCP), poly{9,9-bis[N,N-(trimethylammonium)hexyl]fluorene-co-1,4-phenylene} iodide (PFP-NR3) induces compaction in DNA in an acetonitrile/water mixture, as seen by fluorescence microscopy. At high concentrations of PFP-NR3, some chain structures still appear to exist. However, these are larger than normal DNA coils and have "beads" of enhanced fluorescence along the chain. These structures have also been imaged on freshly cleaved mica surfaces using atomic force microscopy. Images obtained following deposition onto mica from mixtures of DNA and the polyelectrolyte PFP-NR3 in acetonitrile/water 25:75 show both the efficient compaction of DNA induced by the polymer and linking and bridging of DNA/PFP-NR3 aggregates. The strong interaction between DNA and PFP-NR3 results in the formation of DNA/PFP-NR3 networks across the mica surface in which several strands of DNA are linked with aggregated polymer/DNA structures at various points along these chains. The linking of DNA strands is confirmed by a large increase in the apparent length of the DNA, which increases from 775 (+/-82) nm (with no PFP-NR3 present) to 4050 (+/-800) nm in the presence of PFP-NR3. Larger aggregates, believed to be PFP-NR3/compacted DNA, which also link other DNA strands, can also be seen. The fluorescence of PFP-NR3 is quenched by DNA, and this is accompanied by a bathochromic wavelength shift of the CCP emission, indicating complexation. Although the quenching mechanism is not yet clear, it appears to be a consequence of DNA-CCP aggregate formation. These results have implications on the use of CCPs in DNA sensing and, because the particular polymer has a rigid backbone, on the effect of chain rigidity on compaction and on formation of extended and supramolecular structures, which may have implications in DNA nanotechnology.

DNA encapsulation by biocompatible catanionic vesicles.

The encapsulation of DNA by catanionic vesicles has been investigated; the vesicles are composed of one cationic surfactant, in excess, and one anionic. Since cationic systems are often toxic, we introduced a novel divalent cationic amino-acid-based amphiphile, which may enhance transfection and appears to be nontoxic, in our catanionic vesicle mixtures. The cationic amphiphile is arginine-N-lauroyl amide dihydrochloride (ALA), while the anionic one is sodium cetylsulfate (SCS). Vesicles formed spontaneously in aqueous mixtures of the two surfactants and were characterized with respect to internal structure and size by cryogenic transmission electron microscopy (cryo-TEM); the vesicles are markedly polydisperse. The results are compared with a study of an analogous system based on a short-chained anionic surfactant, sodium octylsulfate (SOS). Addition of DNA to catanionic vesicles resulted in associative phase separation at very low DNA concentrations; there is a separation into a precipitate and a supernatant solution; the latter is first bluish but becomes clearer as more DNA is added. From studies using cryo-TEM and small angle X-ray scattering (SAXS) it is demonstrated that there is a lamellar structure with DNA arranged between the amphiphile bilayers. Comparing the SOS containing DNA-vesicle complexes with the SCS ones, an increase in the repeat distance is perceived for SCS. Regarding the phase-separating DNA-amphiphile particles, cryo-TEM demonstrates a large and nonmonotonic variation of particle size as the DNA-amphiphile ratio is varied, with the largest particles obtained in the vicinity of overall charge neutrality. No major differences in phase behavior were noticed for the systems here presented as compared with those based on classical cationic surfactants. However, the prospect of using these systems in real biological applications offers a great advantage.