Volatile-oils composition, and bioactivity of the essential oils of Plectranthus barbatus, P. neochilus, and P. ornatus grown in Portugal.

Volatile-oils chemical composition and bioactivity of the essentail oils from Plectranthus barbatus, P. neochilus, and P. ornatus (Lamiaceae) were assessed. Aerial parts from these three related Plectranthus species were collected from cultivated plants grown in Portugal, during vegetative and flowering phases. Volatiles, isolated by distillation-extraction, were analyzed by GC and GC/MS. Monoterpene hydrocarbons (12-74%) and sesquiterpene hydrocarbons (4-45%) constituted the main fractions in all volatiles. α-Pinene (3; 12-67%), oct-1-en-3-ol (6; traces-28%), ß-pinene (7; 0.1-22%), and ß-caryophyllene (50; 7-12%) dominated P. barbatus volatiles. P. neochilus major volatile components were α-terpenyl acetate (41; traces-48%), α-thujone (2; 2-28%), ß-caryophyllene (50; 2-28%), ß-pinene (7; 1-25%), and α-pinene (3; 1-19%). Oct-1-en-3-ol (6; 13-31%), ß-pinene (7; 11-24%), α-pinene (3; 11-19%), and ß-caryophyllene (50; traces-11%) were the main constituents from P. ornatus volatiles. These chemical compositions were rather different from those previously found for specimens harvested in Africa and Brazil. Moreover, the volatiles from the flowers are herewith reported for the first time. Essential oils, isolated by hydrodistillation from leaves and stems, showed a yellowish color and unpleasant odor, with yields ranging from 0.08% to 0.84% (v/dry weight). Antioxidant and antimicrobial activities of the essential oils were evaluated by DPPH· and TBARS assays, and agar disc-diffusion method, respectively. Results showed low or moderate antioxidant capacity and significant antimicrobial activity against Gram-positive bacteria.

Gel network photodisruption: a new strategy for the codelivery of plasmid DNA and drugs.

In the last 5 years, we have gained further insight on the physical/chemical field of DNA gels. Our expertise on the gel swelling behavior, compaction of DNA by cationic entities, as lipids and surfactants, as well as on the assembly structures of these complexes allow us for the development of novel systems to be used in a variety of biomedical applications. In our previous reports, the physicochemical characterization has been well-established, and now one can evolve to the challenge of using DNA-based carriers in the biological area. Moreover, a new plasmid DNA (pDNA) hydrogel that is porous, is able to swell in the presence of additives, is biocompatible and, thus, is suitable to be used therapeutically was prepared. Here, the dual release of pDNA and solutes with pharmaceutical interest was the main challenge, and thus, we report on the photodisruption of plasmid DNA (pDNA) gels cross-linked with ethylene glycol diglycidyl ether (EGDE) as a strategy for this simultaneous release. The disruption over time, after the irradiation of the gel with ultraviolet light (400 nm), was characterized through the cumulative plasmid DNA release, the evolution in dry weight, the extent of swelling, and also the variations in the gel mesh size. The controlled release of different molecular weight solutes from plasmid DNA gels was investigated, and the influence of both the hydrogel degradation and cross-linker density on the release kinetics were addressed. While the release of lysozyme follows a Fickian process, the release of bovine serum albumin (BSA) and fluoresceinisothiocyanato-dextran (FITC-dextran) is characteristic of a Super Case II release phenomena. In addition, the size of the three solutes partially influences the release behavior; polymer chain mobility and the degree of swelling also play a role. To gain a fundamental understanding of drug release profile from pDNA matrices, in vitro release studies were evaluated using several anti-inflammatory drugs. The quantification of the release mechanism indicates a Super Case II release profile, which can be related with the gel swelling degree. A correlation between the drug release trend and the drug hydrophobicity can be found, with more hydrophobic drugs showing a slower release rate. In brief, this new pDNA gel system is biocompatible, is degradable upon light irradiation, and allows for the controlled and sustained release of plasmid DNA and incorporated solutes. This codelivery of pDNA and drugs would find relevant clinical uses due to the possibility of gene and nongene therapy combination in order to improve the therapeutic efficiency.

Novel biocompatible DNA gel particles.

Surfactants with the cationic functionality based on an amino acid structure have been used to prepare novel biocompatible devices for the controlled encapsulation and release of DNA. We report here the formation of DNA gel particles mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with two different single-chain amino acid-based surfactants: arginine-N-lauroyl amide dihydrochloride (ALA) and N(alpha)-lauroyl-arginine-methyl ester hydrochloride (LAM). The degree of DNA entrapment, the swelling/deswelling behavior, and the DNA release kinetics have been studied as a function of both the number of charges in the polar head of the amino acid-based surfactant and the secondary structure of the nucleic acid. Analysis of the data indicates a stronger interaction of ALA with DNA, compared with LAM, mainly attributed to the double charge carried by the former surfactant compared to the singly charged headgroup of the latter species. The stronger interaction with amphiphiles for ssDNA compared with dsDNA suggests the important role of hydrophobic interactions in DNA. Data on the microstructure of the complexes obtained from small-angle X-ray scattering (SAXS) of the particles strongly suggests a hexagonal packing. It was found that, the shorter the lattice parameter, the stronger the surfactant-DNA interaction and the slower the DNA release kinetics. Complexation and neutralization of DNA on the DNA gel particles was confirmed by agarose gel electrophoresis measurements.

Controlling the morphology in DNA condensation and precipitation.

This work addresses the influence of solution inhomogeneity on conformation, aggregation, and coil/globule and bundle/single chain coexistence of T4 DNA molecules. The inhomogeneity is induced by mixing two solutions containing, respectively, protamine and DNA, with different relative concentrations, but aiming at producing the same final concentrations. The study was conducted by means of fluorescence microscopy (FM), complemented with scanning electron microscopy (SEM). It is shown that the degree of precipitation, the structures formed, and the relative population of compacted and unfolded structures are highly dependent on the method of preparation of the mixtures that contain the DNA/protamine complexes. Most of the structures reported in the literature, that is, overcharged/undercharged globules, toroids, chains internally segregated, and bundles composed of several chains were observed in our different mixtures of fixed final concentration.

Cationic agents for DNA compaction.

Fluorescence microscopy was used to investigate the conformational changes of individual T4 DNA molecules induced by different compacting agents, namely the cationic surfactants, cetyltrimethylammonium bromide (CTAB) and chloride (CTAC), iron(III), lysozyme, and protamine sulfate. A protocol for establishing size estimates is suggested to obtain reproducible results. Observations show that in the presence of lysozyme and protamine sulfate, DNA molecules exhibit a conformational change from an elongated coil structure to compact globules, usually interpreted as a first-order transition. The maximum degree of compaction that is attained when iron(III) or CTAB (CTAC) are used as compacting agents is considerably smaller, and intermediate structures (less elongated coils) are visible even for high concentrations of these agents. Dynamic light scattering experiments were carried out, for some of the systems, to assess the reliability of size estimates from fluorescence microscopy.

Effect of additives on swelling of covalent DNA gels.

The volumetric response of polymer gels on cosolute addition depends on the interaction of the polymer with the cosolute and can be used as a simple and sensitive way of elucidating these interactions. Here we report on DNA networks, prepared by crosslinking double-stranded DNA with ethylene glycol diglycidyl ether (EGDE); these have been investigated with respect to their swelling in aqueous solution containing different additives, such as metal ions, polyamines, charged proteins, and surfactants. The deswelling on addition of metal ions occurs at lower concentrations with increasing valency of the counterion. The collapse of the gels in the presence of trivalent ions seems to follow the same kind of mechanism as the interaction in solution, but addition of these ions leads to DNA denaturation and formation of single-stranded DNA. Striking features were found in the deswelling of DNA gels by chitosan, spermine, spermidine, lysozyme, poly-l-lysine and poly-l-arginine. Chitosan is the most efficient cosolute of those investigated with respect to DNA gel collapse. The effect of the cationic surfactant tail length on the volume phase transition of DNA gels was studied as a function of surfactant concentration. Cationic surfactants effectively collapsed the gel from the critical aggregation concentration (cac), decreasing with increasing length of the hydrophobic tail. In several cases, the deswelling as a function of cosolute concentration shows a pronounced two-step behavior, which is interpreted in terms of a combination of DNA chain condensation and general osmotic deswelling. The studies included investigations on the state of the DNA chain after deswelling, on the reversibility of the deswelling as well as on the kinetics. With the exception for the trivalent lanthanide ions, it appears that the DNA chain always retains a double-helix conformation; with these metal ions, single-stranded DNA is found. The deswelling appears to be reversible as exemplified by addition of anionic surfactant subsequent to gel collapsed by cationic surfactant and addition of sodium bromide to gels collapsed by a polycation. An investigation of the kinetics shows that an increase in the surfactant tail length gives a pronouncedly slower deswelling kinetics.

Responsive polymer gels: double-stranded versus single-stranded DNA.

Cross-linking of polyelectrolytes such as DNA gives gels that are osmotically highly swollen but contract upon addition of electrolytes and, in particular, upon association of oppositely charged cosolutes with the polyelectrolyte chain. The deswelling behavior of cross-linked DNA gels thus reflects the DNA-cosolute interactions and provides a basis for the development of responsive DNA formulations. Gels of both single- and double-stranded DNA have interesting applications, and a comparison between them provides the basis for understanding mechanisms. Denaturation of cross-linked ds-DNA gels was induced by heating them above the melting temperature and then cooling. This process, studied by fluorescence using ethidium bromide, appeared to be reversible when a heating/cooling cycle was performed. The swelling behavior upon addition of different cosolutes, such as metal ions, polyamines, charged proteins, and surfactants, was investigated for different DNA gel samples, including long and short ds-DNA and long and short ss-DNA. The DNA molecular weight was found to have only a slight effect on the deswelling curves, whereas conformation exhibited a pronounced effect. In general, single-stranded DNA gels exhibited a larger collapse in the presence of cations than did double-stranded DNA. This difference was more pronounced with surfactants than with the other cosolutes investigated. The difference between double- and single-stranded DNA was attributed to differences in linear charge density, chain flexibility, and hydrophobicity. For surfactants with different chain lengths, the swelling behavior displayed by ss-DNA can be interpreted in terms of an interplay between hydrophobic and electrostatic interactions, the latter being influenced by polymer flexibility. Increasing hydrophobicity of the network leads to a decreased critical aggregation concentration (cac) for the surfactant/gel complex, as a result of the strengthened hydrophobic attractive force between the surfactant and the gel chain. The swelling of DNA gels appears to be reversible and to be independent of DNA conformation. Surfactant-induced deswelling of DNA gels under some conditions appears to be quite homogeneous, whereas under other conditions, there is a separation into a collapsed region in the outer parts of the gel sample and an inside swollen part. Such "skin" formation is quite different for ss- and ds-DNA, with ss-DNA giving more pronounced skin formation over a wider range of binding ratio, beta. For example, no macroscopic separation into collapsed and swollen regions was observed at intermediate degrees of binding for ds-DNA gels, whereas a dense surfactant-rich surface phase (skin) was found to coexist with a swollen core network for ss-DNA gels with beta>0.5. One explanation for this difference is the large deformation energy required for the compression of the very stiff ds-DNA chains.

Surfactant-DNA gel particles: formation and release characteristics.

Aqueous mixtures of oppositely charged polyelectrolytes undergo associative phase separation, resulting in coacervation, gelation, or precipitation. This phenomenon has been exploited here to form DNA gel particles by interfacial diffusion. We report on the formation of DNA gel particles by mixing solutions of DNA (either single-stranded (ssDNA) or double-stranded (dsDNA)) with solutions of cationic surfactant cetyltrimetrylammonium bromide (CTAB). By using CTAB, the formation of DNA reservoir gel particles, without adding any kind of cross-linker or organic solvent, has been demonstrated. Particles have been characterized with respect to the degree of DNA entrapment, surface morphology, and secondary structure of DNA in the particles. The swelling/deswelling behavior and the DNA release have been investigated in response to salt additions. Analysis of the data has suggested a higher degree of interaction between ssDNA and the cationic surfactant, confirming the stronger amphiphilic character of the denatured DNA. Fluorescence microscopy studies have suggested that the formation of these particles is associated with a conservation of the secondary structure of DNA.

Composition of the essential oils of Thymus and Origanum species from Algeria and their antioxidant and antimicrobial activities.

The composition of the essential oils of Origanum and Thymus species restricted to Algeria and the North Africa region was determined. Antioxidant and antibacterial activities of the isolated essential oils were also determined. The oils of oregano plants were strongly characterized by p-cymene (16.8-24.9%), gamma-terpinene (16.8-24.9%), thymol (8.4-36.0%), and carvacrol (1.1-29.7%), a thymol chemotype for Origanum floribundum and a alpha-terpineol chemotype for Thymus numidicus being described for the first time. The strains of Listeria monocytogenes tested were relatively resistant to the action of essential oils of either Origanum or Thymus species. All essential oils possessed antioxidant activity, but this was dependent on the specific chemical composition and the method employed to determine such activity.

Plasmid DNA hydrogels for biomedical applications.

In the last few years, our research group has focused on the design and development of plasmid DNA (pDNA) based systems as devices to be used therapeutically in the biomedical field. Biocompatible macro and micro plasmid DNA gels were prepared by a cross-linking reaction. For the first time, the pDNA gels have been investigated with respect to their swelling in aqueous solution containing different additives. Furthermore, we clarified the fundamental and basic aspects of the solute release mechanism from pDNA hydrogels and the significance of this information is enormous as a basic tool for the formulation of pDNA carriers for drug/gene delivery applications. The co-delivery of a specific gene and anticancer drugs, combining chemical and gene therapies in the treatment of cancer was the main challenge of our research. Significant progresses have been made with a new p53 encoding pDNA microgel that is suitable for the loading and release of pDNA and doxorubicin. This represents a strong valuable finding in the strategic development of systems to improve cancer cure through the synergetic effect of chemical and gene therapy.

DNA gel particles: an overview.

A general understanding of interactions between DNA and oppositely charged compounds forms the basis for developing novel DNA-based materials, including gel particles. The association strength, which is altered by varying the chemical structure of the cationic cosolute, determines the spatial homogeneity of the gelation process, creating DNA reservoir devices and DNA matrix devices that can be designed to release either single- (ssDNA) or double-stranded (dsDNA) DNA. This review covers recent developments on the topic of DNA gel particles formed in water-water emulsion-type interfaces. The degree of DNA entrapment, particle morphology, swelling/dissolution behavior and DNA release responses are discussed as functions of the nature of the cationic agent used. On the basis of designing DNA gel particles for therapeutic purposes, recent studies on the determination of the surface hydrophobicity and the hemolytic and the cytotoxic assessments of the obtained DNA gel particles have been also reported.

PTEN mediates the antioxidant effect of resveratrol at nutritionally relevant concentrations.

INTRODUCTION: Antioxidant properties of resveratrol have been intensively studied for the last years, both in vivo and in vitro. Its bioavailability after an oral dose is very low and therefore it is very important to make sure that plasma concentrations of free resveratrol are sufficient enough to be active as antioxidant. AIMS: In the present study, using nutritionally relevant concentrations of resveratrol, we aim to confirm its antioxidant capacity on reducing peroxide levels and look for the molecular pathway involved in this antioxidant effect. METHODS: We used mammary gland tumor cells (MCF-7), which were pretreated with different concentrations of resveratrol for 48 h, and/or a PTEN inhibitor (bpV: bipy). Hydrogen peroxide levels were determined by fluorimetry, PTEN levels and Akt phosphorylation by Western Blotting, and mRNA expression of antioxidant genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Resveratrol treatment for 48 h lowered peroxide levels in MCF-7, even at low nutritional concentrations (1 nM). This effect was mediated by the activation of PTEN/Akt pathway, which resulted in an upregulation of catalase and MnSOD mRNA levels. CONCLUSION: Resveratrol acts as an antioxidant at nutritionally relevant concentrations by inducing the expression of antioxidant enzymes, through a mechanism involving PTEN/Akt signaling pathway.

DNA-poly(vinyl alcohol) gel matrices: release properties are strongly dependent on electrolytes and cationic surfactants.

The release of DNA from cryogel PVA-DNA gel matrices to different electrolyte aqueous solutions was investigated. The rate of release and the distribution coefficient of DNA have been quantified by using a first order kinetic law equation, developed in the frame of a partition-based model. The release of DNA from gels to 1:1 sodium and nitrate salts shows that the transport properties are dependent on the ability of anions/cations to solubilise the DNA in the aqueous phase which, with the exception of bromide, can be related to the Hofmeister series; in the presence of multivalent electrolytes, or increasing the ionic strength, the condensation of DNA inside the gel, followed by a phase separation as seen by scanning electron microscopy, induces the retention of DNA inside the polymer matrix. The DNA condensation and/or phase separation, which contribute to a decrease in the water volume fraction inside the gel, determined by swelling degree experiments, also lead to a decrease in the rate constant of DNA release; such decrease can be justified by the difficulty of the molecular aggregate to move through out the polymeric structure. The DNA release is also dependent on the pH of the bulk solution. The effect of uni- and di-valent cationic surfactants on the release properties of DNA was also evaluated. Our findings suggest that the kinetics of DNA release depends on a complex balance between different structural properties of the surfactants, namely charge, bulkiness of the headgroup and alkyl chain length.

Swelling behavior of a new biocompatible plasmid DNA hydrogel.

Chemical plasmid DNA (pDNA) gels were prepared by a cross-linking reaction with ethylene glycol diglycidyl ether (EGDE). Fluorescence microscopy (FM) and scanning electron microscopy (SEM) images of pDNA gels are reported. For the first time, the pDNA gels have been investigated with respect to their swelling in aqueous solution containing different additives, such as metal ions, polyamines, polycations and surfactants. The effect of the cationic surfactant tail length on the volume phase transition of pDNA gels was studied as a function of surfactant concentration; the critical aggregation concentration (cac), is found to decrease with increasing length of the hydrophobic tail. The deswelling appears to be reversible as exemplified by the addition of anionic surfactant subsequent to collapsing the gel by a cationic surfactant. Cell viability assays suggest that the plasmid DNA gels are non-toxic to cells and do not cause any distinct harm to them. This step contributes to the possibility of using these gels, as carriers, in real biological systems.

Plasmid DNA microgels for a therapeutical strategy combining the delivery of genes and anticancer drugs.

Plasmid DNA (pDNA) is encapsulated into biocompatible microgels by an inverse microemulsion polymerization method. Plasmid DNA and doxorubicin are successfully released from pDNA microgels and their release profiles are characterized by appropriate release models. The co-delivery of genes and drugs from the microgels is evaluated as an enhancer of clinical treatment. Moreover, the release of the encapsulated pDNA is capable of transfection in vitro resulting in the expression of p53 protein. As a whole, a novel pDNA-based system is described that may find biomedical uses, especially in the cancer treatment through the combined action of chemotherapy and gene delivery approach.

Swelling properties of cross-linked DNA gels.

This work represents our contribution to the field of physical chemistry of DNA gels, and concerns the synthesis and study of novel chemically cross-linked DNA gels. The use of covalent DNA gels is a very promising way to study DNA-cosolute interactions, as well as the dynamic behaviour of DNA and cationic compacting agents, like lipids, surfactants and polycations. Manipulating DNA in new ways, like DNA networks, allows a better understanding and characterization of DNA-cosolute complexes at the molecular level, and also allows us to follow the assembly structures of these complexes. The use of responsive polymer gels for targeted delivery of toxic and/or labile drugs has, during the past few years, shown to be a promising concept. The features found in the proposed system would find applications in a broader field of gel/drug interaction, for the development of controlled release and targeted delivery devices.

Mixed protein carriers for modulating DNA release.

Aqueous mixtures of oppositely charged polyelectrolytes undergo associative phase separation, resulting in coacervation, gelation, or precipitation. This phenomenon has been exploited in forming DNA gel particles by interfacial diffusion. We report here the formation of DNA gel particles by mixing solutions of double-stranded DNA with aqueous solutions containing two cationic proteins, lysozyme and protamine sulfate. The effect of the lysozyme/protamine ratio on the degree of DNA entrapment, surface morphology, swelling-deswelling behavior, and kinetics of DNA release has been investigated. By mixing the two proteins, we obtain particles that display higher loading efficiency and loading capacity values, in comparison to those obtained in single-protein systems. Examination of the release profiles has shown that in mixed protein particles, complex, dual-stage release kinetics is obtained. The overall release profile is dependent on the lysozyme/protamine ratio. The obtained profiles, or segments of them, are accuratelly fitted using the zero-order and first-order models, and the Weibull function. Fluorescence microscopy studies have suggested that the formation of these particles is associated with the conservation of the secondary structure of DNA. This study presents a new platform for controlled release of DNA from DNA gel particles formed by interfacial diffusion.

DNA gel particles: particle preparation and release characteristics.

Aqueous mixtures of oppositely charged polyelectrolytes undergo associative phase separation, resulting in coacervation, gelation, or precipitation. This phenomenon has been exploited here to form DNA gel particles by interfacial diffusion. We report the formation of DNA gel particles by mixing solutions of DNA (either single-stranded (ssDNA) or double-stranded (dsDNA)) with solutions of cationic surfactant CTAB and solutions of the protein lysozyme. Swelling, surface morphology, and DNA release determinations indicate different interaction of ssDNA and dsDNA with both the surfactant and the protein. By using CTAB and lysozyme as the base material, the formation of a DNA reservoir hydrogel, without adding any kind of cross-linker or organic solvent, was demostrated.

Antimicrobial activity, cytotoxicity and intracellular growth inhibition of Portuguese Thymus essential oils

Thyme essential oils are well recognized by their excellent biological activities and the antimicrobial activity of Portuguese thyme essential oils has been investigated with promising results, particularly against food borne pathogens. In this study the potential antimicrobial activity of the essential oils of five species of Thymus (Lamiaceae), namely Th. caespititius Brot., Th. camphoratus Hoffmanns. & Link, Th. capitellatus Hoffmanns. & Link., Th. carnosus Boiss. and Th. zygis L. was evaluated against Candida albicans, Haemophilus influenza, Helicobacter pylori, Listeria monocytogenes, Salmonella enterica and Streptococcus pneumoniae. H. pylori strains were the most susceptible bacteria, particularly to the essential oils of Th. caespititius (Planalto Central), Th. zygis (Rebordãos) and Th. caespititius (Pico) which minimum inhibitory concentration (MIC) values ranged from 0.05 to 0.08 mg.mL-1. Th. caespititius essential oil from Planalto Central or its main component, carvacrol significantly (p<0.05) inhibited the intracellular growth of H. pylori, and showed no citotoxicity to the gastric cell line. Our results suggest the potential of this essential oil and its main component as a promising tool as anti-Helicobacter agent potentiating the eradication of this important gastroduodenal pathogen.