Detailed H-1 and C-13 NMR structural assignment and relative stereochemistry determination for three closely related sesquiterpene lactones

A complete analysis of H-1 and C-13 NMR spectra of the trypanocidal sesquiterpene lactone eremantholide C and two of its analogues is described. These structurally similar sesquiterpene lactones were submitted to H-1 NMR, C-13 (H-1) NMR, gCOSY, gHSQC, gHMBC, J-resolved and DPFGSE-NOE NMR techniques. The detailed analysis of those results, correlated to some computational calculations (molecular mechanics), led to the total and unequivocal assignment of all H-1 and C-13 NMR data. The determination of all H-1/H-1 coupling constants and all signal multiplicities, together with the elimination of previous ambiguities were also achieved. Copyright (C) 2008 John Wiley & Sons, Ltd.

On the mechanisms of phenothiazine-induced mitochondrial permeability transition: Thiol oxidation, strict Ca(2+) dependence, and cyt c release

Phenothiazines (PTZ) are drugs widely used in the treatment of schizophrenia. Trifluoperazine, a piperazinic PTZ derivative, has been described as inhibitor of the mitochondrial permeability transition (MPT). We reported previously the antioxidant activity of thioridazine at relatively low concentrations associated to the inhibition of the MPT (Brit. J. Pharmacol., 2002;136:136-142). In this study, it was investigated the induction of MPT by PTZ derivatives at concentrations higher than 10 mu M focusing on the molecular mechanism involved. PTZ promoted a dose-response mitochondrial swelling accompanied by mitochondrial transmembrane potential dissipation and calcium release, being thioridazine the most potent derivative. PTZ-induced MPT was partially inhibited by CsA or Mg(2+) and completely abolished by the abstraction of calcium. The oxidation of reduced thiol group of mitochondrial membrane proteins by PTZ was upstream the VIP opening and it was not sufficient to promote the opening of PTP that only occurred when calcium was present in the mitochondrial matrix. EPR experiments using DMPO as spin trapping excluded the participation of reactive oxygen species on the PTZ-induced MPT. Since 117 give rise to cation radicals chemically by the action of peroxidases and cyanide inhibited the PTZ-induced swelling, we propose that VIZ bury in the inner mitochondrial membrane and the chemically generated 117 cation radicals modify specific thiol groups that in the presence of Ca(2+) result in MPT associated to cytochrome c release. These findings contribute for the understanding of mechanisms of MET induction and may have implications for the cell death induced by PTZ. (C) 2010 Elsevier Inc. All rights reserved.

LC-MS-MS Identification and Determination of the Flavone-C-Glucoside Vicenin-2 in Rat Plasma Samples Following Intraperitoneal Administration of Lychnophora Extract

The flavone C-glucoside, vicenin-2, in semi-purified extracts of the leaves of Lychnophora ericoides was quantified in rat plasma samples using a method based on reversed-phase high performance liquid chromatography coupled to tandem mass spectrometry. Vicenin-2 was analyzed on a LiChrospher (R) RP18 column using an isocratic mobile phase consisting of a mixture of methanol: water (30:70, v/v) plus 2.0% glacial acetic acid at a flow rate of 0.8 mL min(-1). Genistein was used as internal standard. The mass spectrometer was operated in positive ionization mode and analytes were quantified by multiple reaction monitoring at m/z 595 > 457 for vicenin-2 and m/z 271 > 153 for internal standard. Prior to the analysis, each rat plasma sample was acidified with 200 mu L of 50 mmol L(-1) acetic acid solution and extracted by solid-phase extraction using a C18 cartridge. The absolute recoveries were reproducible and the coefficients of variation values were lower than 5.2%. The method was linear over the 12.5 - 1500 ng mL(-1) concentration range and the quantification limit was 12.5 ng mL(-1). Within-day and between-day assay precision and accuracy were studied at three concentration levels (40, 400 and 800 ng mL(-1)) and were lower than 15%. The developed and validated method seems to be suitable for analysis of vicenin-2 in plasma samples obtained from rats that receive a single i.p. dose of 200 mg kg(-1) vicenin-2 extract.

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHM`s inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 mu M Ca(2+), DHM (50-250 mu M) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca(2+) efflux and cytochrome c release. DHM (50-250 mu M) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline. (C) 2009 Elsevier Ltd. All rights reserved.

Ca(2+) binding to c-state of adenine nucleotide translocase (ANT)-surrounding cardiolipins enhances (ANT)-Cys(56) relative mobility: A computational-based mitochondrial permeability transition study

The oxidation of critical cysteines/related thiols of adenine nucleotide translocase (ANT) is believed to be an important event of the Ca(2+)-induced mitochondrial permeability transition (MPT), a process mediated by a cyclosporine A/ADP-sensitive permeability transition pores (PTP) opening. We addressed the ANT-Cys(56) relative mobility status resulting from the interaction of ANT/surrounding cardiolipins with Ca(2+) and/or ADP by means of computational chemistry analysis (Molecular Interaction Fields and Molecular Dynamics studies), supported by classic mitochondrial swelling assays. The following events were predicted: (i) Ca(2+) interacts preferentially with the ANT surrounding cardiolipins bound to the H4 helix of translocase, (ii) weakens the cardiolipins/ANT interactions and (iii) destabilizes the initial ANT-Cys(56) residue increasing its relative mobility. The binding of ADP that stabilizes the conformation ""m"" of ANT and/or cardiolipin, respectively to H5 and H4 helices, could stabilize their contacts with the short helix h56 that includes Cys(56), accounting for reducing its relative mobility. The results suggest that Ca(2+) binding to adenine nucleotide translocase (ANT)-surrounding cardiolipins in c-state of the translocase enhances (ANT)-Cys(56) relative mobility and that this may constitute a potential critical step of Ca(2+)-induced PTP opening. (C) 2009 Elsevier B.V. All rights reserved.

Preparation and characterization of chitosan-treated alginate microparticles incorporating all-trans retinoic acid

Chitosan treated alginate microparticles were prepared with the purpose of incorporating all-trans retinoic acid (ATRA) using an inexpensive, simple and fast method, enhancing dermal localization and sustaining the release of ATRA into the skin. Microparticles characterization, drug-polymer interaction, release profile and in vitro skin retention were investigated. Microparticles presented spherical shape and drug loading capacity of 47%. The drug content of these microparticles was affected by ATRA concentration and by the solvent used and it was more weakly affected by chitosan concentration. The release of ATRA was also affected by chitosan concentration. Microparticles prepared with 0.4% chitosan (w/w) resulted in drug release with a more sustained profile. The results of in vitro retention studies showed that chitosan treated alginate microparticles decreased the drug retention in the stratum corneum (SC), where occur the skin irritation, but maintained the ATRA concentration in the deeper skin layers, where occur the pathologies treated with ATRA. Then, the microparticles developed in this work can be a good candidate to improve the topical therapy with retinoid.

THE ROLE OF COLLOIDAL SILICON DIOXIDE IN THE ENHANCEMENT OF THE DRYING OF HERBAL PREPARATIONS IN SUSPENDED STATE

Recently, some research groups have been developing studies aiming to apply spouted beds of inert particles for production of dried herbal extracts. However, mainly due to their complex composition, several problems arise during the spouted bed drying of herbal extracts such as bed instability, product accumulation, particle agglomeration, and bed collapse. The addition of drying carriers, like colloidal silicon dioxide, to the extractive solution can minimize these unwanted effects. The aim of this work was to study the influence of the addition of colloidal silicon dioxide on enhancement of the performance of the drying of hydroalcoholic extract of Bauhinia forficata Link on a spouted bed of inert particles. The physical properties of the herbal extract and of its mixture with colloidal silicon dioxide at several concentrations (20% to 80% related to solids content) were quantified by determination of the surface tension, rheological properties, density, pH, and contact angles with the inert surfaces. Drying performance was evaluated through determination of the elutriation ratio, product recovery ratio, and product accumulation. The product was characterized through determination of the thermal degradation of bioactive compounds and product moisture content. The results indicated that the rheological properties of the extracts and their preparations, the contact angle with inert material, and the work of adhesion play important roles in the spouted bed drying of herbal extracts. Higher concentration of the drying carrier significantly improved the spouted bed drying performance.

Retention of the Enzymatic Activity and Product Properties During Spray Drying of Pineapple Stem Extract in Presence of Maltodextrin

The aim of this work was to investigate the effects of drying parameters on the retention of the enzymatic activity and on the physical properties of spray-dried pineapple stem extract. A Box and Behnken experimental design was used to investigate the effects of the processing parameters on the product properties. The parameters studied were the inlet temperature of drying gas (Tgi), the feed flow rate of the pineapple extract relative to evaporative capacity of the system (Ws /Wmax), and the concentration of maltodextrin added to the extract (MD). Significant effects of the processing parameters on the retention of the proteolytic activity of the powdered extract were observed. High processing temperatures lead to a product with a smaller moisture content, particle size, and lower agglomerating tendency. A product with insignificant losses of the proteolytic activity ( 10%) and low moisture content (less than 6.5%) is obtained at selected conditions.

Brazilian Propolis: Seasonal Variation of the Prenylated p-Coumaric Acids and Antimicrobial Activity

Brazilian green propolis, which is used in food and beverages to improve health and to prevent diseases, demostrates antioxidant, antimutagenic, and antimicrobial activities. Most biological activities are thought to be related to the high levels of drupanin, artepillin C, and baccharin, which are compounds also present in Baccharis dracunculifolia D.C. (Asteraceae). Since propolis chemical composition depends on the region and the period of collection, as well as its plant origin, the effect of seasonal variation on the both content of prenylated p-coumaric acids and in vitro antimicrobial activity of Brazilian propolis from four different sites, was performed. The results showed that MIC values ranged from 100 to 300 mu g/mL against both Staphylococcus aureus and Kocuria rhizophila, while none of the propolis samples was active against Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. HPLC analysis showed that the content of drupanin, artepillin C, and baccharin varied throughout the year, as well as among the different study sites. Also, it is suggested that Baccharis dracunculifolia is the main botanical source of Brazilian propolis in sites I and 2, while in sites 3 and 4, other plant species are also used by bees to produce propolis. All the evaluated propolis samples exhibited similar antibacterial activity, but different contents of prenylated p-coumaric acids throughout the year.

In vitro antioxidant activity and in vivo efficacy of topical formulations containing vitamin C and its derivatives studied by non-invasive methods

Background/purpose: Vitamins C and its derivatives, mainly due to their antioxidant properties, are being used in cosmetic products to protect and to reduce the signs of ageing. However, there are no studies comparing the effects of vitamin C [ascorbic acid (AA)] and its derivatives, magnesium ascorbyl phosphate (MAP) and ascorbyl tetra-isopalmitate (ATIP), when vehiculated in topical formulations, mainly using objective measurements, which are an important tool in clinical efficacy studies. Thus, the objective of this study was to determine the in vitro antioxidant activity of AA and its derivatives, MAP and ATIP, as well as their in vivo efficacy on human skin, when vehiculated in topical formulations. Methods: The study of antioxidant activity in vitro was performed with an aqueous and a lipid system. The in vivo methodology consisted of the application of these formulations on human volunteers` forearm skin and the analysis of the skin conditions after 4-week period daily applications in terms of transepidermal water loss (TEWL), stratum corneum moisture content and viscoelasticity using a Tewameter (R), Corneometer (R) and Cutometer (R), respectively. Results: In vitro experiments demonstrated that in an aqueous system, AA had the best antioxidant potential, and MAP was more effective than ATIP, whereas in the lipid system ATIP was more effective than MAP. In in vivo studies, all formulations enhanced stratum corneum moisture content after a 4-week period daily applications when compared with baseline values; however, only the formulation containing AA caused alterations in TEWL values. The formulations containing MAP caused alterations in the viscoelastic-to-elastic ratio, which suggested its action in the deeper layers of the skin. Conclusion: AA and its derivates presented an in vitro antioxidant activity but AA had the best antioxidant effect. In in vivo efficacy studies, only the formulation containing AA caused alterations in TEWL values and the formulation containing MAP caused alterations in the viscoelastic-to-elastic ratio. This way, vitamin C derivatives did not present the same effects of AA on human skin; however, MAP showed other significant effect-improving skin hydration, which is very important for the normal cutaneous metabolism and also to prevent skin alterations and early ageing.

Antimicrobial activity of the extract and isolated compounds from Baccharis dracunculifolia D. C. (Asteraceae)

Baccharis dracunculifolia D. C. (Asteraceae) is the most important plant source of the Brazilian green propolis. Since propolis is known for its antimicrobial activity, the aim of this work was to evaluate the showed that the leaves extract of B. dracunculifolia (BdE) presents antifungal and antibacterial activities, especially against Candida krusei and Cryptococcus neoformans, for which the BdE showed IC50 values of 65 mu g mL(-1) and 40 mu g mL(-1), respectively In comparison to the BdE, it was observed that the green propolis extract (GPE) showed better antimicrobial activity, displaying an IC50 value of 9 mu g mL(-1) against C krusei. Also, a phytochemical study of the BdE was carried out, affording the isolation of ursolic acid (1), 2 alpha-hydroxy-ursolic acid (2), isosakuranetin (3), aromadendrin-4`-methylether (4), baccharin (5), viscidone (6), hautriwaic acid lactone (7), and the clerodane diterpene 8. This is the first time that the presence of compounds 1, 2, and 8 in B. dracunculifolia has been reported. Among the isolated compounds, 1 and 2 showed antibacterial activity against methicillin-resistant Staphylococcus aureus, displaying IC50 values of 65 mu g mL(-1) and 40 mu g mL(-1), respectively. 3 was active against C neoformans, showing an IC50 value of 15 mu g mL(-1) and a MIC value of 40 mu g mL(-1), while compounds 4-8 were inactive against all tested microorganisms. The results showed that the BdE, similar to the GPE, displays antimicrobial activity, which may be related to the effect of several compounds present in the crude extract.

Development of a Phytopharmaceutical Intermediate Product via Spray Drying

The influence of concentration and incorporation time of different drying excipients on the processing yields and physical properties of Eugenia dysenterica DC spray-dried extracts were investigated following a factorial design. Under the established conditions, the process yield ranged from 57.55 to 89.14%, and in most experiments, the recovered products presented suitable flowability and compressibility, as demonstrated by the Hausner factor, Carr index, and angle of repose. Additionally, in a general way, the parameters related to the dried products` flowability varied over a range acceptable for pharmaceutical purposes. An analysis of variance (ANOVA) proved that both factors and some of their interactions significantly affected most of the investigated responses at different levels. Mannitol proved to be an interesting alternative as an excipient for the drying of herbal extracts, even at low concentrations such as 12.5%. Furthermore, these results imply that the best condition to obtain dry extracts of E. dysenterica with high performance and adequate pharmacotechnical properties involves the lowest concentration and the highest incorporation time of mannitol.

Spray Drying of Extracts from Red Yeast Fermentation Broth

Red yeast rice is a pigmented material that is traditionally used in Asia as a food colorant. In addition to food applications, red yeast rice is known in traditional Chinese medicine for its therapeutic actions. The aim of this work was to study the quality interactions during spray drying of extracts from the Monascus ruber van Tiegham fermentation broth. The quality indicators used for the dry powder properties were the levels of monacolin K, ratio of red to yellow pigments, as well as their antioxidant activity. The experiments followed a Box-Behnken design to study the effects of the adjuvant/drug ratio, adjuvant incorporation time, and oulet drying temperature on the pharmacotechnical, chemical, and biological properties of the dry extract. The influences of these factors on the characteristics of the dry powder were evaluated by the bulk density, tapped density, Carr index, Hausner factor, residual moisture content, water activity, antioxidant activity, monacolin K, yellow-to-red pigment ratio, and antioxidant activity. The analysis of variance (ANOVA) on experimental data revealed that an increase in drying temperature significantly increased the dry powder yield and caused an improvement in powder flow properties, which may be related to lower moisture contents. The drying temperature did not affect the monacolin K content in dry powder but showed a complex influence on its antioxidant activity. The increase in drying adjuvant-to-drug ratio affected the yield and also indicated a protective effect on the monacolin K content. The duration of drying adjuvant incorporation had little or negligible effect on powder properties. The dry extracts of red yeast rice showed adequate properties and the process proposed herein can be used to prepare nutraceutical products.

Characterization and stability study of a water-in-oil microemulsion incorporating quercetin

Background: The effectiveness of a water/oil (w/o) microemulsion containing quercetin against ultraviolet B radiation (UVB) induced damage was recently demonstrated by our group. However, during the development of new pharmaceutical products, the evaluation of percutaneous absorption and in vivo effectiveness should be accompanied by evaluation of stability parameters as an integral part of the process. Objective: The aim was to investigate the stability of the final microemulsion formulation considering the temperature ranges of storage and application. Methods: The physical, chemical, and functional stability of this formulation under different conditions of storage during 12 months and the photostability of quercetin incorporated into this system over UVB exposure for 7 days were evaluated. Results: Although the results indicated a notable physical stability of the w/o microemulsions during the experimental period under all employed conditions, in both, the chemical and functional studies, a significant loss of quercetin content and antioxidant activity was found after 6 months of storage at 30 degrees C/70% relative humidity and after 2 months at 40 degrees C/70% relative humidity. The photostability study results demonstrated that the incorporation of quercetin into the w/o microemulsion maintained the previously demonstrated photostability of this flavonoid under forced exposure to UVB irradiation. Conclusion: Thus, this work demonstrates that special storage conditions (at 4 +/- 2 degrees C) are necessary to maintain the functionality of the w/o microemulsion containing quercetin and mainly emphasizes the importance of studying physical, chemical, and functional parameters at the same time during stability evaluation of active principles.

Role of phospholipase C and protein kinase C in Aspergillus nidulans during growth on pectin or glucose: Effects on germination and duplication cycle

The effects of PLC and Pkc inhibitors on Aspergillus nidulans depend on the carbon source. PLC inhibitors Spm and C48/80 delayed the first nuclear division in cultures growing on glucose, but stimulated it in media supplemented with pectin. Less intense were these effects on the mutant transformed with PLC-A gene rupture (AP27). Neomycin also delayed the germination in cultures growing on glucose or pectin; however, on glucose, the nuclear division was inhibited whereas in pectin it was stimulated. These effects were minor in AP27. The effects of Ro-31-8425 and BIM (both Pkc inhibitors) were also opposite for cultures growing on glucose or pectin. On glucose cultures of both strains BIM delayed germination and the first nuclear division, whereas on pectin both parameters were stimulated. Opposite effects were also detected when the cultures were growing on glucose or pectin in the presence of Ro-31-8425.