Profile of pterostilbene-induced redox homeostasis modulation in cardiac myoblasts and heart tissue.

This study was designed to investigate the effect of pterostilbene (PTS) on cardiac oxidative stress in vitro, as this is a simple and promising methodology to study cardiac disease. Cardiac myoblasts (H9c2 cells) and homogenised cardiac tissue were incubated with the PTS and cyclodextrin (PTS + HPßCD) complex for 1 and 24 h, respectively, at concentrations of 50µM for the cells and 25 and 50µM for cardiac tissue. The PTS + HPßCD complex was used to increase the solubility of PTS in water. After the pretreatment period, cardiomyoblasts were challenged with hydrogen peroxide (6.67µM) for 10 min, while cardiac tissue was submitted to a hydroxyl radical generator system (30 min). Cellular viability, oxidative stress biomarkers (e.g. total reactive oxygen species (ROS), carbonyl assay and lipoperoxidation) and the antioxidant response (e.g. sulfhydryl and the antioxidant enzyme activities of superoxide dismutase, catalase and glutathione peroxidase) were evaluated. In cardiomyoblasts, the PTS + HPßCD complex (50µM) increased cellular viability. Moreover, the PTS + HPßCD complex also significantly increased sulfhydryl levels in the cells submitted to an oxidative challenge. In cardiac tissue, lipid peroxidation, carbonyls and ROS levels were significantly increased in the groups submitted to oxidative damage, while the PTS + HPßCD complex significantly reduced ROS levels in these groups. In addition, the PTS + HPßCD complex also provoked increased catalase activity in both experimental protocols. These data suggest that the PTS + HPßCD complex may play a cardioprotective role through a reduction of ROS levels associated with an improved antioxidant response.

In Vitro Evaluation of Mucosa Permeation/Retention and Antiherpes Activity of Genistein from Cationic Nanoemulsions.

In this report, we described the genistein distribution on excised porcine esophageal mucosa from cationic nanoemulsions, as well as the anti-HSV-1 activity against a viral strain resistant to acyclovir. Genistein-loaded cationic nanoemulsions were prepared by spontaneous emulsification. This procedure yielded monodisperse nanoemulsions exhibiting a mean droplet size of approximately 200-300 nm. Hydroxyethyl cellulose (HEC) was added at the end of the manufacturing process as a thickening agent (at 3%). Such formulations exhibit a non-Newtonian pseudoplastic behavior. The addition of HEC significantly reduces the genistein flux through excised porcine mucosa specimens as compared with values elicited by nanoemulsions before thickening. Furthermore, a significant increase of genistein retention in mucosa was observed as compared to the genistein propylene glycol solution, as illustrated by confocal fluorescence microscopy images. Formulations exhibited antiherpetic activity in vitro against HSV-1 (strain 29R). Taken together, these results suggest that these formulations have promising potential to be used topically for herpes infections.

Antiherpes evaluation of soybean isoflavonoids.

The antiviral effects of soybean isoflavonoids have been investigated recently, especially those of genistein. It has been reported that this isoflavone is able to inhibit herpes simplex virus (HSV) replication, which is associated with skin and epithelial mucosa infections. The treatment of these infections with antiherpes drugs has resulted in the emergence of resistant viral strains. Based on this evidence, the aim of this study was to investigate the anti-HSV effects of soybean isoflavonoids: daidzein, genistein, glycitein, and coumestrol. Genistein and coumestrol inhibited HSV-1 (KOS and 29R strains, which are acyclovir sensitive and acyclovir resistant, respectively) and HSV-2 (333 strain) replication, whereas no antiviral effects were detected for daidzein and glycitein. The mechanisms of action were evaluated by different methodological strategies. Coumestrol affected the early stages of viral infection, and both compounds were able to reduce HSV-1 protein expression, as well as HSV-2 cell-to-cell spread.

Simultaneous quantification of flavonoids from Achyrocline satureioides by a polar-reversed phase LC method--application to skin permeation/retention studies.

A selective and sensitive polar-reversed phase LC method was validated for simultaneous quantification of the main Achyrocline satureioides flavonoids (quercetin, luteolin, and 3-O-methylquercetin) in skin samples after permeation/retention studies from topical nanoemulsions. The method was linear in a range of 0.25 to 10.0 microg/mL exhibiting a coefficient of determination higher than 0.999 for all flavonoids. No interference of the nanoemulsion excipients or skin components was observed in the retention times of all flavonoids. The R.S.D. values for intra- and inter-day precision experiments were lower than 6.73%. Flavonoids recovery from nanoemulsions and skin matrices was between 90.05 and 109.88%. In a permeation/retention study with porcine ear high amount of 3-O-methylquercetin was found in the skin sample (0.92 +/- 0.22 microg/g) after two hours. The proposed method was suitable to quantify the main flavonoids of A. satureioides in skin permeation/retention studies from topical nanoemulsions.

LC analysis of coumestrol incorporated into topical lipid nanoemulsions.

A simple, rapid, and sensitive LC method to determine coumestrol incorporated in the lipid nanoemulsions was validated. The analyses were performed at room temperature on a reversed-phase C18 column using a mobile phase composed of methanol/water with 0.1% trifluoracetic acid (70:30, v/v) at 0.8 mL min(-1). The detection was carried out on a UV detector at 343 nm. The linearity, in the range of 0.1-6.0 microg/mL, presented a determination coefficient (r2) of 0.999, calculated by the least square method. No interferences of the oil core or the gelling excipients were detected. The R.S.D. values for intra- and inter-day precision experiments were lower than 2%. The recovery ranged from 99.42% to 100.72%. Finally, the proposed method was successfully applied to determine coumestrol incorporated in the proposed topical formulations.

Identification and stability of a new bichalcone in Achyrocline satureioides spray dried powder.

A new chemical structure, the 4,2',4",2'''-tetrahydroxy-6',6'''-dimethoxy-4'-O-4'''- bichalcone, named achyrobichalcone was isolated and identified from an Achyrocline satureioides spray-dried powder (SDP80). The thermal and photo stability of this new compound as well as that of the main polyphenols present in the spray dried powder, quercetin, luteolin, 3-O-metylquercetin and the corresponding kinetics of degradation are reported. In the long-term testing (30 +/- 2 degrees C/75 +/- 5% RH, 12 months), the total polyphenols contained in SDP80 demonstrated to be stable, remaining higher than 90% after a 12 month exposure. The photo stability testing revealed that all polyphenols were stable for 48 h when SDP80 was conditioned in amber or transparent flasks and exposed to UV-C radiation (light express LE UV, 254 nm, 30W). In contrast, when unprotected, the polyphenols demonstrated to be sensitive to both, thermal stress testing (80 +/- 2 degrees C), for 14 days and to UV-C radiation. Luteolin showed to be the most stable against UVC light and 3-O-methylquercetin against temperature. The achyrobichalcone demonstrated to be the more unstable against both, temperature and light. The kinetics of polyphenol thermal degradation (80 +/- 2 degrees C, 49 days) and photodegradation (UV-C radiation, 96 h) followed, 2nd and 1st order reaction, respectively.

Development of topical nanoemulsions containing the isoflavone genistein.

We have recently described the incorporation of genistein into topical nanoemulsions. This study describes the physicochemical properties and the genistein permeation profile from these nanoemulsions. Formulations composed of egg lecithin, medium chain triglycerides (MCT) or octyldodecanol (ODD) and water were prepared by spontaneous emulsification. Irrespective of the oil core employed (MCT or ODD), this procedure yielded monodisperse emulsions with mean droplet sizes in the range of 230-280 nm. The addition of genistein in the oil phase, before emulsification, did not alter the properties of nanoemulsions. The amount of genistein incorporated in both formulations was close to 100% (1 mg/mL). Solubility and DSC experiments suggested that egg-lecithin may play an important role on the incorporation of genistein in nanoemulsions. Genistein permeation from formulations was assessed using pig ear skin in Franz type diffusion cells. The overall results showed a slow permeation profile for genistein from both nanoemulsions.Such results open interesting perspectives for the topical administration of genistein.

Development of topical nanoemulsions containing quercetin and 3-O-methylquercetin.

This study describes the physico-chemical properties and the skin permeation profile of quercetin (Q) and 3-O-methylquercetin (MQ) from lipid nanoemulsions. Formulations composed of octyldodecanol, egg lecithin, water (NE) and cetyl trimethyl ammonium bromide (CNE) were obtained by spontaneous emulsification. This procedure yielded monodisperse nanoemulsions exhibiting a mean droplet size of approximately 200-300 nm. Nanoemulsions were further characterized in terms of zeta-potential, surface tension, and morphology by transmission electron microscopy. The amount of flavonoids incorporated into nanoemulsions reached nearly 100% (at 1 mg/mL). The permeation studies were carried out using ear pig skin mounted in Franz diffusion cells. The overall results have shown a slow permeation profile of both Q and MQ from nanoemulsions. However, a higher permeation flux rate of flavonoids from CNE (approximately 0.2 microg/cm2/h) as compared to NE (approximately 0.08 microg/cm2/h) was observed, showing the effect of the positively charged surface of CNE on this parameter. Such results open interesting perspectives for the topical administration of the flavonoids Q and MQ.

Influence of excipients and technological process on anti-inflammatory activity of quercetin and Achyrocline satureioides (Lam.) D.C. extracts by oral route.

The flavonoids quercetin, 3-O-methylquercetin and luteolin play an important role in the anti-inflammatory activity of Achyrocline satureioides ethanol extracts when administered intraperitoneally. The present work describes the oral anti-inflammatory effect of quercetin and A. satureioides extracts and the role played by the solvent concentration, adjuvant and drying processes of freeze-drying (FD) or spray-drying (SD) on the effect. The best anti-edema effect was observed with 250 mg/kg body wt of the freeze-dried powder (FDP), prepared with 40% (v/v) ethanol (FDP40). In contrast, 250 mg/kg body wt of FDP80, prepared with ethanol 80% (ES80), did not significantly inhibit the carrageenan-induced rat paw edema. However, when ES80 was freeze-dried in the presence of polysorbate 80 (FDP80-P80) or spray-dried in the presence of colloidal silicon dioxide (CSD) and P80 (SDP80), both dried extracts became more active. Quercetin suspension in saline did not inhibit paw edema, but the mixture of quercetin with polysorbate 80 was effective in edema inhibition by the oral route. Aqueous extract (ESAQ), freeze-dried (FDPAQ, FDPAQ-P80) or spray-dried (SDPAQ) did not exhibit the edema-inhibition effect. Taken together, the results point to the following order of efficacy (at 4 h, for example): FDP40 > indomethacin > SDP40 > SDP80 = FDP80-80 > Quercetin-P80. Additionally, the FDP40, SDP40 (prepared from 40% v/v ethanol added of CSD) and SDP80 reduced the total leukocyte and polymorphonuclear cell migration in the pleural cavity.

Thermal and photo stability of phenolic constituents of an Achyrocline satureioides spray-dried powder.

Thermal and the photo stabilities of an Achyrocline satureioides powder (SDP40) were evaluated in particular concerning the total polyphenol content as well as the main identified constituents quercetin, luteolin, 3-O-methylquercetin and caffeic acid. SDP40 presented good stability for nine months under normal storage conditions of 25 degrees C temperature and 60% relative humidity (RH). In accelerated term testing, 50 degrees C temperature and 90% RH and also in stress testing, 80 degrees C, caffeic acid and a non-identified constituent P3 were the most instable constituents. Luteolin and 3-O-methylquercetin were the most stable constituents. Quercetin presented an unusual behavior, improving its concentration after 1 month at 50 degrees C or 2 days at 80 degrees C exposition, followed by a decrease in its concentration. The hypothesis that this observation is related to the simultaneous decreasing of a non-identified peak P3 or to the hydrolysis of a non-identified precursor as a quercetin heteroside is being investigated. The SDP40 presented good stability against UV-C light when conditioned in amber or transparent containers, but it suffered degradation when stored in open-dishes. In summary, the total polyphenol content remains within acceptable limits of 10% under normal storage conditions for nine months. However, the LC polyphenol analysis demonstrated that the behavior of individual constituents has still to be enlightened.

Development and validation of a LC method for determination of genistein in topical nanoemulsions.

The aim of this study was to develop and validate an isocratic LC method for the quantification of genistein in topical nanoemulsions. The analyses were performed at room temperature on a reversed-phase C18 column using a mobile phase composed of methanol/water/acetonitrile (70:25:5, w/w/w) at 1.0 ml x min(-1). The detection was carried out on a UV detector at 327 nm. The linearity, in the range of 25-75 microg/ml, presented a determination coefficient (r2) higher than 0.999, calculated by the least square method. No interferences from the excipients (egg-lecithin, octyldodecanol or medium chain triglycerides) were detected. The R.S.D. values for intra- and inter-day precision experiments were lower than 2.3%. The recovery of genistein from nanoemulsions ranged from 96.6% to 106.6%. The excellent performance of the method, its linearity, accuracy and precision, demonstrate that it can be readily used to quantify genistein incorporated in nanoemulsions.

Characterization of different samples of quercetin in solid-state: indication of polymorphism occurrence.

The present work was designed to compare four commercial samples of quercetin, three of them presenting pharmaceutical grade (QPGa, QPGb and QPGc) and the other one pro-analysi grade (QPA) by means of different techniques. Physical and chromatographic characterization of these samples shows different properties following its origin, especially a clear evidence of polymorphism occurrence.

Evaluation of the antiherpetic activity of standardized extracts of Achyrocline satureioides.

Traditionally, Achyrocline satureioides or 'marcela' has been used in South America for the treatment of several disorders. For the present study, three spray-dried extracts (N1, N2 and N3) were used, all of them prepared with 50% of an hydroethanolic extract rich in flavonoid compounds and 50% of blends of different adjuvants. The cytotoxic concentration which causes destruction in 50% monolayer cells (CC50) was 62.5 microg/ml for the three extracts. The antiviral activity was evaluated by using two different strains of herpes simplex virus (HSV-1) and the best results were obtained with KOS strain and N2 extract. Studies concerning the mechanism of the antiherpetic activity demonstrated that N2 extracts showed no virucidal effect or activity on cellular receptors. HSV-1 DNA synthesis was not inhibited. The antiherpetic activity occurred between the second and ninth hour of the virus replication cycle, probably indicating a perturbation on late stages of this cycle.

Antioxidant, a pro-oxidant and cytotoxic effects of Achyrocline satureioides extracts.

In this study we compared the antioxidant properties of five different extracts of different composition obtained from Achyrocline satureioides' inflorescences (Compositae), a widely used Brazilian folk medicinal herb. All of the extracts presented significant antioxidant potential identified by TRAP assay, which increased in the presence of human plasma. Characterization of the content of flavonoids in each extract showed that the FDP80 (ethanol 80%) and FFr (enriched flavonoid fraction) extracts contained a higher content of flavonoids. Cytotoxicity of the extracts as determined in Sertoli cell culture showed that FDP80 and FFr were highly toxic at most concentrations tested. The extracts induced a significant increase in lipid peroxidation levels in Sertoli cells. These results suggest that medicinal herb extracts that contain higher flavonoid concentrations and shows higher antioxidant protection in vitro might not always produce the greatest benefit.

Carbamazepine/betaCD/HPMC solid dispersions. II. Physical characterization.

Solid dispersions containing carbamazepine (CBZ) associated with beta-cyclodextrin (betaCD) and/or hydroxypropyl methylcellulose were prepared by two different methods, spray-drying or physical mixture, and characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), infrared (IR) spectroscopy, and x-ray powder diffraction analysis (XRPD) studies. Scanning electron microscopy pictures showed that spray-drying produced a mixture of hollow, spherical, and partially shrunken microparticles of homogeneous materials, whereas the physical mixtures yielded heterogeneous systems in which all individual components could be identified. Thermal and IR analyses suggest the existence of a strong interaction between CBZ and excipients in spray-dried solid dispersions, but no CBZ polymorphic transition was detected by either IR spectroscopy or XRPD analysis after the spray-drying process.

CNS activities of liquid and spray-dried extracts from Lippia alba-Verbenaceae (Brazilian false melissa).

The CNS activity of Lippia alba liquid and spray-dried extracts, containing the non-volatile fraction from the leaves, was investigated. L. alba liquid extracts were prepared by percolation with EtOH 40, 60 or 80%. The liquid extracts, named ES(40%,) ES(60%) and ES(80%,) were concentrated, the ethanol eliminated and then tested in Swiss mice to evaluate its sedative and anticonvulsant effects. The animals received the extracts, orally, in doses corresponding to 200 mg of dry residue by kilogram of body weight. All mice were evaluated in the barbiturate-induced sleep test. Similarly, other groups of mice were submitted to convulsions induced by pentylenetetrazol (PTZ). The concentrated extract obtained from ES(80%) showed the most significant sedative and myorelaxant effects as well as the highest total flavonoid content (66 mg/100 g, expressed in apigenin). Two spray-dried powders, SDP(1) and SDP(2), were prepared from ES(80%) using as excipients, respectively, colloidal silicon dioxide (CSD) and CSD associated to beta-cyclodextrin. Only SDP(1) showed sedative profile similar to that presented by ES(80). In conclusion, we demonstrated that the non-volatile fraction of L. alba, extracted in ethanol 80% (v/v), presents sedative and myorelaxant effects and that, among the tested extracts, this presents the highest flavonoid content. We demonstrated also the technological feasibility of spray-dried extracts and the influence of the excipient on its sedative properties.

LC determination of flavonoids: separation of quercetin, luteolin and 3-O-methylquercetin in Achyrocline satureioides preparations.

The pharmacological activities of the flavonoids show the interest in quantifying these constituents in phytopharmaceutical preparations, as well as in the validation of the analytical methodologies. LC methods have been reported to quantify isolated flavonoids or these compounds in complex biological matrices, such as herbal raw materials and extractive preparations. This work was designed, therefore, to develop an LC system to separate quercetin, luteolin and 3-O-methylquercetin and to quantify them in extractive solutions from Achyrocline satureioides. The main validation parameters of the method are also determined. The method showed linearity for quercetin and luteolin in the range 1-10 microg/ml. The aqueous and ethanol 80% extractive solutions showed linear response in the range 2.5-20 microl/ml and ethanol 40% extractive solution in the range 2.5-10 microl/ml. Precision and accuracy were determined for ethanol 80% extractive solution, in the concentration of 10 microl/ml. The LC method showed an excellent performance in separating the flavonoids quercetin, luteolin and 3-O-methylquercetin in A. satureioides extracts, since the presence of interference has been previously evaluated.

Influence of adjuvants on the dissolution profile of tablets containing high doses of spray-dried extract of Maytenus ilicifolia.

A 2(3) factorial design was used in order to evaluate the influence of some adjuvants on the dissolution profile of tablets containing high doses of Maytenus ilicifolia spray-dried extract. Tablets were prepared on a single punch tablet press using 15 mm flat punches by individual direct compression of 650 mg from each formulation containing 375 mg of the spray-dried extract. The factors investigated were disintegrant (croscarmellose sodium or sodium starch glycolate), lubricant (colloidal silicon dioxide or magnesium stearate) and filler/binder (microcrystalline cellulose or lactose). The dissolution profiles were analyzed to determine the dissolution kinetics, the dissolution half-lives (t50%), the similarity factor (f2) and the dissolution efficiency (DE %), which was selected as the response criteria to evaluate the factorial design. The results revealed that in spite of the high content of spray-dried powder in the tablets, the dissolution profiles of the extract did depend on the adjuvant used. The filler/binder had the most important effect on the dissolution efficiency of the tablets.

Ofloxacin/beta-cyclodextrin complexation.

Ofloxacin (OFX) is a fluorquinolone characterized by photochemical instability. With the goal to improve its photostability in aqueous solutions, the complexation of ofloxacin with beta-cyclodextrin was investigated. The complexes showed a water solubility enhancement of approximately 2.6 times; nevertheless, the photodegradation of ofloxacin was not reduced. The complexes obtained were characterized by thermal and 1H nuclear magnetic resonance (NMR) analysis, which revealed an interaction between ofloxacin and beta-cyclodextrin. The last analysis indicated that only partial inclusion of the N-methylpiperazinyl moiety occurred, which can explain the fact that photostabilization was not improved. This partial inclusion phenomenon could be explained also by computer-aided molecular modeling.

Polymeric colloidal systems containing ethionamide: preparation and physico-chemical characterization.

The association of ethionamide with different colloidal systems was evaluated. Nanocapsules (NC), nanospheres (NS), and nanoemulsions (NE) were prepared by interfacial deposition and spontaneous emulsification techniques. Ethionamide was incorporated before (B) and after (A) preparation of nanoparticles. Ethionamide was assayed by HPLC, the particle size was determined using a Nanosizer, and the zeta potential using a Zetasizer 4. Free ethionamide was determined using a combined ultrafiltration-centrifugation technique. The drug release was determined by direct dilution of the nanoparticle dispersion in phosphate-buffer pH 7. All preparations retained acceptable particle size distribution (+/- 300 nm), except the NE. The zeta potential of all formulations was between -36.6 mV and -46.1 mV. Percentages of ethionamide associated were: NC (B: 62.4%, A: 56.2%), NS (B: 53.0%, A: 43.2%), and NE (B: 38.5%). After 45 days, the percentage of drug association with NC increased (B: 66.8%, A: 60.6%). The release profiles demonstrated that associated ethionamide was more readily released from the NC and NS prepared by procedure A rather than B. The ethionamide amount not released (B) was greater in NS than NC. The drug is mainly adsorbed onto the surface of nanoparticles. However, approximately 10% of ethionamide is encapsulated into NC and 20% entrapped into NS, respectively.