The role of formulation and follicular pathway in voriconazole cutaneous delivery from liposomes and nanostructured lipid carriers.

In general, colloids provide increased cutaneous permeation of drugs. Still, skin interaction and main pathways for drug diffusion may vary depending on system and formulation characteristics. The knowledge of how different colloidal systems interact with biological membranes and the formulation impact on delivery is especially relevant for drugs that can be encapsulated in multiple nanosystems, as voriconazole (VOR). In here, we compared VOR release and permeation profile from liposomes (LP) and nanostructured lipid carriers (NLC) in aqueous colloidal dispersions and in gel formulations. Despite the controlled drug release provided by gel formulations, formulation only had a significant impact on drug skin accumulation from LP. The reduced mobility in gel formulations compromised follicle deposition and drug retention in the skin. Such a hypothesis was confirmed by permeation experiments evaluating follicle pathway influence. Follicular route also had an influence on delivery from NLC, which was only significant for total drug that reached the acceptor medium. These differences could be attributed to the mechanisms of colloid interaction with the skin and subsequent drug release. Follicle LP deposition and slow drug release leads to higher cutaneous amounts whilst NLC interaction with skin and fast drug release leads to fast drug diffusion and deeper penetration. By the low MIC50 values encountered against Trichophyton rubrum (∼ 0.001 µg/mL), permeated amounts could inhibit fungal growth, regardless the system. In conclusion, both LP and NLC seem to be valuable systems for cutaneous VOR delivery. Fluidic formulations could provide better efficiency for cutaneous drug delivery from LP.

Determinação de ácido rosmarínico em Cordia verbenacea por cromatografia líquida: aplicabilidade em estudo sazonal / Determination of rosmarinic acid in Cordia verbenacea by liquid chromatography: applicability in seasonal study

RESUMO Neste estudo, uma técnica de cromatografia líquida de alta resolução em fase reversa (CLAE-FR) para a determinação de ácido rosmarínico em Cordia verbenacea foi desenvolvida e validada. A análise de regressão foi avaliada, com observação de uma boa linearidade (r = 0,9997). Os valores obtidos para a precisão e exatidão estão de acordo com as diretrizes do ICH e com a legislação brasileira. Os valores de repetibilidade e precisão intermediária foram 2,79% e 4,76%, respectivamente. Os limites de detecção e de quantificação de ácido rosmarínico foram de 1,92 µg/mL e 5,81 µg/mL, respectivamente. Os resultados mostraram que o método desenvolvido é uma técnica por CLAE-FR de confiança para a determinação de ácido rosmarínico em tintura de C. verbenacea. Além disso, essa metodologia foi aplicada em estudo sazonal, que revela uma correlação positiva relativamente forte entre o período de chuvas e o teor de ácido rosmarínico.

ABSTRACT In this study, a reverse phase-high performance liquid chromatography (RP-HPLC) technique for determination of rosmarinic acid in the Cordia verbenacea was developed and validated. A regression analysis was performed, with the observation of good linearity (r =0.999949). The values obtained for precision and accuracy determination are in agreement with ICH guidelines and the Brazilian legislation. The values of repeatability and intermediate precision were 2.79% and 4.76%, respectively. The detection and the quantitation limits of the rosmarinic acid were 1.92 µg/mL and 5.81 µg/mL, respectively. The results demonstrated that the developed method is a reliable RP-HPLC technique for the determination of rosmarinic acid in C. verbenacea tincture. In addition, this methodology was applied at a seasonal study indicating relatively strong positive correlation between the rain period and the rosmarinic acid content.

Liquid-liquid extraction of commercial and biosynthesized nisin by aqueous two-phase micellar systems.

Nisin is a natural additive for conservation of food, and can also be used as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth of a variety of Gram-positive and Gram-negative bacteria. In this paper we present a potentially scalable and cost-effective way to purify commercial and biosynthesized in bioreactor nisin, including simultaneously removal of impurities and contaminants, increasing nisin activity. Aqueous two-phase micellar systems (ATPMS) are considered promising for bioseparation and purification purposes. Triton X-114 was chosen as the as phase-forming surfactant because it is relatively mild to proteins and it also forms two coexisting phases within a convenient temperature range. Nisin activity was determined by the agar diffusion assay utilizing Lactobacillus sake as a sensitive indicator microorganism. Results indicated that nisin partitions preferentially to the micelle rich-phase, despite the surfactant concentration tested, and its antimicrobial activity increases. The successful implementation of this peptide partitioning, from a suspension containing other compounds, represents an important step towards developing a separation method for nisin, and more generally, for other biomolecules of interest.

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions.

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.

Purification and properties of a xylanase from Ceriporiopsis subvermispora cultivated on Pinus taeda.

The production of hemicellulose and cellulose degrading enzymes by the white-rot fungus Ceriporiopsis subvermispora was determined while growing in Pinus taeda wood chips. Enzymes produced by the fungus were extracted after 30 days of cultivation and at least two different xylanases were secreted. An endo-(1,4)-beta-xylanase was purified by means of ultrafiltration, anion exchange chromatography and gel filtration. Its molecular mass was 29 kDa and the pH and temperature optima were 5.0 and 60 degrees C, respectively. The endo-xylanase was able to hydrolyze xylan to principally xylotriose and xylotetraose and it has different activities against different xylans. With birchwood xylan as substrate, the enzyme showed a K(m) of 1.93 mg/ml and specific activity of 538 units/mg protein at 50 degrees C.