Coumestrol/hydroxypropyl-ß-cyclodextrin association incorporated in hydroxypropyl methylcellulose hydrogel exhibits wound healing effect: in vitro and in vivo study.

Several beneficial effects on the skin have been reported for coumestrol (COU), such as protection against photoaging and improvement of skin elasticity and thickness in postmenopausal women. However no reports on the effect of COU on wound healing were found. Nevertheless, COU has low aqueous solubility, which is a crucial limitation for biological tests. The present study was designed as a two-step experiment to evaluate the wound healing effect of COU. First, we used fibroblasts and the experimental in vitro artificial wound model, scratch assay, to compare the effects of COU free, dissolved in dimethyl sulfoxide (DMSO) or Dulbecco's modified Eagle's medium (DMEM), or associated with hydroxypropyl-ß-cyclodextrin (HPßCD). The 50 µM (66.1%) and 10 µM (56.3%) COU/HPßCD association induced cell proliferation and migration in inflicted wounds. Subsequently, the in vivo wound healing experimental model (Wistar rats) revealed that COU/HPßCD incorporated into hypromellose (HPMC) hydrogel had similar efficacy in wound healing in comparison to the positive control (Dersani®), with the advantage that 50% wound healing was achieved within a shorter period. In summary, the results successfully demonstrated, for the first time, the wound healing effect of COU/HPßCD incorporated into HPMC hydrogel and describe the feasibility of the biological tests with the use of HPßCD instead DMSO.

Ilex paraguariensis Pellets from a Spray-Dried Extract: Development, Characterization, and Stability.

Several studies have shown the potential use of Ilex paraguariensis in developing products with the aim to protect biological systems against oxidative stress-mediated damages. In the same way, technological studies have demonstrated the feasibility of obtaining dry products, by spray-drying process, from aqueous extracts of I. paraguariensis in laboratory. The present work was designed to develop pellets by extrusion/spheronization process, from an I. paraguariensis spray-dried powder. The pellets were characterized with respect to their chemical, physical, and technological properties, and the thermal and the photostability of the main polyphenol constituents were investigated. The pellets exhibited adequate size, shape, and high process yield (78.7%), as well as a good recovery of the total polyphenols (>95%) and a good dissolution in water (89.44 to 100.05%). The polyphenols were stable against light when conditioned in amber glass bottles; unstable against heat when the samples were conditioned either in open glass bottles or in hermetically sealed glass bottles and demonstrated to be hygroscopic and sensible to the temperature, especially when stored in permeable flasks. These findings pointed to the relevance of reducing the residual moisture content of pellets as well as of conditioning them in opaque humidity tight packages under low temperatures. The feasibility of obtaining pellets from an I. paraguariensis spray-dried powder using extrusion/spheronization technique was, for the first time, demonstrated. This finding represents a novelty for the herbal products in both pharmaceutical and food fields.

A versatile, stability-indicating and high-throughput ultra-fast liquid chromatography method for the determination of isoflavone aglycones in soybeans, topical formulations, and permeation assays.

There is a growing interest in the pharmaceutical field concerning isoflavones topical delivery systems, especially with regard to their skin care properties and antiherpetic activity. In this context, the present work describes an ultra-fast liquid chromatography method (UFLC) for determining daidzein, glycitein, and genistein in different matrices during the development of topical systems containing isoflavone aglycones (IA) obtained from soybeans. The method showed to be specific, precise, accurate, and linear (0.1 to 5 µg mL(-1)) for IA determination in soybean acid extract, IA-rich fraction obtained after the purification process, IA loaded-nanoemulsions, and topical hydrogel, as well as for permeation/retention assays in porcine skin and porcine esophageal mucosa. The matrix effect was determined for all complex matrices, demonstrating low effect during the analysis. The stability indicating UFLC method was verified by submitting IA to acidic, alkaline, oxidative, and thermal stress conditions, and no interference of degradation products was detected during analysis. Mass spectrometry was performed to show the main compounds produced after acid hydrolysis of soybeans, as well as suggest the main degradation products formed after stress conditions. Besides the IA, hydroxymethylfurfural and ethoxymethylfurfural were produced and identified after acid hydrolysis of the soybean extract and well separated by the UFLC method. The method's robustness was confirmed using the Plackett-Burman experimental design. Therefore, the new method affords fast IA analysis during routine processes, extract purification, products development, and bioanalytical assays.

Isolation of achyrobichalcone from Achyrocline satureioides by high- speed countercurrent chromatography.

Achyrobichalcone is a biflavonoid recently found in Achyrocline satureioides. This substance has unprecedented chemical structure and occurrence, but resembles other bioactive bichalcones, which have important pharmacological properties, such as anticancer activity. The major challenge for evaluation of the physicochemical and biological properties of this new molecule is the isolation step, which affects the purity and yield of the isolated product. Thus, the objective of this work was to develop a semi-preparative method for achyrobichalcone isolation from Achyrocline satureioides by high-speed countercurrent chromatography. The high-speed countercurrent chromatography separation was achieved in two steps. In the first step, an enriched fraction of achyrobichalcone from the freeze-dried extract was obtained, using the solvent system hexane-ethyl acetate-methanol-water 0.8:1:0.8:1, v/v. The purification of achyrobichalcone from the enriched fractions was achieved by further high-speed countercurrent chromatography fractionation with hexane-ethyl acetate-methanol-water 0.9:0.9:0.8:1, v/v. The final isolated product was obtained using preparative thin layer chromatography and crystallization procedure. A yellow semi-crystalline solid with purity close to 90% was obtained as the final product. The mass recovery of achyrobichalcone isolation was near 67%. The structural identification from spectroscopic and chromatographic techniques confirmed the achyrobichalcone structure. This is the first report of achyrobichalcone isolation on a semi-preparative scale by high-speed countercurrent chromatography. This method afforded achyrobichalcone in good yield and purity for further biopharmaceutical studies.

Identification of phenolic compounds in Equisetum giganteum by LC-ESI-MS/MS and a new approach to total flavonoid quantification.

Equisetum giganteum L., commonly called "giant horsetail", is an endemic species of Latin America. Its aerial parts have been widely used in ethnomedicine as a diuretic and in herbal medicine and food supplements as a raw material. The phenolic composition of E. giganteum stems was studied by liquid chromatography coupled to diode array detection (LC-DAD) and liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), which identified caffeic acid derivatives, flavonoids and styrylpyrones. The most abundant glycosilated flavonoids in this sample were kaempferol derivatives. Other rare phenolic components, namely, quercetin-3-O-(caffeoyl)-glucoside and 3-hydroxyhispidin-3,4'-di-O-glucoside, were reported for first time in the Equisetum genus. An LC-UV method for the simultaneous quantification of flavonoid aglycones in E. giganteum obtained after hydrolysis was developed and validated. The method exhibited excellent linearity for all analytes, with regression coefficients above 0.998, LOD ≥ 0.043µg mL(-1), LOQ ≥ 0.158 µg mL(-1) and recovery rates of 96.89-103.33% and 98.22-102.49% for quercetin and kaempferol, respectively. The relative standard deviation for the intra- and inter-day precision was ≤ 3.75%. The hydrolysis process was optimized by central composite rotational design and response surface analysis. The second-order response models for the aglycones contents were as follows: quercetin (µg g(-1))=24.8102+55.2823 × HCl+0.776997 × Time-7.23852 × HCl(2)-7.46528E-04 × Time(2)-0.229167 × HCl × Time; kaempferol (µg g(-1))=-9.66755+974.822 × HCl+11.8059 × Time-130.612 × HCl(2)-0.0125694×Time(2) -3.22917 × HCl × Time, with estimated optimal conditions of 1.18 M HCl and 205 min of hydrolysis. The results obtained with these new methods were compared to those from a spectrophotometric assay used to determine the total flavonoids in the Equisetum arvense monograph (Horsetail, British Pharmacopoeia 2011). For all four species analyzed (E. giganteum, E. arvense, E. hyemale and E. bogotense), the calculated aglycone content was higher using the optimized hydrolysis conditions. Additionally, the LC method was more appropriate and specific for quantitative analysis.

Technological characterization and stability of Ilex paraguariensis St. Hil. Aquifoliaceae (Maté) spray-dried powder.

The present work was designed to produce an Ilex paraguariensis spray-dried powder (SDP), in semi-industrial scale, in order to characterize its technological and chemical properties as well as to evaluate the thermal stability and photostability of the main polyphenol constituents. The yield of the spray-drying process was satisfactory (67%). The resulting SDP showed to be a material presenting spherical particles with a mean size of 19.6 µm, smooth surface, and good flow properties. The four polyphenol compounds previously reported for the species--neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and rutin--were identified. Regarding the photostability test, the polyphenols present in the SDP proved to be stable against ultraviolet C radiation for 48 hours, independently of the packaging material. In the thermal stability test, the polyphenols were demonstrated to be hygroscopic and responsive to temperature (40°C) under an atmosphere of high relative humidity (75%) for 4 months, especially when the SDP was conditioned in permeable flasks. These findings demonstrate that heat and residual moisture content play an important role in the stability of the polyphenols and reinforce the relevance of conditioning SDP in humid tight packages under low temperatures.

Achyrocline satureioides (Lam.) DC., Asteraceae: development of granules from spray dried powder

Achyrocline satureioides (Lam.) DC., Asteraceae, is a herbal specie widely used in folk medicine in the south of Brazil, Uruguay, Argentina and Paraguay. The technological characteristics of an Achyrocline satureioides spray dried extract powder, produced in semi-industrial scale, as well as the feasibility of the granules are reported in the present work. The spray dried powder was characterized as a fine powder consisting of small spherical particles with rough and porous surface. The Hausner's factor, Carr's index, and densification index of the spray dried powder were, respectively, 1,23, 18,9 percent, and 27,2 mL, characterizing it as a poor flow and low density powder. The preparation of granules from this spray dried powder, through dry disaggregation method, yielded irregularly shaped granules, with a rough surface, but with better flow and compactability characteristics. These granules presented a Hausner's factor, a Carr's index, and a densification index of, respectively, 1,09, 8,16 percent, and 12,33 mL. The LC assay of the main polyphenols, quercetin, luteolin, and 3-O-methylquercetin revealed that the granulation process did not changed the quantitative and qualitative profile of these constituents originally present in the spray dried powder. The comparative evaluation of the physical stability of both the spray dried powder and the granules, under relative humidity conditions of 65 percent and 99 percent, showed an expressive reduction in the humidity sorption on the granules as compared to the spray dried powders.

Achyrocline satureioides (Lam.) DC., Asteraceae, é uma planta amplamente utilizada na medicina popular no sul do Brasil, Uruguai, Argentina e Paraguai. As características tecnológicas do extrato seco por aspersão de Achyrocline satureioides, produzido em escala semi-industrial, assim como a viabilidade da produção de granulados são relatadas no presente trabalho. O extrato seco por aspersão foi caracterizado como um pó fino, composto por pequenas partículas esféricas com superfície rugosa e porosa. O fator de Hausner, índice de Carr e o índice de densificação das partículas foram, respectivamente, 1,23, 18,9 por cento e 27,2 mL, caracterizando-o como um pó com fluxo pobre e de baixa densidade. A preparação dos granulados a partir deste extrato seco por aspersão, através do método de desagregação por via seca, originou grânulos com forma irregular, superfície rugosa, mas com melhor fluxo e melhores características de compactabilidade. Estes grânulos apresentaram fator de Hausner, índice de Carr e índice de densificação de 1,09, 8,16 por cento e 12,33 mL, respectivamente. A análise por de CLAE dos polifenóis principais quercetina, luteolina e 3-O-metilquercetina revelou que o processo de granulação não altera o perfil quantitativo e qualitativo dos constituintes inicialmente presentes no extrato seco. A avaliação da estabilidade física do extrato seco por aspersão e do granulado, em condições de unidade relativa de 65 e 99 por cento, mostrou uma expressiva redução na absorção de umidade dos grânulos quando comparados com o extrato seco.

Quantification of saponins in extractive solution of mate leaves (Ilex paraguariensis A. St. Hil.).

Mate (Ilex paraguariensis A. St. Hil.) is a raw material used to prepare popular beverages in South America, whose healthy effects have been pointed out. The leaves and stems of mate contain a complex assemblage of saponins, mainly ursolic acid and oleanolic acid glycosides, and their quantification presents difficulties such as weak cromophorous sites and interference of other compounds. On the other hand, the sugar moieties are a chief character that distinguishes them. We developed and validated a high-performance liquid chromatography method using a refractometer detector for quantifying the major saponins in extractive solution of mate leaves. The validation indicates the suitability of the method, presenting linearity in the concentration range between 102.5 to 854.0 microg/mL to matesaponin 1 and between 103.0 to 515.0 microg/mL to matesaponin 3. The repeatability coefficient of variation was 4.70% and 6.61% for matesaponin 1 and matesaponin 3, respectively. The inter-day coefficient of variation was 6.77% and 7.79% for matesaponin 1 and matesaponin 3, respectively. The analysis of plants from Brazilian States Mato Grosso do Sul and Rio Grande do Sul showed the predominance of matesaponins 1, 2, and 3 in the corresponding extractive solution. The sum of these three saponins ranged between 3 and 10 mg/g for mature leaves in the majority of sampled plants.

HPLC method for the determination of ecdysterone in extractive solution from Pfaffia glomerata.

A RP-LC method was developed and validated to quantify ecdysterone in extractive solution from subterraneous parts of Pfaffia glomerata. The analysis was performed using a RP-18 column with acetonitrile:water isocratic elution and the detection was carried out by UV at 242 nm. The standard curve for ecdysterone was linear over the range of 5.2-41.6 microg/ml (R2=0.9995). The extractive solution showed linear response in the range of 25.05-175.35 microg/ml (R2=0.9977). This method showed excellent repeatability (relative standard deviation, R.S.D.<2.0%), intermediary precision (R.S.D.=2.13%) and accuracy (101.04; R.S.D.=1.51%). The limit of detection (LOD) was 0.036 microg/ml and the limit of quantification (LOQ) was 0.110 microg/ml, demonstrating the sensitivity of the method. This assay can be readily utilized as quality controlled method for P. glomerata preparations.

Mathematical evaluation of in vitro release profiles of hydroxypropylmethylcellulose matrix tablets containing carbamazepine associated to beta-cyclodextrin.

The release kinetics of carbamazepine (CBZ) either complexed or physically mixed with beta-cyclodextrin (betaCD) from hydroxypropylmethylcellulose (HPMC) matrix tablets was investigated using different mathematical equations. The model-dependent approach was compared to the utilization of fit factors. Notwithstanding difference (f1) and similarity (f2) factors allowed the differentiation of the formulations containing CBZ complexed with betaCD from the one containing a simple physical mixture of CBZ and betaCD. The Weibull model was more useful for comparing the release profiles. Weibull parameters were more sensitive to the differences between the two release kinetic data.

Bioavailability of carbamazepine:beta-cyclodextrin complex in beagle dogs from hydroxypropylmethylcellulose matrix tablets.

The bioavailability of a carbamazepine:beta-cyclodextrin (CBZ:betaCD) complex from hydroxypropylmethylcellulose (HPMC) matrix tablets was evaluated in beagle dogs. A solubility study demonstrated the improvement of CBZ aqueous solubility by adding increasing amounts of betaCD. The 1:1 CBZ:betaCD molar ratio was chosen to produce the complex, which was obtained by spray-drying. Matrix tablets were prepared by direct compressing either a CBZ:betaCD complex or a physical mixture of both substances with HPMC. Both matrix formulations displayed a controlled-release profile when compared to the reference formulation (Tegretol CR 200). CBZ presented a significantly higher bioavailability from matrix tablets containing the CBZ:betaCD complex than that obtained from Tegretol CR 200). Although a high inter-subject variability was observed, the results pointed to the feasibility of using betaCD in order to modulate CBZ release and absorption, as well as to reduce the drug dosage maintaining the same plasma levels.

From a medicinal plant to a pharmaceutical dosage form. A (still) long way for Brazilian medicinal plants

Phytopharmaceutical drugs play an important role in the Brazilian health system considering both economic and cultural perspectives. Pondering the richness of the Brazilian flora and the wide use of medicinal plants by the population, there should be sufficient knowledge to apply that information as a suitable starting point to transform medicinal plants into adequate industrial pharmaceutical dosage forms. Problems involving lack of organized knowledge and scientific information are not sufficiently solved to accomplish the modern drug concept, which has to consider efficacy, safety and constant quality. On basis of one of the most used medicinal plants in southern Brazil, Achyrocline satureioides, this paper presents and discusses the problems involving the transformation of medicinal plants into phytopharmaceutical drugs, showing some steps carried out by the academic milieu in order to provide answers to solve this question.