Achyrocline satureioides compounds, achyrobichalcone and 3-O-methylquercetin, induce mitochondrial dysfunction and apoptosis in human breast cancer cell lines.

Natural products are a valuable source of new molecules and are important for drug discovery. Many chemotherapeutics currently in clinical use were originated from natural sources and are effective cytotoxic agents. In this study, we investigated the cytotoxic and pro-apoptotic effects of achyrobichalcone (ACB) and 3-O-methylquercetin (3OMQ), two novel compounds isolated from the Achyrocline satureioides plant. Because extracts from this plant have been shown to have anticancer activity in vitro, we evaluated ACB and 3OMQ using a human breast cancer cell line, MDA-MB-231, and a nontumorigenic human breast epithelial cell line, MCF-12A. We found that ACB demonstrates cytotoxic effects on MDA-MB-231 cells, but not MCF-12A cells. 3OMQ also demonstrated cytotoxic effects on MDA-MB-231 cells, but with lower selectivity compared to treated MCF-12A cells. Cell death by both compounds was associated with caspase-9 and caspase-3/7 activation. Using high-resolution respirometry, we found that ACB and 3OMQ were able to cause acute mitochondrial dysfunction in MDA-MB-231-treated cells. These results suggest that apoptosis in MDA-MB-231 cells is induced through the activation of the mitochondrial-dependent pathway. Collectively, these findings suggest that ACB is a strong candidate for further anticancer in vivo tests.

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.

Development, optimisation and validation of a stability-indicating HPLC method of achyrobichalcone quantification using experimental designs.

INTRODUCTION: Achyrobichalcone is a new biflavonoid found in Achyrocline satureioides. It is structurally similar to other bioactive bichalcones that were proven to exert anti-cancer activity. Recently we isolated several achyrobichalcone batches on a semi-preparative scale, showing the need to assess the quality and stability of this substance by analytical methods. OBJECTIVE: To develop and validate a stability-indicating HPLC method of achyrobichalcone quantification using experimental designs. METHOD: The method was developed and optimised by Box-Behnken design using column temperature, flow rate and acetonitrile content in the mobile phase as factors and system suitability parameters as responses. Validation parameters were determined according to official compendiums. Robustness was determined by Plackett-Burman design. Stability of achyrobichalcone was assessed in alkaline, acid, oxidative, thermal and photolytic stress conditions. RESULTS: The ideal chromatographic conditions were defined from the optimisation: 37 % of acetonitrile, flow rate of 1.2 mL/min and 33°C temperature. All factors were significant for the resolution between achyrobichalcone and impurities peaks and for the retention factor. The mathematical model developed exhibited a good predictive capacity, and the design proved suitable. The HPLC method was successfully validated, being linear, specific, accurate and precise. The robustness test revealed that the flow rate and detection wavelength should be strictly controlled, as they affect achyrobichalcone concentration. The analyte was unstable only in alkaline media. CONCLUSION: The new method developed affords evaluation of the quality of achyrobichalcone obtained by isolation, and indicates the stability of the molecule under various stress conditions.

Quercetin/beta-cyclodextrin solid complexes prepared in aqueous solution followed by spray-drying or by physical mixture.

The present study was designed to investigate the influence of operating conditions (temperature, stirring time, and excess amount of quercetin) on the complexation of quercetin with beta-cyclodextrin using a 2(3) factorial design. The highest aqueous solubility of quercetin was reached under the conditions 37 degrees C/24 h/6 mM of quercetin. The stoichiometric ratio (1:1) and the apparent stability constant (Ks = 230 M(-1)) of the quercetin/beta-cyclodextrin complex were determined using phase-solubility diagrams. The semi-industrial production of a 1:1 quercetin/beta-cyclodextrin solid complex was carried out in aqueous solution followed by spray-drying. Although the yield of the spray-drying process was adequate (77%), the solid complex presented low concentration of quercetin (0.14%, w/w) and, thus, low complexation efficiency. The enhancement of aqueous solubility of quercetin using this method was limited to 4.6-fold in the presence of 15 mM of beta-cyclodextrin. Subsequently, an inclusion complex was prepared via physical mixture of quercetin with beta-cyclodextrin (molar ratio of 1:1 and quercetin concentration of 23% (w/w)) and characterized using infrared spectroscopy, differential scanning calorimetry, nuclear magnetic resonance spectroscopy, and scanning electron microscopy analyses. The enhancement of aqueous solubility of quercetin using this method was 2.2-fold, similar to that found in the complex prepared in aqueous solution before the spray-drying process (2.5-fold at a molar ratio of 1:1, i.e., 6 mM of quercetin and 6 mM of beta-cyclodextrin).

Quercetin and 3-O-methylquercetin in vitro skin layers permeation/retention from hydrogels: why only a methoxy group difference determines different behaviors?

OBJECTIVES: The present study was designed to verify if quercetin (QCT), a flavonoid with antioxidant and antiviral activity, and 3-O-methylquercetin (3OMQ), a quercetin C3-methoxylated derivative, present differences in their behavior against complexation with ß-cyclodextrin (ß-CD) and the corresponding permeation/retention trhough porcine ear skin, when incorporated into hydroxypropyl methylcellulose (HPMC) or chitosan (CS) hydrogels. METHODS: The influence of ß-CD on the skin permeation/retention of QCT and 3OMQ from hydrogels is comparatively evaluated for both flavonoids using porcine ear skin in Franz cells model. The properties of the two flavonoids using the semi-empirical method Recife Model was studied. KEY FINDINGS: Quercetin presented higher skin retention compared with its C3-methoxy derivative 3OMQ. The best permeation/retention of QCT was observed when it was incorporated into CS hydrogel containing 5% ß-CD, whereas, for 3OMQ, the HPMC hydrogel containing 5% ß-CD was the best formulation. The flavonoids complexation with ß-CD in water occurred preferentially with the insertion of the B ring through the secondary OH rim. CONCLUSIONS: The dynamic molecular modeling revealed that the methyl group at C3 in 3OMQ molecule determined significant difference in its complexation with ß-CD, in comparison to its analogous QCT and that difference is coincident with the permeation behavior of these flavonoids, denoting a possible relationship with their molecular dynamics.

A novel, simplified and stability-indicating high-throughput ultra-fast liquid chromatography method for the determination of rosmarinic acid in nanoemulsions, porcine skin and nasal mucosa.

Currently, there is an increasing interest on the development of topical formulations containing rosmarinic acid (RA) due to its well-documented antioxidant activity. This study aimed to develop and validate a stability-indicating ultra-fast liquid chromatography (UFLC) method for the determination of RA in nanoemulsions, porcine skin and nasal mucosa intended to be applied in permeation/retention studies and for development of topical nanoemulsions. Chromatographic separation was carried out using a C18 column packed with 2.6 µm particle size in isocratic conditions using as mobile phase water:acetonitrile (83:17, v/v), acidified with 0.1% trifluoracetic acid (v/v), with a total time of analysis of 3.5 min and detection at 330 nm. RA analysis was specific in the presence of both non-biological (blank nanoemulsion and receptor fluid) and biological matrices (porcine ear skin and porcine nasal mucosa). No interference of degradation products of RA was verified after different stress conditions such as acidic, alkaline, oxidative, light exposure (UV-A and UV-C) and thermal demonstrating the method stability-indicating property. The analytical (0.1-10.0 µg·mL-1) and bioanalytical (0.5-10.0 µg·mL-1) linearity was proved by analysis of the calibration curves of RA and no matrix effect was observed. The method was sensitive, precise and accurate, and showed recovery higher than 85%. The method was considered robust as evaluated by a Plackett-Burman experimental design. In the validated conditions, the RA was determined in the nanoemulsions obtained by spontaneous emulsification procedure (1.007 ±â€¯0.040 mg·mL-1), porcine ear skin (1.13 ±â€¯0.19 µg·cm-2) and nasal mucosa (22.46 ±â€¯3.99 µg·cm-2) after retention/permeation studies. Thus, a highly sensitive, simple, fast and stability-indicating method was developed for RA analysis during the development of topical nanoemulsions and bioanalytical assays in complex matrices.

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.

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.

A bioanalytical HPLC method for coumestrol quantification in skin permeation tests followed by UPLC-QTOF/HDMS stability-indicating method for identification of degradation products.

Coumestrol is present in several species of the Fabaceae family widely distributed in plants. The estrogenic and antioxidant activities of this molecule show its potential as skin anti-aging agent. These characteristics reveal the interest in developing analytical methodology for permeation studies, as well as to know the stability of coumestrol identifying the major degradation products. Thus, the present study was designed, first, to develop and validate a versatile liquid chromatography (HPLC) method to quantify coumestrol in a hydrogel formulation in different porcine skin layers (stratum corneum, epidermis, and dermis) in permeation tests. In the stability-indicating test coumestrol samples were exposed to stress conditions: temperature, UVC light, oxidative, acid and alkaline media. The degradation products, as well as the constituents extracted from the hydrogel, adhesive tape or skin were not eluted in the retention time of the coumestrol. Hence, the HPLC method showed to be versatile, specific, accurate, precise and robust showing excellent performance for quantifying coumestrol in complex matrices involving skin permeation studies. Coumestrol recovery from porcine ear skin was found to be in the range of 97.07-107.28 µg/mL; the intra-day precision (repeatability) and intermediate precision (inter-day precision), respectively lower than 4.71% and 2.09%. The analysis using ultra-performance liquid chromatography coupled to a quadrupole time-of-flight high definition mass spectrometry detector (UPLC-QTOF/HDMS) suggest the MS fragmentation patterns and the chemical structure of the main degradation products. These results represent new and relevant findings for the development of coumestrol pharmaceutical and cosmetic products.

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.

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.

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.

Influence of beta-cyclodextrin complexation on carbamazepine release from hydroxypropyl methylcellulose matrix tablets.

The in vitro release profiles of carbamazepine and beta-cyclodextrin either complexed or simply mixed and subsequently incorporated in hydrophilic matrix tablets containing 15 or 30% hydroxypropyl methylcellulose were evaluated. Solubility studies revealed a linear relationship between the increase in carbamazepine solubility and the increase in beta-cyclodextrin concentration. Drying methods (spray-drying and freeze-drying) were used to obtain carbamazepine/beta-cyclodextrin solid complexes in order to prepare tablets. The results demonstrated that matrix tablets containing carbamazepine/beta-cyclodextrin solid complexes displayed faster carbamazepine and beta-cyclodextrin release compared to that containing simple physical mixture. Gelling and matrix formation was impaired in formulation containing 15% hydroxypropyl methylcellulose and spray-dried complex. The comparison of spray-drying and freeze-drying revealed no significant influence of both drying methods on carbamazepine and beta-cyclodextrin dissolution rate when carbamazepine/beta-cyclodextrin complexes were incorporated in 30% hydroxypropyl methylcellulose matrix tablets. The results point to the possibility of modulating carbamazepine release using a hydroxypropyl methylcellulose matrix associated to the drug complexed with beta-cyclodextrin.

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.

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.

Multiple complexation of cyclodextrin with soy isoflavones present in an enriched fraction.

In the present study we evaluated the complexation of daidzein/genistein/glycitein, present in an isoflavone enriched fraction (IEF), with ß-cyclodextrin and 2-hydroxypropyl-ß-cyclodextrin (HPßCD). Based on the increased solubility and higher complexation efficiency, IEF and HPßCD solid complexes were prepared by kneading, freeze-drying, co-evaporation, spray-drying and microwave. The solid complexes were characterized using Fourier transformed-infrared spectroscopy, differential scanning calorimetry, X-ray diffraction, scanning electron microscopy, and nuclear magnetic resonance spectroscopy, and the isoflavone content and solubility were determined by liquid chromatography. The results suggest that the isoflavones daidzein, genistein and glycitein may be externally associated to HPßCD as well as that isoflavones/HPßCD inclusion complexes are formed through the insertion of B-ring into the cyclodextrin cavity. Except for the freeze-dried IEF/HPßCD solid complex, all complexes showed similar content and solubility. In conclusion, the three isoflavones showed to be able to simultaneously complex with HPßCD.

Optimization of headspace solid-phase microextraction for analysis of ß-caryophyllene in a nanoemulsion dosage form prepared with copaiba (Copaifera multijuga Hayne) oil.

Recent studies have shown the anti-inflammatory activity of Copaiba oils may be addressed to the high content of ß-caryophyllene, the most common sesquiterpene detected, especially in the Copaifera multijuga Hayne species. In the present study, nanoemulsions were proposed as a delivery system for copaiba oil in view to treat locally inflamed skin. This article describes the optimization and validation of a stability-indicating SPME-GC method, for ß-caryophyllene analysis in the nanoemulsions produced by high pressure homogenization. SPME methods are performed with PDMS (polydimethylsiloxane) fiber (100 µm). Three SPME parameters were evaluated by a three-level-three-factor Box-Behnken factorial design as potentially affecting the technique efficiency. According to the results obtained, the best conditions to extract ß-caryophyllene were: (i) sampling temperature of 45°C, (ii) sampling time of 20 min and (iii) no NaCl addition. Results coming from the forced degradation tests showed a reduction of ß-caryophyllene peak area when both caryophyllene methanolic solution and nanoemulsions were exposed to acid hydrolysis, UV-A irradiation, oxidative (H(2)O(2)) and thermolitic (60°C) conditions. Such reduction occurred in lower extent in the nanoemulsions, suggesting a protective effect of the formulation to ß-caryophyllene content. Since no degradation products were detected in the same retention time of ß-caryophyllene, the specificity of the method was demonstrated. The method was linear in the range of 0.14-0.68 µg mL(-1) of ß-caryophyllene (r(2)>0.999), and was also validated for precision (R.S.D.≤5.0%), accuracy (97.85-101.87%) and robustness. Finally, the method was applied to quantification of ß-caryophyllene content in the developed formulations.

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.

Isoflavone-aglycone fraction from Glycine max: a promising raw material for isoflavone-based pharmaceutical or nutraceutical products

ABSTRACT The present work was designed to obtain a fraction containing high concentration of isoflavone-aglycones from Glycine max (L.) Merr., Fabaceae, named isoflavone enriched-fraction, from a dry extract containing isoflavones-glycosides. A simple and low cost method was proposed: extraction of isoflavone glycosides from the G. max dry extract with a proper solvent, hydrolysis of the glycosides, recovery of the aglycones, and purification of the fraction containing high isoflavone-aglycones concentration. All the extraction and purification parameters were optimized based on the isoflavones yields, which were analyzed by liquid chromatography and expressed as total isoflavone aglycones. The optimization of the process conditions was accomplished using the classical one-variable-at-a-time method. The identity and purity of the isoflavones contained in this enriched fraction were determined by LC/UV/ESI/MS analysis, Fourier transformed-infrared spectroscopy, and 1H and 13C nuclear magnetic resonance spectroscopy. The physicochemical properties of the isoflavone enriched-fraction were evaluated by scanning electron microscopy, X-ray diffraction and differential scanning calorimetry. The moisture content, particle size, equilibrium solubility and thermal and photostability were also determined. The high isoflavone-aglycone content (daidzein, 489.35 mg/g; glycitein, 251.02 mg/g and genistein, 158.96 mg/g) as well as the high purity obtained (90% of total isoflavones) make this fraction a promising novel raw material for the production of isoflavone-aglycones based pharmaceuticals or functional foods.

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.