Pod storage with roasting: A tool to diversifying the flavor profiles of dark chocolates produced from 'bulk' cocoa beans? (part I: aroma profiling of chocolates).

The impact of pod storage (PS) and roasting temperature (RT) on the aroma profiles of dark chocolates were evaluated. Cocoa liquor samples comprised of ten different combinations of PS and RT, whilst keeping the roasting time fixed at 35 min. Additionally, commercial cocoa liquors from renowned origins (Ecuador, Madagascar, Venezuela, Vietnam, Ivory Coast and Ghana) were acquired for comparison. From these, 70% dark chocolates were produced under the same conditions after which they were subjected to headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis. Although both PS and RT were found to influence the aroma volatile concentrations, the impact of RT over PS seemed to be greater. An agglomerative hierarchical clustering (AHC) of all chocolates on the basis of their aroma profiles revealed a similar impact as earlier observed, where major clustering of the chocolates was in accordance with the intensity of the roasting process applied. However, within each group, the dissimilarities owing to PS among the chocolates was clearly depicted. Comparatively, chocolates with low (100-120 °C), instead of moderate to high (135-160 °C) RT's, rather showed a low dissimilarity with those from the commercial cocoa liquors of the different origins. Although from the same beans, the diversity of aroma profiles of these chocolates as well as the similitude of some treatments to some chocolates from commercial grade cocoa liquors, unequivocally underscores the possibility for steering diverse distinct flavors from 'bulk' cocoa through PS and roasting, with beneficial implications, both from an application and an economic point of view.

Capillary zone electrophoresis method to assay tipranavir capsules and identification of oxidation product and organic impurity by quadrupole-time of flight mass spectrometry.

Tipranavir (TPV) is one of the most recently developed protease inhibitors (PI) and it is specially recommended for treatment-experienced patients who are resistant to other PI drugs. In this work, a simple and friendly environmental CZE stability-indicating method to assay TPV capsules was developed and two TPV organic impurities were identified by high resolution mass spectrometry (HRMS). The optimized analytical conditions were: background electrolyte composed of sodium borate 50mM, pH 9.0 and 5% of methanol; voltage + 28kV; hydrodynamic injection of 5s (100mbar), detection wavelength 240nm, at 25°C. The separation was achieved in a fused silica capillary with 50µm × 40cm (inner diameter × effective length), using furosemide as internal standard. All the validation parameters were met and the method was specific, even in the presence of degradation products and impurities. Oxidation was indicated as the main degradation pathway among those evaluated in this study (acidic, alkaline, thermal, photolytic and oxidative) and it showed a second order degradation kinetic, under the conditions used in this study. The main oxidation product and an organic impurity detected in the standard were characterized by Q-TOF, and both of them correspond to oxidation products of TPV.

Capillary Electrophoresis as Tool for Diastereomeric Separation in a Trichilia catigua Fraction.

INTRODUCTION: The tree Trichilia catigua, popularly known as "catuaba", shows several biological activities and has emerged as a potential source of new drugs. Considering that more than 10 species are known under the same popular name, regulatory agencies require more rigorous quality control of this medicinal plant. OBJECTIVE: To develop and validate a methodology using capillary electrophoresis (CE) with ultraviolet (UV) detection for analysing polyphenols in the ethyl-acetate fraction (EAF) of Trichilia catigua. METHODOLOGY: Different electrophoretic conditions (such as wavelength of UV detection, voltage, buffer concentration and pH, cyclodextrin type and concentration) were investigated. After optimisation, borate buffer 80 mmol/L at pH 8.80 with 2-hydroxypropyl-ß-cyclodextrin 10 mmol/L was selected as background electrolyte. A voltage reduction was used to improve the separation of a diastereomeric pair of cinchonains. RESULTS: The method proved to be simple, sensitive, accurate, linear, precise and reproducible. For the first time in natural products analysis, a voltage reduction and hydroxypropyl-ß-cyclodextrin were used to improve the separation of diastereomeric pairs. Until now, this is the only described methodology able to separate catechin, epicatechin, cinchonains Ia, Ib, IIa, and IIb from Trichilia catigua samples on the same run in less than 12 min. When compared to the high performance liquid chromatography with photo-diode array detection (HPLC-PDA) method previously developed by our research group, the CE method was more efficient, faster, less expensive and less polluting. CONCLUSION: Our results demonstrate that this method could be employed in a quality-control laboratory for the quantification of polyphenols in EAF of Trichilia catigua. Copyright © 2016 John Wiley & Sons, Ltd.

Multiresponse Optimisation Applied to the Development of a TLC Autography for the Detection of Tyrosinase Inhibitors.

INTRODUCTION: Autographic methods are useful tools to detect bioactive compounds in complex matrixes. Experimental design and optimisation techniques were implemented for the development of an autographic assay suitable for the detection of tyrosinase inhibitors. OBJECTIVES: To develop an autographic assay to detect tyrosinase inhibitors using gel entrapped enzyme, experimental design and response surface methodology (RSM) to optimise conditions with a minimum number of experiments. METHODS: Gel entrapment was used for the assay and the effects of four factors on the sensitivity and the detection limit for known inhibitors of the enzyme were evaluated. The factors were: tyrosinase amount (TA), L-tyrosine amount (LTA), incubation time and incubation temperature. RESULTS: The assay allowed the detection of kojic acid in an extract of Calamagrostis viridiflavescens (Poir.) Steud spiked with 0.1% w/w. CONCLUSION: The developed assay is able to detect tyrosinase inhibitors present in complex matrixes in a reproducible way.

Characterization and evaluation of coconut aroma produced by Trichoderma viride EMCC-107 in solid state fermentation on sugarcane bagasse

Background Sugarcane bagasse was shown to be an adequate substrate for the growth and aroma production by Trichoderma species. In the present work the ability of Trichoderma viride EMCC-107 to produce high yield of coconut aroma in solid state fermentation (SSF) by using sugarcane bagasse as solid substrate was evaluated. The produced aroma was characterized. Results Total carbohydrates comprised the highest content (43.9% w/w) compared with the other constituents in sugarcane bagasse. The sensory and gas chromatography-mass spectrometric (GC-MS) analysis revealed that the highest odor intensity and maximum yield of volatiles were perceived at the 5th d of induction period. The unsaturated lactone, 6-pentyl-α-pyrone (6-PP), was the major identified volatile compound. Saturated lactones, δ-octalactone, γ-nonalactone, γ-undecalactone, γ-dodecalactone and δ-dodecalactone, were also identified in the coconut aroma produced during the induction period (12 d). A quite correlation was found between the composition and odor profile of the produced aroma. The effect of varying the concentration of sugarcane bagasse on 6-PP production and biomass growth was evaluated. The results revealed high 6-PP production at 4.5 g sugarcane bagasse whereas the biomass showed significant (P < 0.05) increase by increasing the concentration of sugarcane bagasse. Conclusion The concentration of 6-PP, the most contribution of coconut aroma, produced in present study (3.62 mg/g DM) was higher than that reported in previous studies conducted under the same fermentation conditions. The significant increase in biomass with increasing the concentration of sugarcane bagasse may be attributed to the increase in sugar content that acts as carbon and energy source.

Mariculture and natural production of the antitumoural (+)-discodermolide by the Caribbean marine sponge Discodermia dissoluta.

Biotechnological research on marine organisms, such as ex situ or in situ aquaculture and in vitro cell culture, is being conducted to produce bioactive metabolites for biomedical and industrial uses. The Caribbean marine sponge Discodermia dissoluta is the source of (+)-discodermolide, a potent antitumoural polyketide that has reached clinical trials. This sponge usually lives at depths greater than 30 m, but at Santa Marta (Colombia) there is a shallower population, which has made it logistically possible to investigate for the first time, on ways to supply discodermolide. We thus performed in situ, 6-month fragment culture trials to assess the performance of this sponge in terms of growth and additional discodermolide production and studied possible factors that influence the variability of discodermolide concentrations in the wild. Sponge fragments cultured in soft mesh bags suspended from horizontal lines showed high survivorship (93 %), moderate growth (28 % increase in volume) and an overall rise (33 %) in the discodermolide concentration, equivalent to average additional production of 8 µg of compound per millilitre of sponge. The concentration of discodermolide in wild sponges ranged from 8 to 40 µg mL(-1). Locality was the only factor related to discodermolide variation in the wild, and there were greater concentrations in peripheral vs. basal portions of the sponge, and in clean vs. fouled individuals. As natural growth and regeneration rates can be higher than culture growth rates, there is room for improving techniques to sustainably produce discodermolide.

Validation and evaluation of an HPLC methodology for the quantification of the potent antimitotic compound (+)-discodermolide in the Caribbean marine sponge Discodermia dissoluta.

The sponge Discodermia dissoluta is the source of the potent antimitotic compound (+)-discodermolide. The relatively abundant and shallow populations of this sponge in Santa Marta, Colombia, allow for studies to evaluate the natural and biotechnological supply options of (+)-discodermolide. In this work, an RP-HPLC-UV methodology for the quantification of (+)-discodermolide from sponge samples was tested and validated. Our protocol for extracting this compound from the sponge included lyophilization, exhaustive methanol extraction, partitioning using water and dichloromethane, purification of the organic fraction in RP-18 cartridges and then finally retrieving the (+)-discodermolide in the methanol-water (80:20 v/v) fraction. This fraction was injected into an HPLC system with an Xterra RP-18 column and a detection wavelength of 235 nm. The calibration curve was linear, making it possible to calculate the LODs and quantification in these experiments. The intra-day and inter-day precision showed relative standard deviations lower than 5%. The accuracy, determined as the percentage recovery, was 99.4%. Nine samples of the sponge from the Bahamas, Bonaire, Curaçao and Santa Marta had concentrations of (+)-discodermolide ranging from 5.3 to 29.3 microg/g(-1) of wet sponge. This methodology is quick and simple, allowing for the quantification in sponges from natural environments, in situ cultures or dissociated cells.

A validated higher-performance liquid chromatography method for quantification of cinchonain Ib in bark and phytopharmaceuticals of Trichilia catigua used as Catuaba.

The hydroalcoholic extract, prepared from authentic chopped barks of Trichilia catigua, was evaluated by high-performance liquid chromatography using a diode array detector (200-400 mn). The crude extract was purified by rotation locular counter-current chromatography and the chloroform fraction obtained was clean-up by solid-phase extraction. With the aim of getting preliminary structure information on-line, the methanol fraction thus obtained was analyzed by gradient elution using the diode array detector coupled to a mass spectrometer. The presence of flavalignan in this extract was inferred by the chromatographic band, in the total ion current trace, that had an [M-H](-) = 451. With this information, cinchonain Ib was isolated as a pure compound from the crude hydroalcoholic extract using a solid-phase extraction procedure for the sample clean-up followed by a semi-preparative separation using the reverse mode of elution. The isolated compound, after complete characterization, was used as an external standard for the development and validation of a method for the analysis of this compound in herbal medicines using the ultraviolet as the detector. The validated method has been successfully applied for quantification of cinchonain Ib in commercialized herbal medicines sold as Catuaba in Brazil and also in standard chopped barks of T. catigua.

An HPLC-PAD method to analyse flavonoid glycosides and styrylpyrones from Cryptocarya species (Lauraceae).

An HPLC-PAD method has been developed in order to evaluate simultaneously the main secondary metabolites, flavonoid glycosides and styrylpyrones, of leaves of Cryptocarya moschata. The sample preparation, consisting of extraction, liquid-liquid extraction and centrifugation, requires minimum sample manipulation but produces high yields with reproducibility, selectivity and simplicity. HPLC on a C18 column presents each class of metabolites grouped and with good resolution of the main compounds. The experimental conditions can be used to study inter- and intra-specific variability of secondary metabolites in Cryptocarya spp.