Gerardo F. Barbero

Ultrasound-assisted extraction of capsaicinoids from peppers

The development of a rapid, reproducible and simple method of extraction of the majority capsaicinoids (nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin and homodihydrocapsaicin) present in hot peppers by the employment of ultrasound-assisted extraction is reported. The study has covered four possible solvents for the extraction (acetonitrile, methanol, ethanol and water), the optimum temperature for extraction (10-60 degrees C), the extraction time (2-25 min), the quantity of sample (0.2-2 g), and the volume of solvent (15-50 mL). Under the optimum conditions of the method developed, methanol is employed as solvent, at a temperature of 50 degrees C and an extraction time of 10 min. The repeatability and reproducibility of the method (R.S.D.<3%) have been determined. The capsaicinoids extracted have been analysed by HPLC with fluorescence detection and using monolithic columns for the chromatographic separation. The method developed has been employed for the quantification of the various capsaicinoids present in different varieties of hot peppers cultivated in Spain.

Supercritical carbon dioxide extraction of capsaicinoids from malagueta pepper (Capsicum frutescens L.) assisted by ultrasound.

Extracts from malagueta pepper (Capsicum frutescens L.) were obtained using supercritical fluid extraction (SFE) assisted by ultrasound, with carbon dioxide as solvent at 15MPa and 40°C. The SFE global yield increased up to 77% when ultrasound waves were applied, and the best condition of ultrasound-assisted extraction was ultrasound power of 360W applied during 60min. Four capsaicinoids were identified in the extracts and quantified by high performance liquid chromatography. The use of ultrasonic waves did not influence significantly the capsaicinoid profiles and the phenolic content of the extracts. However, ultrasound has enhanced the SFE rate. A model based on the broken and intact cell concept was adequate to represent the extraction kinetics and estimate the mass transfer coefficients, which were increased with ultrasound. Images obtained by field emission scanning electron microscopy showed that the action of ultrasonic waves did not cause cracks on the cell wall surface. On the other hand, ultrasound promoted disturbances in the vegetable matrix, leading to the release of extractable material on the solid surface. The effects of ultrasound were more significant on SFE from larger solid particles.

Optimization of the ultrasound-assisted extraction of anthocyanins and total phenolic compounds in mulberry (Morus nigra) pulp.

New ultrasound-assisted extraction methods for the determination of anthocyanins and total phenolic compounds present in mulberries have been developed. Several extraction variables, including methanol composition (50-100%), temperature (10-70°C), ultrasound amplitude (30-70%), cycle (0.2-0.7s), solvent pH (3-7) and solvent-solid ratio (10:1.5-20:1.5) were optimized. A Box-Behnken design in conjunction with a response surface methodology was employed to optimize the conditions for the maximum response based on 54 different experiments. Two response variables were considered: total anthocyanins and total phenolic compounds. Extraction temperature and solvent composition were found to be the most influential parameters for anthocyanins (48°C and 76%) and phenolic compounds (64°C and 61%). The developed methods showed high reproducibility and repeatability (RSD<5%). Finally, the new methods were successfully applied to real samples in order to investigate the presence of anthocyanins and total phenolic compounds in several mulberry jams.

Evolution of total and individual capsaicinoids in peppers during ripening of the Cayenne pepper plant (Capsicum annuum L.)

The evolution of total capsaicinoids and the individual contents of the five major capsaicinoids: nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin and homodihydrocapsaicin present in the Cayenne pepper (Capsicum annuum L.), during fruit ripening, has been established. Capsaicinoids begin to accumulate gradually in the peppers from the beginning of its development up to a maximum concentration (1,789 μmol/Kg FW). From this time there is initially a sharp decrease in the total capsaicinoid content (32%), followed by a gradual decrease until day 80 of ripening. The two major capsaicinoids present in the Cayenne pepper are capsaicin and dihydrocapsaicin, which represent between 79% and 90%, respectively, of total capsaicinoids depending on fruit ripening. The relative content of capsaicin differs from the evolution of the other four capsaicinoids studied.

Application of Hansch’s Model to Capsaicinoids and Capsinoids: A Study Using the Quantitative Structure−Activity Relationship. A Novel Method for the Synthesis of Capsinoids

We describe a synthetic approach for two families of compounds, the capsaicinoids and capsinoids, as part of a study of the quantitative relationship between structure and activity. A total of 14 capsaicinoids of increasing lateral chain lengths, from 2 to 16 carbon atoms, were synthesized. In addition, 14 capsinoids with identical lateral chains, as well as capsiate and dihydrocapsiate, have been synthesized, and a new method for the synthesis of these compounds has been developed. The yields range from 48.35 to 98.98%. It has been found that the synthetic capsaicinoids and capsinoids present a lipophilia similar to those of the natural compounds and present similar biological activity. The bioactivity of the synthetic capsaicinoids and capsinoids decreases proportionally to the degree of difference in lipophilia (higher or lower) compared to the natural compounds. Biological activity was determined using the etiolated wheat (Triticum aestivum L.) coleoptiles bioassay and by comparing results of the synthesis with those presented by their counterpart natural compounds. The bioactivities found correlated directly to the lipophilic properties of the synthesized compounds.

Fast analysis of curcuminoids from turmeric (Curcuma longa L.) by high-performance liquid chromatography using a fused-core column.

The recent development of fused-core technology in HPLC columns is enabling faster and highly efficient separations. This technology was evaluated for the development of a fast method for the analysis of main curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin) present in extracts of turmeric (Curcuma longa L.). A step-by-step strategy was used to optimize temperature (40-55 °C), flow rate (1.0-2.5 mL min(-1)), mobile phase composition and equilibration time (1-5 min). A gradient method was developed using acidified water and acetonitrile combined with high column temperature (55 °C) and flow rate (2.5 mL min(-1)). Optimized conditions provided a method for the separation of these three curcuminoids in approximately 1.3 min with a total analysis time (sample-to-sample) of 7 min, including the clean-up and the re-equilibration of the column. Evaluation of chromatographic performance revealed excellent intraday and interday reproducibility (>99%), resolution (>2.23), selectivity (>1.12), peak symmetry (1.24-1.42) while presenting low limits of detection (<0.40 mg L(-1)) and quantification (<1.34 mg L(-1)). The robustness of the method was calculated according to the concentration/dilution of the sample and the injection volume. Several combinations of methanol and ethanol with water as sample solvents were evaluated and the best chromatographic results and extraction rate were obtained using 100% methanol. Finally, the developed method was validated with different extracts of turmeric rhizome and products that use turmeric in their formulation.

Recovery of anthocyanins from residues of Rubus fruticosus, Vaccinium myrtillus and Eugenia brasiliensis by ultrasound assisted extraction, pressurized liquid extraction and their combination

This work investigated the extraction efficiency of polyphenols (anthocyanins) from blackberry, blueberry and grumixama residues using combined ultrasonic assisted extraction (UAE) and pressurized liquid extraction (PLE) (UAE+PLE). The performance of UAE+PLE was compared to those achieved by the isolated PLE and UAE methods and conventional Soxhlet extraction. The effects of the extraction methods and solvents (acidified water pH 2.0, ethanol+water 50% v/v and ethanol+water 70% ethanol v/v) on total phenolics content, anthocyanin composition and antioxidant capacity of extracts were investigated by a full factorial design. The extraction efficiency for total phenolics and antioxidant capacity in decreasing order was: UAE+PLE>PLE≈Soxhlet>UAE, and for anthocyanins it was: Soxhlet≈UAE>UAE+PLE>PLE, using hydroethanolic mixtures as solvents. Extractions with acidified water and ultrasound were not effective to recover phenolics. Two, four and fourteen anthocyanins were identified in the extracts from grumixama, blackberry and blueberry, respectively.

Capsaicinoid Contents in Peppers and Pepper-Related Spicy Foods

The quantification of the major capsaicinoids, namely nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin, present in spicy foods made from peppers has been performed. The capsaicin content is directly related to the pungency properties of foods that contain hot peppers. The samples studied included 10 different dried hot peppers, 19 hot sauces, 4 kinds of paprika, and 4 different ketchups. The range of concentrations of capsaicinoids found were as follows: dried hot peppers (554.1–1705.9 mol kg−1), paprikas (582.0–665.0 mol kg−1), spicy ketchups (4.0–12.4 mol kg−1), and hot sauces (4.6–843.8 mol kg−1). Variability in the capsaicinoid content was found, with capsaicin and dihydrocapsaicin present at the highest levels. The capsaicin content was generally higher than the dihydrocapsaicin content.

Determination of Ignitable Liquids in Fire Debris: Direct Analysis by Electronic Nose

Arsonists usually use an accelerant in order to start or accelerate a fire. The most widely used analytical method to determine the presence of such accelerants consists of a pre-concentration step of the ignitable liquid residues followed by chromatographic analysis. A rapid analytical method based on headspace-mass spectrometry electronic nose (E-Nose) has been developed for the analysis of Ignitable Liquid Residues (ILRs). The working conditions for the E-Nose analytical procedure were optimized by studying different fire debris samples. The optimized experimental variables were related to headspace generation, specifically, incubation temperature and incubation time. The optimal conditions were 115 °C and 10 min for these two parameters. Chemometric tools such as hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA) were applied to the MS data (45–200 m/z) to establish the most suitable spectroscopic signals for the discrimination of several ignitable liquids. The optimized method was applied to a set of fire debris samples. In order to simulate post-burn samples several ignitable liquids (gasoline, diesel, citronella, kerosene, paraffin) were used to ignite different substrates (wood, cotton, cork, paper and paperboard). A full discrimination was obtained on using discriminant analysis. This method reported here can be considered as a green technique for fire debris analyses.

Determination of Melatonin in Rice (Oryza sativa) Grains by Pressurized Liquid Extraction

Melatonin provides a number of benefits for human health. The study reported here concerns the optimization, validation, and application of analytical pressurized liquid extraction and high-performance liquid chromatography coupled to a fluorescence detector for the determination of melatonin in rice grains. The factors that are most likely to affect the extraction efficiency were optimized with a 2IV7-2 fractional factorial design. The optimum extraction conditions were achieved by applying 70% EtOAc in MeOH at 200 °C and 200 atm for a static time of 5 min in two cycles with 50% flushing and a 60 s purge to extract a 2.5 g rice sample. The method validation ensured excellent linearity, limit of detection, limit of quantification, precision, and recovery. Furthermore, the method was applied to various rice products composed of polished, whole grain, aromatic, black, black glutinous, red, and parboiled rice. All kinds of pigmented rice grains showed high levels of melatonin (>100 μg kg -1), and the highest levels were found in red rice.