Desarrollo y aplicación de técnicas y análisis de flavonoides en frutas

La uva (Vitis vinifera L.), una de las frutas con mayor producción a nivel mundial, presenta una elevada concentración en compuestos fenólicos y especialmente en flavonoides. Estos compuestos fenólicos son de gran interés a nivel de salud de los consumidores y de calidad de la materia prima y sus productos. Su consumo reduce la probabilidad de desarrollar enfermedades crónicas, como problemas cardiovasculares, diabetes y cáncer. Además, son uno de los parámetros de calidad más importantes en los vinos debido a su influencia en propiedades sensoriales, como el color, la estabilidad, el amargor y la astringencia. Por ello, el conocimiento sobre la composición fenólica de las uvas y sus derivados es un gran desafío en la actualidad. La cromatografía líquida de alta resolución (HPLC) ha sido la técnica más empleada en los últimos años para el análisis de compuestos fenólicos en matrices naturales. En este trabajo, se propone el empleo de la cromatografía líquida de ultra eficacia (UHPLC) ya que proporciona separaciones en un menor tiempo, mayor resolución y sensibilidad. Concretamente, se ha desarrollado un nuevo método cromatográfico para la determinación y cuantificación de 27 de los principales compuestos fenólicos (entre ellos flavonoides) presentes en la uva mediante detección de matriz de fotodiodos y fluorescencia (UHPLC-DAD-FLR). Los compuestos analizados han sido: ácido gálico, ácido protocatéquico, ácido clorogénico, ácido vainíllico, ácido cafeico, ácido siríngico, ácido p-cumárico, ácido ferúlico, ácido p-hidroxibenzoico, ácido caftárico, miricetina, rutina, quercetina 3-O-rhamnósido, quercetina 3-O-glucósido, quercetina 3-O-galactósido, kaempferol, quercetina, catequina, epicatequina (EC), epicatequina galato (ECG), epigalocatequina galato (EGCG), trans-resveratrol, ε-viniferina, trans-piceido, procianidina B1, procianidina B2 y procianidina A2. El método se validó mediante el estudio de la linealidad, límites de detección y cuantificación, precisión, selectividad y robustez. El método se aplicó con éxito a distintas variedades de uva, permitiendo analizar y cuantificar 17 compuestos fenólicos. Los resultados obtenidos demuestran que los compuestos fenólicos permiten clasificar las uvas, lo cual es de gran interés para la enología. Además, también se demuestra que indistintamente de la variedad, los compuestos fenólicos se clasifican en familias con características semejantes.69 página

Influence of Fruit Ripening on the Total and Individual Capsaicinoids and Capsiate Content in Naga Jolokia Peppers (Capsicum chinense Jacq.)

"Naga Jolokia" (Capsicum chinense Jacq.) is a hot pepper variety native to India which has received the attention of the global scientific community due to its high capsaicinoid concentration. The present study evaluated the influence of fruit ripening on the total and individual capsaicinoids, as well as capsiate content. The aim was to determine the optimal moment to harvest the peppers depending on their pungent properties. Ultrasound-assisted extraction (UAE) using methanol as the extraction solvent and reverse-phase ultra-high-performance liquid chromatography (UHPLC-photodiode array (PDA)) were employed. Capsaicinoids gradually accumulated in the peppers from the moment they started growing until they reached a maximum concentration (7.99 +/- 0.11 mg g(-1) of fresh weight (FW)) at 33 days postanthesis (dpa). For this reason, based on its content of pungent compounds, as it is one of the main attributes of this variety, the optimal time for collection would be on day 33. From then on, there was a sharp decrease (96.35% of the total concentration) due to the peroxidase enzymes. The evolution of the principal capsaicinoids in "Naga Jolokia" peppers had a different behavior with respect to literature reports. After this investigation, these changes in content can be attributed to each pepper genotype. Capsiate content reached it maximum value at 19 dpa (0.27 +/- 0.01 mg g(-1) of FW). Then, there was a gradual drop due to the activities of different peroxidases. Given the important biological activity of capsaicinoids and capsinoids, the information described here allows for determining the ideal time to harvest "Naga Jolokia" peppers

Optimization and Comparison of Ultrasound and Microwave-Assisted Extraction of Phenolic Compounds from Cotton-Lavender (Santolina chamaecyparissus L.)

The interest in natural phenolic compounds has increased because of their attractive use especially as antioxidant and antimicrobial agents in foods. The large content in phenolic compounds of interest in Santolina chamaecyparissus L. (S. chamaecyparissus) makes this plant a target source that is worthy of note. In this work, new extraction technologies comprising ultrasound (UAE) and microwave (MAE) assisted extraction of the phenolic compounds in S. chamaecyparissus have been developed, optimized, and compared. Several extraction factors have been optimized based on a Box-Behnken design. Such optimized factors include the percentage of methanol in water (25-75%), the temperature (10-70 degrees C), the ultrasound amplitude (20-80%), the ultrasound cycle (0.2-1 s), the solvent pH (2-7) and the solvent-sample ratio (5/0.2-15/0.2 mL/g) with regard to UAE, while the percentage of methanol in water (50-100%), the temperature (50-100 degrees C), the pH (2-7) and the solvent-sample ratio (5/0.2-15/0.2 mL/g) were optimized for MAE. The solvent composition was the most influential parameter both on MAEs (64%) and UAEs (74%). The extraction optimum time was established as 15 min for MAE and 25 min for UAE. Five major phenolic compounds were detected and identified by Ultra-High-Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry (UHPLC-QToF-MS) in the extracts: chlorogenic acid, quercetin 3-O-galactoside, quercetin 3-O-glucoside, isoorientin, and cynarin. With the exception of chlorogenic acid, the other four compounds have been identified for the first time in S. chamaecyparissus. The findings have confirmed that MAE is a significantly more efficient extraction method than UAE to extract phenolic compounds from S. chamaecyparissus

Changes in Capsiate Content in Four Chili Pepper Genotypes (Capsicum spp.) at Di erent Ripening Stages

Interest in the consumption of the fruits of pepper (Capsicum spp.) is not only due to its organoleptic characteristics, but also due to its bioactive compounds content, which are reported to provide essential benefits to human health. However, the amount and diversity of these compounds in each fruit specimen depend on its genotype and on a number of environmental factors. This work describes the quantitative ultra-high-performance liquid chromatography coupled to photodiode-array (UHPLC-PDA) analysis of the capsinoids content in four varieties of pepper ('Habanero', 'Habanero Roxo', 'Bode', and 'Malagueta') grown until different development stages in a greenhouse under controlled conditions. In all the varieties analyzed, capsiate was the only capsinoid found. The accumulation of capsiate, in all the pepper varieties, started from the 10th to the 20th day post-anthesis (dpa), and increased during the first days (between the 20th and the 27th dpa). From that moment a drastic reduction took place until the end of the ripening stage, except for 'Bode' peppers, where the capsiate content increased from the first harvest point on the 20th dpa up to the 76th dpa. The capsiate accumulation patterns over the development of the fruit has been related to the capsaicionoids accumulation patterns in the same samples of the four varieties of pepper. According to our results, the content evolution of both families of compounds will vary depending on each fruit's genotype, as well as on environmental conditions. No clear trends have been established and, therefore, an in-depth analysis under controlled conditions should be carried out

Content of Capsaicinoids and Capsiate in “Filius” Pepper Varieties as A ected by Ripening

Peppers are fruits with wide genetic variability and multiple ways of being consumed that hold a relevant position in the human diet. Nowadays, consumers are interested in new gastronomic experiences provided by pepper cultivars that present new shapes, colors, and flavors while preserving their bioactive compounds, such as their capsaicinoids and capsinoids. However, numerous changes take place during their development that may alter their biological properties. Therefore, this work evaluates the capsaicinoid and capsiate contents in two traditional varieties of ornamental peppers ("Filius Blue" and "Filius Green'") during fruit maturation. The aim is to determine the ideal harvesting moment depending on the farmer's objective (e.g., achieving a specific color, shape, or flavor; achieving the maximum concentrations of bioactive compounds). The capsaicinoid contents followed different patterns in the two varieties analyzed. The "Filius Blue" variety exhibited increasing concentrations of capsaicinoids up to the 41st day post-anthesis (dpa), from which point on this trend was reversed. The concentrations in the "Filius Green" variety increased and decreased several times, reaching maximum concentrations on the 69th dpa. Regarding capsiate contents, both varieties varied in the same way, reaching maximum concentrations on the 34th dpa and then decreasing

Optimizing and Comparing Ultrasound- and Microwave-Assisted Extraction Methods Applied to the Extraction of Antioxidant Capsinoids in Peppers

Capsinoids are very similar antioxidant compounds to capsaicinoids, but less irritating, non-pungent and more palatable, and can thus be used in greater concentrations for food applications. To date, three capsinoids (capsiate, dihydrocapsiate, and nordihydrocapsiate) have been isolated from the pepper fruits. Due to its substantial commercial importance, it would be convenient to determine which pepper varieties have a richer content. Ultrasound- (UAE) and microwave- (MAE) assisted extraction have been implemented and analyzed using multivariate statistical methods. Firstly, di erent solvents were tested individually. The three best solvents were used in a set mixture design, where 42% methanol and 58% ethyl acetate were determined as the optimum combination for UAE, and 100% methanol for MAE. Subsequently, a Box–Behnken experimental design with four variables for both UAE and MAE (time, temperature, pH and sample mass:solvent volume “ratio”) was performed. The sample mass:solvent volume was the most influential variable in UAE; while for MAE no variable was any more influential than the others. Finally, both optimized extraction methods were successfully applied to di erent varieties of peppers. Besides, to demonstrate the e ciency of both extraction methods, a recovery study was performed. The results prove the potential of both techniques as highly adequate methods for the extraction of capsinoids from peppers

Alternative Ultrasound-Assisted Method for the Extraction of the Bioactive Compounds Present in Myrtle (Myrtus communis L.)

The bioactive compounds in myrtle berries, such as phenolic compounds and anthocyanins, have shown a potentially positive effect on human health. Efficient extraction methods are to be used to obtain maximum amounts of such beneficial compounds from myrtle. For that reason, this study evaluates the effectiveness of a rapid ultrasound-assisted method (UAE) to extract anthocyanins and phenolic compounds from myrtle berries. The influence of solvent composition, as well as pH, temperature, ultrasound amplitude, cycle and solvent-sample ratio on the total phenolic compounds and anthocyanins content in the extracts obtained were evaluated. The response variables were optimized by means of a Box-Behnken design. It was found that the double interaction of the methanol composition and the cycle, the interaction between methanol composition and temperature, and the interaction between the cycle and solvent-sample ratio were the most influential variables on the extraction of total phenolic compounds (92.8% methanol in water, 0.2 s of cycle, 60 ◦C and 10:0.5 mL:g). The methanol composition and the interaction between methanol composition and pH were the most influential variables on the extraction of anthocyanins (74.1% methanol in water at pH 7). The methods that have been developed presented high repeatability and intermediate precision (RSD < 5%) and the bioactive compounds show a high recovery with short extraction times. Both methods were used to analyze the composition of the bioactive compounds in myrtle berries collected from different locations in the province of Cadiz (Spain). The results obtained by UAE were compared to those achieved in a previous study where microwave-assisted extraction (MAE) methods were employed. Similar extraction yields were obtained for phenolic compounds and anthocyanins by MAE and UAE under optimal conditions. However, UAE presents the advantage of using milder conditions for the extraction of anthocyanins from myrtle, which makes of this a more suitable method for the extraction of these degradable compounds

Development of a Rapid UHPLC-PDA Method for the Simultaneous Quantification of Flavonol Contents in Onions (Allium cepa L.)

Onion, one of the most consumed vegetables in the world, is also known to contain high levels of antioxidant compounds, with protective effects against different degenerative pathologies. Specifically, onion is rich in flavonols, mainly quercetin derivatives, which are compounds with high antioxidant and free radical scavenging power. For this reason, it is of the utmost importance to count on optimal analytical methods that allow for the determination and quantification of these compounds of interest. A rapid ultra-high performance liquid chromatography (UHPLC)-photo-diode array (PDA) method for the separation of the major flavonols in onions was developed using a Box-Behnken design in conjunction with multiresponse optimization on the basis of the desirability function. The conditions that provided a successful separation were 9.9% and 53.2% of phase B at the beginning and at the end of the gradient, respectively; 55 degrees C column working temperature; and 0.6 mL min(-1) flow rate. The complete separation was achieved in less than 2.7 min with excellent chromatographic characteristics. The method was validated, and its high precision, low detection and quantification limits, good linearity, and robustness were confirmed. The correct applicability of the method improves the analysis of the raw material, increasing the quality of onions and its subproducts in terms of bioactive compounds and functional characteristics for consumers

Flavonol Composition and Antioxidant Activity of Onions (Allium cepa L.) Based on the Development of New Analytical Ultrasound-Assisted Extraction Methods

The onion is one of the most cultivated and consumed vegetables, and is a very valuable source of antioxidant substances. Every onion variety is rich in flavonols-mainly quercetin derivatives which makes onions the main dietary source of these compounds. This study intends to develop an ultrasound-assisted extraction method (UAE), an economical, green, and efficient technique, that allows us to determine the quality of onion extracts in terms of flavonol composition and antioxidant activity. For this purpose, an ultrasound-assisted extraction method has been optimized to obtain extracts with a high amount of flavonols, as well as with a high antioxidant activity, not only separately using a Box-Behnken design, but also simultaneously, based on multi-response optimization. Multiple response optimization has not only demonstrated an efficacy level similar to that of the individual ones, but it also represents a considerable reduction in terms of cost, time and work. The optimal conditions for simultaneous extractions were determined as follows: 76.8% methanol as an extraction solvent at pH 2 and 58.5 degrees C temperature, 85% amplitude, 0.9 s cycle, and 0.2:13 g:mL sample mass/solvent volume ratio. Furthermore, the developed method exhibited a high precision level and great recoveries in a rather short extraction time (2 min). These results, together with the development of a fast and simple UHPLC analysis method, lead us to consider the developed UAE method as a suitable technique for the extraction of bioactive compounds from onion matrices.This work has been supported by the project "EQC2018-005135-P" (Equipment for liquid chromatography by means of mass spectrometry and ion chromatography), of the State Subprogram of Research Infrastructures and Technical Scientific Equipment. This research was funded by University of Cadiz, by the INIA (National Institute for Agronomic Research) and FEDER (European Regional Development Fund, within the framework of the Operational Programme under the Investment for Growth 2014-2020) for their financial support (Project RTA2014-00083-C03-03), and by the Ministry of Science and Innovation of Spain (FPU grant AP-2018-03811 to Ana Velasco Gonzalez de Peredo)

Discrimination of Myrtle Ecotypes from Di erent Geographic Areas According to Their Morphological Characteristics and Anthocyanins Composition

Myrtus communis L. is an evergreen shrub that produces berries with a high content in antioxidant compounds. Since these compounds have demonstrated a positive e ect on human health, the interest on berries and their usages has increased. However, environmental conditions may a ect the productivity of these species and consequently the quality of wild myrtle. Ecotypes from diverse geographical origins may result in significant variations in terms of bioactive compounds content as well as in chemical traits. For this reason, in this work ecotypes from two di erent localizations have been studied to determine if their di erences in morphological and anthocyanins traits can be attributed to their origin and the environmental characteristics of these locations. For this, chemometric analyses such as Hierarchical Cluster Analysis and Principal Component Analysis, were employed. The results showed di erences between the ecotypes depending on their location. In particular, myrtle berries from maritime zones present greater fruit size and amount of bioactive compounds, which means an improvement in the quality of the final product based on this raw material. It can be concluded that both morphological and anthocyanins traits are related to the location of the ecotype and allow selecting the best ecotype for the required applications