Bilberry (Vaccinium myrtillus L.) can be considered a functional food, as it has been recognized to provide several health-protecting attributes. The wide spectrum of its therapeutic effects has been suggested to be related to the high concentrations and wide variety of phenolic compounds, especially anthocyanins. The development of effective extraction procedures for these compounds can be difficult due to their structural diversity and to their potent antioxidant activity, leading to a rapid reaction with other constituents in the matrix. Mechanical Solvent Extraction (MSE) is an established and widely used procedure for the extraction of bioactive compounds from natural materials. Recently, Microwave-Assisted Extraction (MAE) and Ultrasonic-Assisted Extraction (UAE) have been considered as potential alternatives to the conventional MSE technique. Since many and significantly interacting factors are involved in the development of any extraction procedure, a multivariate strategy has been devised in order to carry out its optimization in a rational and scientific way. In this study, a Quality by Design (QbD) approach was applied in order to systematically compare the performances of MSE, MAE and UAE for maximizing the extraction efficiency of polyphenolic compounds from bilberry. QbD is a risk management-oriented methodology, which has been progressively integrated with analytical method development. To the best of our knowledge, this approach has not yet been reported for the development of extraction procedures. The QbD approach successfully allows the quality to be built into the process from the early stages of its development, returning a deep understanding of the whole procedure. In the first step, the screening phase, the three techniques were investigated through screening matrices, selecting extraction time, kind of solvent, percentage of organic phase and sample/extractant ratio as critical method parameters (CMPs). In the case of MAE, temperature was also taken into account. The critical method attributes (CMAs) were chosen as the total phenolic content (TPC), the total monomeric anthocyanin (TMA), the antioxidant activity evaluated by 2,2-diphenyl-1-picrylhydrazyl assay (DPPH), the 2,2'-azinobis( 3-ethylbenzothiazoline-6-sulphonic acid) assay (ABTS), and Ferric Reducing Antioxidant Power Assay (FRAP). Graphic analysis of the effects made it possible to effectively compare the different extraction techniques and to select UAE for further optimization. The best performances obtained by UAE could be due to the ultrasound waves increasing mixing and micro-mixing, promoting solvent penetration into the sample matrix and increasing the mass transfer rate of the antioxidant compounds into the extraction solvent. Through the same procedure, methanol was selected as solvent and a new experimental domain for the other CMPs was set on the basis of the screening results. A Response Surface Study was carried out and a Box-Behnken design was employed to find the coefficients of the quadratic models relating the CMPs to the CMAs. In this phase, further detailed information on the extraction process was obtained by means of HPLC-MS/MS analysis of the extracts, in order to evaluate the effect of the CMPs on the extraction of the most abundant polyphenolic compounds. A threshold value was set for each CMA and the sweet spot plots were drawn, highlighting the multidimensional zone where the requirements for all the CMAs were satisfied according to their predicted values. Hence, the method operable design region (MODR) was calculated by Monte-Carlo simulations, propagating the predictive error by using the model equations to the CMAs and computing the probability to reach the desired objectives. The threshold for the risk of failure was set to 10% and this risk was graphically plotted in the probability surfaces. Finally, the MODR was validated by verification points and an optimum working point was selected. This study pointed out how the great potential of a simple and fast extraction technique such as UAE can be effectively strengthened towards the production of phenolic natural extracts, through the implementation of a QbD approach.

Quality by Design compliant strategy for the optimization of the extraction of phenolic compounds from Vaccinium myrtillus berries / O. Ochkur, S. Orlandini, L. Marzullo, O. Koshovyi, L. Renai, C.V.A. Scordo, S. Furlanetto, M. Del Bubba. - ELETTRONICO. - (2019), pp. 1-2. (Intervento presentato al convegno XXVIII Congress of the Analytical Chemistry Division tenutosi a Bari nel 22-26 Settembre 2019).

Quality by Design compliant strategy for the optimization of the extraction of phenolic compounds from Vaccinium myrtillus berries

S. Orlandini;MARZULLO, LUCA;L. Renai;C. V. A. Scordo;S. Furlanetto;M. Del Bubba
2019

Abstract

Bilberry (Vaccinium myrtillus L.) can be considered a functional food, as it has been recognized to provide several health-protecting attributes. The wide spectrum of its therapeutic effects has been suggested to be related to the high concentrations and wide variety of phenolic compounds, especially anthocyanins. The development of effective extraction procedures for these compounds can be difficult due to their structural diversity and to their potent antioxidant activity, leading to a rapid reaction with other constituents in the matrix. Mechanical Solvent Extraction (MSE) is an established and widely used procedure for the extraction of bioactive compounds from natural materials. Recently, Microwave-Assisted Extraction (MAE) and Ultrasonic-Assisted Extraction (UAE) have been considered as potential alternatives to the conventional MSE technique. Since many and significantly interacting factors are involved in the development of any extraction procedure, a multivariate strategy has been devised in order to carry out its optimization in a rational and scientific way. In this study, a Quality by Design (QbD) approach was applied in order to systematically compare the performances of MSE, MAE and UAE for maximizing the extraction efficiency of polyphenolic compounds from bilberry. QbD is a risk management-oriented methodology, which has been progressively integrated with analytical method development. To the best of our knowledge, this approach has not yet been reported for the development of extraction procedures. The QbD approach successfully allows the quality to be built into the process from the early stages of its development, returning a deep understanding of the whole procedure. In the first step, the screening phase, the three techniques were investigated through screening matrices, selecting extraction time, kind of solvent, percentage of organic phase and sample/extractant ratio as critical method parameters (CMPs). In the case of MAE, temperature was also taken into account. The critical method attributes (CMAs) were chosen as the total phenolic content (TPC), the total monomeric anthocyanin (TMA), the antioxidant activity evaluated by 2,2-diphenyl-1-picrylhydrazyl assay (DPPH), the 2,2'-azinobis( 3-ethylbenzothiazoline-6-sulphonic acid) assay (ABTS), and Ferric Reducing Antioxidant Power Assay (FRAP). Graphic analysis of the effects made it possible to effectively compare the different extraction techniques and to select UAE for further optimization. The best performances obtained by UAE could be due to the ultrasound waves increasing mixing and micro-mixing, promoting solvent penetration into the sample matrix and increasing the mass transfer rate of the antioxidant compounds into the extraction solvent. Through the same procedure, methanol was selected as solvent and a new experimental domain for the other CMPs was set on the basis of the screening results. A Response Surface Study was carried out and a Box-Behnken design was employed to find the coefficients of the quadratic models relating the CMPs to the CMAs. In this phase, further detailed information on the extraction process was obtained by means of HPLC-MS/MS analysis of the extracts, in order to evaluate the effect of the CMPs on the extraction of the most abundant polyphenolic compounds. A threshold value was set for each CMA and the sweet spot plots were drawn, highlighting the multidimensional zone where the requirements for all the CMAs were satisfied according to their predicted values. Hence, the method operable design region (MODR) was calculated by Monte-Carlo simulations, propagating the predictive error by using the model equations to the CMAs and computing the probability to reach the desired objectives. The threshold for the risk of failure was set to 10% and this risk was graphically plotted in the probability surfaces. Finally, the MODR was validated by verification points and an optimum working point was selected. This study pointed out how the great potential of a simple and fast extraction technique such as UAE can be effectively strengthened towards the production of phenolic natural extracts, through the implementation of a QbD approach.
2019
XXVIII Congress of the Analytical Chemistry Division-Book of Abstracts
XXVIII Congress of the Analytical Chemistry Division
Bari
O. Ochkur, S. Orlandini, L. Marzullo, O. Koshovyi, L. Renai, C.V.A. Scordo, S. Furlanetto, M. Del Bubba
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1172279
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