Microwave-assisted extraction of phenolic acids and flavonoids and production of antioxidant ingredients from tomato: A nutraceutical-oriented optimization study

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Highlights

  • A 5-level full factorial design was successfully implemented for MAE optimization.

  • Time, temperature, ethanol concentration and solid/liquid ratio were relevant variables.

  • Antioxidant extracts rich in functional phenolic acids and flavonoids were obtained.

  • Non-significant differences between experimental and predicted values were found.

  • Tomato extracts presented potential as ingredients in the design of functional foods.

Abstract

The production of natural extracts requires suitable processing conditions to maximize the preservation of the bioactive ingredients. Herein, a microwave-assisted extraction (MAE) process was optimized, by means of response surface methodology (RSM), to maximize the recovery of phenolic acids and flavonoids and obtain antioxidant ingredients from tomato. A 5-level full factorial Box–Behnken design was successfully implemented for MAE optimization, in which the processing time (t), temperature (T), ethanol concentration (Et) and solid/liquid ratio (S/L) were relevant independent variables. The proposed model was validated based on the high values of the adjusted coefficient of determination and on the non-significant differences between experimental and predicted values. The global optimum processing conditions (t = 20 min; T = 180 °C; Et = 0%; and S/L = 45 g/L) provided tomato extracts with high potential as nutraceuticals or as active ingredients in the design of functional foods. Additionally, the round tomato variety was highlighted as a source of added-value phenolic acids and flavonoids.

Introduction

Phenolic compounds are a group of secondary metabolites widely spread throughout the plant kingdom. Tomato (Lycopersicon esculentum Mill.) fruits, apart from being a functional food rich in carotenoids, vitamins and minerals [1], [2], is also an important source of phenolic compounds, including phenolic acids and flavonoids [3]. As antioxidants, these functional molecules play an important role in the prevention of human pathologies [4], [5] and found many applications in nutraceutical, pharmaceutical and cosmeceutical industries [6]. Therefore, obtaining added-value functional compounds from natural sources, such as tomatoes, is highly desirable by the food industrial sector. Furthermore, the global nutraceutical market has grown in the last decade and a large percentage of the developed nutraceuticals and functional foods are driven by plant-based products [7].

Tomato is a key element of the Mediterranean diet [8] and the second most important vegetable crop worldwide, being consumed either fresh or in the form of processed products. In Trás-os-Montes, North-eastern Portugal, native population’s lifestyle has highlighted the importance of local tomato varieties, which are grown using extensive farming techniques and considered as very tasty and healthy foods [9]. Among them, the common variety of tomato, locally known as “tomate Redondo” (round tomato), was reported as a source of p-coumaric acid and quercetin derivatives, as well as of the non-phenolic compound benzyl alcohol dihexose [3]. The p-coumaric acid has antioxidant, antilipidemic, antihypertrophic and cardioprotective properties [10], [11]. Quercetin shows a wide range of biological and pharmacological effects, including antioxidant, anti-inflammatory, antitumor and antibacterial activities, as well as neuroprotective, hepatoprotective, cardioprotective, anti-atherosclerotic, anti-thrombotic and antihypertensive effects [12], [13], [14], [15]. In tomato, quercetin is commonly found in the glycoside, i.e., esterified with rutinose. Rutin, known as vitamin P, also display a remarkable array of health-promoting effects and is widely used in the industry [16]. In turn, benzyl alcohol, an aromatic alcohol, is used in cosmetic formulations, as local anesthetic, and as a flavoring substance in foods and beverages [17]. Furthermore, epidemiological studies support the protective effect of tomatoes against certain degenerative diseases associated to oxidative stress, including cardiovascular diseases and various types of cancer [18]. Meanwhile, there has been an increasing concern to develop and include phenolic-rich functional foods in the diet in order to improve the nutritional and health status.

Extraction is an important analytical step in the isolation of compounds from plant materials prior to chromatographic identification, or from a preparative point of view, to produce functional ingredients to use in new formulations [7], [19]. Today, microwave-assisted extraction (MAE) is gaining many merits due to the higher extraction rate and superior products quality at lower cost. In fact, this novel green technology is considered as a potential alternative to conventional solid–liquid extraction of bioactive compounds from plant matrices [20]. However, the MAE efficiency depends on several variables which may not be generalized for all plant materials due to the diverse nature of existing bioactive phytochemicals, being necessary to select and optimize the processing conditions as a function of the used matrix and taking into account the desired responses.

Apart from the large amounts of industrial by-products derived from tomato processing, sometimes a surplus production of this fruit occur, which can be sustainably used for functional ingredients recovery. In a previous study conducted by Li et al. [20], optimal extraction conditions were determined based on the ferric reducing antioxidant power (FRAP) and oxygen radical absorption capacity (ORAC) assays. These optimized conditions were then used in the analysis of phenolic compounds. However, non-phenolic compounds can influence antioxidant responses. Therefore, an RSM optimization based on chromatographic analysis is more accurate and desired, once the optimal conditions obtained from antioxidant responses may not match the conditions for the extraction of individual compounds. In addition, the low range of extraction time (⩽3.68 min) originated non significant results. Our study aimed at determining the optimal MAE conditions for maximizing the recovery of functional phenolic compounds and the antioxidant capacity of extracts from tomato. Different variables (processing time, temperature, ethanol concentration, microwave power, and solid/liquid ratio) were investigated and the extraction process optimized using a central composite design coupled with response surface methodology (RSM). The content of the major phenolic compounds (two phenolic acids: benzyl alcohol dihexose and a cis p-coumaric acid derivative; and two flavonoids: quercetin pentosylrutinoside and quercetin-3-O-rutinoside) and the antioxidant activity (DPPH free-radical scavenging activity and reducing power) were evaluated as responses.

Section snippets

Standards and reagents

HPLC-grade acetonitrile was from Fisher Scientific (Lisbon, Portugal). Formic acid was purchased from Prolabo (VWR International, France). The phenolic compound standards (p-coumaric acid, caffeic acid and rutin) were from Extrasynthese (Genay, France). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was from Sigma (St. Louis, MO, USA). All the other chemicals were of analytical grade and

Response criteria for the RSM analysis

Fig. 1 shows the phenolic profile of the tomato extract obtained under the run no. 9 of the RSM design, whose processing conditions are presented in Table 2. Benzyl alcohol dihexose (P1) and cis p-coumaric acid derivative (P2) were the major phenolic acids, while quercetin pentosylrutinoside (F1) and quercetin-3-O-rutinoside (F2) were the main flavonoids, in agreement to Barros et al. [3]. These compounds were identified by comparison of their UV spectra and retention time with those of

Conclusions

The combined effects of the independent variables of t, T, Et and S/L on the extraction of phenolic compounds and production of antioxidant extracts from tomato were investigated. A 5-level full factorial Box–Behnken design of 25 combinations and 7 replicates at the center of the experimental domain was successfully implemented for MAE optimization by RSM. The MAE conditions were optimized for each response, as well as for the set of all responses. Under the global optimum conditions (t = 20 min, T

Abbreviations

tProcessing time
TProcessing temperature
EtEthanol concentration
S/LSolid/liquid ratio
P1Benzyl alcohol dihexose
P2cis p-Coumaric acid derivative
F1Quercetin pentosylrutinoside
F2Quercetin-3-O-rutinoside

Acknowledgments

The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support to CIMO (PEst-OE/AGR/UI0690/2014), REQUIMTE (UID/QUI/50006/2013 - POCI/01/0145/FERDER/007265), J. Pinela (SFRH/BD/92994/2013) and L. Barros (SFRH/BPD/107855/2015); FCT/MEC and FEDER under Programme PT2020 for financial support to LSRE (UID/EQU/50020/2013), and to QREN, ON2 and FEDER (NORTE-07-0162-FEDER-000050); to the Xunta de Galicia for financial support for the post-doctoral

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