Abstract
A green process, both in terms of solvent (natural deep eutectic solvents, NADES) and extraction method (microwave-assisted extraction, MAE) for the recovery of bioactive components from sunflower pomace (SFP, a by-product of oil extraction process), was designed to contribute to their sustainable valorization. For the extraction of valuable phenolics from sunflower pomace, nine potential NADES were prepared. Among them, choline chloride-urea-water (CC-U-W) at a molar ratio of 1:2:4 was selected for extraction, showing better yield and physicochemical properties. Operational parameters (extraction temperature, time, water ratio, and solvent-to-solid ratio) for MAE were optimized and modeled utilizing response surface methodology. Under optimal conditions, the antioxidant properties of SFP extract were evaluated by CUPRAC, ABTS, and DPPH methods. The total phenolic contents of extracts were evaluated by the Folin–Ciocalteu method. Phenolics were characterized by using the HPLC–PDA system. The results showed that SFP NADES extract had potential antioxidant activity which was higher than that of traditional solvents. In order to better evaluate SFP extract as a valuable food ingredient, SFP extracts at a ratio of 5, 10, and 20% added smoothie-like beverages with strawberries and yogurt were prepared. Fortification of beverages with NADES extract enhanced antioxidant efficiency, increasing total antioxidant capacity in a range of 12.4 to 68.6% and free radical scavenging capacity between 0.4 to 67.9% during both the initial and the in vitro gastrointestinal digestion stages. The addition of SFP NADES extract to food samples made a positive contribution in terms of the antioxidant activity of the final product. The results of this study revealed that sunflower by-products can be evaluated as a potential antioxidant source which is easily accessible, and the proposed extraction process has an important potential to recover bioactive compounds with high efficiency.
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1 Introduction
Sunflower (Helianthus annuus L.) is an important oilseed crop known as a first-class source of oil and dietary fiber, and cultivated worldwide, especially in the Russian Federation, Ukraine, Argentina, and Türkiye [1]. Sunflower has high nutritional value as a good source of nutritious bioactive compounds, unsaturated fats, proteins, inorganic compounds, and phytochemicals [2]. In addition, it exhibits significant biological activity with its anticancer, antioxidant, analgesic, anti-inflammatory, antibacterial, and antihypertensive properties [3]. Sunflower seeds contain a high amount of phenolic substances, especially chlorogenic acid (CGA), which occurs in complex or protein-bound forms [2]. By-products of sunflowers are formed in large quantities and are left in the fields after the seed harvest [4]. The disposal of wastes left over from sunflowers used by edible oil industries has become an important environmental problem [5]. Evaluation of these wastes, whose antioxidant properties are known from previous studies, is valuable in terms of both functional food production and sustainable valorization.
Today, interest in extraction techniques (i.e., ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE)) for the recovery of phenolics with important biological activities from various natural sources is increasing in terms of both yield and compatibility with green chemistry. The closed-vessel MAE system offers advantages such as allowing the extraction solvent to operate at temperatures well beyond its boiling point, extraction efficiency, extraction speed, low solvent requirement, and the ability to use multiple samples. Solvent selection is a critical problem; although ionic liquids (ILs) have been a first choice in the last two decades, their high cost, difficult synthesis, viscosity, and corrosion problems arising from their susceptibility to hydrolysis have limited their use [6]. Deep eutectic solvents (DESs), especially their natural ones (NADES), have largely replaced ILs. DESs form a hydrogen-bonded network between their hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) components, incorporating carboxylic acids, sugars, amino acids, and alcohols; DESs have a lower melting point than those of their components [7]. NADES preserve the physicochemical properties of their constituents due to the absence of a chemical reaction between them; these properties may be regulated by changing the molar ratios of HBA and HBD components and sometimes water. The study by Liu et al. [8] showed that curcuminoids have higher antioxidant activity and better stability in NADES extracts containing organic acids and sugars compared to organic solvent extracts, along with obtaining higher extraction yields with the usage of NADES. Similarly, the study by Barbieri et al. [9] revealed that choline chloride–based DESs provided better stabilization capacity for phenolic compounds extracted from Rosmarinus officinalis L. based on the kinetic degradation assay compared to ethanol, and this observed stabilization capacity of DESs was attributed to the intermolecular interactions formed by hydrogen bonds between the phenolic acids in the extracts and the solvents. In addition to these examples, the solubility of rutin and its bioavailability were investigated by Faggian et al. [10], who found that the solubility of rutin in proline/glutamic acid (2:1) NADES was better than that in conventional solvents and 20 times higher than that in water. A significant improvement in the bioavailability of rutin was also observed in proline/glutamic acid (2:1) NADES compared to that in water after oral administration. NADES has also been reported to increase the bioavailability of hydroxysafflor yellow A and anhydrosafflor yellow B [11], anthocyanins [12], and puerarin [13]. Therefore, NADES are green solvents featured to increase the solubility, stability, and bioavailability of bioactive components.
The extraction of bioactive compounds with NADES as a green solvent makes it possible to produce ready-to-use extracts for the design of new functional foods rich in bioactive compounds, since NADES is mainly composed of biological metabolites such as organic acids, amino acids, sugars, choline derivatives, and water, which are found in living cells and organisms and already used in food products as well as their display of other properties such as high purity, easy preparation, low cost, biodegradability, non-toxicity, and sustainability [14]. DESs, which offer advantages for the efficient recovery of polyphenols from agricultural food by-products, contribute to a circular and sustainable economy [15, 16]. Chocolate/cocoa drinks were fortified with the addition of NADES extracts from cocoa by-products, and their sensory acceptability was investigated by Manuela et al. [17]. The results of electronic tongue analysis showed that fortification with the addition of 1–10% NADES extracts was sensory acceptable except with the addition of 10% NADES composed of choline chloride to glucose (1:1). To our best knowledge, the effect of NADES addition to the food matrix regarding antioxidant activity, stability, solubility, and bioavailability of bioactive compounds has not been investigated before, which represents a major gap in the development of new formulations.
In this study, nine different NADES were prepared for antioxidant extraction from sunflower pomace, and the choline chloride (CC)-urea-water (CC-U-W) mixture was chosen as the most suitable solvent combination in terms of both yield and physicochemical properties. The operational factors for the process, such as extraction time, extraction temperature, solvent-to-solid ratio, and water ratio, were optimized and modeled by response surface methodology (RSM). The antioxidant properties of the extract obtained under optimal conditions, total antioxidant capacity (TAC), free radical scavenging capacity (FRC), total phenolic content (TPC), and ABTS radical scavenging capacity (ARC) were measured and compared with the results of the extracts obtained with the most commonly used classical solvent mixtures (80% ethanol in water and 80% methanol in water). The chromatographic analysis of phenolics in NADES extracts of sunflower pomace was performed in this study. In order to evaluate the potential use of sunflower pomace extract obtained with NADES as a valuable food component and to examine its effects on the nutritional value of food, smoothie-like beverages containing strawberry and yogurt were prepared at different weight percentages. The outputs of this study can provide new contributions to industrial waste valorization in terms of the development of new solvents and new processes for the extraction of antioxidants from sunflower by-products and their use in product applications.
2 Materials and methods
2.1 Chemicals and instrumentations
All chemicals of analytical purity were obtained from the relevant suppliers listed: urea, ethylene glycol, 1,2-butanediol, 1,2-propylene glycol, DL-malic acid, levulinic acid, trolox, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 3-O-caffeoylquinic acid (3-CQA), 1,3-di-O-caffeoylquinic acid (1,3-DCQA), 1,5-di-O-caffeoylquinic acid (1,5-DCQA), potassium persulfate, neocuproine, methanol, and ethanol from Sigma-Aldrich (Steinheim, Germany); copper(II) chloride dihydrate, copper(II) sulfate, sodium carbonate, sodium potassium tartrate, choline chloride (CC), and ammonium acetate from Merck (Darmstadt, Germany); 2,2′-azino-bis(3-ethylbenzothiazoline-6 sulphonic acid) (ABTS) from Fluka (Buchs, Switzerland). Sunflower pomace was supplied from Olin Edirne Yaş Sanayi ve Ticaret A.Ş.
The instruments used in the study and their suppliers are listed below: Shimadzu IRTracer-100 spectrometer (Japan), Shimadzu UV-1900i UV–vis spectrophotometer (Japan), Milestone ETHOS Easy microwave system (USA), Anton Paar density meter and RXA 170 refractometer combined measuring system (Anton Paar, Graz, Austria), AND SV-10 viscometer (Japan). In chromatographic analysis, a Waters Breeze 2 Model HPLC device (Milford, MA, USA), 1525 model binary pump, column thermostat, 2998 model photo-diode array (PDA) detector (Chelmsford, MA, USA), Hamilton 50 μL-syringe, and Waters C18 analytical column (250 mm × 4.6 mm, 5 μm) were used.
2.2 NADES preparation and characterization
The abbreviations used for NADES, the names of the components, and their molar ratios are given in Table 1. NADES used in the study were prepared by heating and mixing method [18]. First, their amounts, calculated according to the molar ratio corresponding to each component, were precisely weighed on an analytical balance with an accuracy of ± 0.0001 g and placed in a 50-mL flask. The prepared mixture was heated with a temperature-controlled mixer in a water bath at 80 °C at a stirring speed of 800 rpm for 2 h until a clear liquid was obtained.
FTIR analysis was performed to examine the NADES formation mechanism as a result of the interaction between the components. The spectral acquisition took place through a single attenuated total reflectance (ATR) attachment with a diamond prism, spanning the scanning range of 4000 to 400 cm−1. Densities and refractive index values of NADES were measured with high precision, and the viscosity of NADES was measured using the tuning-fork vibration method at room temperature (25 °C).
2.3 MAE of sunflower pomace samples
Sunflower pomace (released as a result of the oil extraction process) was powdered by passing through a mechanical grinder and used in research after passing through analytical sieves with a diameter of 350 µm. The operational parameters such as extraction time, extraction temperature, solvent-to-solid ratio, and water ratio for the extraction of antioxidant components are 18 min, 78 °C, 11 mL/g-solid (DS), and 38% (v/v). The temperature in the closed microwave system was controlled by the system. The final extracts were first filtered through a Buchner funnel to the flask under vacuum and then passed through 0.45-μm Chromafil PET-45/25 syringe filters and stored in the refrigerator at + 4 °C.
2.4 Preparation of smoothie-like beverages with the addition of NADES extract
Strawberry and yogurt-based smoothie-like beverages enriched with bioactive compounds were prepared by adding selected NADES extract at different ratios with the aim of valorizing the sunflower pomace as a valuable food additive and observing the effects of the NADES solution. The strawberries and yogurt used were purchased from a local market for the production of homemade smoothie-like beverages. Firstly, the supplied strawberries were washed, separated from the green parts, chopped, and pureed using a household blender. A smoothie-like beverage containing strawberries and yogurt was then prepared as a control sample by mixing the strawberry puree, yogurt, and water in a 2:2:1 ratio by weight. For the production of fortified beverages, sunflower pomace extract obtained using a selected NADES solution was incorporated into the beverage formulation at concentrations of 5%, 10%, and 20% (w/w). This was achieved by proportionally reducing the water content in the control sample formulation. In addition, to make a detailed comparison and to examine the effect of the added NADES extract, another control sample was prepared by mixing strawberry puree and NADES extract obtained from sunflower pomace at a 2:1 ratio by weight. The codes and formulations of the prepared products are given in Table 2.
2.5 Extraction of polyphenols from smoothie-like beverages
The solvent extraction prior to the in vitro digestion procedure of the polyphenols in beverage samples was carried out with an 80% aqueous ethanol solution. Briefly, 5 mL of 80% aqueous ethanol solution was added to 2 g of sample and mixed for 15 min in a cooled ultrasonic bath. The obtained mixture was then centrifuged at 4 °C and 4000 rpm for 10 min, and the supernatant was collected. These extraction steps were performed twice for each sample. The combined extracts were stored at − 20 °C until further analysis and passed through a 0.45-µm filter (Chromafil PET-45/25) before spectrophotometric analysis.
2.6 In vitro digestion procedure
An in vitro gastrointestinal digestion model, including the mouth, stomach, and small intestine, based on the method described by Minekus et al. [19], was applied to all test and control samples to simulate the conditions of food digestion after ingestion in the human body. The temperatures of all solutions used were adjusted to 37 °C prior to the experiment, and the incubation of all samples was performed at that temperature during the experiment.
In the mouth phase, 5 g of sample was mixed with 3.5 mL of simulated saliva fluid (SSF), 0.5 mL of α-amylase stock solution (1500 U/mL), 25 μL of 0.3 M CaCl2 solution, and 975 μL of Milli-Q water sequentially, and the final mixture (pH 7.0) was incubated for 2 min in an incubator shaker.
In the stomach phase, 10 mL of sample from the mouth phase was sequentially mixed with 7.5 mL of simulated gastric fluid (SGF), 1.6 mL of porcine pepsin stock solution (25,000 U/mL), and 5 μL of 0.3 M CaCl2 solution, and the pH of obtained mixture was adjusted to 3.0 by adding of 0.2 mL of 1 M HCl solution. Then, 695 μL of Milli-Q water was added and the final mixture was incubated for 2 h in an incubator shaker. At the end of incubation, 5 mL of sample was collected.
In the small intestine phase, 15 mL of sample from the stomach phase was sequentially mixed with 8.25 mL of simulated intestinal fluid (SIF), 3.75 mL of pancreatin stock solution (800 U/mL), 1.875 mL of fresh bile solution (160 mM in fresh bile salts), and 30 μL of 0.3 M CaCl2 solution, and the pH of obtained mixture was adjusted to 7.0 by adding 1 M NaOH solution. Then, 9825 μL of Milli-Q water was added and the final mixture was incubated for 2 h in an incubator shaker. As in the stomach phase, 5 mL of sample was collected at the end of incubation.
After all stages of the in vitro gastrointestinal digestion procedure were completed, the samples collected from the stomach and small intestinal phases were centrifuged at 4 °C and 4000 rpm for 30 min for removal of any large particles. Then, all collected supernatants were stored at − 20 °C until further analysis.
2.7 Total antioxidant capacity (TAC) assay
The TAC of sunflower pomace extract was measured by the CUPRAC (cupric reducing antioxidant capacity) method [20]. In this method, 1-mL volumes of Cu(II) (10 mM), Nc (7.5 mM), and buffer (1 M NH4Ac) solutions were mixed in a tube, and after adding x mL of extract, the total volume was made to 4.1 mL with distilled water. Thirty minutes after the addition of the extract, absorbance was recorded against the reagent blank at 450 nm. TAC was expressed as trolox equivalents (mmol TR/g-dry sample (DS)) based on the absorbance/concentration standard curve of trolox.
2.8 ABTS radical scavenging capacity (ARC) assay
ARC of sunflower pomace extract was determined by the assay based on the reduction of the chromogenic ABTS radical cation (ABTS•+) with antioxidants [21]. ABTS was prepared by dissolving it in 50 mL of water at a final concentration of 7.0 mM, and persulfate was added to this solution at a final concentration of 2.45 mM. The resulting ABTS•+ solution was incubated at room temperature and in the dark for 12–16 h. ABTS•+ solution was diluted 1:10 with ethanol. In this method, x mL of extract, (4-x) mL of methanol, and 1 mL of ABTS•+ solution were mixed in the tube. The absorbance of the mixture against ethanol at 734 nm was recorded after 6 min of incubation. Corrected absorbance values (ΔA) were used to calculate the ARC of the extracts. ΔA was calculated from the following equation (Eq. 1):
AABTS was the absorbance of ABTS•+ reagent without sample and AE was the absorbance of the extract. ARC was expressed as TR equivalents (mmol TR/g-DS) based on the absorbance/concentration standard curve of TR.
2.9 Free radical scavenging activity (FRC) assay
The FRC value of the sunflower pomace extract was determined by measuring its ability to scavenge DPPH free radicals [22]. In this method, x mL of extract, (2-x) mL of methanol, and 2 mL of DPPH free radical solution (0.2 mM) were mixed in the tube. Absorbance values were recorded at 515 nm against methanol after 30 min of waiting. Corrected absorbance values (ΔA) were used to calculate the FRC of the extracts. ΔA was calculated from the following equation (Eq. 2):
ADPPH was the absorbance of DPPH radical without sample and AE was the absorbance of the extract. FRC was expressed as TR equivalents (mmol TR/g-DS) based on the absorbance/concentration standard curve of TR.
2.10 Determination of total phenolic content (TPC)
Total phenolic content (TPC) of the sunflower pomace (SFP) extracts was determined using the Folin–Ciocalteu (FC) method as described by Singleton et al. [23]. Lowry C used in the assay was prepared as a mixture of 50 mL Lowry A (2% aqueous Na2CO3 in 0.1 M NaOH) and 1 mL Lowry B (0.5% CuSO4 aqueous solution in 1% NaKC4H4O6) solutions. The FC reagent was diluted with H2O at a volume ratio of 1:3 prior to use. The SFP extract was diluted 1:20 with distilled water or 80% aqueous ethanol or 80% aqueous methanol. A volume of x mL of diluted extract, (1-x) mL of distilled water, and 2.5 mL of Lowry C solution was added into a test tube. Then, 0.25 mL of FC reagent was added and mixed. After 30 min, the absorbance of the reaction solution was measured against a blank at 750 nm. Since FC assay simultaneously measures total phenolics as well as total antioxidant capacity, the result was converted to trolox equivalent (mmol TR/g of sample) unit based on the standard curve obtained with trolox. The assays were carried out in triplicate and the results were expressed as mean value ± standard deviation.
2.11 Chromatographic analysis of sunflower pomace extract
Phenolics were determined by performing the HPLC analysis method [24]. Working conditions are as follows: methanol (mobile phase A) and 0.2% phosphoric acid in bidistilled water (mobile phase B). The flow rate was 0.8 mL min−1, and the column temperature was 30 °C. The details of the gradient elution program were as follows: 0 min 10% A–90% B; 8 min 30% A–70% B (slope 6.0); 30 min 52% A–48% B (slope 6.0); 35 min 10% A–90% B. Detection wavelength was selected at 340 nm.
2.12 Statistical analysis
Face-centered composite design (FCCD) (Design-Expert® Software, Version 11 (Stat-Ease, Inc., Minneapolis, USA)) was used to determine optimal conditions and determine the four variables (A, extraction time (min); B, extraction temperature (°C); C, solvent-to-solid ratio (mL/g-DS); and D, water ratio (%)) that were used to investigate the effect of extracts on TAC values. Table 3 summarizes the factors and levels investigated in the experimental design. Analysis of variance (ANOVA) test was used in the Design-Expert program to examine the relationship between operational factors and responses (Table 4).
3 Results and discussion
3.1 Characterization of NADES and selection of suitable solvent for antioxidant extraction
In this study, a total of nine potential NADES were used for the extraction of valuable phenolics from SFP by-product. FTIR analyses were performed to show that the mixtures used in the study formed an eutectic mixture and to elucidate the formation mechanisms. The obtained spectra are presented in Fig. 1 (a-i).
FTIR spectra of NADESs and their constituents: a CC-U-EG, b CC-U-BD, c CC-U-PG, d CC-U-MA-W, e CC-LA-W, f CC-PG-W, g CC-BD, h CC-U-W, i CC-EG
Spectra of CC-U-EG are presented in Fig. 1a. Among the specific absorption peaks of urea, the IR band observed at 1589 cm−1 represents the C = O functionality. This peak shifts to 1664 cm−1 in the DES spectrum, indicating that urea is incorporated into the composition. It is seen that the OH vibration peaks for urea at 3419 cm−1, for CC at 3319 cm−1, and for EG at 3288 cm−1 shifted and showed band broadening in the NADES spectrum at 3307 cm−1. The characteristic peaks of these single components in the CC-U-EG NADES spectrum indicate hydrogen bonding interactions [25].
Similarly, the characteristics of CC and urea are also seen in the CC-U-BD NADES. The observation of characteristic (–CH) sp3 groups of BD in the range of 2875–2970 cm−1 shows that BD also participates in the NADES composition. It is seen that CC-U-PG NADES has similar FTIR spectra due to the fact that both BD and PG are classified as diols. OH peaks of CC and BD in the composition of CC-BD, which is another NADES containing diol in its composition, shifted from 3319 cm−1 and 3321 cm−1 to 3315 cm−1, respectively. C–H bonding of BD was observed in the NADES spectrum in the range of 2964–2875 cm−1. This indicates that a hydrogen bridge is formed between the components, as in the interpretations of DESs containing similar structures [25, 26]. In the CC-PG-W NADES, which contains water in its composition, asymmetric H–O–H bending (of hydrated protons) was observed at 1637 cm−1. This bending appears to fade at 1649 cm−1, indicating that water has joined the composition by forming a hydrogen bond [26]. It is seen that OH stretching in pure components expands in the range of 3000–3500 cm−1 in the NADES spectrum and shows characteristic split behavior. The C = O absorption peak of urea, monitored at 1589 cm−1, and the H–O–H bending of water, monitored at 1637 cm−1, merged and deepened and shifted to 1608 cm−1 in the NADES spectrum. The vibrations from 1450 to 800 cm−1 as representative of C–O stretching, O–H bending, and CH stretching from the hydroxyl and alkyl group in the acid structure shifted and widened in the CC-U-MA-W spectrum. Additionally, the characteristic carboxyl peak of the acid was observed at 1678 and 1734 cm−1. It can be said that this peak of DES composition combines with the H–O–H peaks of water and the C = O peaks of urea. When the spectrum of CC-LA-W DES, which contains another carboxylic acid, was examined, it was seen that the carboxyl peak recorded at 1701 cm−1 shifted to 1705 cm−1. In addition, the CC and OH stretches of water observed in the range of 3000–3500 cm−1 expand and dampen in the same range [27]. The O–H vibration bands of CC and EG were monitored around 3319 and 3288 cm−1, respectively. It is seen that these bands shifted to 3302 cm−1 after DES formation. This broad band is a representation of the O–H functional group, confirming the formation of hydrogen bonds between ChCl and EG. This band can become stronger as the mole ratio of EG in the DES composition increases. The bands between 2850 and 3000 cm−1 are defined as stretching modes of aliphatic C–H bonds (CH2 and CH3) [28]. Some physical and rheological properties of NADES obtained by interactions between pure components (such as viscosity, refractive index, and density) were measured and presented in Table 1.
Although the physicochemical properties of solvents are important in the extraction process, it would not be right to use a suitable extraction solvent based on these features alone. For this reason, the extraction process was carried out in the presence of both NADES samples and frequently used conventional solvents. A general extraction procedure was applied for solvent selection, as it has not yet been made into the optimization process. In the MAE process, operational factors were used such as the extraction time of 30 min, extraction temperature of 75 °C, and solvent-to-solid ratio of 15 mL/g-DS. When looking at the TAC values (Fig. 2), it was observed that the most suitable NADES was CC-U-W. Antioxidant extraction efficiency was found to be higher in the presence of certain NADES media compared to conventional solvents. In addition, a higher TAC value was obtained in an aqueous medium compared to conventional solvents. Therefore, the efficiency of the water ratio in the most appropriate NADES medium was also examined. The CC-U combination is one of the first developed NADES and is frequently used for the extraction of phenolics from natural sources. Extraction of bioactive components from onion peel wastes using CC:U:W-based NADES with the aid of the MAE system had been reported [29]. In another study, the efficiency of NADES for the extraction of phenolics from Agrimonia eupatoria plant was investigated, and CC:U and CC:glycerol became prominent in terms of extraction efficiency [30]. CC-based NADES were investigated for the extraction of bioactive components from Achillea millefolium L., and the highest scavenging activity against ABTS radical was reported for CC-U-based NADES [31].
Effect of solvents on antioxidant extraction from sunflower pomace (N = 3)
The mechanism behind the high efficiency of CC-U-W combination (also known as hydrated reline) to extract polyphenols is probably the reduced redox potential of phenolic compounds (Ar-OH) in DES solvents [32] because of the enhanced stabilization of aryloxyl radicals (ArO•, produced as a result of one-electron oxidation of H-bonded phenolics by TAC reagents) through proton-coupled electron transfer (PCET) in DES media [33], because low redox potential means facile electron transfer leading to enhanced antioxidant activity. The H-bonded stabilization of phenolics in DES media is a well-established fact [34]. However, hydrated reline should have a different property to explain its high extraction power for phenolic antioxidants thanks to the simultaneous H-bond donor and H-bond acceptor properties of urea, in that reline can form neutral, ionic, and doubly ionic hydrogen bonds [35]. The H bonds in reline cover a wide variety (e.g., OH…O = C, NH…O = C, OH…Cl, NH…Cl, OH…NH, CH…Cl, CH…O = C, NH…OH, and NH…NH), together with the plausible formation of complex anions of [Cl(urea)2]− and cations of [urea(choline)]+, the latter potentially stabilizing phenolate anions with choline for facile electron transfer. Since phenols can be more easily oxidized by dissociation into phenolate, this additional electrostatic property may also be held responsible for the enhanced TAC values observed in the CC-urea-water combination.
3.2 Modeling and optimization of the MAE process for the extraction of phenolics from sunflower pomace
A p < 0.0001 was obtained for the model corresponding to the TAC response, indicating a significant relationship between the response and the independent variables. Extraction temperature is the most important operational factor affecting TAC among all factors. The significance of each coefficient was determined using the F-test and p-value, summarized in Table 5. Increasing the absolute F-value and decreasing the p-value make the variable significant. From this, it is understood that the model is statistically significant at the 95% confidence level. According to the results of ANOVA, the use of quadratic models was found appropriate to predict the response. The quadratic model obtained using the coded independent variables in the TAC estimation determination is summarized in Eq. 3.
The predicted and the adjusted R2 values for TAC values were 0.7989 and 0.9188, respectively. A difference of less than 0.2 indicates that the predicted R2 values are reasonably consistent with the adjusted R2 values. A sufficient sensitivity value measures the signal-to-noise ratio, and it is desired that this value be greater than 4. The signal-to-noise ratio for TAC was found to be 16.808, and the proposed model was found to be usable in the design. Figure 3 shows the correlation between the independent variables and the estimated and actual TAC value obtained. Figure 3 allows us to comment that the values estimated from the model are in sufficient agreement with those obtained from the real data. The highest TAC (0.25 mmol TR/g-DS) yield was obtained at the following conditions: A = 18 min, B = 78 °C, C = 10 mL/g-DS, and D = 38% water.
The correlation between the predicted versus actual TAC values of the sunflower pomace extract
3.3 Effect of the operational parameters on MAE
The effects of operational factors on MAE of phenolics from sunflower pomace were investigated. Figure 4 shows the effects of operational parameters on antioxidant extraction in the form of three-dimensional (3-D) graphs.
3-D graphs for the TAC of the sunflower pomace extract as a function of operational factors
Extraction temperature is the most important operational factor for the MAE process in phenolic extraction from sunflower pomace, and its selection is critical. Increasing temperature decreases solvent viscosity and surface tension and increases the solubility of phenolics, thereby wetting the sample more and increasing matrix penetration [36]. An increase in extraction temperature positively affects efficiency, but at high temperatures (usually after 80 °C), the decomposition of phenolics begins. Furthermore, high temperatures are not preferred in terms of both safety and economy. As a result, optimization of the extraction temperature is important, and 78 °C was found as the optimal extraction temperature for the extraction of phenolics in sunflower pomace.
Figure 4a reveals the relationship between exposure time and antioxidant extraction. During the process, extraction basically takes place in three stages: wetting the sample with the solvent, dissolving the components, and releasing the target components from the matrix together with the solvent. Antioxidant extraction increases with increasing time, but decreases slightly with prolonged extraction times (Fig. 4a). The structural degradation of sensitive bioactive compounds with prolonged thermal exposure is the main reason for this decline [37]. In addition, it is known that long extraction times are not preferred for economic reasons and workload.
Another investigated parameter for the extraction process is the solvent-to-solid ratio. In this process, it was observed that the solvent-to-solid ratio did not have a significant effect on the extraction yield in the studied range. With the increase of the solvent-to-solid ratio, the extraction efficiency slightly increased in line with the mass transfer principles (Fig. 4b). When the amount of solvent is low compared to the solid, the transfer cannot be completed [38]. It is generally undesirable to use high volumes of solvents due to both cost and environmental factors.
The effect of water ratio on extraction efficiency was also investigated. Adding water to NADES reduces viscosity, increases polarity, and increases extraction efficiency. On the other hand, increasing the water ratio excessively may reduce the efficiency by weakening or breaking the intermolecular hydrogen bond structure of NADES components [39]. Figure 4c shows the effect of water ratio ranging from 0 to 40% on antioxidant extraction. The optimal water content is about 38%. However, no significant increase in TAC was observed with the increase of the water ratio, and this is thought to be due to the water added during the NADES preparation stage.
3.4 Antioxidant efficacies of sunflower pomace
Antioxidant activities of sunflower pomace extract under optimal conditions were determined by using various antioxidant tests. In addition, the antioxidant properties of the extract prepared with NADES were compared with those obtained using traditional solvent mixtures, such as the frequently used ethanol-to-water mixture (80:20 (v/v)) and methanol-to-water mixture (80:20 (v/v)). The TAC, ARC, FRC, and TPC values of the extracts prepared under optimized conditions are summarized in Fig. 5. The reagent of the Folin–Ciocalteu (FC) method has a high redox potential in an alkaline environment where most phenolic compounds are deprotonated and open to oxidative attack, and therefore, its TPC values were found to be higher than others; the FC reagent may non-selectively respond to certain amino acids, sugars, and other phenolic species actually devoid of antioxidant activity [40]. The possible reason for the higher antioxidant capacity obtained with the CUPRAC reagent compared to those with ARC and FRC tests is that the CUPRAC chromophore, Cu(I)-neocuproine complex, dissolves well in both hydrophilic and hydrophobic solvent environments, and therefore with DES solvents forming an efficient combination of these two solvent environments. CUPRAC displays a superior performance.
Effect of type of solvent on the MAE of antioxidant constituents
3.5 The chromatographic analysis of phenolics in NADES extracts of sunflower pomace
Phenolics were determined by performing the HPLC analysis method [24]. The quinic acid derivatives in sunflower pomace extracts were identified by spiking known amounts of standards and/or by matching retention times and UV spectra (Figs. 6 and 7). The major component in the extract was identified as 3-CQA. Due to the fact that not all standards were supplied, the total phenolic content of sunflower pomace extract was calculated as mg/g 3-CQA equivalent by summing up peak areas of individual components in the related chromatograms. In other studies, caffeoyl quinic acid derivatives as phenolic constituents were found in sunflower seed, flower, and pomace which was proved by HPLC analysis [41]. According to the findings, extracts prepared in DES medium showed higher phenolic content (29 mg/g) compared to alcoholic extracts (approximately 1.4-fold). It was shown that the selected NADES medium provides an efficient extraction capacity of phenolics in sunflower pomace.
Chromatogram of 1:20 (v/v) sunflower pomace DES extract detected at 340 nm (inset, UV spectra of constituents)
Chromatograms of diluted sunflower pomace DES extract spiked with A 1,3-DCQA and 1,5-DCQA and B 3-CQA solutions detected at 340 nm
3.6 Effects of fortification with NADES extracts on antioxidant activity of smoothie-like beverages
At this stage, the effects of fortification with SFP NADES extracts on the bioactivity of natural antioxidants in smoothie-like beverages before and after in vitro gastrointestinal digestion were investigated (Table 6). Based on the CUPRAC assay, the presence and the amount of NADES extract provided a considerable improvement in the TAC of final products for both before and after in vitro gastrointestinal digestion compared to the control sample. The highest TAC value was detected in the 20% N-B sample with 476.2; 339.9; and 442.3 mg TR/100 g FW in the initial, gastric, and intestinal phases, respectively, and 68.6% was the most efficient improvement for the 20% N-B sample. The highest TAC value was also followed by the 10% N-B, 5% N-B, and B (control) samples in decreasing order. A similar trend was seen for the FRC values in each beverage sample before and after in vitro gastrointestinal digestion. 67.9% was the highest improvement in the antioxidant capacity obtained with the addition of 20% NADES extract according to the DPPH assay. Interestingly, the higher ARC value was found in B (control) sample, and decreasing values were obtained with NADES extracts added to beverage samples in the intestinal phase, although similar results to the TAC and FRC values were obtained for beverages fortified with NADES in the initial and gastric phases based on the ABTS assay. These ARC values observed in the intestinal phase may be obtained due to the pH value and digestive enzymes of the intestinal phase and/or the interaction of food components, phenolic compounds, NADES solution, and reagents used in the ABTS assay. Naturally, there may be differences in the results depending on the parameters of the analytical method such as pH, time, different interactions of the chemical reagents with the food components, or the interactions among food components themselves. Therefore, it is not sufficient to choose a single antioxidant assay to evaluate the antioxidant activity of bioactive compounds in foods being multifunctional or complex multiphase systems. Different antioxidant analysis methods should be performed to better understand the interaction effects on the antioxidant activity and bioaccessibility of the investigated compounds [42].
Considering the results of antioxidant assays performed, the added amounts of NADES extracts contributed to the increased antioxidant capacity of final beverages. Fortification with NADES extract in beverages improved the antioxidant capacity by 12.4–68.6% and 0.4–67.9% in the initial, gastric, and intestinal phases for TAC and FRC values, respectively, and by 26.5–58.2% in the initial and gastric phases for ARC values. This positive effect of the presence of NADES extract in smoothie-like beverage samples can be attributed to the solubilization and protection ability of NADES with the formation of hydrogen bonds between the NADES solution and the phenolic compounds, resulting in a reduction in degradation and improvement in bioaccessibility of phenolic compounds [9, 43]. The increase in antioxidant activity of fortified beverages was supported by previous studies investigating the effects of in vitro gastrointestinal digestion on phenolic compounds in NADES extracts. For example, the use of NADES solvents was shown to increase the bioaccessibility and stability of phenolic compounds from blueberry [12], borage flowers [43], bitter melon [44], and olive oil by-products [45] during in vitro gastrointestinal digestion.
On the other hand, strawberry puree as another control sample and the mixture of strawberry puree and NADES extract (2:1 w/w) were also prepared for the detailed observation of the effects of NADES extracts within the food matrix, since the prepared smoothie-like beverages have a more complex matrix due to the presence of yogurt and its ingredients. As expected, the N-S.P sample had higher TAC by 34.4–88.7%, FRC by 29.1–62.0%, and ARC by 6.0–65.3% values than that of the S.P (control) sample in each phase. When comparing the N-S.P sample and the 5, 10, and 20% N-B samples, different results were obtained using different assays. The results of the DPPH assay showed that the N-S.P sample had higher FRC values than the 5, 10, and 20% N-B samples in each phase. However, samples with 5, 10, and 20% N-B had higher TAC and ARC values compared to the N-S.P sample in both gastric and intestinal phases while their values were lower than the N-S.P sample in the initial phase. The reason for this situation may be due to the presence of yogurt proteins in the formulation of the beverage samples. It is known that phenolic compounds and proteins interact with each other. Also, this interaction is influenced by the individual structure and concentration of the proteins and phenolic compounds as well as by environmental parameters such as temperature and pH. Thus, the interactions between phenolics and proteins may cause some positive or negative effects on the antioxidant capacity, solubility, bioaccessibility, and bioavailability of both compounds in food, especially during in vitro gastrointestinal digestion [46].
These variable results of fortified smoothie-like beverages and strawberry puree with SFP NADES extract may be due to the presence of yogurt protein in the beverage samples, the presence and different levels of NADES extract, the possible interactions of the phenolic compounds, NADES solutions and proteins, the environmental conditions of the in vitro gastrointestinal digestion process, and/or the use of different reagents and the possible reactions between the reagent and the phenolic compounds in different assays. In addition, Kehili et al. reported that DES solvent solubilized carbohydrates, mainly mannose and manno-oligosaccharides, during polyphenol extraction from defatted date seeds [47]. The use of NADES/DES solvents can therefore have an effect on the solubility, quantity, and stability of other food components. These effects may also promote various interactions between phenolic compounds and other food components during the extraction process and/or in the final food products. Related to these effects, the DES/NADES solvent itself may exert a distinct influence on the gastrointestinal fate of bioactive compounds and/or the final antioxidant activity.
Moreover, when comparing the antioxidant activity of smoothie-like beverages fortified with 5, 10, and 20% NADES extract after in vitro gastrointestinal digestion phases, higher antioxidant activity was determined in the intestinal phase than that of the gastric phase for each sample. According to the results of the CUPRAC assay, the antioxidant capacity was 23%, 19%, and 30% higher in the intestinal phase than in the gastric phase for 5, 10, and 20% N-B samples, respectively. In addition, a 2.8- to 3.4-fold and 3.4- to 4.6-fold higher antioxidant capacity was obtained in fortified beverages in the intestinal phase compared to the gastric phase, based on the DPPH and ABTS assays, respectively. Similar observations were obtained about the antioxidant activity in strawberry yogurt [48] and yogurt enriched with tamarillo powder [49] during in vitro gastrointestinal digestion. These results were attributed to the effects of the yogurt matrix providing possible protection of bioactive compounds against degradation, enhancement of bioaccessibility, and increase in the absorption in the small intestine [48, 49], due to the release of bound polyphenols from complex formed by the natural binding affinity between plant polyphenols having medium-polarity and proteins rich in proline [50] with the protein hydrolysis by the activity of hydrolytic enzymes and change in pH during digestion [49, 51]. Additionally, the study by Türker and Doğan [52] concluded that α-casein or β-casein can provide protection for bioactive compounds such as anthocyanin in microencapsulated extract obtained by NADES from black carrots during simulated digestive systems. Furthermore, in smoothie-like beverages fortified with NADES extract, the presence of NADES solution right along with the presence of yogurt proteins may also have contributed to the solubilization and protection for polyphenols due to the formation of hydrogen bonds with polyphenols during in vitro gastrointestinal digestion. For example, da Silva et al. [12] stated that NADES solution provided an increase in the stability of blueberry phenolic compounds with the delay of the gastric chyme neutralization during in vitro digestion in their study.
Overall, the aim of this part was to produce functional foods with SFP NADES extract and to study the effects of fortification with an understanding of possible interactions between NADES extract and food components. The results showed that the fortification with NADES extracts rich in sunflower pomace phenolics at different ratios improved the antioxidant capacity of smoothie-like beverages containing strawberry and yogurt. Furthermore, the amount of NADES extract added to the final products was highly effective for improving the antioxidant activity, as was the presence of NADES extract, since an increase in the antioxidant capacity was observed in parallel with the amount of NADES extract added, both before and after the in vitro gastrointestinal digestion phases. Thus, the 20% N-B sample with the highest antioxidant activity was found to give the best result. The phenolic-rich NADES extract from sunflower pomace can be considered a natural food additive and has great potential for the design of new functional food formulations with high antioxidant activity. However, there is a large gap in the literature on the consumption level of NADES extracts and its toxicity in the human body [17]. The addition of 20% SFP NADES, which can be accepted as a high-level of fortification, was only used to understand the effects of the amount of additive and possible interactions with food ingredients although the choline chloride and urea used in this study to produce the NADES solvent are accepted as “generally recognized as safe” (GRAS) chemicals [47]. In accordance with the GRAS status of NADES-building components, carefully selected NADES were used for efficient extraction of polyphenols from cocoa by-products and the obtained NADES extracts were used for the fortification of food industrial products, such as chocolate/cocoa drinks fortified with NADES extracts without removal of extraction solvent, since they are proven safe and estimated as sensory acceptable [17]. However, there is an urgent need for toxicity assessment and/or in vivo studies and, with the results of these studies, regulations may be adopted for the daily intake and thus the use of NADES extracts for fortification in the food industry.
4 Conclusions
In this study, MAE conditions of NADES prepared with CC-U-W combination and phenolics from sunflower pomace were optimized and modeled. It was observed that the antioxidant properties of the extracts obtained with the prepared NADES were stronger than those of the extracts prepared with traditional solvent systems, and plausible mechanisms for this observation were discussed. The use of NADES also provides advantages over conventional solvents with its low cost, toxicity, and biocompatibility. In addition, the MAE system used provides opportunities such as multiple sample processing, short extraction time, low solvent usage, and automation. Also, strawberry and yogurt-based smoothie-like beverages fortified with the addition of selected NADES extracts rich in phenolic compounds of sunflower pomace at different ratios were produced. The fortification with NADES extracts increased the antioxidant activity of final beverages compared to the control sample based on the CUPRAC, DPPH, and ABTS assays. The positive effects of fortification with NADES extracts showed that sunflower pomace extracts obtained with NADES as a ready-to-use extract can be a valuable and natural food additive for the production of functional foods. However, there is a need for further studies on the effects of using NADES extract on antioxidant capacity, bioaccessibility, and bioavailability of bioactive compounds within the food matrix as well as sensory acceptability, regulation of daily intake, and toxicity of NADES. The procedure presented with this study presents an effective and sustainable procedure that can potentially be used for the extraction of phenolics from sunflower pomace.
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Acknowledgements
The authors thank the Application & Research Center for the Measurement of Food Antioxidants, Istanbul University-Cerrahpaşa, for sharing its research infrastructures.
Funding
Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK). Reşat Apak expresses his gratitude to TUBITAK (Turkish Scientific and Technical Research Council) for the 2247-A National Leading Researchers Program Project (Project No:120C134). Mustafa Bener and Yavuz Selim Aşçı also express their gratitude to the Istanbul University Research Fund, Bilimsel Araştırma Projeleri (BAP) Yurutucu Sekreterligi (Project No: FBG-2022–38594). Mustafa Bener was supported by the Turkish Academy of Sciences (TÜBA) in part by the TÜBA Outstanding Young Scientist Award Programme (GEBİP).
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Furkan Burak Şen: investigation, methodology, formal analysis. Elifsu Nemli: investigation, methodology, formal analysis. Burcu Bekdeşer: investigation, methodology, formal analysis. Saliha Esin Çelik: conceptualization, methodology. Melisa Lalikoglu: investigation, resources, methodology. Yavuz Selim Aşçı: investigation, resources, methodology. Esra Capanoglu: conceptualization, supervision. Mustafa Bener: conceptualization, software, writing-original draft preparation. Reşat Apak: conceptualization, supervision, writing—reviewing and editing.
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Şen, F.B., Nemli, E., Bekdeşer, B. et al. Microwave-assisted extraction of valuable phenolics from sunflower pomace with natural deep eutectic solvents and food applications of the extracts. Biomass Conv. Bioref. (2024). https://doi.org/10.1007/s13399-024-05942-2
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DOI: https://doi.org/10.1007/s13399-024-05942-2