Phytochemical and functional characterization of phenolic compounds from Cowpea (Vigna unguiculata (L.) Walp.) obtained by green extraction technologies

agronomy Article Phytochemical and Functional Characterization of Phenolic Compounds from Cowpea (Vigna unguiculata (L.) Walp.) Obtained by Green Extraction Technologies M. Victoria Avanza 1,†, Gerardo Álvarez-Rivera 2,† , Alejandro Cifuentes 2, José A. Mendiola 2,* and Elena Ibáñez 2   Citation: Avanza, M.V.; Álvarez-Rivera, G.; Cifuentes, A.; Mendiola, J.A.; Ibáñez, E. Phytochemical and Functional Characterization of Phenolic Compounds from Cowpea (Vigna unguiculata (L.) Walp.) Obtained by Green Extraction Technologies. Agronomy 2021,11, 162. https:// doi.org/10.3390/agronomy11010162 Received: 11 December 2020 Accepted: 14 January 2021 Published: 16 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Instituto de Química Básica y Aplicada del Nordeste Argentino (IQUIBA-NEA), Facultad de Ciencias Exactas y Naturales y Agrimensura-UNNE and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Avenida Libertad 5470, Corrientes 3400, Argentina; [email protected] 2Foodomics Laboratory, Institute of Food Science Research (CIAL, CSIC), C/Nicolás Cabrera 9, 28049 Madrid, Spain ; gerardo.alvar[email protected] (G.Á.-R.); [email protected] (A.C.); [email protected] (E.I.) *Correspondence: [email protected]; Tel.: +34-91-0017-900 † Both authors contributed equally. Abstract: This work presents a green, downstream process, from extraction to phytochemical characterization and bioactivity testing, to obtain and evaluate the functional properties of phenolic compounds from cowpea (Vigna unguiculata (L.) Walp.) seeds and pods. Phenolic-rich extracts were obtained by pressurized liquid extraction (PLE). The main factors affecting the extraction conditions (temperature and solvent) were optimized in order to attain extracts with the highest extraction yield, antioxidant capacity, and total phenolic content. The optimal extraction conditions were 1:1 ethanol:water at 170 ◦ C with one extraction cycle for seeds and three extraction cycles for pods. Phenolic compounds of optimal extract were analyzed by UHPLC-q-TOF-MS/MS (quadrupole-time of flight tandem MS). The obtained PLE-extracts exhibited higher phenolic content and antioxidant activity compared to conventional extraction procedures. The in vitro anti-neurodegenerative potential of extracts was measured through Acetylcholinesterase (AChE) inhibition assay. The results revealed the higher bioactivity observed in cowpea pod samples compared to seed extracts, which might be related to higher levels of quercetin and quercetin glycosides, kaempferol diglucoside, and other tetrahydroxylated flavones and flavonols identified in these samples. These results also provide an added-value benefit to the cultivation of this legume, considering the high potential of cowpea phenolic extracts as nutraceutical and functional ingredients in food formulations. Keywords: pressurized liquid extraction; green extraction; cowpea; phenolic compounds; antioxidant capacity; neuroprotective properties; acetylcholinesterase 1. Introduction From a nutritional standpoint, legumes are an important source of proteins, calories, minerals, and vitamins [ 1 ]. Although the consumption of pulses in many Western countries is relatively low as compared to many other countries like India, legumes contain significant amounts of polyphenols such as flavonoids, phenolic acids, and lignins, which are considered natural antioxidants [ 2 ]. Cowpea (Vigna unguiculata (L.) Walp.) is a legume that is mainly cultivated in Africa, Asia, and Latin America. In the northeast region of Argentina, the cultivation of cowpea is run by smalland medium-sized producers. Cowpea seeds are used for human food and animal feed, while pods are either discarded or burned. Cowpea contains high levels of polyphenols with a unique profile that are mainly concentrated in the tegument and give the seeds their typical color [ 3 ]. The main polyphenols common to all varieties of cowpea are phenolic acids and flavonol glycosides. Some varieties also contain anthocyanins and/or flavan-3-ols [ 4 ]. Cowpea polyphenols’ extracts Agronomy 2021,11, 162. https://doi.org/10.3390/agronomy11010162 https://www.mdpi.com/journal/agronomy Agronomy 2021,11, 162 2 of 18 have been reported to have significant anti-inflammatory activity, with beneficial effects against diabetes, cancer, and cardiovascular diseases [ 4 ]. Cowpea pods can be considered lignocellulosic biomasses that do not contain starch and in which the carbohydrates are present as cellulose and hemicellulose [ 5 ]. This biomass also contains polyphenols, whose content increases as the plant reaches maturity while the tannin content decreases [6]. Many bioactive compounds derived from agricultural residues have been proven to be potentially useful in the food and pharmaceutical industry [ 7 ]. Nonetheless, the use of agricultural wastes as a rich, low-cost, and bio-renewable resource for the production of bioactive compounds is crucial; however, the bulk of knowledge of the field remains scarce [8]. The harmful effects of free radicals, together with the toxic effects produced by synthetic antioxidants used as food preservatives, have promoted the search for molecules with antioxidant properties to be applied in food, cosmetic, and pharmaceutical industries. In this sense, legumes are a potential source of antioxidants mainly because they synthetize a great variety of secondary metabolites with free radical scavenging capacity in response to either artificial or environmental stimuli [2]. Among the different important diseases associated with oxidative stress processes, cancer and cardiovascular diseases have been highlighted and are among the main causes of death in Argentina and in the world. These diseases are based on the accumulation of highly reactive free radicals or the impairment of the defense systems that protect biomolecules from oxidative damage [ 9 ]. Epidemiological studies show a relationship between the consumption of legumes, fruits, cereals, and vegetables and low incidence values of these syndromes [10]. Moreover, a therapeutic alternative for patients with Alzheimer disease, which is featured by oxidative stress [ 11 ], is focused on the inhibition of the acetylcholinesterase (AChE) [ 12 ]. To date, several plant species have proven to have acetylcholinesterase inhibitory (AChEI) activity [ 13 ]. Among the natural phytochemicals, flavonoids and phenolic acids represent an interesting class of biologically active compounds in this regard. In addition, there is evidence suggesting that these compounds have antioxidant capacity [14]. In recent years, there has been an increasing interest in the development of environmentally clean processes for obtaining high-added-value extracts and compounds with biological activity from natural sources. In this line, pressurized liquid extraction (PLE) has been consolidated as a high throughput and green extraction technique that may be used for the extraction of medium and polarity bioactive compounds, depending on the extraction solvent employed [ 15 ]. Green extraction processes, such as those based on PLE, make use of GRAS solvents (generally recognized as safe), which guarantees the absence of toxic solvents in the final ingredients and products obtained. While PLE can be performed using a wide range of solvents, ethanol and water are preferred for green extractions. Considering that cowpea polyphenolic compounds are mainly obtained by conventional extraction methods (extraction with organic solvents) [ 16 , 17 ], an alternative green extraction procedure, followed by chemical and functional characterization, was proposed in this work in order to obtain phenolic-enriched extracts from cowpea and waste semifeedstocks (cowpea pods) that could be used as ingredients in food formulation. 2. Materials and Methods 2.1. Samples and Chemicals Seeds and pods of two cowpea varieties, named as Cuarentón (CUA) and Colorado (COLO), were provided by Estación Experimental El Sombrero, province of Corrientes (Instituto Nacional de Tecnología Agropecuaria (INTA)), Argentina (2018 harvest). Both cultivars are registered with the number 1981-01-13 at Registro Nacional de Cultivares (RNC) from the Instituto Nacional de Semillas de Argentina Registration. They are not native species from Argentina but foreign varieties that have been selected by humans for use since the colonial period. Therefore, they have different record numbers (colorado: Agronomy 2021,11, 162 3 of 18 N ◦ de registro 914 and cuarenton: registry number: 909) but the same registration number. Shrunken, discolored, and insect-infested seeds were discarded. Seeds and pods were sun-dried and stored in a hermetic vessel at 10 ◦ C until used. Cowpea seeds and pods were ground (Braun KSM2, coffee grinder, Mexico) and sifted through a 500µ m sieve. Ultrapure water obtained from a Millipore purification system (Billerica, MA, USA) and ethanol (99.5%) provided by VWR Chemicals (Fontenay-sous-Bois, France) were used for PLE. DPPH (2,2-Diphenyl-1-picrylhydrazyl hydrate, free radical, 99%), gallic acid, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, ≥ 97%), and ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid, ≥ 99%) were acquired from SigmaAldrich. Folin-Ciocalteu reagent for total phenolic analysis was purchased from Merck (Darmstadt, Germany). 2.2. Maceration Extraction (ME) Seeds’ and pods’ flour dispersions (5 g in 50 mL) were prepared in different solvent mixtures: ethanol, water, water + 2% v/vformic acid, water + 5% v/v formic acid, and 70% v/vacetone + 1% v/vformic acid. Mixtures were macerated for 24 h with shaking at 230 rpm. Dispersions were then centrifuged (Hettich 380R) at 5000 rpm for 15 min and the resulting supernatant was filtered. Extracts were then evaporated using nitrogen stream and/or by lyophilization (Labconco Corporation, Kansas City, Mo, USA), depending on the extraction solvent. The yield, the polyphenols’ content, and the antioxidant activity were determined in the dried sample extracts. 2.3. Pressurized Liquid Extraction (PLE) These extractions were carried out in a pressurized liquids extractor (ASE 200, Dionex, Sunnyvale, CA, USA). Ethanol, ultrapure water, and the mixture ethanol:water (50:50) were used as solvents, as described below. To eliminate dissolved air, at the beginning of the working day the solvents were sonicated for 10 min. For each extraction, 1 g of dried seed or pod sample was mixed with 2 g of dispersive agent (sea sand). A sandwich scheme was used inside the 11-mL stainless-steel extraction cell. This mixture was placed in the center among 2 g of sea sand and cellulose filters (Restek, Bellefonte, PA, USA) located at both ends. Briefly, the extraction was done as follows. Initially, the selected solvent filled the extraction cell and the pressure rose. Then it was heated at extraction conditions. Secondly, the static extraction conditions were maintained for 10 min, with the system closed to keep a constant pressure (1500 psi, 10.34 MPa). Finally, the extract was collected in a glass vial, the extraction cell was rinsed with fresh solvent (60% of cell volume), and pure nitrogen for 120 s was used for purging. To avoid carryover effects, a thorough rinse was done between the consecutive extractions. After extraction, solvents were eliminated using nitrogen stream, and water was removed by freeze-drying (Labconco Corporation, Kansas City, Mo, USA). The dried extracts were stored at − 20 ◦ C and protected from light until using. 2.4. Experimental Design For optimization of PLE from cowpea seed and pods, a three-level factorial design in 16 runs (Box-Behnken) was performed to study the effects of percentage of ethanol in the mixture solvent (0–100%) (v/v), temperature (50–170 ◦ C), number of cycles (1–3) on the extraction yield (%), total phenolic content (mg gallic acid equivalents, GAE/g extract), antioxidant capacity (TEAC, mmol of Trolox equivalents/g extract), and DPPH radical scavenging activity (EC 50 , µ g/mL: concentration required to obtain a 50% antioxidant effect). All the experiments (16), including four center points, were carried out in random order. Response surface methodology (Statgraphics Centurion XVII, StatPoint Technologies, Inc., Warrenton, VA, USA) was used for experimental design and data analysis. In the extraction process, the influence of independent variables on the response variables was Agronomy 2021,11, 162 4 of 18 assessed using the pure error. A confidence level of 95% was considered for all the variables. The quadratic model proposed for each response variable (Yi) was: Yi=a+bA+cB+dC+eA2+ fB2+ gC2+ hAB + iAC + jBC + ε(1) where A is the temperature; B is the solvent composition (percentage of ethanol in the mixture); C is the number of cycles; a is the intercept; b, c, and d are the linear coefficients; e, f, and g are quadratic coefficients; h, i, and j are the interaction coefficients; and ε is the error variable. This quadratic model (Equation (1) was estimated considering R 2 (percent variation explained by model) and RSD (residual standard deviation). Additionally, lackof-fit test was done for the models from the analysis of variance (see Tables S1 and S2 in Supplementary Material) as the significance measures. For each of the response variables the effect of each factor and their statistical significance was analyzed from the standardized Pareto chart. Graphically, a response surface of the individual mathematical model was also obtained, and significant differences were considered with p ≤ 0.05. Additionally, multiple response optimization was done combining different responses to maximize all of them at the same time. 2.5. In Vitro Experiments 2.5.1. Total Phenolic Content (TPC) The TPC was determined in ME and PLE extracts spectrophotometrically by the Folin–Ciocalteu’s method with some modifications, as previously described [ 18 ]. Briefly, a 10µ L aliquot of the extract (10 mg mL −1 ) and 600 µ L of ultrapure water were mixed. Fifty µ L of undiluted Folin–Ciocalteu reagent were subsequently added. After 1 min, 150 µ L of 20% (w/v) Na 2 CO 3 were added and the volume was adjusted to 1.0 mL with ultrapure water. Samples were incubated for 2 h at 25 ◦ C in the darkness. Three hundred µ L of each reaction mixture were transferred to a 96-well microplate. The absorbance was measured at 760 nm in a microplate reader (Synergy HT, BioTek Instruments, Winooski, VT, USA). All analyses were done in triplicate. For calibration, standard curves constructed with serial dilutions of gallic acid (0.031–2 mg mL −1 ) were done and mg of gallic acid equivalents (GAE) per g of extract was used to express the TPC. 2.5.2. Trolox Equivalents of Antioxidant Capacity (TEAC) TEAC was determined in ME and PLE extracts using the method described by Re et al. [ 19 ] with some modifications. Briefly, the ABTS •+ radical was generated through the reaction of 7 mM ABTS with 2.45 mM potassium persulfate in the absence of light, at room temperature, for 16 h before using it. This aqueous ABTS •+ solution was diluted with phosphate buffer 5 mM (pH 7.4) to an absorbance of 0.7 UA ( ± 0.02) at 734 nm. Samples (10 µ L, at five different concentrations, from 0.625 to 10 mg mL −1 ) and 1 mL of ABTS •+ solution were mixed in an Eppendorf vial. After incubation for 45 min, 300 µ L of the mixture were transferred to a 96-well microplate. The endpoint absorbance was measured in a microplate reader (Synergy HT, BioTek Instruments, Winooski, VT, USA) at 734 nm. The results were expressed as TEAC values (mmol of Trolox/g extract), with Trolox used as reference standard. All analyses were done in triplicate. The values shown were obtained using five concentrations of each extract, chosen to obtain a linear response between 20% and 80% of the absorbance of the blank. 2.5.3. DPPH Radical Scavenging Assay ME and PLE extracts were analyzed by the DPPH radical scavenging method adapted from the methodology described by Brand-Williams et al. [ 20 ]. A working solution containing 2.35 mg of DPPH in 100 mL of methanol was used. The extracts were tested in a range between 0.625 to 10 mg mL −1 . In each well 10 µ L of each extract solution or methanol (blank) and 290 µ L of DPPH solution were placed to make up the final reaction volume of 300 µ L. After 4 h in the dark at room temperature, the absorbance was measured in a microplate reader (Synergy HT, BioTek Instruments, Winooski, VT, USA) at Agronomy 2021,11, 162 5 of 18 516 nm. The remaining concentration of DPPH was calculated from a calibration curve. Measurements were made in triplicate. The data shown consist of the concentration of the extract (expressed in µ g mL −1 ) necessary for a half initial concentration of DPPH, that is, the EC50 value. 2.5.4. In Vitro AChE Inhibition Assay The acetylcholine esterase inhibitory (AChE) activity was measured by the Ellman’s method [ 21 ] with minor modifications [ 22 ]. Kinetic parameters (Km, Michaelis-Menten constant, and V max , maximum velocity) values were derived from the Michaelis–Menten plot. The reaction mixture contained 100 µ L of 0.15 M Tris-HCl buffer (pH 8.0), 25 µ L of 0.8 U/mL acetylcholine esterase (type VI-S from Electrophorus electricus, Sigma-Aldrich, St Louis, Mo, USA), 100 µ L of each extract at different concentrations in 50% ethanol (1 and 0.5 mg/mL), and 25 µ L of 4 mM of DTNB (5,5-dithio-bis-(2-nitrobenzoic acid). Mixtures were incubated for 30 min at 37 ◦ C. After 30 min, the reaction was initiated by the addition of 50 µ L of 1.2 mM acetylthiocholine (substrate). The hydrolysis of acetylthiocholine was monitored as the Vmax at 412 nm for 5 min each 15 s. Galanthamine was used as positive control. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = [(E −S)/E] ×100 (2) where E is the Vmax of the enzyme without sample and S is the Vmax of enzyme with the test sample. 2.6. Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-ESI-q-TOF-MS/MS) For the phytochemical characterization of cowpea seeds and pods extracts, an Agilent 1290 UHPLC system (Ultrahigh Performance Liquid Chromatography) coupled to an Agilent 6540 quadrupole-time-of-flight mass spectrometer (q-TOF MS) equipped with an orthogonal ESI source was employed, all from Agilent Technologies (Santa Clara, CA, USA. A Zorbax Eclipse Plus C18 column (2.1 mm diameter, 100 mm length, 1.8µ m particle diameter) was employed for chromatographic separation at 30 ◦ C. The mobile phase was composed of two solvents: solvent A water (0.01% formic acid) and solvent B acetonitrile (0.01% formic acid). Five microliters of the sample were injected using a flow rate of 0.5 mL/min in gradient elution. The gradient timetable was as follows: 0 min, 100% A; 7 min, 70% A; 9 min, 20% A; 11 min, 0% A; 13 min, 0% A; 14 min, 100% A. The mass spectrometer was operated in MS and tandem MS modes for the structural analysis of all compounds. MS parameters were the following: nebulizer pressure, 40 psi; capillary voltage, 4000 V; drying gas temperature, 350 ◦ C at a flow rate of 10 L/min; skimmer voltage, 45 V; and fragmentor voltage, 110 V. A scan rate of 5 spectra per second between 50–1100 and 50–800 m/z for MS and Auto MS/MS modes was used. 2.7. Statistical Analysis Each treatment was performed at least in triplicate, as stated above. Values are shown as average ± standard deviation. One-way analysis of variance was employed. Additionally, Fisher Least significant difference (LSD) test with α = 95% (confidence interval) was used to contrast the means. The statistical analysis was performed using the Infostat software (https://www.infostat.com.ar/ Universidad Nacional de Córdoba, Argentina). 3. Results and Discussion 3.1. Maceration Extraction (ME) Table 1shows the results of the maceration of cowpea (COLO variety) seeds and pods obtained with ethanol, water, water-formic acid (2% and 5% v/v), and acetone-formic acid (1%). The highest yield, polyphenols’ content’ and antioxidant activity was obtained by extracting with the mixture of 70% v/vacetone and 1% v/vformic acid in both seeds’ and pods ‘samples, though values were higher for seed preparations. The extraction of phenolic compounds is favored by polar organic solvents or by mixtures of polar organic Agronomy 2021,11, 162 6 of 18 solvents with water. Acetone is a dipolar aprotic solvent with great solubilization capacity of organic compounds. Addition of 1% v/vformic acid increased acetone solubility of phenolic compounds, leading to an increase of the radical scavenging capacity. Acetone 1% v/vformic acid was used to obtain relatively high levels of proanthocyanidins present in cowpea varieties of cowpea, as reported by Ojwang et al. [ 16 ]. The extraction performed with water rendered pods extract with a higher polyphenols’ content and antioxidant activity while these values were lower when ethanol was used, mainly in seeds’ samples. The content of polyphenols in the extracts of water and formic acid at 2 and 5% v/v was higher in pods than in seeds; however, the antioxidant activity was lower in pods. These results may be due to the different compounds extracted that varied according to the extraction solvent polarity and the antioxidant capacity of extracted compound. Vijayalaxmi et al. [ 23 ] reported high polyphenols’ contents in agricultural wastes such as wheat bran, peanut shell, and rice bran. Avanza at al. [ 3 ] reported similar values of polyphenols’ content in methanol extracts of cowpea seeds of different varieties. The polyphenols’ content obtained in this work are comparable to those reported by other researchers for V. unguiculata seeds [ 24 ] and are higher than those obtained in seed extracts of lentil and chickpea cultivars from Pakistan [ 25 , 26 ]. Previous reports have associated the TPC to the observed antioxidant activity [27,28]. Table 1. Yields, polyphenols’ content, and antioxidant activity (DPPH and ABTS) of cowpea seed and pods’ extracts from Colorado variety obtained by maceration. Samples Solvent Yield (%) TPC (mg GAE g−1) TEAC (mmol TE g−1) EC50 (µg/mL) COLO seed ethanol 1.67 ±0.16 c17.70 ±0.40 b0.198 ±0.006 c93.86 ±1.63 c water 5.55 ±1.41 b15.32 ±0.25 c0.460 ±0.004 b268.92 ±4.01 a water-2% v/vformic acid 7.26 ±0.81 b12.88 ±0.37 d0.121 ±0.002 c271.57 ±3.99 a water-5% v/vformic acid 7.36 ±1.45 b17.40 ±0.24 b0.115 ±0.001 c191.53 ±3.60 b acetone-1% v/vformic acid 12.61 ±1.13 a65.68 ±0.31 a3.650 ±0.267 a21.82 ±0.46 d COLO pods ethanol 1.18 ±0.12 b16.70 ±0.49 d0.442 ±0.010 c479.64 ±8.34 b water 4.54 ±0.95 a43.84 ±0.26 a2.283 ±0.028 a326.74 ±4.87 c water-2% v/vformic acid 5.45 ±1.02 a30.20 ±0.67 c0.086 ±0.001 d566.12 ±8.32 a water-5% v/vformic acid 4.63 ±0.83 a31.50 ±0.41 b0.066 ±0.005 d293.50 ±5.52 d acetone-1% v/vformic acid 5.44 ±0.75 a44.35 ±0.96 a1.327 ±0.045 b142.45 ±3.02 e The values are means ± sd. Different superscripts (a, b, c, d) indicate significant differences (p ≤ 0.05) within columns for each type of sample (seed or pods). Two blocks used for pairwise comparison, seed or pod. 3.2. Pressurized Liquid Extraction (PLE) In spite of the good results obtained with acetone in the maceration experiments, only green solvents that could be directly used in the formulation of food ingredients were tested on PLE. As mentioned, the experimental design was based on a response surface, where the factors were the number of extraction cycles (1, 2, and 3), ethanol–water percentage (0–100%) , and the temperature (50, 110, and 170 ◦ C). Those solvents were chosen considering their low toxicity and high extraction capacity. The rest of the extraction variables (a static extraction time of 10 min and an extraction pressure of 10.3 MPa) remained constant, based on reference values obtained from previous works by our research group [ 29 ]. The three-level factorial design allowed assessing the effect of the extraction parameters in four different response variables, i.e., the extraction yield, the polyphenols’ content, the TEAC value (ABTS), and the EC 50 value (DPPH). The experimental matrix and the results obtained for each extraction condition for the COLO seeds and pods are shown in Tables 2 and 3. The results of the ANOVA obtained for each response are provided as supplementary data (Tables S1 and S2). Results showed that the behavior of the response variables changed with the extraction condition. Extraction yield increased both with temperature and with the solvent percentage, achieving a peak value at 170 ◦ C. For both seeds and pods, the highest yield was obtained with 50% ethanol, as compared to pure ethanol or water. Agronomy 2021,11, 162 7 of 18 Table 2. Experimental matrix design conditions (factor levels between parentheses) and results for each response variable studied for the optimization of the PLE of COLO cowpea seeds. Results are expressed as mean ±sd. Exp. Run Extraction Conditions Response Variables Temp. (◦C) Ethanol (%) Cycles Yield (%) TPC (mg GAE g−1)a ABTS (mmol TE g−1)b DPPH EC50 (µg mL−1) 1 50 0 2 17.70 ±0.37 13.83 ±0.15 0.207 ±0.017 390.08 ±5.61 2 50 50 1 9.15 ±0.19 22.29 ±0.35 0.468 ±0.015 88.26 ±1.45 3 50 50 3 14.56 ±0.30 24.86 ±0.10 0.568 ±0.033 73.46 ±1.01 4 50 100 2 1.14 ±0.02 13.26 ±0.20 0.073 ±0.002 429.10 ±6.14 5 110 0 1 16.24 ±0.34 9.79 ±0.15 0.164 ±0.012 389.43 ±6.50 6 110 0 3 19.28 ±0.40 12.73 ±0.23 0.275 ±0.010 283.06 ±2.32 7c110 50 2 20.70 ±0.43 13.89 ±0.21 0.324 ±0.009 159.24 ±1.90 8c110 50 2 21.44 ±0.44 11,33 ±0.21 0.287 ±0.011 160.40 ±3.29 9c110 50 2 20.69 ±0.43 11.03 ±0.25 0.282 ±0.011 160.39 ±3.28 10 c110 50 2 21.46 ±0.44 12.83 ±0.21 0.297 ±0.012 159.74 ±1.28 11 110 100 1 2.60 ±0.05 12.73 ±0.06 0.158 ±0.002 318.09 ±5.28 12 110 100 3 3.81 ±0.08 13.09 ±0.25 0.214 ±0.007 307.72 ±5.11 13 170 0 2 65.09 ±1.35 19.86 ±0.17 0.275 ±0.010 150.55 ±2.32 14 170 50 1 35.33 ±0.73 42.76 ±0.78 0.926 ±0.055 49.31 ±1.42 15 170 50 3 44.57 ±0.92 33.63 ±0.38 0.724 ±0.040 64.22 ±1.88 16 170 100 2 9.30 ±0.19 40.36 ±0.26 0.895 ±0.031 67.01 ±1.93 Superindex meaning; a: GAE (gallic acid equivalents), b: TE (Trolox equivalents), c: experimental design center points. Table 3. Experimental matrix design conditions (factor levels between parentheses) and results for each response variable studied for the optimization of the PLE of COLO cowpea pods. Results are expressed as mean ±sd. Exp. Run Extraction Conditions Response Variables Temp. (◦C) Ethanol (%) Cycles Yield (%) TPC (mg GAE g−1)a ABTS (mmol TE g−1)b DPPH EC50 (µg mL−1)c 1 50 0 2 8.97 ±0.20 49.19 ±0.42 1.126 ±0.013 409.75 ±7.19 2 50 50 1 5.35 ±0.12 53.29 ±0.35 2.067 ±0.046 212.24 ±4.54 3 50 50 3 7.99 ±0.18 52.32 ±0.95 1.565 ±0.051 160.56 ±1.62 4 50 100 2 1.76 ±0.04 9.02 ±0.32 0.209 ±0.002 579.42 ±7.24 5 110 0 1 13.11 ±0.30 39.09 ±0.45 0.758 ±0.024 375.30 ±5.93 6 110 0 3 24 ±0.55 33.86 ±0.26 0.588 ±0.021 398.72 ±5.90 7c110 50 2 13.26 ±0.30 50.32 ±1.20 0.916 ±0.032 187.62 ±3.13 8c110 50 2 13.54 ±0.31 49.02 ±0.49 0.865 ±0.006 188.47 ±3.37 9c110 50 2 13.99 ±0.32 46.06 ±1.22 0.898 ±0.046 193.59 ±3.17 10 c110 50 2 13.46 ±0.31 49.52 ±0.72 0.906 ±0.038 189.60 ±3.91 11 110 100 1 1.51 ±0.03 44.36 ±0.53 0.996 ±0.060 188.26 ±3.41 12 110 100 3 3.54 ±0.08 54.99 ±0.51 1.363 ±0.026 168.49 ±2.33 13 170 0 2 34.01 ±0.77 79.62 ±0.65 0.796 ±0.019 62.03 ±1.33 14 170 50 1 21.36 ±0.49 124.46 ±2.52 3.037 ±0.135 32.84 ±0.50 15 170 50 3 29.56 ±0.67 107.42 ±2.05 2.442 ±0.043 35.21 ±0.58 16 170 100 2 7.86 ±0.18 174.62 ±1.58 4.549 ±0.179 27.08 ±0.43 Superindex meaning; a: GAE (gallic acid equivalents), b: TE (Trolox equivalents), c: experimental design center points. Agronomy 2021,11, 162 8 of 18 High temperatures also caused an increase in the TPC, with the difference between 110 ◦ C and 170 ◦ C being more marked than that observed between 50 ◦ C and 110 ◦ C. The highest polyphenols’ content was obtained with 50% ethanol at 170 ◦ C for both seeds and pods (42.76 mg GAE g −1 and 107.42 mg GAE g −1 , respectively). According to the results presented in this study, the mixture ethanol:water (1:1) was the most suitable solvent for the extraction of total polyphenols. Regarding the antioxidant capacity, the most active seed extracts were obtained at 50 ◦ C with three extraction cycles using 50% ethanol (TEAC = 0.568 mmol TE g −1 and EC 50 = 73.46 µ g mL −1 ) and at 170 ◦ C with one extraction cycle with 50% ethanol (TEAC = 0.926 mmol TE g −1 and EC 50 = 49.31 µ g mL −1 ). The most active pod extracts were also obtained at 50 ◦ C and 170 ◦ C, employing 50% ethanol, but performing one extraction cycle at the lowest temperature (TEAC = 2.067 mmol TE g −1 EC 50 = 212.24 µ g mL −1 ) and with two extraction cycles carried out at the highest temperature (TEAC = 4.549 mmol TE g −1 and EC 50 = 27.08 µ g mL −1 ). For these response variables, for both seeds and pods, the temperature was the main critical variable, detecting a significant increase between 50 ◦ C and 170 ◦ C for 50% ethanol and a decrease for an intermediate temperature (110 ◦ C). On the other hand, it was not possible to obtain a mathematical model based only on the studied variables (temperature, solvent composition, and number of cycles) to predict the antioxidant capacity measured as DPPH radical scavenging within the tested range. Figures 1and 2show the standardized Pareto charts for the three response variables analyzed, as well as their corresponding response surfaces for one and three extraction cycles (seeds and pods, respectively). The vertical line indicates the 95% confidence, whereas the colors in the bars indicate positive and negative effects. The temperature along with its quadratic effect were the main significant factors for all response variables, both in seeds and pods. As expected, an increase in temperature provided the highest yield due to an increase in the solubility of the target compounds with the temperature and to a decrease in the viscosity of the solvent that allowed improving the mass transfer of the solvent to the solid matrix. Moreover, response surfaces were similar for both polyphenols’ content and TEAC antioxidant activity in both seeds and pods. High and low temperature values proved to have a positive effect on these variables, while intermediate temperature values had a negative effect. For the three response surfaces, the ethanol-water 50% mixture had a positive effect in both seeds and pods. Agronomy2021,11,xFORPEERREVIEW9of18   ExtractionYield.  TotalPhenolsContent.  Antioxidantactivity—TEAC  Figure1.StandardizedParetochartsandresponsesurfacesforthethreeresponsevariablesstudiedintheexperimental design(blueandgraybarsshownegativeorpositiveeffects,respectively)obtainedwithoneextractioncycleofColorado (COLO)cowpeaseeds. ExtractionYield.  TotalPhenolsContent.  Antioxidantactivity—TEAC. Figure 1. Cont. Agronomy 2021,11, 162 9 of 18 Agronomy2021,11,xFORPEERREVIEW9of18   ExtractionYield.  TotalPhenolsContent.  Antioxidantactivity—TEAC  Figure1.StandardizedParetochartsandresponsesurfacesforthethreeresponsevariablesstudiedintheexperimental design(blueandgraybarsshownegativeorpositiveeffects,respectively)obtainedwithoneextractioncycleofColorado (COLO)cowpeaseeds. ExtractionYield.  TotalPhenolsContent.  Antioxidantactivity—TEAC. Figure 1. Standardized Pareto charts and response surfaces for the three response variables studied in the experimental design (blue and gray bars show negative or positive effects, respectively) obtained with one extraction cycle of Colorado (COLO) cowpea seeds. Agronomy 2021, 11, x 9 of 18 Antioxidant activity—TEAC Figure 1. Standardized Pareto charts and response surfaces for the three response variables studied in the experimental design (blue and gray bars show negative or positive effects, respectively) obtained with one extraction cycle of Colorado (COLO) cowpea seeds. Extraction Yield. Total Phenols Content. Antioxidant activity—TEAC. Figure 2. Standardized Pareto charts and response surfaces for the three response variables studied in the experimental design (blue and gray bars show negative or positive effects, respectively) obtained with three extraction cycles of COLO cowpea pods. Total Phenols Content. Figure 2. Standardized Pareto charts and response surfaces for the three response variables studied in the experimental design (blue and gray bars show negative or positive effects, respectively) obtained with three extraction cycles of COLO cowpea pods. Agronomy 2021,11, 162 16 of 18 herein reveal that cowpea could be used as an ingredient in the development of functional foods. Supplementary Materials: The following are available online at https://www.mdpi.com/2073-439 5/11/1/162/s1, Table S1: Coefficients of regression of third order model for the variables response of factorial design for the analysis of RSM for COLO seed PLE-extracts; Table S2: Coefficients of regression of third order model for the variables response of factorial design for the analysis of RSM for COLO pods PLE-extracts. Author Contributions: Conceptualization, M.V.A, J.A.M., E.I.; Data curation, M.V.A. and G.Á.-R.; Formal analysis, M.V.A., J.A.M., G.Á.-R.; Funding acquisition, A.C. and E.I.; Investigation, M.V.A. and G.Á.-R.; Methodology, J.A.M. and E.I.; Software, J.A.M.; Supervision, J.A.M., A.C. and E.I.; Writing—original draft, M.V.A., G.Á.-R.; Writing—review & editing, M.V.A., G.Á.-R., J.A.M. and E.I. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by Spanish Ministry of Research (AGL2017-89417-R). 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