Comprehensive Analysis of Antioxidant Compounds from Lippia citriodora and Hibiscus sabdari a Green Extracts Attained by Response Surface Methodology

antioxidants Article Comprehensive Analysis of Antioxidant Compounds from Lippia citriodora and Hibiscus sabdariffaGreen Extracts Attained by Response Surface Methodology María del Carmen Villegas-Aguilar 1,2 , Francisco Javier Leyva-Jiménez 2, María de la Luz Cádiz-Gurrea 1,2,* , Antonio Segura-Carretero 1,2,†and David Arráez-Román1,2,*,† 1Department of Analytical Chemistry, University of Granada, 18071 Granada, Spain; [email protected] (M.d.C.V.-A.); [email protected] (A.S.-C.) 2Research and Development of Functional Food Centre (CIDAF), 18016 Granada, Spain; [email protected] *Correspondence: [email protected] (M.d.l.L.C.-G.); [email protected] (D.A.-R.) †These authors shared author co-seniorship. Received: 22 October 2020; Accepted: 23 November 2020; Published: 25 November 2020   Abstract: Phenolic compounds have shown to have a high bioactive potential against various pathologies, postulating as an interesting alternative to manage some diseases. In this sense, both Lippia citriodora and Hibiscus sabdariffaare two botanical sources with a demonstrated high bioactive potential, in which their antioxidant capacity stands out. In this work, the optimization of the extraction conditions for the recovery of phytochemicals from L. citriodora leaves and H. sabdariffa calyces has been carried out using Response Surface Methodologies (RSM) considering their total polar compounds measured by HPLC-ESI-TOF/MS and Folin-Ciocalteu assay, and its antioxidant capacity evaluated by Ferric Reducing Antioxidant Power (FRAP) and Trolox Equivalent Antioxidant Capacity (TEAC) assays. The results showed that to maximize the antioxidant capacity in H. sabdariffa, a moderate temperature and high ethanol percentage are needed, while a low temperature and a high percentage of ethanol are needed in L. citriodora. In addition, with the results obtained in the multiple response analysis, it is possible to affirm the importance of this type of analysis to develop functional ingredients, taking into account both total content of phenolic compounds and their bioactivity. Furthermore, as confirmed in this study, these analyses can be extrapolated in different techniques and in different matrices, with phenolic compounds from different families being important to develop new high added value products for food, pharmaceutical or cosmetic industries. Keywords: phenolic compounds; response surface methodology; Lippia citriodora;Hibiscus sabdariffa; antioxidant; Microwave-Assisted Extraction (MAE); Pressurized Fluid Extraction (PLE) 1. Introduction In recent years, both nutraceuticals and functional food have received increasing attention from the scientific community, consumers and food manufacturers [ 1 ]. This growing interest is related to their composition and health benefits. These benefits are attributed by the source used during their manufacturing, which are, generally, botanicals. In fact, there is a huge variety of botanicals that have been reported to exhibit biological properties such as antioxidant or anti-inflammatory [ 2 – 4 ]. In this sense, plants such as Hibiscus sabdariffaand Lippia citriodora stand out among an extensive list, since both have been traditionally used to prepare herbal drinks with numerous health benefits. Several of these beneficial properties have been related to the bioactive compounds present in their composition, mainly, phenolic compounds [5,6]. Antioxidants 2020,9, 1175; doi:10.3390/antiox9121175 www.mdpi.com/journal/antioxidants Antioxidants 2020,9, 1175 2 of 16 Several reports of H. sabdariffa have demonstrated its potential effects such as antioxidant [ 6 ], anti-obesity [ 7 ], hypotensive [ 8 ], antidiabetic [ 7 ], hypocholesterolemic [ 9 ], immunomodulatory [ 10 ], hepatoprotective[ 11 ]anddiuretic[ 10 ]. InthecaseofL.citriodora,itsantioxidant[ 10 ],anti-inflammatory[ 12 ], antimicrobial [ 13 ] and anticancer [ 10 ] properties, among others, have been also highlighted. These biological properties are especially related to the phenolic compounds, which are secondary metabolites contained in their leaves, stems or flowers. In spite of having great benefits on the human health, the amount of these compounds are usually reduced, being necessary an adequate extraction technique to solve several issues: (i) to avoid or decrease their degradation during the extraction processes and (ii) to concentrate them or to perform selective extractions. There are numerous extraction techniques to attain enriched extracts in phenolic compounds from botanical sources, but currently the non-conventional or advanced techniques stand out, due to the fact that they have fewer extraction times, lesser consumption of solvents (which may be Green and Generally Recognized As Safe, GRAS) and energy. For these reasons, they have been recognized as environmentally friendly techniques, being more efficient than conventional ones [ 14 , 15 ]. Some examples of advanced extraction techniques are Microwave-Assisted Extraction (MAE) and Pressurized Fluid Extraction (PLE). Regardless of the extraction technique selected, during an extraction process, the adjustment of the extraction parameters is an important step since, depending on the targets desired, the best extraction conditions may have a determinant influence. In the last decade, the use of Response Surface Methodologies (RSM) has been applied with the purpose of optimizing the extraction conditions considering the aims desired, enhancing the efficiency of advanced extraction technologies [ 14 , 16 , 17 ]. There is a large number of studies that propose different extraction methods to obtain enriched extracts in specific bioactive compounds by RSM and its possible incorporation in functional food or in nutraceuticals [ 14 , 17 – 19 ]. However, the health benefits of the extracts lie in their bioactivity, which is influenced by synergies and antagonism between compounds. Due to this, innovative researches are focused not only on optimizing the extraction and composition performance; rather, they use variables that measure bioactivity [20,21]. The aim of this study was to optimize the extraction conditions for the recovery of phytochemicals from L. citriodora leaves and H. sabdariffacalyces, considering their total polar content and antioxidant capacity, by different methods in order to obtain ingredients with high antioxidant potential. In addition, the optimization of extraction processes by MAE and PLE was compared, considering, on one hand, the total content of polar compounds measured by high-performance liquid chromatography coupled to mass spectrometry (HPLC-ESI-TOF/MS) and, on the other hand, the bioactivity measured by different in vitro tests. The optimization was carried out using an RSM based on Central Composite Design 2 3 (CCD) model with 16 experiments including center and star points. The independent variables were temperature, extraction time and percentage of solvent (ethanol and water) and the response variables were total phenolic compounds measured by HPLC-ESI-TOF/MS and by Folin-Ciocalteu assay, antioxidant capacity evaluated by Ferric Reducing Antioxidant Power (FRAP) assay and Trolox Equivalent Antioxidant Capacity (TEAC). 2. Materials and Methods 2.1. Chemicals and Reagents For extractions and solutions, ultrapure water was obtained with a Milli-Q system Millipore (Bedford, MA, USA) and absolute ethanol was purchased from VWR chemicals (Radnor, PA, USA). To measure the antioxidant capacity and total phenolic content, the following reagents were provided from the indicated suppliers: ABTS (2,2 0 -azinobis (3-ethylbenzothiazoline-6-sulphonate)), ferric sulfate, Folin–Ciocalteu reagent, potassium persulfate, TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) from Sigma-Aldrich (St. Louis, MO, USA). Antioxidants 2020,9, 1175 3 of 16 Acetic acid and methanol were purchased from Fluka (Sigma-Aldrich, Steinheim, Germany) and Lab-Scan (Gliwice, Poland), respectively, whereas gallic acid, sodium acetate, ferric chloride, hydrochloric acid, trihydrated sodium acetate and sodium carbonate were obtained from Panreac (Barcelona, Spain). The analytical procedures were performed using LC-MS grade methanol and acetic acid, which were purchasedfromFisherChemicals(Waltham,MA,USA)andSigma-Aldrich(Steinheim,Germany),respectively. 2.2. Sample Preparation H. sabdariffacalyces and L. citriodora leaves were provided by Monteloeder (Alicante, Spain) whose calyces and leaves were milled using an ultra-centrifugal mill ZM 200 (Retsch GmbH, Haan, Germany). The resulting powder was stored in darkness and kept at room temperature until extraction. 2.3. Extraction of Phenolic Compounds and Other Polar Compounds from H. sabdariffa Calyces MAE was carried out in according to the methodology used by Pimentel-Moral et al. [ 17 ] in a modular microwave extraction system Multiwave 3000 SOLV (Anton Paar GmbH, Graz, Austria). Briefly, 3 g of H. sabdariffadried calyces were added into the vessels together with 30 mLof water-ethanol mixtures. After cooling, samples were centrifuged at 17,000 × gfor 15 min in a centrifuge (Sorvall ST 16 R, Thermo Scientific, Leicestershire, UK) and the supernatants were dried under vacuum in a SavantTM SpeedVac Concentrator SC250 EXP (Thermo analysis Scientific, Sunnyvale, CA, USA). The extracts were stored at − 20 ◦ C until further use. Prior to use, the dry extracts were reconstituted in the same extraction solvent mixture to a concentration of 10 mg/mL and filtered with single-use syringe filters (0.20 µm pore size). 2.4. Extraction of Phenolic Compounds and Other Polar Compounds from L. citriodra Leaves PLE was run according to the methodology used by Leyva-Jim é nez et al. [ 14 ] with a pressurized liquid extractor (ASE ™ 350 system, Dionex, Sunnyvale, CA, USA). Briefly, for each extraction, 5 g of sample were mixed with 10 g of sea sand and loaded onto 33 mL stainless-steel extraction cells. After extraction, the extracts obtained were ice-cold to achieve a temperature of 20–25 ◦ C and centrifuged at 17,000 × gfor 15 min in a centrifuge (Sorvall ST 16 R, Thermo Scientific, Leicestershire, UK). The supernatants were dried under vacuum in a Savant TM SpeedVac Concentrator SC250 EXP (Thermo analysis Scientific, Sunnyvale, CA, USA) and they were stored at − 20 ◦ C until further use. Prior to use, the dry extracts were reconstituted with the same extraction solvent mixture to a concentration of 10 mg/mL and filtered with single-use syringe filters (0.20 µm pore size). 2.5. Experimental Design For both MAE and PLE extractions, the experimental conditions were defined according to an RSM using a CCD 2 3 model with axial and center points. RSM was designed with Statgraphics Centurion XVI software (Statpoint Technologies (Warrenton, VA, USA). In the MAE of H. sabdariffa calyces, the independent variables were temperature (50, 100, 150 ◦ C), extraction time (5, 12.5, 20 min) and percentage of ethanol in the hydro-alcoholic mixture (15%, 45%, 75%). Conditions were selected based on Pimentel-Moral et al. [ 17 ]. A total of 16 experimental conditions were obtained (Table S1) that were carried out in a randomized order. In the PLE of L. citriodora leaves, the independent variables were temperature (40, 110, 180 ◦ C), extraction time (5, 12.5, 20 min) and percentage of ethanol in the hydro-alcoholic (15%, 50%, 85%). Conditions were selected based on Leyva-Jim é nez et al. [ 14 ]. A total of 16 experimental conditions were obtained (Table S2) that were carried out in a randomized order. Total polar compounds were quantified by HPLC-ESI-TOF/MS according to Pimentel-Moral et al. in H. sabdariffaextracts and adapted from Leyva-Jim é nez et al. in the case of L. citriodora extracts; total phenolic compounds were measured by Folin-Ciocalteu assay, whereas antioxidant capacity was evaluated using FRAP and by TEAC. The results of each assay were chosen as response variables in Antioxidants 2020,9, 1175 4 of 16 both models. The following second-order polynomial model was used to fit the experimental data (Equation (1)): Y=β0+ k X i=1 βiXi+ k X i=1 βii X2 i+ k X i=1 k X j=i+1 βij XiXj(1) Yrepresents the predicted response; β0 is a constant coefficient that fixes the response at the central point of the experiments, and βi , βii and βij are the regression coefficients of the linear, quadratic and interaction terms, respectively; X i and X j represent the value of the independent variables. The parameters were considered to evaluate the model adequacy: regression coefficient (R 2 ), coefficient of variation (CV) and model value. 2.6. Total Phenolic Content by Folin Ciocalteu The total phenolic content (TPC) was measured by Folin-Ciocalteu method with some modifications [ 22 ]. L. citriodora and H. sabdariffaextracts were dissolved in ethanol–water (1:1) (different concentrations were tested). The absorbance measurement was carried out on a Synergy Mx Monochromator-Based Multi-Mode Micro plate reader (Bio-Tek Instruments Inc., Winooski, VT, USA) using 96-well polystyrene microplates. Phenol content was calculated based on the calibration curves of Gallic Acid and expressed as mg Gallic acid equivalents/g of dry extract. The experiments were made in triplicate. 2.7. Antioxidant Activity Assays The antioxidant activity of L. citriodora and H. sabdariffaextracts were evaluate using two methods based on single-electron transfer (SET): FRAP and TEAC assays. In both, fluorescence was measured with the same equipment mentioned above. 2.7.1. Ferric Reducing Antioxidant Power The FRAP assay was carried out following the method described by Benzie and Strain [ 23 ]. FRAP values were calculated measuring the absorbance at 593 nm in a microplate reader. A standard curve of FeSO4·7H2O was assessed and results were expressed as mmol FeSO4equivalents/g of dry extract. The experiments were made in triplicate. 2.7.2. Trolox Equivalent Antioxidant Capacity The TEAC assay was originally described by Miller et al. and was performed with some modifications according to C á diz-Gurrea et al. [ 24 , 25 ]. TEAC values were calculated using Trolox as standard and reading absorbance at 734 nm in a microplate reader. The results were expressed in mmol Trolox equivalents/g of dry extract. The experiments were made in triplicate. 2.8. HPLC-MS Analysis The qualitative characterization of H. sabdariffa and L. citriodora extracts was carried out using a RRPC 1200 series (Agilent Technologies, Palo Alto, CA, USA) following the methods reported by Pimentel-Moraletal.[ 17 ]andLeyva-Jim é nezetal.[ 14 ], respectively, inorder toensure thereproducibility. 3. Results and Discussion 3.1. Total Polar Compounds, Total Phenolic Contents and Antioxidant Activities of H. sabdariffa by Microwave-Assisted Extraction (MAE) The analysis of the beneficial properties of plant extracts can be approached from different points of view. For instance, attention can be focused on the composition of bioactive compounds as evaluating a wide group of compounds as polar compounds or more specific as phenolic compounds, since these are responsible for the vast majority of beneficial properties reported. On the other hand, the studies can Antioxidants 2020,9, 1175 5 of 16 be focus on the bioactivity, such as antioxidant capacity of the extracts that may be mostly associated with the phenolic profile of this type of polar extractions, but taking into account the positive and negative synergies that have influence in the antioxidant properties. It is necessary to consider that the results of each applied assay to evaluate this bioactivity may change according to the chosen technique. For these reasons, this work pretends to approach the relationship between phytochemicals contained in the extracts and their antioxidant activity in order to establish optimal conditions for the obtainment of functional ingredients. In this sense, Table 1shows the results of the total content of polar compounds adapted from Pimentel-Moral et al. [ 17 ], the TPC measured by the Folin-Ciocalteu and the antioxidant activity obtained by the FRAP and TEAC assays for each of the experimental conditions of MAE extraction of H. sabdariffacalyces. In this case, the extraction yield has been used to ensure the reproducibility of the extractions with those made by Pimentel-Moral et al. [ 17 ], noting that there were no non-significant differences (data not shown). Table 2shows the major compounds tentatively identified in H. sabdariffa calyces by HPLC-ESI-TOF-MS following the method reported by Pimentel-Moral et al. [17]. As can be seen in Table 1, there is no condition in which all the response variables reach the maximum; however, when attention is paid to the condition with the lowest value for the response variables, it is observed that condition 10 has the lowest value for the four response variables. These results indicate that the composition of the H. sabdariffaextracts have an important influence on the antioxidant activity. The results of Total Polar Compounds variable ranged from 201.466 ± 5.356 mg of total polar compounds/g of dry extract (run 11) to 50.027 ± 1.107 mg total polar compounds/g of dry extract (run 10); the extraction conditions under these conditions were 164 ◦ C, 12.5 min and 45% ethanol, and 150 ◦ C, 20 min and 15% ethanol, respectively. For Folin-Ciocalteu response, the condition with the highest value was run 8 with 78.679 ± 2.591 mg Gallic acid equivalents/g of dry extract under the conditions 150 ◦ C, 5 min, 75% ethanol; for FRAP, conditions 5 and 15 with 0.995 ± 0.034 and 0.995 ± 0.018 mmol FeSO 4 equivalents/g of dry extract under 100 ◦ C, and 45% ethanol, and for TEAC, condition 11 gave 0.285 ± 0.010 mmol Trolox equivalents/g of dry extract. The results achieved by condition 11 offered higher values for total polar compounds as well as TEAC assay, which may highlight the synergic effect of polar compounds in the antioxidant activity. Some examples of this synergistic effect on antioxidant activity between phenolic compounds was evaluated by Skroza et al., in which they observed that in different tests to measure antioxidant capacity there was a synergistic effect between resveratrol and other phenols such as catechin and caffeic acid [ 26 ]. Also in this sense, in the case of the TEAC assay, Sanchez-Marzo et al. tested different fractions of compounds present in L. citriodora, checking the fraction with a combination of Apigenin-7-diglucuronide, Verbascoside, Isoverbascoside and Forsythoside A presented higher values in both FRAP and TEAC, compared to other fractions with different combinations or with isolated compounds. Thus, this demonstrates the synergistic effect of the compounds present in this fraction [ 27 ]. As mentioned above, condition 10 gave the lowest values for all responses evaluated. Antioxidants 2020,9, 1175 6 of 16 Table 1. Results of the response variables Total Polar Compounds, TPC, FRAP and TEAC for each condition in H. sabdariffaMAE extracts. Experimental Design Condition Total Polar Compounds a,1 TPC bFRAP cTEAC d 1 92.514 ±4.727 41.114 ±1.832 0.935 ±0.036 0.254 ±0.009 2 76.171 ±1.501 57.953 ±2.591 0.959 ±0.044 0.260 ±0.002 3 56.469 ±3.547 39.822 ±0.006 0.767 ±0.024 0.213 ±0.004 4 96.682 ±1.676 72.634 ±5.393 0.781 ±0.062 0.268 ±0.009 5 83.807 ±1.365 52.772 ±4.487 0.995 ±0.034 0.284 ±0.008 6 84.987 ±1.934 43.273 ±3.957 0.757 ±0.043 0.216 ±0.004 7 58.774 ±1.767 25.570 ±1.832 0.651 ±0.027 0.182 ±0.005 8 87.112 ±4.804 78.679 ±2.591 0.711 ±0.054 0.228 ±0.013 9 81.339 ±1.511 45.864 ±1.496 0.799 ±0.016 0.233 ±0.003 10 50.027 ±1.107 26.002 ±1.496 0.524 ±0.011 0.133 ±0.006 11 201.466 ±5.356 61.408 ±2.991 0.819 ±0.024 0.285 ±0.010 12 53.368 ±1.331 40.682 ±1.496 0.537 ±0.024 0.166 ±0.003 13 56.314 ±0.951 44.136 ±3.957 0.607 ±0.024 0.190 ±0.001 14 77.399 ±0.682 74.361 ±5.983 0.968 ±0.022 0.280 ±0.006 15 71.110 ±0.937 54.499 ±2.991 0.995 ±0.018 0.261 ±0.001 16 69.784 ±1.262 42.407 ±0.003 0.903 ±0.043 0.232 ±0.003 a Expressed in mg total polar compounds/g of dry extract; b Expressed in mg Gallic acid equivalents/g of dry extract; c Expressed in mmol FeSO 4 equivalents/g of dry extract; d Expressed in mmol Trolox equivalents/g of dry extract; 1 Adapted from Pimentel Moral et al. [ 17 ]; TPC: Folin-Ciocalteu; FRAP: Ferric Reducing Antioxidant Power; TEAC: Trolox Equivalent Antioxidant Capacity. Table 2. Major compounds tentatively identified in H. sabdariffacalyces by high-performance liquid chromatography coupled to mass spectrometry (HPLC-ESI-TOF-MS). Molecular Formula m/zCalculated Compound C6H8O8207.0146 Hydroxicitric acid C6H6O7189.0041 Hibiscus acid C8H12O8235.0459 Hibiscus acid hydroxyethylester C8H10O7217.0354 Hibiscus acid dimethylester C16H18O9353.0878 Neochlorogenic acid C16H18O9353.0878 Chlorogenic acid C16H18O9353.0878 Cryptochlorogenic acid C15H12O9335.0409 Methyl digallate C16H18O8337.0929 Coumaroilquinic acid C16H20O10 371.0984 Dihydroferulic acid-4-O-glucuronide C26H28O16 595.1305 Quercetin-3-sambubioside C17H30O16 489.1461 Quercetin-3-rutinoside C16H16O8335.0772 5-O-caffeoylshikimic acid C21H20O12 463.0882 Quercetin-3-glucoside C18H22O9381.1191 Ethylchlorogenate C18H22O9381.1191 Ethylchlorogenate isomer II C16H16O7319.0823 Methylepigallocatechin C15H10O8317.0303 Myricetin C18H19NO4312.1241 N-feruloyltiramine C15H10O7301.0354 Quercetin C15 H10O6285.0405 Kaempferol In spite of run 11 revealing the highest total polar compounds in the evaluated design, the value reached in Folin-Ciocalteu by condition 8 was the highest value. This unexpected value may be due to the fact that Folin-Ciocalteu reagent not only reacts with phenolic compounds, which are included within polar compounds, but also reacts with others such as proteins, thiols, many vitamin derivatives and inorganic ions Fe 2+ , Mn 2+ , I − and SO 32− [ 28 ]. According to these results, higher percentage of ethanol from 45% (run 11) to 75% (run 8) allowed greater extraction of pigments such as chlorophyll, carotenoids and xanthophylls [ 29 ], which can be conjugated with proteins and have some of the aforementioned ions in their structure, reacting with the Folin-Ciocalteu reagent. Additionally, Antioxidants 2020,9, 1175 7 of 16 the lowest and least maintained temperature over time of condition 8 compared to 11 prevents the degradation of certain proteins and vitamins, explaining the high value for Folin-Ciocalteu of condition 8 [30]. Considering the response variables to evaluate the antioxidant capacity, condition 11 showed the highest TEAC value and the greatest total polar compounds. Moreover, the values of organic acids, phenolic acids, flavonoids and anthocyanins showed the highest, so that in relation to TEAC the greatest concentration of compounds is directly related to the highest antioxidant activity. However, in the case of FRAP, the results differ, since there were two conditions that revealed greater values than conditions 11, 5 and 15 that were performed with 45% of ethanol. Moreover, the temperature had an important relevance since, while in condition 11 the extraction temperature was 164 ◦ C, in 5 and 15 it was 100 ◦ C. These results may be occasioned by the thermal degradation of compounds like flavonoids. Quercetin and its derivatives are flavonoids that were found in H. sabdariffaextracts, and their abilities to reduce iron in the FRAP test compared to other flavonoids have been reported [ 31 ]. According to Pimentel-Moral et al., in conditions 5 and 15, the content of quercetin-3-rutinoside was greater than condition 11, which could explain the higher value in FRAP of these two conditions [17]. 3.2. Total Polar Compounds, Total Phenolic Contents and In Vitro Antioxidant Activities of L. citriodora Pressurized Fluid Extraction (PLE) Table 3shows the results attained after evaluating the PLE L. citriodora extracts, where the results of total content of polar compounds were adapted from Leyva-Jim é nez et al. [ 14 ], whereas the TPC was measured applying the Folin-Ciocalteu assay and the antioxidant activity performing FRAP and TEAC assays. In the same way that was mentioned previously for H. sabdariffa, the extraction yield has been the parameter used to compare the extractions carried out in this study with those made by Leyva-Jim é nez et al. [ 14 ], noting that there were no non-significant differences (data not shown). Table 4shows the major compounds tentatively identified in L. citriodora leaves by HPLC-ESI-TOF-MS following the method described by Leyva-Jiménez et al. [14]. Table 3. Results of the response variables for each condition in L. citriodora. Response variables: Total Polar Compounds, TPC, FRAP and TEAC. Experimental Design Condition Total Polar Compounds a,1 TPC bFRAP cTEAC d 1 304.734 ±21.965 181.442 ±9.098 1.720 ±0.080 0.64 ±0.028 2 369.613 ±24.362 253.117 ±1.496 2.402 ±0.131 0.553 ±0.054 3 213.096 ±13.480 168.489 ±5.393 2.095 ±0.048 0.580 ±0.068 4 220.119 ±11.880 195.259 ±4.487 1.997 ±0.063 0.524 ±0.005 5 199.962 ±9.621 159.853 ±10.786 1.745 ±0.098 0.653 ±0.053 6 268.933 ±17.803 235.846 ±7.915 1.802 ±0.058 0.553 ±0.019 7 228.959 ±17.110 197.850 ±2.591 1.875 ±0.120 0.438 ±0.022 8 221.533 ±12.409 227.211 ±12.779 2.313 ±0.055 0.574 ±0.029 9 141.768 ±9.291 145.173 ±7.479 1.685 ±0.153 0.555 ±0.049 10 252.724 ±10.062 219.439 ±3.957 1.615 ±0.125 0.553 ±0.031 11 329.635 ±20.249 193.532 ±3.957 2.252 ±0.085 0.532 ±0.085 12 226.461 ±8.706 189.214 ±1.496 1.728 ±0.111 0.420 ±0.006 13 155.232 ±28.764 211.667 ±1.496 1.877 ±0.069 0.493 ±0.024 14 282.828 ±16.771 242.755 ±6.520 2.318 ±0.031 0.665 ±0.002 15 125.646 ±5.013 185.760 ±7.915 1.230 ±0.048 0.453 ±0.022 16 96.830 ±4.096 163.307 ±1.496 1.590 ±0.056 0.558 ±0.088 a Expressed in mg total polar compounds/g of dry extract; b Expressed in mg Gallic acid equivalents/g of dry extract; c Expressed in mmol FeSO 4 equivalents/g of dry extract; d Expressed in mmol Trolox equivalents/g of dry extract; 1 Adapted from Leyva-Jim é nez et al. [ 14 ] with some modifications; TPC: Folin-Ciocalteu; FRAP: Ferric Reducing Antioxidant Power; TEAC: Trolox Equivalent Antioxidant Capacity. Antioxidants 2020,9, 1175 8 of 16 Table 4. Major compounds tentatively identified in L. citriodora leaves by high-performance liquid chromatography coupled to mass spectrometry (HPLC-ESI-TOF-MS). Molecular Formula m/zCalculated Compound C6H11O7195.0510 Gluconic acid C16H21O10 373.1140 Gardoside C16H19O11 387.0933 Ixoside C20H29O12 461.1664 Verbasoside C21H27O13 487.1457 Cistanoside F C16H21O11 389.1089 Theveside C18H27O9387.1661 Tuberonic acid glucoside C29H37O16 641.2087 βHydroxyverbascoside derivative C29H37O16 641.2087 βHydroxyisoverbascoside derivative C29H35O16 639.1931 βHydroxyverbascoside C27H25O18 637.1140 Luteolin-7-diglucoronide C29H35O16 639.1931 βHydroxyisoverbascoside C27H25O17 621.1097 Apigenin-7-diglucoronide C25H29O13 537.1614 Lippioside I C30H37O16 653.2087 Campneoside I C28H27O18 651.1355 Chrysoeriol-7-diglucuronide C29H35O15 623.1981 Verbascoside C26H33O11 521.2028 Lariciresinol glucopyranoside C29H35O15 623.1981 Isoverbascoside C29H35O15 623.1981 Forsythoside A C25H29O12 521.1664 Hydroxycampsiside C26H31O13 551.1770 Durantoside I C30H37O15 637.2138 Leucoseptoside A C28H27O17 635.1254 Acacetin-7-diglucoronide C31H39O15 651.2294 Martynoside C29H35O13 591.2083 Osmanthuside B C16H11O6299.0561 Dimethyl kaempferol C17H13O7329.0667 Dimethyl quercetin Concerning the FRAP response variable, the condition with the highest value coincided with Total Polar Compounds and Folin-Ciocalteu, pointing out a positive relationship between the amount of phenoliccompounds and theabilitytoreduce theironionthrough thetransferofelectrons. Furthermore, in accordance with Leyva-Jim é nez et al., run 2 showed the highest amount of verbascoside, which may explain the antioxidant results of this run. This could be corroborated by various studies that show the positive relationship between the amount of verbascoside in the matrix and the highest capacity in the FRAP test [27,32]. Nevertheless, for the TEAC response variable, the condition run 14 exhibited the highest value. These results may be explained due to synergistic relationships between the phytochemicals contained in this extract and showing that the amounts of polar/phenolic compounds were not related with reduction of the ABTS cation radical. There are studies that show that there is not always a direct relationship between the relative concentration of a compound or certain compounds with the bioactivity, but in many situations, synergistic or antagonist relationships are determinant to the bioactive potential of the extracts obtained from plants [33]. Lastly, it is necessary to note that the minimum value of all responses evaluated was achieved after applying higher temperatures (above 180 ◦ C). This may be due to the fact that there were compounds that are thermolabile at high temperatures and therefore could be degraded [12]. Antioxidants 2020,9, 1175 9 of 16 3.3. RSM Analysis of H. sabdariffa Calyces MAE and L. citriodora Leaves PLE Conditions 3.3.1. Model Fitting Parameters To maximize the response variables for each model, an analysis of variance (ANOVA) was carried out for each response to discern the adjustment of the results and optimize the statistical model. To achieve this goal, several fitting parameters were used, which were evaluated to determine the adequacy of the model. The results were fitted to a quadratic polynomial model (Equations (S1) and (S2)) and regression coefficients were generated using the least squares method (MLS). The first parameter to evaluate the adequacy of the model was the regression coefficient (R 2 ), in which values above 70% are considered acceptable [ 34 ]. This value explains the variability of the data which may be explained with the proposed model. The coefficient of variation (CV) was also used, a lower value (<10%) indicates a good reproducibility of the investigated systems and a value between 11% and 20%, indicates an acceptable variation; hence, CV shows the dispersion of data and a small value indicated high reproducibility between the predicted and experimental values [ 35 ]. Finally, the adequacy of the model (model value) was also used as approach to discern the good choice of the design. Table 5 shows the parameters mentioned to validate the model in H. sabdariffaand Table 6the results of the ANOVA for this matrix. Table 7shows the parameters mentioned to validate the model in L. citriodora and Table 8the results of the ANOVA for this matrix. 3.3.2. H. sabdariffaCalyces MAE Optimization Regarding the evaluated fitting parameters (Table 5), Folin-Ciocalteu, FRAP and TEAC response variables showed R 2 values greater than 70% (93.7%, 78.4% and 74.0%, respectively), hence revealing a good explanation of the obtained results. Moreover, the adequacy of the model of each response gave significant values (p ≤ 0.05) demonstrating a good adequacy, and the CV was below 20 and close to 10, revealing an acceptable reproducibility of the results. Nevertheless, Total Polar Compound did not present a good approximation to the experimental conditions since neither the result of R 2 model value nor CV was acceptable. Considering the effects of the independent variables, it is noteworthy that the % ethanol had a significant influence for the Folin-Ciocalteu, FRAP and TEAC response. Furthermore, the optimization of these responses (Table 6) was achieved when the ethanol used was between 67% and 83%. This could be explained because water, despite having higher dielectric constant, has a dissipation factor significantly lower than ethanol. In this sense, a high dissipation factor improves the passage of microwave energy through the solvent and the dissipation of microwave energy into heat, as is the case with ethanol [ 36 ]. Despite this characteristic, its ability to absorb energy is low. This causes less heat transfer from the solvent to the sample [ 37 ], requiring water in the solvent mixture to improve this situation. With these observations, a higher extraction yield of phenolic compounds were obtaining using 80% of ethanol in the mixture, comparing with water and other mixtures of ethanol-water and methanol-water, which coincide with the results obtained in the present study [36]. Furthermore, Total Polar Compounds and Folin-Ciocalteu showed the optimal temperatures at 164 ◦ C and the extraction time was 13 and 3 min, respectively. Nevertheless, the optimal temperature for FRAP was 62 ◦ C, while for TEAC it was 118 ◦ C, and the optimal extraction time was 12 and 13 min, respectively. In these cases, the temperature necessary to obtain the optimal values of FRAP and TEAC enabled the extraction of thermo sensitive compounds as flavonoids. These flavonoids had a greater affinity for reducing iron in FRAP compared to the TEAC test based on the ABTS radical. These flavonoids had a higher affinity for reducing iron in FRAP compared to the ABTS radical-based TEAC test. This result may be explained considering the results attained by Bahorun et al. and Csepregi et al., which conducted FRAP and TEAC tests for different types of flavonoids. The results of their studied revealed that particularly quercetin and quercetin-3-O-rutinoside, very abundant in H. sabdariffa, had higher values for FRAP than for TEAC [ 38 , 39 ]. This can be explained since the Antioxidants 2020,9, 1175 16 of 16 34. 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