Box-Behnken Design for DPPH Free Radical Scavenging Activity Optimization from Microwave-Assisted Extraction of Polyphenolic Compounds from Agave lechuguilla Torr. Residues

Abstract

The guishe is a by-product of the fiber extraction from Agave lechuguilla. This material has no commercial value, although it contains metabolites that could be used as a resource for producing high-value products. This study optimized the DPPH^• (2,2-diphenyl-1-picrylhydrazyl) antioxidant activity through microwave-assisted extraction (MAE) of polyphenolic compounds from Agave lechuguilla residues. The MAE process was optimized using a Box-Behnken design, with extraction time (5–15 min), temperature (40–50 °C), and solvent: sample ratio (1:20–1:30 m/v) as independent variables. In contrast, the dependent variable was DPPH^• free radical scavenging activity. As a result, the highest antioxidant activity was at 8 min of irradiation, extraction temperature of 45 °C, and solvent: sample ratio 1:30 w/v, obtaining a total flavonoid content of 19.25 ± 0.60 mg QE/g DW, a total polyphenol content of 6.59 ± 0.31 mg GAE/g DW, a DPPH^• free radical scavenging activity of 73.35 ± 1.90%, and an ABTS^+• ([2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate)]) free radical scavenging activity of 91.93 ± 0.68%.

1. Introduction

Polyphenolic compounds are secondary metabolites of plants, low molecular weight compounds composed of an aromatic ring and a hydroxyl group [1]. They can be involved in various biological functions, including plant development, flower coloration, adaptation to the environment, the attraction of insects for pollination and seed dispersal, and have various bioactive effects, including antibacterial anti-inflammatory, antiarthritic activities, anti-viral, cardioprotective, anti-diabetic, anti-cancer and antioxidants [2,3,4,5].

Polyphenols include flavonoids, phenolics, and lignans [6]. Phenolic compounds are natural bioactive compounds that contain multiple hydroxyl groups as the main reactive active groups [4]. The hydroxyl group positions are closely correlated to their antioxidant activity by ending the chain reaction of free radicals to form stable semiquinone radicals after losing a hydrogen atom [7,8]. These compounds play a crucial role in human health, as they inhibit or delay oxidation reactions and thus prevent oxidative stress related to diseases such as high blood pressure, neurodegenerative disorders, or cancer [8,9]. Additionally, phenolic compounds are valued in cosmeceutical industries because they exhibit antioxidant, anti-aging, anti-inflammatory, and photoprotective properties [10,11], and their addition in cosmetic formulations incentivizes sustainable cosmetic demand [12]. For this reason, new sources for extracting these compounds are being sought.

Extracts from different species of agaves have shown antioxidant [13,14,15,16,17], antimicrobial (bacteria and fungi) [18,19,20,21], antiparasitic (nematodes and insects) [22,23], anti-inflammatory [24,25], and anticancer activities [17]. In some studies, these biological activities were attributed to the presence of phenolic compounds and flavonoids. An option for the extraction of phenolic compounds is the use of Lechuguilla. Currently, there is a great boom in the use of this plant due to its great potential. The Agave lechuguilla Torr. is one of the most abundant plants in Mexico’s arid and semiarid ecosystems. The lechuguilla is used by the local population to extract fiber (Ixtle) through manual or mechanical processes [26]. Fiber extraction produces 15% of ixtle and 85% of guishe [27]. Studies have shown the feasibility of using A. lechuguilla to produce biofuels [26,28,29,30], in wastewater treatment process [31], for uses in construction [32] and phytochemicals extraction [27], achieving great interest due to possessing phytochemicals as phenolic compounds. Moreover, Anguiano-Sevilla et al. (2018) [17] reported the presence of kaempferol, quercetin, and a flavonoid dimer formed by afzelechin-4β-8-quercetin with cytotoxic activity present in A. lechuguilla leaves; while Morreeuw et al. (2021) [33] reported the presence of isorhamnetin, flavanone, hesperidin, delphinidin, quercetin, kaempferol, cyanidin, apigenin and catechin in A. Lechuguilla’s guishe is a by-product that has been shown to possess antioxidant, antimicrobial, and anti-inflammatory activities and may be valuable in the cosmeceutical industry.

The extraction process is critical in recovering phytochemicals from plant material. Conventional methods, such as Soxhlet, maceration, and reflux extraction, require excessive solvents, time, and energy [34]. Ultrasonic-assisted extraction and microwave-assisted extraction (MAE) are considered emergent technologies, which can be carried out in a shorter time, have higher efficiency and less power consumption than conventional extraction techniques, and it is environmentally friendly [35,36]. Amongst these methods, MAE is emerging as a potentially outstanding extraction process due to its advantages compared to conventional methods [37,38,39]. Therefore, the present study aims to DPPH^• free radical scavenging activity optimization from microwave-assisted extraction of polyphenolic compounds from A. lechuguilla residues using Box-Behnken design, which could be of interest to the cosmeceuticals industry for its antioxidant activity.

2. Materials and Methods

2.1. Agave lechuguilla Residue Collection

The residue (guishe) was obtained after carving cogollos for fiber production from the Cosme Coahuila community of the Ramos Arizpe municipality (25°51′44″ N, 101°20′39″ W). The collection was carried out in February 2022. The material was later dried with a hot air dehydrator at 50 °C for 24 h (Koleff, Mod. KL-10), and a milling process was carried out in a cutting mill (Retsch-SM100 Industrial Mill, Haan, Germany) to obtain an average particle size of 2 mm.

2.2. Characterization from guishe of Agave lechuguilla

The chemical composition of guishe was determined following the National Renewable Energy Laboratory (NREL) protocols. Cellulose, hemicellulose, and lignin content were determined by the sulfuric (72%) method described by Rios-González et al., 2018 [40]. The extractive content was determined by water and ethanol recirculating using the analytical method NREL/TP-510-42619 [41], and the ash content by the protocol NREL/TP-510-42622 [42]. The pectin content was determined by the method of (NaPO₃)6 (2%) according to the procedure described by Contreras et al. (2006) [43], the protein content was quantified by CuSO₄/H₂SO₄ Kjeldahl digestion solution described by Wang et al. (2016) [44], and the ethereal extract was determined by hexane recirculation according to the method (2003.06) described by Thiex et al., (2003) [45].

2.3. DPPH^• Free Radical Scavenging Activity Optimization from Microwave-Assisted Extraction of Polyphenolic Compounds

The extraction parameter conditions were set according to experimental values determined in a previous study (data unpublished), using a single-factor design with parameters of irradiation time, temperature, solvent-to-sample ratio, and solvent concentration (ethanol) to simplify the selection of the most effective ranges in the experiment. A Box-Behnken Design (BBD) [2] with three factors were used to optimize through the DPPH^• antioxidant assay where the independent variables and their levels (

Table 1. Box-Benkhen design for the DPPH^• free radical scavenging activity.

Independent Variable	Variable Level
	Low (−1)	High (1)
Irradiation time (min)	5	15
Temperature (°C)	40	50
Solvent to sample ratio (w/v)	1:20	1:30

) were as follows: irradiation time (5–15 min), temperature (40–50 °C) and solvent (water/ethanol 3:7) to sample ratio (1:20, 1:30 w/v). Fifteen experiments, including three center points (

Table 2. DPPH^• free radical scavenging activity used in the Box–Behnken design for response surface methodology.

Run	Parameters
	Irradiation Time (min) X1	Temperature (°C) X2	Solvent-to-Sample Ratio (g/mL) X3
1	15	40	1:25
2	5	45	1:30
3	10	45	1:25
4	10	50	1:20
5	10	40	1:20
6	10	40	1:30
7	15	50	1:25
8	10	45	1:25
9	15	45	1:20
10	5	40	1:25
11	5	45	1:20
12	5	50	1:25
13	10	50	1:30
14	15	45	1:30
15	10	45	1:25

X₁: Irradiation time, X₂: Temperature, X₃: Solvent-to-sample ratio.

), were carried out according to the BBD using the Minitab^® 19 (64-bit) Statistical Software. The DPPH^• free radical scavenging activity was used as the response variable. Analyses and extraction processes were carried out in triplicate. The same experimental matrix (fifteen experiments) analyzed other variables: total polyphenolic content, total flavonoid content, and ABTS^+• antioxidant assays. The extraction yield was calculated using Equation (1).

$$Extraction yield \left(\%\right)={\displaystyle \frac{Weight of extracted yield \left(w\right)}{Weight of dry sample used \left(w\right)}}\times 100$$

(1)

2.4. Total Polyphenol Content (TPC)

The TPC was evaluated using the Folin-Ciocalteu method according to the protocol of Morreuw et al. (2021) [33] with some modifications. In test tubes, 20 µL of diluted extract was added in a test tube (10 mg/mL), followed by 800 µL of distilled water and 60 µL of Folin-Ciocalteu reagent (Sigma–Aldrich, St. Louis, MO, USA). The samples were incubated for 5 min in the dark, and 160 µL of Na₂CO₃ (20%) were added. The mixture was incubated at room temperature for 2 h in the dark, and absorbance was measured at 765 nm using a UV-VIS Spectrophotometer (VARIAN, Cary 50, Santa Clara, CA, USA). Ethanol was used as the analysis blank, and gallic acid was used as the standard for TPC quantifying (0–500 mg/L). TPC was quantified using the equation obtained (y = 0.0023x − 0.0291, R² = 0.9999) from the standard calibration curve (Figure S1). and was expressed in terms of gallic acid equivalent per gram of dry weight (mg GAE/g DW). The TPC of the extract was calculated based on Equation (2).

$$HPC={\displaystyle \frac{ Sample concentration \left(mg/L\right)\ast Volume of solvent \left(L\right)}{Weight of dry sample \left(g\right)}}$$

(2)

where the Sample concentration is the result of the calibration curve, the Volume of solvent is the volume of solvent used in the extraction process, the Weight of the dry sample is the dry weight of sample used in the extraction process.

2.5. Total Flavonoid Content (TFC)

The TFC was quantified according to the protocol of Morreuw et al. (2021) [33], with some modifications. In a test tube, 200 µL of extract dilution (10 mg/mL) were added, followed by 75 µL of NaNO₂ at 5%, 375 µL of 2% AlCl₃ solution, 500 µL of NaOH at 4%, and 500 µL of distilled water with homogenization between each addition and incubating for 5 min in the dark. Ethanol was used as the analysis blank. Absorbance was measured at 765 nm using a UV-VIS Spectrophotometer (VARIAN, Cary 50, Santa Clara, CA, USA), and quercetin was used as the standard for total flavonoid quantification (0–100 mg/L). TFC was determined using the resulting equation (y = 0.0009x − 0.022, R² = 0.996) from the standard calibration curve (Figure S2) and was expressed in terms of quercetin equivalents per gram of dry weight (mg of quercetin/g DW). The TFC of the extract was calculated based on Equation (3).

$$TFC={\displaystyle \frac{ Sample concentration \left(mg/L\right)\ast Volume of solvent\left(L\right)}{Weight of dry sample \left(g\right)}}$$

(3)

where the Sample concentration is the result of the calibration curve, the Volume of solvent is the volume of solvent used in the extraction process, the Weight of the dry sample is the dry weight of sample used in the extraction process.

2.6. Antioxidant Activity Assays

2.6.1. DPPH^• Free Radical Scavenging Activity

The free radical scavenging activity was determined using 2,2-Diphenyl-1-picrylhydrazyl (DPPH^•) according to the procedure described by Alara et al. (2020) [38] with some modifications. Firstly, 20 µL of extract dilution (10 mg/mL) was added at a 96-well microplate; afterward, 180 µL DPPH^• ethanolic solution (25 mg/L). The absorbance was measured at 540 nm in a microplate reader (Thermo Scientific, MULTISKAN SkyHigh) after incubating in the dark for 30 min at room temperature. Water was used as the blank, DPPH^• ethanolic solution was used as the control, and Trolox was used as the standard. The percentage of the DPPH^• radical inhibition was evaluated using Equation (4).

$${DPPH}^{•} free radical scavenging activity \left(\%\right)={\displaystyle \frac{A_{Control}-A_{sample}}{A_{control}}} \ast 100$$

(4)

where A_control is the absorbance of the DPPH^• radical, and the absorbance of the A_sample is the sample with the addition of the DPPH^• radical after thirty minutes of incubation.

2.6.2. ABTS^+• Free Radical Scavenging Activity

The radical scavenging capacity using ABTS^+• assay was determined according to the procedure described by Alara et al. (2020) [38] with some modifications. The stock solutions of 7 mM ABTS^+• and 2.45 mM potassium persulfate (K₂S₂O₈) were separately prepared by dissolving them in distilled water. The working solution was prepared by mixing equal parts of the two stock solutions and allowed to rest for 12 h in a dark place at room temperature. It later mixed 1 mL of ABTS^+• solution diluting in 60 mL of methanol to obtain a reference absorbance of 0.7 ± 0.02 at a wavelength of 734 nm in a microplate reader (Thermo Scientific, MULTISKAN SkyHigh). Afterward, 10 µL of extract dilution (5 mg/mL) was added to a 96-well microplate and 190 µL of ABTS^+• solution. The absorbance was measured at 734 nm using a UV-VIS Spectrophotometer after incubating in the dark for 2 h at room temperature. Methanol was used as the assay blank, ABTS^+• solution was used as the control, and Trolox was used as the standard. The free radical scavenging activity was calculated using Equation (5).

$${ABTS}^{+•} free radical scavenging activity \left(\%\right)={\displaystyle \frac{A_{control}-A_{sample}}{\begin{array}{c}{A}_{control}\\ .\end{array}}} \ast 100$$

(5)

where A_control is the absorbance of the ABTS^+• radical, and the absorbance of the A_sample is the sample with the addition of the ABTS^+• radical.

2.7. Statistical Analysis

The total polyphenol content, total flavonoid content, and antioxidant capacity by ABTS^+• free radical scavenging activity were expressed as mean standard deviation. All the treatments were conducted in triplicate and compared by Tukey multiple comparison test at a significance level of p < 0.05, using Minitab 19 Statistical Software.

3. Results

3.1. Chemical Composition from guishe of Agave lechuguilla

The proximal characterization of A. lechuguilla guishe is shown in

Table 3. Chemical composition of guishe from Agave lechuguilla.

Component	% (Dry Basis)
Glucans	12.11 ± 3.99
Xylan	2.30 ± 0.38
Lignin	8.97 ± 0.25
Extractives	42.72 ± 0.45
Protein	4.78 ± 0.10
Lipids	2.17 ± 0.08
Pectin	7.43 ± 0.09
Ash content	10.78 ± 0.50

. The extractives (material soluble in either water or ethanol that include non-structural components of biomass samples: chlorophyll, waxes, nitrate/nitrites, sucrose) were the main component (42.72%), followed by the carbohydrates: glucan and xylan (14.41%), ashes (10.78%), Lignin (8.97%), pectin (7.43%), protein (4.78%) and lipids (2.17%).

3.2. Total Polyphenols Content from guishe of Agave lechuguilla

The ethanolic extracts showed a TPC from 5.12 ± 0.04 to 7.91 ± 0.23 mg GAE/g DW (Table S1). A higher concentration of total polyphenols was obtained with 10 min of irradiation, a temperature of 50 °C, and a mass: volume ratio of 1:30 m/v. However, the comparison of means of the concentration of total polyphenols by Tukey determined that there is a significant difference (p < 0.05) in the means about the parameters used. At the same time, the coefficient of determination (R²) was 0.8912 (Figure 1).

3.3. Total Flavonoid Content from guishe of Agave lechuguilla

The ethanolic extracts evaluated showed a TFC from 10.40 ± 0.31 to 15.30 ± 0.44 mg QE/g DW (Table S2). A higher concentration of total flavonoid was obtained with 10 min of irradiation, a temperature of 40 °C, and a mass: volume ratio of 1:30 m/v. However, the comparison of means of the concentration of total flavonoid by Tukey determined that there is a significant difference (p < 0.05) in the means about the parameters used. At the same time, the coefficient of determination (R²) was 0.8652 (Figure 2). Finally, a similar behavior was observed in the extraction process of flavonoids and polyphenols with the parameters and levels used for the extraction process.

3.4. Antioxidant Activity by DPPH^• and ABTS^+• from guishe of Agave lechuguilla

The ethanolic extracts showed DPPH^• free radical scavenging activity from 36.35 ± 0.59 to 68.27 ± 3.02%, and the standard (trolox) 83.80 ± 0.33%. The comparison of means of the DPPH^• free radical scavenging activity by Tukey determined that there is a significant difference (p < 0.05) in the means of the studied parameters. Meanwhile, the coefficient of determination (R2) was 0.9434. However, an increase was observed in the antioxidant activity by ABTS^+• (74.40 ± 1.40 to 86.45 ± 0.67%) and the Trolox standard (100%). The comparison of means of the ABTS^+• free radical scavenging activity by Tukey determined that there is a significant difference (p < 0.05) in the means about the parameters used (

Table 4. Antioxidant activity in extracts from Agave lechuguilla guishe using DPPH^• and ABTS^+• free radical scavenging.

Run	DPPH• Free Radical Scavenging Activity (%)	ABTS+• Free Radical Scavenging Activity (%)
1	57.44 ± 2.58 CD	83.07 ± 2.05 ab
2	67.12 ± 0.87 AB	78.96 ± 3.89 abc
3	62.90 ± 0.28 D	86.45 ± 0.67 a
4	46.80 ± 2.98 E	85.72 ± 3.43 a
5	61.46 ± 2.63 ABCD	80.47 ± 1.86 abc
6	68.27 ± 3.02 A	85.18 ± 0.47 ab
7	36.35 ± 0.59 F	81.65 ± 1.71 abc
8	62.90 ± 0.28 EF	79.82 ± 4.74 abc
9	58.59 ± 3.31 BCD	79.90 ± 2.22 abc
10	41.91 ± 2.07 EF	82.13 ± 1.96 ab
11	65.78 ± 1.22 ABC	82.54 ± 1.37 ab
12	59.45 ± 4.48 BCD	80.83 ± 1.47 abc
13	59.16 ± 5.24 BCD	80.22 ± 2.33 abc
14	58.39 ± 2.41 CD	77.50 ± 2.32 bc
15	62.90 ± 0.28 ABCD	74.40 ± 1.40 c

Distinct letters indicated a significant difference (A–F p < 0.05), (a–c p < 0.05).

).

3.5. Statistical Analysis

The statistical analysis for the various variables was carried out as a complementary analysis, but no conditions were obtained for process optimization. Therefore, only the statistical analysis of antioxidant activity by DPPH^• is presented.

The optimization for antioxidant activity was investigated using response surface methodology; the parameters were irradiation time (X₁), temperature (X₂), and solvent-to-sample ratio (X₃). The response variable for the Analysis of variance (ANOVA) was DPPH^• free radical scavenging activity (Table 4).

The regression equation in coded level neglecting insignificant terms was generated Equation (6):

$$\begin{gathered} {DPPH}^{•} free radical scavenging activity \left(\%\right)=2452 {+ 22.9X}_{1 }{+ 28.2X}_2 {- 5037X}_{3 } \\ \hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}{ - 0.2114X}_1^2-0.3532X_2^2 + 1941X_3^2 \\ \hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em} - 0.3863X_1{X}_2- 1.53X_1{X}_3+5.6X_2{X}_3 \end{gathered}$$

(6)

The ANOVA for the DBB for optimization of antioxidant activity parameters is presented in

Table 5. ANOVA analysis of MAE process optimization for antioxidant activity.

Source	Sum of Squares	Df	Mean Square	F Value	p-Value (Prob > F)
Model	1090.13	9	121.125	6.03	<0.031
X1	68.94	1	68.94	3.43	0.123
X2	93.29	1	93.29	4.64	0.084
X3	51.62	1	51.62	2.57	0.17
X1X2	373.06	1	373.06	18.58	<0.008
X1X3	0.59	1	0.59	0.03	0.871
X2X3	7.73	1	7.73	0.38	0.562
X12	103.09	1	103.09	5.13	0.073
X22	287.92	1	287.92	14.34	<0.013
X32	86.95	1	86.95	4.33	0.092
Residual	100.42	5	20.084
Lack of fit	100.42	3	33.473
Pure error	0	2	0
Cor total	1190.55	14
R2	0.91
Adjusted R2	0.76
Coefficient of variation	4.48

. The predicted model’s determination coefficient (R²) was 0.91, showing that only 0.09% of the variation cannot be explained by the present model. The Model F-value is 6.03, and the p-value is lower (0.031), which implies the model is significant. For the model coefficients, the ANOVA results show that the interaction X₁X₂ was significant (0.013), and only the quadratic effect X² was significant (0.008). The other terms were not significant (p > 0.05).

3.5.1. Analysis of Response Surface Plots

Figure 3 shows the effects of irradiation time, temperature, and solvent: sample ratio on DPPH^• free radical scavenging activity. Figure 3a shows that DPPH^• free radical scavenging activity was increased with high temperatures and irradiation time, with a decrease in antioxidant activity with increasing temperature and irradiation time greater than 45 °C and 8 min, respectively. The effects of solvent: sample ratio and irradiation time of the DPPH^• free radical scavenging activity were shown in Figure 3b, an increase in DPPH free radical scavenging activity at an irradiation time of 8 min and solvent to sample ratio of 1:30 m/v, with a decrease as irradiation time increases at a fixed temperature (45 °C). Finally, Figure 3c shows a higher DPPH^• free radical scavenging activity at a temperature of 45 °C with a decrease as temperature increases, a solvent-to-sample ratio of 1:30 m/v at a fixed irradiation time. Increasing the mass-to-volume ratio can increase DPPH^• activity by flavonoids in the extracts.

3.5.2. Model Validation

The model was verified based on the results obtained from response surface analysis; the optimal extraction conditions were 8 min of irradiation time, 45 °C of temperature, and 1:30 m/v of solvent to sample ratio. Under these optimal conditions, the antioxidant capacity (DPPH^• free radical scavenging activity) was 70.83 ± 2.89%. The validation was conducted by getting 73.35 ± 1.90% DPPH^• free radical scavenging activity, which agreed with the predicted value since there are no differences between the experimental and predicted values. Therefore, the model was adequate and accurate for this study.

4. Discussion

4.1. Chemical Composition from guishe of Agave lechuguilla

The proximal characterization of A. lechuguilla guishe is like those reported by Carmona et al. (2017) and Ortíz et al. (2017) [28,29], where chemical characterization of A. lechuguilla cogollos was also made. However, the guishe biomass presented lower cellulose and hemicellulose content, attributed to fiber removed from the cogollos during extraction (15%) [2].

4.2. Total Polyphenol and Total Flavonoid Content from guishe of Agave lechuguilla

The hydrolyzable polyphenol content in the extracts is superior to that reported by Guayat et al. (2020) [46] and lower than that reported by Anguiano-Sevilla et al. (2018) [17] and Morreeuw (2021) [47] in extracts from the guishe of A. lechuguilla through conventional extraction, enzymatic, ultrasound-assisted, and supercritical fluids. This is attributed to the time and place of sampling because environmental factors influence the polyphenol content in plants. After all, the polyphenol content varies with geographical origin [33].

Total flavonoid content was superior to those reported by Guayat et al., 2020 and Morreeuw et al., 2021 [46,47] in extracts from A. lechuguilla guishe through conventional extraction, enzymatic, ultrasound-assisted, and supercritical fluids. This is attributed to the energy generated during the irradiation process causing accumulation of heat in the solvent due to the absorption of microwave energy [48]; this causes the rupturing of cells by the swelling of plant material by increasing the moisture content, experiencing thermal stress and the analytes have a greater tendency to leave from plant matrix into the solvent by dissolution and diffusion [48,49,50].

Also, the content in the microwave is heated simultaneously in a volumetric way; compared to conventional techniques, the extraction is by batches, which causes a lower heating rate; with microwave-assisted extraction, the heating is several times higher, which induces a higher extraction, because the electromagnetic waves of the microwave facilitate the damage in the cell wall, releasing more phytochemicals by the rupture of the cell, mainly by the mass transfer between the solid and the solvent by the high pressure that is accumulated by the microwaves [48].

4.3. Effect of the Parameters on Antioxidant Activity

It is important to know the results of the different combinations of the levels of each factor that cause the same response because they help to visualize the relationship of the response variable.

Microwave power contributes to reducing the extraction time needed to extract phenolic compounds and antioxidants because the microwaves penetrate directly to plant material, extracting the metabolites of interest [51]; increasing the time can cause the extraction of unwanted compounds and the degradation of phenolic compounds of the plant material, affecting the quality of the extract and causing a reduction of bioactive compounds and DPPH^• activity [52]. The extraction of phenolic compounds increases with increasing temperature due to the solubility of the plant material analytes; however, high temperature decreases the extraction efficiency of phenolics with antioxidant properties; therefore, increasing the temperature after 45 °C and irradiation longer than 8 min can cause degradation of phenolic compounds, decreasing the DPPH^• activity [53]. This is attributed to higher contact of the plant biomass with the solvent, releasing the compounds due to mass transfer principles. However, increasing the ratio can impede the extraction of bioactive compounds by preventing diffusion [53,54]. In addition, ethanol as a solvent improves the solubility of phenolic compounds, leading to higher DPPH^• activity because ethanol can dissolve polar compounds [55].

The results obtained demonstrate an antioxidant activity in A. lechuguilla guishe extracts, which increases with the content of phenolic compounds and the number of hydroxyl groups, which can react with free radicals in the oxidation process, acting as hydrogen donors, interrupting the reaction of free radical chains, by stabilizing them through intramolecular hydrogen bonds and semiquinone radicals [56].

4.4. Antioxidant Activity from guishe of Agave lechuguilla

Results of the antioxidant activity of A. lechuguilla guishe extracts by DPPH^• assay were higher than those of Guayat et al., 2020 and Morreeuw et al., 2021 [46,47] in guishe of A. lechuguilla extract through conventional extraction, enzymatic, ultrasound-assisted, and supercritical fluids. This is attributed to the higher amount of flavonoids in extracts, which contain multiple hydroxyl groups as the main antioxidant active groups [4]; their positions closely correlate to their antioxidant activity. However, the results were lower than those reported by Carmona et al. (2017) [28], attributed to other compounds that are co-extracted, such as saponins and free sugars, which also have antioxidant potential that can interfere with the DPPH^• test [57].

The variation in antioxidant capacity can be attributed to the difference in the diversity and concentration of phytochemicals (tannins, flavonoids, and anthocyanins) present in extracts, as well as their total content of phenolic compounds because these bioactive compounds are mainly responsible for antioxidant capacity. Not only does the content of phenolic compounds influence the antioxidant activity, but also the type of phenolic compound and the synergy that can originate between them [58], the compositions of non-polar tannins [17], and the process of obtaining the extracts because the extraction process is related to the type and amount of metabolites extracted [59].

On the other hand, the antioxidant activity of A. lechuguilla guishe extracts by ABTS^+• assay was higher than that of the DPPH^• assay. The difference between the assays is attributed to the oxidizing compounds present in the extracts because the reactivity depends on the reaction mixture and the type of radical used. It has been determined that some antioxidant compounds can react rapidly with peroxyl radicals and react slowly or not at all against the DPPH^• radical, which is associated with steric hindrance [17]. DPPH^• and ABTS^+• compounds have proton free radicals that significantly decrease when exposed to proton radical scavengers. Additionally, most plant compounds show better antioxidant activity against ABTS^+• radicals than DPPH^• radicals. The sensitivity of ABTS^+• makes the kinetic reaction faster, resulting in higher antioxidant activity [36].

Finally, optimal extraction conditions obtained a total polyphenol content of 6.59 ± 0.31 mg GAE/g DW, a total flavonoid content of 19.25 ± 0.60 mg QE/g DW, 73.35 ± 1.90% and 91.93 ± 0.68 of DPPH^• and ABTS^+• free radical scavenging activity, respectively and an extraction yield of 25.30 ± 0.89%.

5. Conclusions

MAE process of A. lechuguilla’s guishe presented a greater extraction of flavonoids (19.25 mg QE/g DW), increasing antioxidant activity (DPPH^•: 73.35% and ABTS^+•: 91.93 ± 0.68%) compared to other reported methods. The results potentialize this residue as a viable option for obtaining bioactive compounds of interest for the cosmeceuticals industry for their antioxidant activity. Therefore, further research will focus on the use of the extracts for the elaboration of a cosmeceutical product for topical use, as well as the optimization of all extraction parameters. Furthermore, using the guishe as a raw material to obtain other products could provide added value, generate extra economic activity for those dedicated to fiber extraction, and incorporate guishe into a value chain.