Optimization of Ultrasonication Probe-Assisted Extraction Parameters for Bioactive Compounds from Opuntia macrorhiza Using Taguchi Design and Assessment of Antioxidant Properties

Abstract

Opuntia macrorhiza, commonly referred to as red prickly pear, is a type of cactus fruit. The Opuntia macrorhiza (OM) fruit is rich in polyphenols and contains a high amount of ascorbic acid and betalains. The fruit peels have demonstrated many biological abilities, including antioxidant, antifungal, and antibacterial activities. Ultrasound probe-assisted extraction (UPAE) is a highly promising method for efficiently extracting valuable molecules from natural sources. The objective of this study is to optimize the parameters of UPAE, including the appropriate solvent, liquid-to-solid ratio, extraction duration, and pulsation level. The aim is to maximize the yield of bioactive compounds (polyphenols, betalains, and ascorbic acid) from OM fruits (pulps and peels) and assess their antioxidant activities using Taguchi design. The optimal extraction conditions through the partial least squares method for OM pulp were determined to be aqueous extraction for 12 min with a liquid-to-solid ratio of 60 mL/g and 48 pulses/min, while for OM peels they were determined to be aqueous extraction for 20 min with a liquid-to-solid ratio of 60 mL/g and a pulsation of 48 pulses/min. The optimum UPAE conditions were compared with the values obtained from the optimum extraction under stirring extraction (STE). Overall, UPAE exhibited higher yields than STE. The obtained total polyphenol content ranged from 10.27 to 13.07 mg gallic acid equivalents/g dry weight, while the betalain content ranged from 974 to 1099 μg/g dry weight. Overall, these fruits demonstrated potential as new components for food and medicinal uses due to their good health effects and lack of toxicity.

1. Introduction

Opuntia ssp. is the primary genus of the Cactaceae family, which comprises nearly 1500 species worldwide [1]. Of these, Opuntia macrorhiza, commonly called red prickly pear, is approximately 2–5 m in height, producing green cladodes with numerous long spines and crimson, red-purple fruit. The plant is extensively distributed, originating from Mexico and various regions, including South America and the Mediterranean basin. Recent research has indicated that a mature O. macrorhiza (OM) fruit contains a significant amount of dietary fiber, organic acids, minerals, phenolic compounds, and carbohydrates [2]. The juice of OM fruit exhibited favorable technological attributes, including a low pH value (3.6–3.8) and total soluble solids (about 10.5 °Brix), alongside elevated viscosity (approximately 58 mPa·s at 20 °C) [3]. The slightly lower pH value renders OM fruits a viable alternative to Opuntia ficus-indica fruits for organoleptic properties and microbiological deterioration; nonetheless, the juice production is minimal.

OM fruit peels have demonstrated a variety of biological properties, including antioxidant, antifungal, and antimicrobial properties. In general, these fruits have shown the potential to be novel ingredients for food applications due to their non-toxicity and health benefits [4]. In this context, Opuntia spp. appears to be one of the most suitable raw materials for the production of dietary supplements, as they are widely recognized for their numerous health benefits, including the prevention of cardiovascular disease, cancer, and diabetes, as well as the inhibition of inflammation and viral infections [5,6]. These advantages are principally attributable to the substantial quantities of nutrients, including phenolic compounds, polysaccharides, vitamins, minerals, and betalains [5]. Moreover, the betalains in these fruits and their peels, which are responsible for their color, are widely used as a red colorant in foods [3]. Betalain pigments can also enhance the body’s redox equilibrium and diminish lipid oxidation through their antioxidant properties that scavenge free radicals. This also provides health benefits as they are hepatoprotective and regulate gene expression [7,8,9].

In recent years, there has been an increasing trend toward using sustainable methods to mitigate their environmental footprint [10]. Consequently, green extraction techniques are being utilized with growing frequency [11]. Ultrasonication induces negative pressure within the liquid medium, resulting in acoustic cavitation, which emerges as bubbles due to the inability of gases to remain dissolved under such conditions [12]. There are two operating ultrasound systems, the ultrasound bath and the ultrasound probe. Between them, the probe system is more convenient in the extraction sectors [13]. The probe system provides increased energy intensity over a reduced surface area, specifically at the tip of the ultrasound probe. Consequently, it can reduce energy dissipation, thereby improving the efficacy of ultrasonic treatment in the extraction process [13,14]. Moreover, in the probe system, the energy imparted is targeted on a designated sample area, resulting in a more effective cavitation effect [15].

Several studies explored the recovery of Opuntia spp. with the use of ultrasonication baths. Hernández-Carranza et al. [16] investigated the valorization of O. ficus-indica and its mucilage, whereas Melgar et al. [17] recovered bioactive compounds from O. engelmanii Valencia peels. Vázquez-Espinosa et al. [18] isolated betalains from hydroethanolic O. dillenii extract as food colorants. Given the nutritional importance of OM, its potential benefits, its significant applications in the food and pharmaceutical industries, and the scarcity of studies using ultrasonication probe-assisted extraction (UPAE), the main objective of this study was to investigate the use of the specific extraction technique. In this research, both OM pulp and peels were analyzed to ensure which part has a higher concentration of bioactive compounds (i.e., polyphenols, ascorbic acid, and betalains). The Taguchi design of experiments was utilized to assess the impact of various extraction conditions of OM pulp and peel. Optimization with the specific design of the experiment provides low cost and solvent waste, as a limited number of experiments within a robust and innovative approach could be achieved [19].

2. Materials and Methods

2.1. Chemicals and Solvents

Anhydrous sodium carbonate, citric acid, disodium hydroxyphosphate, Folin-Ciocalteu reagent, 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH^•), gallic acid, and formic acid (98% w/v) were all purchased from Penta (Prague, Czech Republic). Methanol, acetonitrile, iron (III) chloride, hydrochloric acid, trichloroacetic acid (TCA), ascorbic acid, and all standards used for the chromatographic quantification of polyphenols were purchased from Sigma-Aldrich (Steinheim, Germany). A deionizing column was employed to generate deionized water for the experiments.

2.2. Instrumentation

A freeze-dryer (lyophilizer), BK-FD10P, from Biobase (Jinan, China), was utilized to freeze-dry the pulp and peels of OM. An Analysette 3 PRO (Fritsch GmbH, Oberstein, Germany) was employed for the sieving process. A Biobase UCD-150 ultrasonic (US) cell disrupter from Jinan, China, with a 150 W maximum nominal power and a 6 mm diameter probe tip (emitting surface), was utilized for all ultrasonication-assisted extractions. A Heidolph stirring hotplate (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) was employed to conduct the stirring process. In order to conduct the assays, the samples were heated in an Elmasonic P70H ultrasonic bath (Elma Schmidbauer GmbH, Singen, Germany). A Shimadzu UV-1900i UV/Vis spectrophotometer (Kyoto, Japan) was used for photometric analyses. A Lovibond CAM-System 500 colorimeter (The Tintometer Ltd., Amesbury, UK) was used to measure CIELAB coordinates (L*, a*, and b*) from the extracts. The supernatant was extracted from the liquid samples by centrifuging them using a NEYA 16R (Remi Elektrotechnik Ltd., Palghar, India). Individual polyphenols were quantified through high-performance liquid chromatography (HPLC) by employing a Shimadzu CBM-20A liquid chromatograph and a Shimadzu SPD-M20A diode array detector (Shimadzu Europa GmbH Duisburg, Germany). The compounds were separated through a Phenomenex Luna C18(2) column (Torrance, CA, USA) maintained at 40 °C (100 Å, 5 μm, and 4.6 mm × 250 mm).

2.3. Raw Material Collection and Handling

Fruits of the red prickly pear variety, Opuntia macrorhiza (OM), were sourced from local sellers in the Kavala region of Eastern Macedonia, Greece, and stored at 4 °C prior to the commencement of the experiments. The fruits were of comparable maturity and weight. The thorns were manually removed from the fruit, rinsed with tap water to remove any surface impurities, and then peeled with a steel knife, where peels and pulp were separated. Additionally, the seeds were manually removed from the pulp. The samples were frozen and left overnight to freeze-dry at −54 °C. The dried OM peels and pulp were ground to a fine powder (<400 μm diameter), sieved, and stored at −40 °C until further analysis.

2.4. Ultrasonication Extraction Procedure

Optimal extraction of OM peels and pulp was achieved using a multifactorial extraction procedure using a US probe chamber cell, the details of which are shown in

Table 1. The actual and coded levels of the independent variables used to optimize the process.

Independent Variables	Code Units	Coded Variable Level
		1	2	3
Liquid-to-solid ratio (R, mL/g)	X1	20	40	60
Ethanol in water concentration (C, % v/v)	X2	0	50	100
Pulsation (ON/OFF) (P, pulses/min)	X3	12	30	48
Extraction time (t, min)	X4	4	12	20

below. These parameters include the investigated liquid-to-solid ratio (R, mL/g), duration (t, min), and the concentration of ethanol in water (C_EtOH, % v/v). The pulsation process (P, pulses/min) was also investigated, as it is considered to have a vast impact on the extraction process. All extractions were conducted utilizing the highest amplitude (i.e., 80%, which was equal to 120 W) with a temperature <40 °C using an ice bath to prevent depigmentation of extracts. The temperature ranges were recorded before, during, and after the procedure. The samples underwent centrifugation at 3600× g for 10 min to isolate the supernatant, after which the solid portion was removed and preserved at –40 °C. To compare it with UPAE, a 2-h conventional extraction with a stirring hotplate was conducted at 40 °C with a 20 mL/g liquid-to-solid ratio with water as the extraction solvent, using the optimized extraction technique by Prakash Maran and Manikandan [20]. Preliminary experiments revealed that extraction at temperatures as high as 80 °C could lead to the decomposition of betalains and the depigmentation of the extracts. The stirring hotplate was set at 500 rounds per minute (rpm) of stirring.

2.5. Experimental Design

Total and individual polyphenolic compounds and betalains extraction, which constitute the bioactive compounds from OM peels and pulp, along with antioxidant activity through ferric reduction and radical scavenging, were optimized. The Taguchi method has gained widespread use in disciplines such as technology and engineering due to its ability to enhance processing quality, decrease the number of experiments needed, minimize processing variation, and promote quality stability [21]. This method has evolved into a typical procedure for assessing the effects of interactions among various controllable parameters during ranking and screening operations. The Taguchi technique may address many challenges by utilizing qualitative, discrete, and continuous design elements [22]. Orthogonal arrays are structures in which the columns are utilized for factors and the rows are used for the levels selected for each factor [23]. A three-level Taguchi orthogonal (L9) orthogonal array design, as shown in Table 1, was utilized to identify significant factors among four independent variables: solvent composition (ethanol and water mixtures), liquid-to-solid ratio, extraction time, and pulsation of the US probe. The rationale behind the choice of these conditions was to explore their extreme values in a reasonable range. For instance, UPAE is capable of achieving sufficient bioactive compound recovery swiftly; the time was chosen in the specific range to provide a low, moderate, and high duration using a reasonable time interval (i.e., an 8 min time interval). The same rationale lies beneath the other conditions, in which we explored extreme values using as little as nine experiments. These are deemed critical parameters for extraction. Table 1 details these independent variables and their coded units across three levels. The Taguchi method applied necessitated nine design points to evaluate the three levels of each variable, which are elaborated in

Table 2. The four variables that Taguchi design involves along with their corresponding measured response values.

Design Point	Independent Variables
	X1 (R, mL/g)	X2 (C, % v/v)	X3 (P, P/min)	X4 (t, min)
1	3 (60)	2 (50)	1 (12)	3 (20)
2	1 (20)	2 (50)	2 (30)	2 (12)
3	1 (20)	3 (100)	3 (48)	3 (20)
4	1 (20)	1 (0)	1 (12)	1 (4)
5	3 (60)	1 (0)	3 (48)	2 (12)
6	2 (40)	1 (0)	2 (30)	3 (20)
7	2 (40)	2 (50)	3 (48)	1 (4)
8	2 (40)	3 (100)	1 (12)	2 (12)
9	3 (60)	3 (100)	2 (30)	1 (4)

.

2.6. Bioactive Compounds of OM Extracts

2.6.1. Spectrophotometric Determination of Total Polyphenols

The assays to evaluate the total polyphenol content (TPC) and individual polyphenols were comprehensively described in our previous study [24]. Utilizing the Folin-Ciocalteu assay, we determined TPC. Briefly, 200 μL of the sample and 200 μL of Folin-Ciocalteu reagent were mixed, and, after 2 min, 1600 μL of aqueous sodium carbonate solution (5% w/v) was added in a 2 mL Eppendorf tube. Immediately afterward, incubation of the samples was conducted in a water bath at 40 °C for 20 min, and the absorbance at 740 nm was immediately recorded. As several protocols use a solvent in which gallic acid has the highest solubility (i.e., methanol) [25], the total polyphenol concentration (C_TP) was calculated (Equation (1)) through a calibration curve ranging from 10–100 mg/L of gallic acid (R² = 0.9996) in methanol, with the results being expressed as mg of gallic acid equivalents (GAE) per g of dried weight (dw).

$$\mathrm{TPC} (\mathrm{mg} \mathrm{GAE}/\mathrm{g} \mathrm{dw})={\displaystyle \frac{{\mathrm{C}}_{\mathrm{TP}} \times V}{w}}$$

(1)

In Equation (1), V (expressed in L) and w (expressed in g) denote the volume of the extraction medium and the dry weight of the sample, respectively.

2.6.2. Chromatographic Quantification of Individual Polyphenols

The identification of individual polyphenols was performed using an HPLC system. The mobile phase consisted of 0.5% formic acid in acetonitrile (B) and 0.5% formic acid in aqueous solution (A). The gradient program involved gradual initiation from 5% and an increase to 12% B in 12 min, followed by 55% B in 35 min, 100% B in 1 min, a constant value for 3 min, and then 5% B in 40 min. The mobile phase flow rate was kept constant at 1 mL/min. To simultaneously quantify individual polyphenols (i.e., 2,4-dihydroxybenzoic acid (β-resorcylic acid), 4-hydroxybenzoic acid, pyrogallol, 2,3-dihydroxybenzoic acid (pyrocatechuic acid), caffeic acid, homovanillic acid, syringic acid, and m-coumaric acid), calibration curves of excellent linearity (R² > 0.99) were used. More details about the polyphenol quantification are provided in

Table 3. Linear equation, detection limits of quantified polyphenols, and coefficient of determination (R²) in chromatographic quantification of individual polyphenols.

Phenolic Compound	Linear Regression Equation	LOD a (mg/L)	LOQ b (mg/L)	R2 c
β-Resorcylic acid	y = 73,173x − 374,752	6.38	19.35	0.9960
4-Hydroxybenzoic acid	y = 82,756x − 322,848	4.30	13.02	0.9978
Pyrogallol	y = 57,521x − 598,596	12.93	39.18	0.9919
Pyrocatechuic acid	y = 189,392x − 1,430,809	9.92	30.06	0.9904
Caffeic acid	y = 83,601x − 606,104	9.03	27.36	0.9942
Homovanillic acid	y = 15,054x − 30,731	8.34	25.27	0.9951
Syringic acid	y = 172,124x − 1,804,823	14.08	42.66	0.9881
m-Coumaric acid	y = 110,557x − 583,252	7.37	22.34	0.9954

^a Limit of Detection; ^b Limit of Quantification; ^c Coefficient of Determination.

.

2.6.3. Ascorbic Acid Determination

The determination of ascorbic acid content (AAC) was conducted as previously established [26], with results expressed as mg of AA/g dw. A quantity of 100 μL of properly diluted sample was mixed with 900 μL of aqueous trichloroacetic acid solution (10% w/v) and 500 μL of aqueous Folin–Ciocalteu reagent (10% v/v) in an Eppendorf tube. The absorbance (760 nm) was measured after a 10 min span. A calibration curve using 10% TCA (50–500 mg/L of ascorbic acid, R² = 0.9980) was also employed to assess AAC.

2.7. Assessment of Antioxidant Capacity of OM Extracts

Reducing power and radical scavenging assays were conducted to assess the antioxidant activity of OM extracts, as established in our previous study [18]. The procedure demanded 100 μL of properly diluted sample and 100 μL of FeCl₃ solution (4 mM in 0.05 M HCl) in a 2-mL Eppendorf tube. Incubation of the mixture took place in a water bath at 37 °C for 30 min, in which 1800 μL of TPTZ solution (1 mM in 0.05 M HCl) was immediately added to the mixture right after, with the absorbance (620 nm) being measured after 5 min. The ferric-reducing power (P_R) was conducted with molecule TPTZ as the ligand within a calibration curve of 50–500 μM of ascorbic acid in 0.05 M HCl (R² = 0.9997), with the results being expressed as μmol ascorbic acid equivalents (AAE)/g dw, as indicated in Equation (2):

$$P_R (\mathsf{\mu }\mathrm{mol} \mathrm{AAE}/\mathrm{g} \mathrm{dw})={\displaystyle \frac{C_{\mathrm{A}\mathrm{A}} \times V}{w}}$$

(2)

In Equation (2), V (in L) and w (in g) indicate the volume of the extraction medium and the dried weight of the material, respectively.

In addition, the DPPH^• inhibition calibration curve ranged from 100–1000 μM of ascorbic acid in methanol (R² = 0.9926) and was calculated as μmol AAE/g dw. The initial absorbance (515 nm) of a solution consisting of 50 μL of methanol and 1950 μL of a methanolic 100 μM DPPH^• solution was measured immediately. Properly diluted sample volume of 50 μL was mixed with 1950 μL of the methanolic DPPH^• solution, with the mixture being stored for 30 min in ambient temperature with the absence of light and the absorbance being recorded in the same wavelength. The percentage of inhibition calculation was done using Equation (3):

$$\mathrm{Inhibition} \left(\%\right)={\displaystyle \frac{A_{515\left(\mathrm{i}\right) }-A_{515\left(\mathrm{f}\right)}}{A_{515\left(\mathrm{i}\right)}}} \times 100$$

(3)

Antiradical activity (A_AR) was evaluated using an ascorbic acid calibration curve which was expressed as μmol AAE per gram of dw, as shown in Equation (4):

$$A_{\mathrm{AR}}\left(\mathsf{\mu }\mathrm{mol} \mathrm{AAE}/\mathrm{g} \mathrm{dw}\right)={\displaystyle \frac{C_{\mathrm{AA}} \times V}{w}}$$

(4)

where V (expressed in L) and w (expressed in g) denote the volume of the extraction medium and the dry weight of the sample, respectively.

2.8. Pigment Analysis

2.8.1. Colorimetric Analysis

A previously established methodology [27] was used to measure the pigment of the extracts. A colorimeter was used to measure the CIELAB parameters (L*, a*, and b*) for the OM extracts. Color can be described using three coordinates: The L* value, which represents the brightness of each color, can take values between 0 (representing black) and 100 (representing white). The a* value quantifies the degree to which a color is green (negative values) or red (positive values). The hue of a color is indicated by the b* value, which can be either negative or positive, quantifying its tendency towards blue or yellow, respectively.

2.8.2. Betalains Content (BC)

Betalains were measured in triplicate and quantified as the total of betacyanins and betaxanthins, utilizing the molar extinction coefficients (ε) of betanin and indicaxanthin, respectively. This was accomplished using a McIlvaine buffer at pH 6.5, prepared by dissolving 0.2 M disodium hydrogen phosphate and 0.1 M citric acid in deionized water, and the extracts were diluted appropriately to obtain absorption values between 0.1 and 1.0. Based on the equation proposed by Stinzing et al. [28], BC was determined using the following equation:

$$\mathrm{BC} \left(\mathsf{\mu }\mathrm{g}/\mathrm{g} \mathrm{dw}\right)={\displaystyle \frac{A\times DF\times V\times MW\times {10}^6}{\epsilon \times l\times w}}$$

(5)

where, A represents the absorption at 535 nm for betacyanins and at 480 nm for betaxanthins. DF stands for the dilution factor, l denotes the path length of the 1 cm cuvette, V is the volume of the extraction medium in L, and w is the dry weight of the material in g. The molecular properties of betanin (with an ε of 60,000 L/mol·cm in water, a maximum absorption wavelength (λ_max) of 535 nm, and a molecular weight (MW) of 550 g/mol) and indicaxanthin (with an ε of 48,000 L/mol·cm in water, λ_max of 480 nm, and MW of 308 g/mol) are also taken into account.

2.9. Statistical Analysis

The results were presented as average values ± standard deviation (SD) from triplicate analyses. A one-way analysis of variance (ANOVA) was performed to assess the statistical significance of the differences between the means, and a Tukey HSD multiple comparisons test was applied to determine significance at p < 0.05. The experimental design for the Taguchi design and all associated statistics were conducted using JMP^® Pro 16 (SAS, Cary, NC, USA).

3. Results and Discussion

3.1. Optimization of US-Assisted Extraction Conditions Through Taguchi Design

3.1.1. TPC and Antioxidant Capacity of OM Peel and Pulp Extracts

The optimization of extraction parameters was crucial to enhance efficacy and ensure a more sustainable extraction process [29,30]. The solvent composition was considered essential, as its features significantly influence chemical extraction [31]. Water is an ecological solvent, renowned for its remarkable ability to extract polar molecules (including the water-soluble pigments betalains), as well as for its affordability and non-toxic properties for human consumption [27]. On the other hand, ethanol, which is also a food-grade solvent, was utilized to extract betaxanthins [32]. Water was compared with ethanol as a solvent for the optimal extraction of these compounds, therefore endorsing the food-grade applicability of the extracts [33]. The Taguchi design was employed to examine the extraction conditions using as few as nine design points to identify the optimal approach to generate high-value extracts. In addition, a conventional STE (water as solvent at 40 °C for 2 h) was used as a means of comparing the effectiveness of the UPAE technique. The STE conditions were obtained from a previous study by Prakash Maran et al. [20]. The authors optimized the pigment extraction procedure of Opuntia species in which critical extraction parameters, such as temperature and duration, were studied. They found that the optimum condition to extract total betalains was to use water as a solvent for 115 min at 40 °C. Moussa-Ayoub et al. [34] also stated that water was found to be beneficial in the extraction of bioactive compounds from several Opuntia species, including O. macrorhiza, when compared to a 50% hydroalcoholic mixture. Regarding the extraction temperature, several studies [2,17,18] have emphasized that the ideal extraction temperature should not exceed 40 °C, since higher temperatures may result in the disintegration of betalains and the depigmentation of the extracts, an observation confirmed in our preliminary experiments. Finally, the rationale for this comparison between UPAE and STE was that the target molecules can be successfully extracted using cavitation, in addition to the environmental sustainability of the method compared to conventional extraction [35].

The results of the TPC, FRAP, and DPPH assays across OM peels and pulp are presented in

Table 4. Total polyphenol content (TPC) and antioxidant assays such as FRAP and DPPH radical scavenging activity were measured in both the pulp and peel of OM at various design points.

Design Point	TPC (mg GAE/g dw)		FRAP (μmol AAE/g dw)		DPPH (μmol AAE/g dw)
	Pulp	Peel	Pulp	Peel	Pulp	Peel
1	10.34 ± 0.23	10.21 ± 0.25	47.95 ± 2.83	31.16 ± 1.93	30.93 ± 2.07	28.65 ± 2.01
2	11.38 ± 0.60	12.44 ± 0.54	37.80 ± 1.21	38.93 ± 2.49	30.88 ± 1.70	42.98 ± 2.71
3	6.14 ± 0.42	4.44 ± 0.33	24.81 ± 0.94	16.41 ± 0.80	27.23 ± 1.88	20.74 ± 0.64
4	6.29 ± 0.43	3.34 ± 0.19	26.21 ± 0.58	12.41 ± 0.72	26.00 ± 1.22	13.09 ± 0.43
5	11.83 ± 0.52	11.66 ± 0.59	45.36 ± 1.45	39.06 ± 1.60	47.59 ± 0.94	41.50 ± 1.58
6	10.53 ± 0.26	11.34 ± 0.32	40.58 ± 2.52	38.41 ± 1.34	42.59 ± 2.51	42.58 ± 1.62
7	13.47 ± 0.96	10.15 ± 0.38	49.22 ± 3.30	31.43 ± 0.94	54.73 ± 4.05	30.53 ± 1.68
8	5.51 ± 0.21	2.95 ± 0.16	22.00 ± 1.21	9.14 ± 0.43	23.56 ± 0.80	17.36 ± 0.76
9	6.39 ± 0.21	2.77 ± 0.17	25.04 ± 1.85	6.16 ± 0.18	26.93 ± 1.24	19.21 ± 0.54

Values represent the mean of triplicate determinations ± standard deviation.

. The vast impact of each extraction parameter was observed in all assays. The TPC results revealed a fivefold difference among the samples, as it ranged from 2.77–12.44 in peels and 5.51–13.47 mg GAE/g in pulps, with design points 8 and 9 having the lowest values in both cases. Similar results were observed in the study by Moussa-Ayoub et al. [34]. The authors used pure water or 70% aqueous methanol solution as extraction solvents, using a US bath to extract bioactive compounds from OM fruit (peels and pulp). The results revealed that water was a more efficient solvent than the hydromethanolic solution to recover polyphenols, as peels yielded 10.9 and pulp yielded 12.1 mg GAE/g dw. In a more recent study by Aruwa et al. [36], bioactive total polyphenols from Southern African O. ficus-indica peels and pulp were explored using acidified water, ethanol, acidified methanol, or hexane. The yielded polyphenols widely ranged in terms of both pulp (4.25–14.83 mg GAE/g dw) and peels (9.10–16.51 mg GAE/g dw). Water acidification was not found preferable as it yielded the lowest possible total polyphenols in both cases.

Concerning the antioxidant capacity, the range of both OM peels and pulp in the FRAP assay varied from 6.16–39.06 and 22.00–49.22 μmol AAE/g dw, respectively. The corresponding values in the DPPH assay had a range of 13.09–42.98 and 23.56–54.73 μmol AAE/g dw, respectively. It could be observed that pulp had higher values not only in TPC but also in both antioxidant capacity assays. This interesting trend may be attributed to the antioxidant compounds present in the inner part of the OM fruit.

3.1.2. Pigment Analysis of OM Extracts

Pigments can enhance the appeal of an extract, rendering it more desirable to consumers [37]. The pigmentation process of the fruit could be the reason why peels and pulp have differences in terms of total pigments, wherein pulp is initially pigmented and the pigments are diffused externally to peels [38]. In the context of OM extracts, the pigments present are betalains. A yellow-green color means that betaxanthins are dominant, whereas betacyanins give a red-purple hue to fruit. It is stated that betalain content (BC) mostly contributes to the color of O. ficus-indica rather than anthocyanins [39]. The same trend applies to O. macrorhiza, which also has higher BC than O. ficus-indica, as stated by Moussa-Ayoub [34]. Figure 1A,B depict the absorption spectra of OM extract from peels and pulp, indicating that design point 2 had the maximum absorption values at 535 nm. The absorbances strongly correlate with the extraction solvent; betalains are water-soluble, resulting in purple extracts, whereas ethanolic extracts appear yellow-green. Consequently, it is evident that design points 3, 8, and 9 were ethanolic extracts, but the other points included an evident amount of water. The amount of BC in OM peel and pulp extracts is illustrated in Figure 1C,D, wherein the pigments could be quantified. Interestingly, design points 5 and 7 showed the highest BC in OM pulp, as design points 1 and 2 had the highest BC in OM peels. This pattern could be a matter of how the pigments are distributed throughout the fruit. In the previously mentioned study [34], the authors quantified total betacyanins of ~4–5 mg/g dw in both peels and pulp using pure water or a hydromethanolic mixture. It is possible to distinguish between the examined extracts using this chromatic variation, since the lowest absorbance was observed in design point 9, the color of which was much paler. Similar results to ours were obtained by Valero-Galván et al. [38], who quantified the total betalains in peels (0.18–1.19 mg/g) and pulp (0.17–1.35 mg/g) from several Opuntia spp.

Table 5. Color analysis was measured in both the pulp and peel of Opuntia macrorhizas at various design points.

Design Point	L*		a*		b*		Color
	Pulp	Peel	Pulp	Peel	Pulp	Peel	Pulp	Peel
1	49.5 ± 0.2	48.6 ± 0.7	46.2 ± 0.5	44.6 ± 0.9	−5.9 ± 0.8	−2.2 ± 0.9
2	41.9 ± 0.5	39.7 ± 0.6	34.4 ± 0.9	31.3 ± 0.5	2.0 ± 1.3	3.5 ± 1.4
3	74.8 ± 1.4	73.8 ± 0.2	−9.8 ± 0.8	−11.4 ± 0.1	38.6 ± 3.6	51.1 ± 1.2
4	49.1 ± 0.9	50.2 ± 1.4	46.7 ± 1.6	47.7 ± 0.9	−3.8 ± 0.9	−4.3 ± 1.4
5	51.7 ± 0.5	52.2 ± 1.2	45.9 ± 0.8	44.6 ± 2.4	−2.0 ± 0.8	−6.2 ± 0.9
6	51.9 ± 0.6	48.1 ± 1.2	45.1 ± 0.8	45.4 ± 2.4	−3.3 ± 1.2	−2.2 ± 0.9
7	45.9 ± 0.4	44.6 ± 0.5	45.4 ± 0.9	39.6 ± 1.4	0.4 ± 1.4	2.8 ± 1.3
8	77.4 ± 0.9	76.9 ± 0.4	−5.6 ± 0.5	−7.2 ± 0.5	22.3 ± 2.1	33.3 ± 0.8
9	78.4 ± 0.7	76.4 ± 0.5	−3.5 ± 0.1	−5.6 ± 0.5	15.8 ± 0.5	24.2 ± 0.9

Values represent the mean of triplicate determinations ± standard deviation. The measured L*, a*, and b* values were utilized to populate the table cells with the extract’s corresponding color, represented by the suitable HEX code.

provides further information on the color of the extracts, including the precise values from the CIELAB colorimeter analysis and an illustration of the color using the HEX code. Higher values of the a* coordinate result in more reddish extracts, whereas higher b* values produce more yellowish extracts. Similar CIELAB values were obtained from Salem et al. [40], who used methanol as a solvent and generated greenish extracts from O. ficus-indica fruit. Finally, an examination of the pigments was conducted to assess their potential antioxidant properties (vide infra).

3.2. Multiple Factor Analysis (MFA) and Multivariate Correlation Analysis (MCA)

An MFA was performed to reveal the similarities and differences between the measured variables. In Figure 2, the results of the MFA are depicted, in which the factor scores of each measurement are variable on the first two dimensions, which explain 86.40% and 7.41% of the total variance, respectively. In this figure, the examined variables (X₁–X₄) are also incorporated, which are distinguished according to their levels. The plot reveals blocks of items that are similar (or familiar) to one another, determined by their proximity within the factor space. It appears that factors X₁ and X₃ (liquid-to-solid ratio and pulsation) positively affect the extraction of bioactive compounds from OM pulp, along with factor X₄ (i.e., extraction duration). The latter factor was found to have a significant impact on OM peels rather than pulp (vide infra). On the other hand, factor X₂ (i.e., solvent composition) seems to have a negative correlation to the extraction of all bioactive compounds. This factor was observed to have a negative impact on both OΜ peels and pulp. It is safe to conclude that an aqueous solvent favors the extraction yield for both OM pulp and peels, as further analyzed below. A more comprehensive overview of the results is provided by the MCA in Figure 3. Considering OM pulp, a strong positive association appears among all variables, except for BC and DPPH, which seemingly have less impact on one another. Regarding the OM peels, a strong correlation is present across all variables. The DPPH value exhibits some variance, indicating an average association with all other examined variables.

3.3. Partial Least Squares (PLS) Analysis

PLS analysis was employed to investigate the impact of the main variables (X₁, X₂, X₃, and X₄) of extraction in both OM pulp and OM peels. The PLS results are presented in Figure 4. Plots A and C refer to the OM pulp, and plots B and D refer to OM peels. Figure 4A shows the effect of each parameter on extraction from OM pulp. Starting with parameter X₁, it is obvious that the higher value of the liquid-to-solid ratio favors extraction, and a similar pattern is observed for parameter X₃, where once again the higher pulsation value seems to be the most appropriate. On the contrary, parameter X₂ seems to favor extraction when it is at its minimum value, i.e., the ideal solvent in this case seems to be water. Finally, the extraction duration (X₄) required to obtain the maximum yield is an intermediate extraction duration. Therefore, the optimal conditions for UPAE of OM pulp are established as follows: aqueous extraction for 12 min with a liquid-to-solid ratio of 60 mL/g and 48 pulses/min. The desirability value is 0.94, indicating that this model presents an excellent fit to the experimental data. Figure 4C provides a Variable Importance Plot (VIP) graph, highlighting how important the effect of each parameter is on the extraction of bioactive compounds from OM pulp. The significance value of 1.44 indicates that the most critical parameter is the solvent selection. This is followed by pulsation, whereas the liquid-solid ratio and the duration of extraction do not have a significant impact on the extraction efficiency. Similarly, Figure 4B shows the optimum extraction conditions for OM peels, which are aqueous extraction for 20 min with a liquid-to-solid ratio of 60 mL/g and a pulsation of 48 p/min. The desirability value is 0.95, indicating once again the excellent fit of the statistical model on the results. The VIP (Figure 4D) values concerning OM peels reveal that the only parameter not having a significant impact on the extraction efficacy is the liquid-to-solid ratio, where again it is the effect of the solvent that appears to have the greatest impact on the performance, followed by pulsation and extraction duration. Finally, in

Table 6. The partial least squares (PLS) prediction profiler determined the maximum desirability for all variables under each optimal extraction condition for the pulp and peel of Opuntia macrorhiza extracts.

Opuntia macrorhiza	Independent Variables				PLS Model Values 1
	X1 (R, mL/g)	X2 (C, % v/v)	X3 (P, P/min)	X4 (t, min)	TPC	BC	FRAP	DPPH
Pulp	3 (60)	1 (0)	3 (48)	2 (12)	13.21	1064.30	50.95	50.80
Peel	3 (60)	1 (0)	3 (48)	3 (20)	14.38	1015.66	47.62	47.72

¹ Total polyphenol content (TPC) in mg GAE/g dw; betalain content (BC) in μg/g dw; ferric-reducing antioxidant power (FRAP) in μmol AAE/g dw; 2,2-Diphenyl-1-picrylhydrazyl (DPPH) in μmol AAE/g dw.

, the predicted maximum values according to the PLS model are provided, highlighting the importance of PLS analysis in the extraction optimization process.

The experimental outcomes and the predictions of the PLS model show remarkable concordance, demonstrated by the high correlation coefficients of 0.9984 and 0.9991, and the substantial determination coefficients (R²) of 0.9968 and 0.9983 for the pulp and peel of OM extracts, respectively. The p-values of less than 0.0001 for both pulp and peel suggest that the differences between the observed and predicted values are not statistically significant.

3.4. Analysis of the Optimal Extracts

The extractions were carried out under these optimum UPAE conditions and were compared to the optimized conventional extraction method [20] under stirring, with water as the solvent at 40 °C for 2 h, as previously mentioned, to determine which one was more efficient. The results of the extractions are presented in

Table 7. Various parameters were evaluated under optimal extraction conditions for both the pulp and peel of Opuntia macrorhiza extracts. The study compared the ultrasound probe-assisted extraction (UPAE) technique with the stirring extraction (STE) technique.

Parameters	Pulp		Peel
	UAE	STE	UAE	STE
TPC (mg GAE/g dw)	12.43 ± 0.26 a,b	10.27 ± 0.33 c	13.07 ± 0.78 a	11.11 ± 0.52 b,c
BC (μg/g dw)	1076 ± 62 a	1090 ± 51 a	974 ± 41 a	1099 ± 81 a
FRAP (μmol AAE/g dw)	50.79 ± 2.44 a,b	54.61 ± 1.97 a	45.56 ± 3.24 b	48.76 ± 2.44 a,b
DPPH (μmol AAE/g dw)	51.32 ± 2.05 a	49.49 ± 3.22 a	49.25 ± 1.38 a	48.53 ± 2.09 a
AAC (mg/g dw)	5.07 ± 0.10 a	4.45 ± 0.33 b	3.57 ± 0.11 c	2.15 ± 0.05 d
L*	50.1 ± 1.7 a,b	45.6 ± 0.8 c	50.7 ± 1.9 a	46.8 ± 0.5 b,c
a*	47.7 ± 2.0 a	46.7 ± 2.1 a	46.2 ± 2.3 a	45.4 ± 0.5 a
b*	−7.5 ± 0.8 b	1.1 ± 0.1 a	−7.0 ± 0.9 b	0.5 ± 0.1 a
Color 1

Statistical significance (p < 0.05) is denoted by lowercase letters (e.g., a–d) within each row. ¹ The measured L*, a*, and b* values were utilized to populate the table cells with the extract’s corresponding color, represented by the suitable HEX code.

, while

Table 8. Phenolic compounds in mg/g dw under optimal extraction conditions for both the pulp and peel of Opuntia macrorhiza extracts. The study compared the ultrasound-assisted extraction (UPAE) technique with the stirring extraction (STE) technique.

Phenolic Compound(mg/g dw)	Pulp		Peel
	UAE	STE	UAE	STE
β-Resorcylic acid	0.29 ± 0.02 b	0.32 ± 0.02 a,b	0.32 ± 0.01 a,b	0.34 ± 0.02 a
4-Hydroxybenzoic acid	0.26 ± 0.01 a	0.26 ± 0.02 a	0.25 ± 0.01 a	0.25 ± 0.01 a
Pyrogallol	0.65 ± 0.03 b	0.68 ± 0.05 b	0.88 ± 0.02 a	0.66 ± 0.02 b
Pyrocatechuic acid	0.46 ± 0.03 b	0.48 ± 0.02 b	0.49 ± 0.02 a,b	0.53 ± 0.02 a
Caffeic acid	0.58 ± 0.03 a	0.48 ± 0.02 b	0.52 ± 0.02 b	0.51 ± 0.02 b
Homovanillic acid	0.47 ± 0.01 b	0.39 ± 0.02 c	0.50 ± 0.01 b	0.67 ± 0.05 a
Syringic acid	0.64 ± 0.01 a	0.67 ± 0.04 a	0.65 ± 0.03 a	0.70 ± 0.03 a
m-Coumaric acid	0.51 ± 0.03 b	0.37 ± 0.02 c	0.48 ± 0.03 b	0.60 ± 0.04 a
Total identified	3.85 ± 0.18 a,b	3.65 ± 0.20 b	4.07 ± 0.14 a,b	4.26 ± 0.21 a

Statistical significance (p < 0.05) is denoted by lowercase letters (e.g., a–c) within each row.

summarizes the results of the identification and quantification of the individual polyphenols through HPLC. Compared to Table 6, most of the experimental values do not deviate much from the predicted values. More specifically, the experimental TPC values of OM pulp and peels in Table 7 were ~6% and ~9% lower than predicted by the PLS values in Table 6. Moreover, both OM peel and pulp resulted in higher TPC values when subjected to UPAE rather than stirring. A possible mechanism could be due to acoustic cavitation bubbles that burst close to or even touch the solid surface. Shockwave damage and high-velocity jets of liquid into the surface are the results of a burst bubble. Localized erosion and the breaking apart of friable materials are possible outcomes of such impacts. These factors contribute to a decrease in particle size, which in turn increases the mass transfer of solid particles and generally improves media reactivity [29]. Benattia and Arrar [41] studied the optimal TPC of Opuntia ficus-indica fruits through Soxhlet and maceration, and the obtained TPC values were 1.46 and 1.15 mg GAE/g, significantly lower than the ones obtained in our study. Moreover, Yeddes et al. [42] utilized ultrasonication bath extraction for 15 min, using a mixture of methanol and acetic acid as a solvent to assess the TPC on Opuntia stricta. Their results yielded TPC 3.72 and 4.15 μg/g for pulp and peels, respectively; once again, significantly lower than in our study. These support the statement that Opuntia macrorhiza might serve as a valuable substitute for Opuntia ficus-indica in food industries, and the use of ultrasound is not only beneficial for the environment but also for extraction efficiency. As for the BC, OM pulp yielded a ~1.1% higher value than the predicted one, while OM peels yielded a ~5.3% lower value. In both extraction techniques, the BC recovery from pulp and peels did not display statistically significant differences (p > 0.05). In a similar study, García-Cayuela et al. [43] studied the total betalains of two different red prickly pear varieties from Spain, Sanguinos, and Mexico, Vigor. The extraction technique employed was an ultrasonication bath, utilizing 50% v/v aqueous methanol as solvent. The BC values for peel ranged from 740 to 810 μg/g dw, and for the pulp ranged from 490 to 790 μg/g dw. The experimentally derived FRAP and DPPH values were slightly higher than the PLS model, except for the pulp DPPH value, which was slightly lower. This means that UPAE was successful in ensuring maximum antioxidant capacity (as defined by these two protocols) after optimization. It is worth noting that the values obtained from the optimized STE did not exhibit significant differences (p > 0.05) with those of the UAE. In the previously mentioned study [38], the authors also quantified AAC from several Opuntia spp. Pulp yielded 0.67–1.92 mg/g and peels yielded 1.19–1.72 mg/g, indicating that the several species had vast differences in terms of AAC. Finally, the color of the optimal UPAE peel and pulp extracts appeared slightly lighter than the STE ones.

According to Table 8, the most abundant polyphenol in both OM pulp and peel appears to be pyrogallol, followed by syringic acid. Some polyphenolic compounds determined by HPLC did not differ significantly (p > 0.05) between peel and pulp or between the two techniques. However, it was expected and observed that OM peels had higher polyphenolic content than pulp, regardless of the extraction technique. Serra et al. [44] also determined syringic acid in various Opuntia species. Moreover, Kivrak et al. [45] determined 4-hydroxybenzoic acid and caffeic acid in Opuntia robusta and Opuntia ficus-barbarica. They also determined syringic acid in O. robusta, with a value of 0.46 mg/kg, significantly lower than OM in our study. Finally, the relationship between the identified polyphenols and antioxidant capacity could not be fully declared since we quantified ~30–40% of total polyphenols in both OM peels and pulp. A possible future study quantifying most of the polyphenols could shed more light on the relationship between antioxidant capacity and the structure of polyphenols.

The scatter plot (Figure 5) described is a classic visualization in discriminant analysis, effectively showing the separation of groups based on their distinct characteristics. Plotting the data points in color-coded clusters makes it visually apparent how well the different groups (TPC, BC, DPPH, FRAP, and AAC) are distinguished from each other based on their characteristics. Canonical axes serve as a reference for the linear separation, and minimal overlap between clusters strongly suggests the efficiency of the method. This plot is a graphical representation of the results concerning both the bioactive compounds and the antioxidant capacity of OM pulp and peels. In particular, it is possible to examine which technique is most efficient for each part of the fruit. For example, it was observed that for total polyphenol recovery from OM, it is more efficient to use UPAE than STE, whereas the peels would be preferable as a raw material to pulp. On the contrary, for the most efficient ferric-reducing power, STE in OM pulp is required. Finally, the use of STE in OM peel is more efficient for the extraction of total betalains. Such a clear delineation helps in accurately classifying and understanding the underlying data. However, in the discriminant analysis presented in Figure 5, the AAC lacks scoring coefficients. This could indicate multicollinearity, where AAC is highly correlated with other variables, such as DPPH. In such cases, its unique contribution to the discriminant function is minimal.

4. Conclusions

Ultrasound probe-assisted extraction serves as a sustainable technique for extracting valuable bioactive compounds from fruit byproducts. This method not only facilitates the efficient extraction of antioxidants such as polyphenols and betalains but also meets the increasing demand for environmentally friendly production methods. Particularly, the high levels of betalains provide a viable alternative to artificial dyes in the food, pharmaceutical, and cosmetic sectors, responding to the consumer and regulatory push for healthier, more natural products. The study provided a foundation for several extraction pathways to recover bioactive compounds from OM peels and pulp using UPAE or STE; however, the efficiency of each procedure depends on the duration, environmental sustainability, and total cost. Additionally, the utilization of water as a solvent not only highlights the food-grade applicability but also ensures the environmental advantages of this method, presenting an appealing option for industries seeking to minimize their ecological impact while improving the nutritional and visual quality of their products.