The Opuntia genus, part of the Cactaceae family, encompasses over 1500 species, many producing edible fruits. These fruits are characterized by a thick outer skin, often covered with small prickles and come in various colours, including red, purple, yellow, or white. The flesh is sweet and juicy, containing numerous small seeds (El Kossori et al., 1998; Saenz, 2000; Trejo-González et al., 1996). The prickly pear cactus (Opuntia ficus-indica) is globally distributed and is an important nutrition source. They are utilized in various food products, such as juices, jams and natural sweeteners. Beyond their culinary uses, many parts of the cactus plant have been traditionally employed for medicinal purposes. In Mexico, both the leaves and fruits of Opuntia have been used medicinally, including for treating arteriosclerosis, diabetes, gastritis and hyperglycemia (El Kossori et al., 1998; Galati et al., 2002; Gurrieri et al., 2000; Ibanez-Camacho et al., 1983). The Taif Governorate, located in the Kingdom of Saudi Arabia (KSA), is renowned for cultivating a variety of prickly pear cactus. Efforts are underway to expand the production of prickly pear cactus and incorporate it into more common food products. This development has driven the need to valorize the by-products of the skin of prickly pear fruit (Abdel-Hameed et al., 2014).
       
The efficient utilization of by-products and raw materials has become a critical focus globally, aligning with the United Nations’ 2030 agenda for reducing food waste and promoting sustainability (Hadidi et al., 2022). Agricultural and food waste, collectively termed agri-food waste, are valuable sources of high-value products and represent an opportunity to move toward a zero-waste economy. Globally, food waste poses a significant challenge to food security and the Kingdom of Saudi Arabia (KSA) is no exception. With an average of 427 kilograms of food wasted per person annually, KSA ranks among the highest in food wastage (Baig et al., 2019). Agro-industrial processes, particularly those involving fruits, generate substantial amounts of organic waste, such as peels, seeds, stems and malformed fruits, which can account for more than 50% of the total fruit mass (Castro et al., 2022; Dias et al., 2015). These wastes are produced in high volumes but are often considered low-value materials (Rodríguez-Félix et al., 2022). Agri-food by-products have significant potential for use in various economic and industrial sectors (Anwar et al., 2023; Couto and Estevinho, 2024). By integrating circular economy principles into agro-industrial systems, strategies can be developed to mitigate environmental issues while transforming waste into value-added products (Poponi et al., 2023). This study explores the utilization of Opuntia ficus indica by-products to create high-value formulations with potential applications in food and nutraceutical industries, leveraging their known health benefits (Ziemlewska et al., 2021).
       
Extraction is fundamental in analysing plant phytochemicals to isolate compounds from plant materials. The extraction method plays a critical role in determining the isolated compounds’ quantity, type and activity, including their antioxidant capacity and other biological activities (Boeing et al., 2014; Santas et al., 2008). Although several extraction conditions are described in the literature, there is no universally accepted standard method (Cacace and Mazza, 2003; Chemat et al., 2017). Factors such as the chemical nature of compounds, the extraction technique, sample particle size and the presence of interfering substances can all influence the efficiency of the extraction process (Naczk and Shahidi, 2004). Solid-liquid extraction with various solvents is the most commonly used approach for isolating compounds from plant sources (Alothman et al., 2009; Chanioti et al., 2014). Crude extracts typically contain a complex mixture of different classes of phenols and lipids, which exhibit selective solubility in various solvents. Therefore, solvent polarity plays a crucial role in enhancing the solubility of phenolic and lipid compounds (Naczk and Shahidi, 2006; Saini et al., 2021).
       
This study focuses on the valorization of agricultural by-products from Opuntia ficus-indica cultivated in Taif, Saudi Arabia, by employing various solvents to extract phenolic and lipid compounds and evaluating their antioxidant activity.

Plant materials and sample preparation
 
Ripe cactus (Opuntia ficus indica Mill) fruits were sourced from a farms in the Taif governorate. The cultivar selected for this study had green-coloured fruits. After harvest, the fruits were stored at 4^oC until sample preparation. They were washed three times with tap water and the peels were manually removed. The peels were then oven-dried at 60^oC, ground into a powder and stored at -20^oC.
 
Extraction procedures
 
The extraction procedure was adapted and modified from the method described by Michiels and his coworkers (Michiels et al., 2012). This study extracted 4 g of oven-dried cactus pear peels using 50 mL of solvent under sonication for 20 minutes at 40^oC. After the first extraction, the residue was washed with 50 mL of the same solvent, followed by a second sonication step under the same conditions. The mixture was filtered through Whatman filter paper, except for the aqueous extract, which was centrifuged at 5000 RPM for 10 minutes. The supernatants from both extractions were combined into a 100-mL volumetric flask and the final volume was adjusted using the same solvent. All extracts were stored at 4^oC and analysed within three days. The solvents used included three organic solvents (acetone, methanol and ethanol), distilled water and organic solvent-water mixtures in two ratios: 50:50 (v/v) and 70:30 (v/v). Furthermore, the study incorporated organic solvents containing 0.5% acetic acid (99.5:0.5, v/v) and organic solvent-water mixtures with 0.5% acetic acid in ratios of 70:29.5:0.5 (v/v/v) and 50:49.5:0.5 (v/v/v). These extracts were directly used to measure total phenolic content and assess antioxidant capacity via DPPH• assays.
 
Estimation of total phenolic content (TPC)
 
The TPC of the samples was measured using the Folin-Ciocalteu (FC) method, following the procedure described earlier (Abutaha et al., 2023). In this assay, 2 µL of each sample was mixed with 20 µL of FC reagent. The mixture was incubated at room temperature (25^oC) for 5 minutes. Subsequently, 80 µL of sodium carbonate solution (7.5%) was added and then incubated in the dark for one hour. After the incubation, the absorbance of the reaction mixture was recorded at 760 nm using a spectrophotometer (ChroMate, USA). A standard calibration curve was generated using gallic acid and the TPC was expressed as milligrams of gallic acid equivalents per gram of dry plant material (mg GAE/g DW).
 
Radical scavenging activity (DPPH•)
 
The radical scavenging activity of the samples was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH•) assay, a widely used method for evaluating antioxidant capacity. DPPH• is a stable, violet-coloured free radical that undergoes reduction and decolonisation in the presence of antioxidant compounds. The radical scavenging activity of the samples was determined as reported earlier (Abutaha et al., 2023). Briefly, 190 µL of a 0.004 w/v DPPH• solution prepared in methanol was mixed with 10 µL of each extract (200 mg/mL). The reaction mixtures were incubated in the dark for 30 minutes, after which the absorbance (517 nm) was recorded.
 
Soxhlet extraction of lipids

A Soxhlet extraction was performed using 10 g of powdered sample, which lasted 6 hours at 100^oC. After the extraction, the solvent was evaporated. Two different solvent schemes were employed for lipid recovery: 500 mL of n-hexane alone and a mixture of 375 mL n-hexane and 125 mL ethanol, each conducted in triplicate (n=3). The resulting oily extracts were weighed to determine the oil content as a percentage of the dry weight (% DW). Subsequent fatty acid analyses were then conducted using GC-MS.
 
Folch method
 
Ten grams of plant powder was performed using a modified Folch method with a total solvent volume of 100 mL. A 2:1 (v/v) mixture of chloroform (66.67 mL) and methanol (33.33 mL) was prepared and added to the plant powder. The mixture was thoroughly homogenised (IKA, Germany) for 1 minute to ensure complete suspension of the plant material in the solvent. The homogenate was subsequently sonicated (Wise Clean, Korea) for 30 minutes and filtered to remove solid residues and the filtrate was transferred to a separatory funnel. For separation phase, 25 mL of distilled water was added and the mixture was shaken before settling into two distinct layers. The lower chloroform layer containing the extracted lipids, was carefully collected. The solvent was then evaporated using a rotary evaporator, yielding the lipid extract, which was stored at -20^oC for subsequent analysis.
 
Profiling of phytocompounds by gas chromatography-mass spectrometry (GC-MS)
 
A 1 µL sample was analysed using an Agilent Technologies 7890B GC-MS system (Santa Clara, CA, USA) equipped with the NIST MS database software (version XX) for component identification. Compounds were separated on an Agilent DB-5 MS capillary column (30 m × 0.25 mm, 0.25 µm film thickness) with helium as the carrier gas at a flow rate of 1 mL/min. The injection was conducted in split mode (ratio 50:1) at an inlet temperature of 250^oC. The oven temperature was programmed from 50^oC to 250^oC at a ramp rate of X^oC/min, with a total runtime of 71 minutes. The mass spectrometer operated in scan mode, covering a mass range of 40-500 g/mol, with a scan speed of 1.56, a solvent delay of 4 minutes and an ion source temperature of 230^oC. Data acquisition and compound identification were performed using the NIST MS library, ensuring accurate characterisation of the sample components.

Effect of solvent polarity on phenolic extraction
 
The relationship between the extraction method, yield, total TPC and DPPH radical scavenging activity was analysed, uncovering significant patterns influenced by solvent polarity and composition (Table 1). The highest extraction yields were observed with methanol/water/acetic acid (M5) (2.52%) and methanol/acetic acid (M4) (2.43%), outperforming single solvents such as acetone (0.064%) and ethanol (0.59%), as well as solvent-water mixtures. The inclusion of water improved yields by enhancing compound solubility, while the addition of acetic acid facilitated cell wall disruption and solute release. These results underscore the superior efficiency of mixed solvent systems, which combine polar and non-polar components to optimise the extraction process. The recovery of phenolic compounds is significantly influenced by the choice of solvent and its polarity (Alothman et al., 2009; Taghizadeh et al., 2018). This is demonstrated by the TPC results shown in Table 1. The polarity of the solvent had an impact on TPC values, with the highest TPC observed in methanol/water/acetic acid (M5) (23.9 mg GAE/g) and methanol/water (M2) (23.2 mg GAE/g). Solvent mixtures generally yielded higher TPC than single solvents, highlighting the importance of optimising solvent polarity. Methanol-based mixtures proved effective, likely due to methanol’s broad capacity to dissolve a wide range of phenolic compounds. The choice of solvent strongly influenced the antioxidant capacities of the extracts, as different solvents extract compounds with varying antioxidant potentials based on their polarity, solubility properties and ability to dissolve specific bioactive compounds (Nawaz et al., 2020; Taghizadeh et al., 2018). Numerous studies have reported the use of various solvent combinations, including water, acetone, methanol, ethanol and their aqueous mixtures, with or without added acids, to extract antioxidants from vegetables, fruits and other foodstuffs  (Boeing et al., 2014; Michiels et al., 2012; Nawaz et al., 2020). Acetone/water (A2) was the most efficient solvent for extracting phenolic compounds, achieving the highest TPC values. Methanol/water/acetic acid (70/29.5/0.5) produced the best results for anthocyanin content. These findings are consistent with previous reports emphasizing the importance of solvent polarity in maximizing phenolic compound recovery.


 
Antioxidant activity
 
The antioxidant activity of plant extracts is highly dependent on the concentration and composition of their phytochemical constituents, which dictate their effectiveness in scavenging free radicals (Kumar et al., 2023; Kumari et al., 2017; Choudhary et al., 2015).
       
In the present study, the highest DPPH activity was observed for acetone/water (50/50, v/v) (28.3%), followed by methanol/water/acetic acid (M5) (27.8%) and methanol/water (M3) (22.5%). These results highlight the importance of mixed solvent systems, especially those involving water, in maximising phytocompound extraction and antioxidant potential. When combined with solvents like acetone, methanol, or ethanol, water enhanced extraction efficiency by improving solubility. In contrast, single solvents such as acetone (TPC: 8.1 mg GAE/g; DPPH: not detected) and ethanol (TPC: 10.0 mg GAE/g; DPPH: 7.8%) exhibited significantly lower activity, further emphasising the superior antioxidant potential of mixed solvent systems. A prior study by Alothman et al., (2009) identified acetone-water mixtures as the most effective solvent combination for antioxidant extraction.
       
The acidification of the extraction solvent showed no significant improvement in the yield of antioxidant compounds across all tested combinations, aligning with earlier findings  (Boeing et al., 2014; Michiels et al., 2012) except for methanol (Table 1).
 
Lipid content and fatty acid profile
 
Soxhlet extraction using hexane-ethanol mixtures yielded higher lipid recovery compared to hexane alone (Fig 1). Subsequent GC-MS analysis revealed diverse fatty acid profiles, demonstrating the potential of solvent mixtures to enhance lipid solubility and recovery. The Venn diagram (Fig 2) shows the distribution of elements across three extracts. n-hexane contributed the most unique elements (26.3%), followed by n-hexane: ethanol (21.1%) and chloroform: methanol (14%). Shared compounds included 19.3% expected to all three lists, with smaller overlaps between pairs of extracts. This highlights unique and shared contributions among the extracts (Fig 2). The solvent polarity was pivotal in determining the types of compounds extracted. Hexane, a non-polar solvent, primarily extracted non-polar compounds, such as hydrocarbons, fatty acids and short-chain alcohols (e.g., 2-Heptanol and Octacosane). In contrast, the n-hexane-ethanol mixture, with intermediate polarity, facilitated the extraction of a broader range of compounds, including moderately polar phenols (2,4-Di-tert-butylphenol), aldehydes (2-Undecenal) and fatty alcohols (1-Hexadecanol). Chloroform-methanol, a more polar solvent system, demonstrated the highest capacity for dissolving different compounds. This system extracted sterols (Stigmast-7-en-3-ol), alkaloids (e.g., Aspidospermidin-17-ol) and fatty acid esters, showcasing its ability to isolate diverse bioactive molecules. These patterns highlight that non-polar solvents effectively extract simple lipids and hydrocarbons, whereas polar solvents excel in isolating complex lipids and bioactive polar compounds (Table 1, 2 and 3). This further reinforces that selecting an optimal solvent or solvent mixture is critical for maximizing yields and targeting specific bioactive molecules. While Soxhlet extraction remains a popular technique for biological samples, its drawbacks, including its time-consuming nature and potential for thermal degradation of heat-sensitive compounds like ω-3 fatty acids, limit its applicability (De Castro and Garcýa-Ayuso, 1998; De Castro and Priego-Capote, 2010; Farag et al., 2021; Zhang et al., 2018). Alternative methods are necessary to balance extraction efficiency with compound integrity, especially for heat-sensitive compounds used in nutraceuticals, cosmetics and biopesticides. The chemical composition analysis revealed notable variations among the three extracts. n-hexnae was rich in carboxylic acids (48.71%), steroids (29.79%) and esters (12.56%), with minor contributions from alcohols, phenols, aldehydes, hydrocarbons and terpenes. n-hexane: ethanol showed steroids (34.06%) and carboxylic acids (32.36%) as dominant, along with esters (13.56%), alcohols (9.47%), vitamins (5.54%) and smaller amounts of other classes. Chloroform: methanol primarily contained carboxylic acids (47.44%), steroids (28.14%) and esters (15.31%), alongside minor classes such as alkaloids and alkenes, reflecting broader diversity (Table 4).

This study highlights the critical role of solvent polarity in optimizing the extraction of bioactive compounds. Mixed solvent systems, particularly methanol-based mixtures, demonstrated superior efficiency in phenolic recovery and antioxidant activity. For lipid extraction, solvent mixtures like hexane-ethanol outperformed singles solvents, extracting a broader range of compounds. These findings emphasize the importance of tailored solvent selection to maximize yields and preserve compound integrity for applications in nutraceuticals, cosmetics and biopesticides.

The authors express their sincere appreciation to the Ongoing Research Funding program, (ORF-2026-1513), King Saud University, Riyadh, Saudi Arabia.
 
Funding
 
The present study was supported by the Ongoing Research Funding program, (ORF-2026-1513), King Saud University, Riyadh, Saudi Arabia.

The author declares no competing financial interests.