Extraction, Chemical Modification, and Assessment of Antioxidant Potential of Pectin from Pakistani Punica granatum Peels

Abstract

The conversion of agro-industrial waste into value-added products has attracted the attention of the scientific community. Pectin is an extensively used by-product of agricultural waste and has many applications. The present research used pomegranate peel for the extraction of pectin and explored its antioxidant properties. Pectin from Punica granatum peel was extracted with the help of a feasible, low-cost, and ecofriendly acidified extraction method using ethanol as an extraction solvent. The yield of the pectin with ethanol was found to be 19.1%. The extracted pectin was chemically modified using the amidation method. The structural characterization of the extracted and modified pectin was carried out using the SEM (for morphology), FTIR (for chemical bond and functional groups), EDX (for an elemental analysis), and XRD (for crystallinity) techniques. After confirming the modification of pectin, both the native and modified pectin were assessed for their antioxidant potential. The antioxidant properties of natively extracted pectin and modified pectin were evaluated against different types of free radicals with the help of a hydroxyl radical antioxidant assay, a DPPH radical scavenging assay, ferric-reducing antioxidant power, and a phosphomolybdenum assay. All the performed antioxidant assays revealed that the antioxidant activity of pectin was increased after modification through amidation. The findings could be very useful in obtaining pectin from the peel waste of Punica granatum and obtaining pectin with more bioactive potential via its chemical modification through an optimized method. This is also a step forward in achieving the goal of a sustainable environment. This study contributes to sustainable development by making use of the wasted peels of pomegranate and extracting bioactive pectin at the same time.

1. Introduction

Pectin is a high-molecular-weight complex polysaccharide present in the primary cell walls of dicotyledons and cereal grains. It is a biocompatible, non-toxic, and anionic branched nanostructured composite [1,2]. Pectin also controls numerous properties of the cell wall, such as porosity, surface charge, elasticity, pH, ion balance, cell–cell relation, and biotic stress protection in higher plants [3,4,5].

Naturally, pectin exists in two primary forms. The typical class of pectin is high-methyl ester (HME) pectin with a high percentage (>50 percent) of carboxyl groups existing as methyl ester [6]. Another prevalent form of pectin is low-methyl ester (LME) pectin. In this class, less than 50 percent of the carboxyl groups exist as an alternative methyl ester [7]. Typically, LME pectin is extracted from HME pectin by treatment with an alkali or acid [8].

The molecular structure of pectin fluctuates with the plant’s physiological condition and plant tissues [9]. Pectin, with linear-structure polysaccharides consisting of d-galacturonic acid, is polymerized by an alpha-1,4-glycosidic linkage [10]. The average molecular weight of pectin ranges from 50,000 g/mol to 150,000 g/mol [11]. Pure galacturonan portions of pectin are distinguished from its high-molecular-weight portions by decontaminating them via chemical or enzymatic actions [12].

Apple pomade, pomegranate peels, citrus peels, sugar beets, sunflower beads, mango debris, guava, papaya, coffee, and cocoa are raw materials used to produce pectin. Pectin is utilized in the food industry to replace fat and/or sugar in low-calorie foods, such as jams, jellies, and frozen foods, and more recently, in various products. It is used in the pharmaceutical industry to lower blood cholesterol and treat digestive issues. Pectin is also used in edible films, as a paper substitute, and in foams and plasticizers, among other things [13]. Pectin is regarded as soluble dietary fiber that has a number of advantageous physiological and gastrointestinal effects from a health and nutritional perspective. These effects include a reduction in the absorption of glucose, an increase in fecal mass, and a delay in gastrointestinal emptying, which shorten the time spent in the gastrointestinal tract [14]. Being very useful as a source of dietary fiber and having the ability to form gels, jams, and low-fat dairy products, pectin is used in the food and pharmaceutical industries [15]. It is considered a healthy additive with no restrictions on reasonable daily use [16,17].

However, pectin, when used in special environments, has certain intrinsic limitations [18]. In addition to its slow digestion rate, natural-source pectin can cause stomach cramps, diarrhea, gas, and loose stools. Exposure to pectin dust may also cause asthma in individuals. The usefulness of pectin is affected by its structure, degree of methylation, solubility, pH, and temperature and by the appearance of soluble solids [19].

The presence of pectin in pomegranate peel has not been studied much [20]. Pomegranate (Punica granatum) is a fruit shrub belonging to the Lythraceae family. It is considered an outstanding fruit as it not only provides social benefits but also is inexpensive for achieving natural balance, has ornate value, and improves welfare due to being rich in starch, calcium, vitamins, amino acids, pectin, fatty acids, and polyphenols [21]. Pomegranate is used mainly for the processing of fresh foods, juice, and wine in the food industry. Its main by-product, peel residue, is rich in pectin, which can be used as an effective emulsifier [22]. A range of 6~10 g of natural pectin is present in every 100 g of pomegranate peel residue. It is a very common practice worldwide to throw away food and agricultural waste after consuming the foods and agricultural products. These wasted materials cause contamination and imbalances in the ecological systems of the environment. It is therefore necessary to find approaches to eliminate such issues. The use of these wastes in producing value-added products is a very efficient approach that will provide useful products and a clean environment, thus promoting sustainable development. The current research focuses on the introduction of a green program through the use of agricultural waste for beneficial purposes. Keeping in mind the demand and growing cost of pectin on the global market, it is productive to obtain renewable resources from pomegranate peel. Modest processes are used to extract pectin to meet its rising demand. In our work, pectin was modified using the amidation method because its execution was feasible within our labs and the obtained product has wide applications. The aim of this work was to extract and chemically modify pectin from Punica granatum peel extract; characterize the modified and native pectin using the FTIR, SEM, EDX, and XRD techniques; and study the antioxidant properties of both the native and modified pectin. The expected outcome was to more efficiently obtain pectin with enhanced antioxidant potential and contribute to a clean and sustainable environment.

2. Materials and Methods

The research work designed in this study was completed according to standard operational procedures and is explained under the following headings.

2.1. Chemicals and Reagents

All the chemicals and reagents used in this research were of an analytical grade and purchased from reputed companies, Sigma-Aldrich, St. Louis, MO, USA, and Duksan, Ansan-si, Korea.

2.2. Collection and Pretreatment of Plant Material

Punica granatum fruits and peels were collected from various local juice shops in Faisalabad, Pakistan, by using a random sampling approach. The fruits and peels were taxonomically identified and authenticated by Dr. Muhammad Qasim, Associate Professor in the Department of Botany, Government College University Faisalabad, Pakistan. After authentication, peels were separated, thoroughly washed several times with tap water, and finally rinsed with distilled water. Punica granatum peel wastes were then soaked for an hour to prevent discoloration, after which they were dried in shade at room temperature. After two months of drying, peels were ground and converted into powder using a grinding mill. The peel powder was stored in sealed polythene zip bags at room temperature.

2.3. Extraction and Precipitation of Crude Pectin

The extraction and precipitation of crude pectin from Punica granatum peels were carried out by using a previously reported method [23,24] with minor modifications. Optimized extraction conditions were applied. Initially, 40 g of dried peel powder was dissolved in 2 L of distilled water and solutions’ pH was adjusted to 2.0 by adding an appropriate amount of HCl. The mixture was heated to 85 °C for 60 min and the hot acidic extract was filtered through a muslin cloth. To optimize the best-suited solvent for the extraction of pectin, 40 g of peel powder was dried and then dissolved separately in 95% ethanol, acetone, and n-hexane. Pigments were removed at 50 °C under the reflux condition with continuous stirring, and other ethanol, acetone, and n-hexane soluble impurities were removed using a Soxhlet apparatus. Treated peel powder was dried and 40 g of peel powder was used for the extraction of crude pectin. Powder was added to 1 L of distilled water and heated to 87 °C for two hours with constant stirring. Afterwards, the peel was removed by a filtration process. The extract was concentrated in a hot water bath and pectin was precipitated at room temperature by adding an equal volume of 95% ethanol, acetone, and n-hexane separately. The pectin was washed multiple times with a respective analytical-grade solvent. The pectin was then dried in an oven at 50 °C for one to two days, ground into powder form, and weighed to estimate the yield for each solvent. The dried pectin was appropriately labeled and stored for a further analysis. The pectin extraction yield was estimated by following the below equation (Bhat SA & Singh ER, 2014) [23].

$$\mathrm{Extraction}\mathrm{yield}(\%)={\displaystyle \frac{Weightofpectin}{weightofpeelpowder}}\times 100$$

(1)

2.4. Modification of Pectin

The chemical modification of pectin was performed using an alcoholic ammonia method referred to as amidation [25], which targets the carboxylic and ester group present in pectin. About 3% pectin solution (3 g/100 mL or 6 g/200 mL) was prepared in distilled water and homogenized. Separately, a solution was prepared by mixing three parts by volume of 7% ammonia solution with 200 mL of 95% ethanol to achieve the desired ammonia concentration. The mixture was coagulated in 200 mL of ethanol at 10 °C for 4 h. After 4 h, the precipitated modified pectin was collected and thoroughly washed with alcohol until the pH of the washing liquid became neutral [26]. Finally, the modified pectin was dried at 50 °C for two days and ground into powder form for storage and a further analysis.

2.5. Characterization of Pectin

Native and fully modified pectin was characterized by using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), energy dispersive spectroscopy (EDX), and X-ray diffraction (XRD) analyses.

2.5.1. SEM Analysis

The extracted native and modified pectin, previously ground into fine powder, were subjected to a morphological analysis using an SEM Nova Nano 450 FE (Thermo Fisher Scientific, Waltham, MA, USA) by following the already described [27] method. Imaging was performed at various magnification levels like 10,000×, 25,000×, 50,000×, and 100,000. Images were captured for both the native and modified pectin by adjusting the accelerating voltage of 10 kV.

2.5.2. FTIR Analysis

The FTIR analysis of native and modified pectin was performed by using a Perkin Elmer spectrum two spectrometer (Perkin Elmer, Shelton, CT, USA) following the protocol already described [28]. The spectra were recorded over a wavelength range of 400 cm⁻¹ to 4000 cm⁻¹ and analyzed accordingly.

2.5.3. EDX Analysis

Energy dispersive X-ray spectroscopy (EDX) was utilized for an elemental analysis of native and modified pectin, providing detailed information about their chemical composition. The EDX analysis developed a correlation between native and modified pectin and their high-resolution images as described in earlier reports [29].

2.5.4. XRD Analysis

The X-ray diffraction (XRD) analysis was performed to determine crystallinity of native and modified pectin. Interaction between X-rays and pectin resulted in a constructive interference at 2d sin θ. The diffraction angles and lattice spacing of the pectin were subsequently analyzed. The diffracted rays were detected, counted, and processed as described in earlier methods [30].

2.6. Antioxidant Activities

Antioxidant activity was evaluated using various methods including the hydroxyl radical scavenging assay, DPPH radical scavenging assay, ferric-reducing antioxidant power assay, and phosphomolybdenum assay by following the reported protocols [31].

2.6.1. Hydroxyl Radical Antioxidant Assay

Hydroxyl radical scavenging antioxidant activity of both the native and modified pectin was assessed by following the already described method [32]. A reaction mixture was prepared by combining 3 mL of ferrous sulfate (FeSO₄), 2 mL of hydrogen peroxide (H₂O₂), and 1 mL of a pectin solution at different concentrations. Subsequently, 2 mL of salicylic acid was added and the volume was adjusted to 10 mL with distilled water in test tubes containing pectin concentrations ranging from 20 µg/mL to 100 µg/mL. The mixture was incubated at 37 °C for 30 min, after which the absorbance was measured at 510 nm by the spectrophotometer using a sample blank and control blank as references. The same procedure was applied for ascorbic acid as a standard. All experiments were performed in triplicate and the percentage activity was calculated by using the following formula:

$$\mathrm{Scavenging}\mathrm{activity}(\%)=1-{\displaystyle \frac{Asample}{Acontrol}}\times 100$$

(2)

where the “A” control is absorbance of the control reaction without pectin and the “A” sample is the absorbance of all reagents and pectin.

2.6.2. DPPH Radical Antioxidant Assay

The scavenging activity of 2,2 diphenyl-1-1-picrylhydrazyl was determined by using both the native and modified pectin following the previously reported [33] method. One milliliter of a pectin solution at different concentrations was mixed with one milliliter of 0.0002 M solution of DPPH. The test tube was then filled up to 10 mL with an appropriate amount of distilled water and a pectin concentration ranging from 20 µg/mL to 100 µg/mL. The mixture was incubated for 30 min and absorbance was measured at 517 nm against a blank. The same procedure was repeated for ascorbic acid as a standard and all the analyses were conducted in triplicate. The calculation of percent scavenging activity was conducted using the following formula:

$$\mathrm{Scavenging}\mathrm{activity}(\%)=1-{\displaystyle \frac{Asample-Breference}{Acontrol}}\times 100$$

(3)

where the “A” control is the absorbance of the control reaction, “A” sample is the absorbance of DPPH and test pectin, and reference is absorbance of a pectin solution without DPPH.

2.6.3. Ferric-Reducing Antioxidant Power

Ferric-reducing antioxidant power of both the native and modified pectin was determined by the method in [34]. In this assay, 2.5 mL of a phosphate buffer and 2.5 mL of 1%-weight-by-volume potassium ferric cyanide [K₃Fe(CN)₆] were added to 1 mL of a pectin solution at varying concentrations. The mixture was incubated at 50 °C for 30 min. After incubation, 2.5 mL of trichloroacetic acid and 0.1% ferric chloride solution were added. The volume was adjusted to 10 mL with distilled water and the test tubes were labeled with pectin concentrations ranging from 20 µg/mL to 100 µg/mL. The absorbance was measured at 700 nm by using the spectrophotometer. The higher the absorbance, at 700 nm, the higher the reducing potential of pectin. The same procedure was repeated to calculate the reducing potential of ascorbic acid. All these analyses were performed in triplicate.

2.6.4. Phosphomolybdenum Antioxidant Power

The phosphomolybdenum assay was performed using ascorbic acid as the standard. For this, 1 mL of a sample solution at different concentrations was mixed with 3 mL of a reagent solution consisting of 0.6 M sulfuric acid, 4 mM ammonium molybdate, and 28 mM sodium phosphate. The test tubes were incubated for 90 min. After incubation, the samples were cooled, and the absorbance of the aqueous solution was measured at 695 nm against a blank solution. A typical blank solution was incubated under the same conditions. The antioxidant capacity was expressed as mm of ascorbic acid equivalents/g of pectin [35]. All the analyses were performed in three replicates. Percent inhibition was calculated by the following formula.

$$\%\mathrm{inhibition}=1-{\displaystyle \frac{Absorbanceofsample}{Absorbanceofcontrol}}\times 100$$

(4)

2.7. Statistical Analysis

The data obtained from the chemical analysis and antioxidant activities were reported as the mean ± SD. Furthermore, OriginPro 9 was used to conduct the statistical analysis. All the data were presented with ±SD as well as standard error bars to highlight the statistical significance of the data.

3. Results and Discussion

The research was designed to investigate the antioxidant properties of pectin extracted from Punica granatum peels. In this study, the antioxidant activities of pectin extract were studied. Phytochemical screening was performed, through which the presence of pectin was confirmed. Additionally, pectin was modified in order to enhance the antioxidant activities. The increase in antioxidant activity was confirmed through the characterization of both native and modified pectin.

3.1. Yield of Extract

The yield of crude pectin extract was found to be 7.64 g/40 g, resulting in a % yield of 19.1%. The peels of Punica granatum showed maximum yield when ethanol was used as a solvent. Pectin was also extracted by using hexane and acetone as a solvent and yield was also calculated accordingly [23]. Hexene is a nonpolar solvent so the yield obtained with hexane was less than that obtained with ethanol and acetone (polar solvents) [36]. The yield of pectin obtained in hexane was in the range of 1 g/40 g, resulting in a percentage yield of 2.5%. The yield recorded with acetone was 3.04 g/40 g, resulting in a % yield of 7.6% as shown in

Table 1. Percentage yield of crude extract of pectin.

Plant Name	Part Used	Solvent Used	Yield of Pectin (g/100 g)
Punica granatum	Peels	Ethanol	19.1 ± 0.1
		Hexane	2.5 ± 0.05
		Acetone	7.6 ± 0.05

.

3.2. Modification of Pectin

The chemical modification of pectin was performed through amidation using the alcoholic ammonia method. The percentage yield of modified pectin from Punica granatum peel was obtained after precipitation with ethanol [37]. The ethanol was characteristically chosen as a precipitating solvent for antioxidant materials because of its excellent features in many aspects [26]. The data regarding the yield of modified pectin were obtained, which were 4.4/6 g and percentage yield being 73.3%. Pectin showed maximum yield when ethanol was used as a solvent.

3.3. Characterization of Pectin

The characterization of native and modified pectin was performed using Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) analyses.

3.3.1. Scanning Electron Microscopy (SEM)

The scanning electron microscopic images of native and modified pectin are presented in Figure 1 and Figure 2. The samples were analyzed by SEM with an accelerating voltage of 10 KV. Significant differences were observed in the surface structure and morphological characteristics of native and modified pectin [27]. The SEM photographs revealed that native pectin particles exhibited an irregular, granular rough surface. Similar irregular and rough surfaces were reported in previous studies on pectin extracted from apple pomace and mango [27].

The SEM analysis of modified pectin showed an indistinct appearance with a smooth surface and clotted formations after modification [38] likely due to removal of impurities, reduced particle size, and the aggregation of modified pectin. A comparison with commercial pectin also shows similar results due to small particle size in the observed morphology. The clear structural distinction between native and modified pectin highlights the impact of the modification process. While native pectin displays a rough fibrillar structure, the modification transformed it to a granular, smooth form, reflecting substantial morphological alterations.

From the analysis of the surface morphology of native and modified pectin, two key observations were made. First, native pectin, when dissolved in water, forms a less soluble viscous solution. This is likely due to impurities and the rough particle surface. Second, modified pectin, after amide group modification, becomes highly soluble, exhibiting a soft texture with homogenous micelle-like particles and a smooth, non-porous surface. This modification results in a decrease in viscosity of the aqueous solution. Thus, the SEM analysis clearly demonstrates that the rough surface structure of native pectin can be transformed into a smooth morphology after modification. A comparison of the SEM images across different figures confirms that significant structural changes have occurred due to the modification process.

3.3.2. Fourier Transform Infrared Spectroscopy (FTIR)

Information related to the molecular structures as well as presence of different functional groups can be obtained from the FTIR analysis. It also offers insights into the vibrations of groups in pectin segments [28]. The FTIR spectrum of native pectin, presented in Figure 3, reveals its chemical structure and characteristic wave numbers. In the FTIR spectrum of native pectin, a broad peak in the range of 3250 cm⁻¹ corresponds to hydrogen bonding and O-H vibrational stretching of pectin as reported in previous studies [28]. Previous studies have also reported that an FTIR analysis was effective to determine the configuration of pectin bands in the typical region between 890 and 2000 cm⁻¹ [39]. Key bands observed near 1537 cm⁻¹ and 1710 cm⁻¹ represented major chemical and functional groups. Additionally, the absorbance bands corresponding to C-O-C bonds were detected at 1011 cm⁻¹ and 775 cm⁻¹. Peaks at 775 and 1257 cm⁻¹ represented glycosidic linkages including C, C-O, C-O-H, and C-O-C stretching modes. The findings provide a detailed understanding of the structural features of native pectin.

The FTIR spectrum of modified pectin showed peaks at 1231 cm⁻¹ and 1018 cm⁻¹ attributed to glycosidic linkages and C-O-C stretching. These findings are further supported by a peak at 1715 cm⁻¹ due to the C=O stretching associated with water molecules, which also resulted in a band at 1610 cm⁻¹ in the spectrum [40].

In modified pectin, sharpening of the absorption band at 2352 cm⁻¹ indicates an increased association with water molecules, providing evidence of solubility of modified pectin as compared to the native pectin. The peaks observed in the spectra between 1569 cm⁻¹ and 2863 cm⁻¹ were associated with the presence of N-H bonds, confirming the incorporation of amide groups on modified pectin chains [41]. This demonstrated that the modification was completely achieved by the amidation process. The FTIR spectrum of modified pectin is shown in Figure 4.

3.3.3. Energy Dispersive X-ray Spectroscopy (EDX)

Energy dispersive X-ray spectroscopy was employed in order to determine the elemental composition of pectin. The qualitative results obtained through EDX were based on the single dataset, serving as a reference to understand the surface characteristics and morphology of both native and modified pectin [42]. The elemental composition of native pectin in the form of peaks is shown in Figure 5, obtained through EDX.

The EDX analysis showed that carbon was the predominant element, indicating the long carbon chains in pectin. Carbon content of the native pectin was found to be 57.16% by weight while oxygen was found to be 42.02% by weight, indicating the successful formation of COO– groups. Moreover, trace amounts of elements like chlorine, potassium, and calcium were also detected in native pectin [43]. The concentration and percentages of different elements in native pectin are shown in

Table 2. Concentration and percentage of different elements in native pectin.

Element	Line Type	Apparent Concentration (%)	k Ratio	Weight%
C	K series	64.2	0.64201	57.16 ± 0.01
O	K series	55.71	0.18747	42.02 ± 0.02
Cl	K series	0.73	0.00638	0.45 ± 0.05
K	K series	0.18	0.00151	0.1 ± 0.03
Ca	K series	0.44	0.0039	0.26 ± 0.05

below.

The elemental composition of modified pectin in the form of peaks is shown in Figure 6, obtained through EDX.

The EDX analysis of modified pectin showed increased carbon content as compared to native pectin, confirming the successful modification. The carbon concentration of modified pectin was found to be 58.25% by weight, while the oxygen content was 40.14% by weight. Additionally, a sodium content of 0.63 % by weight was applied to the further modification process [44]. Trace amounts of elements like chlorine, potassium, and calcium were also detected in modified pectin like in native pectin. The concentration and percentages of different elements in modified pectin are shown in

Table 3. Concentration and percentage by weight of different elements in modified pectin.

Element	Line Type	Apparent Concentration (%)	k Ratio	Weight %
C	K series	61.36	0.61364	58.25 ± 0.02
O	K series	48.2	0.1622	40.14 ± 0.04
Na	K series	1.12	0.00473	0.63 ± 0.05
Cl	K series	0.48	0.0042	0.32 ± 0.03
K	K series	0.59	0.00503	0.37 ± 0.02
Ca	K series	0.45	0.00403	0.29 ± 0.04

.

3.3.4. X-ray Diffraction (XRD) Analysis

The X-ray diffractograms for native and modified pectin are presented in Figure 7 and Figure 8, respectively. The acquisition of data was documented in the range of 2θ = 10–80° with a scan step of 0.01°. With the help of XRD, the scattering angle of 17.4628° was observed for native pectin, corresponding to a basal spacing of 5.09157 nm. Another scattering angle at 20.4234° was observed, with a basal spacing of 4.5435 nm, and intensities ranging from 50.18 up to 1010, respectively. These findings confirm the presence of amorphous and irregular arrangements in native pectin. Previous studies have also reported scattering angles of native pectin in the same ranges. For example, [45] observed a scattering angle of 20.05° at 2θ while [30] reported a scattering angle of native pectin at 19.95° at 2θ. These findings align with the findings of previous reports.

The X-ray diffractogram of the modified pectin showed a number of peaks at different angles and positions, 10.8278, 11.0873, 12.5674, 13.9878, 14.2938, 18.6854, 19.8943, 20.8175, 24.931, and 28.6789, with the relative intensities of 50, 93, 87, 102, 56, 66, 84, 101, 56, and 111, respectively, as shown in Figure 7. These peaks indicate significant structural changes due to the modification of pectin.

The X-ray diffractogram of modified pectin also showed diffraction peaks at 2θ = 10.2034, 11.0910, 12.2349, 13.0250, 14.9578, 15.0223, 16.5678, 17.4356, 18.9085, and 19.4523 with the relative intensities of 106, 89, 90, 103, 65, 87, 63, 87, 93, and 101. These results confirm the increase in crystallinity after modification as reported earlier [46]. The comparative analysis of native and modified pectin showed distinguished reflection peaks, with the modified pectin showing a greater number of peaks. This increase in crystalline peaks in the modified pectin was attributed to the incorporation of amide groups. The results of the X-ray diffractogram clearly demonstrate the structural changes caused by the modification.

3.4. Antioxidant Activities

Antioxidant activity of native and modified pectin was assessed by the hydroxyl radical antioxidant assay, DPPH radical scavenging assay, ferric-reducing antioxidant power, and phosphomolybdenum assay following the already reported methods [31].

3.4.1. Hydroxyl Radical Antioxidant Assay

The hydroxyl radical antioxidant assay was performed for both native and modified pectin by using ascorbic acid as a standard. The results of hydroxyl radical antioxidant activity confirmed that modified pectin performed excellent hydroxyl radical antioxidant activities as compared to native pectin. This enhanced activity was due to electron-donating and hydrogen-donating properties of amide groups introduced during the modification process [32]. Native pectin also showed hydroxyl radical antioxidant activity due to hydrogen-donating groups like COOH, CH₃, and OH. A comparison of the hydroxyl radical antioxidant activity of native and modified pectin with standard ascorbic acid is shown in Figure 9. All the results are in line with previous findings [47].

3.4.2. DPPH Radical Scavenging Assay

The antioxidant capacity of native and modified pectin was assessed by using the DPPH free radical scavenging assay. This method is preferred because of its sensitivity at very low concentration and its ability to analyze multiple samples in a short period of time [33]. The DPPH radical scavenging assay examined the ability of native and modified pectin to act as donors of hydrogen atoms or to transfer electrons to DPPH and reduce it. The increase in free radical scavenging capacity of native pectin and modified pectin was also investigated by increasing their concentration in a dependent manner. The DPPH radical assay firstly assessed the antioxidant property, which was confirmed when a color change occurred after reduction from purple to yellow [48]. DPPH radical scavenging activity was noticed for native and modified pectin.

When DPPH radical scavenging activity of native and modified pectin was compared, it was noticed that the radical capacity of modified pectin was greater than the native pectin.

This superior performance is attributed to its enhanced ability to donate hydrogen atoms or transfer electrons to DPPH and reduce it efficiently. DPPH radical scavenging activity of native and modified pectin compared with standard ascorbic acid is shown in Figure 10.

3.4.3. Ferric-Reducing Antioxidant Power

The ferric-reducing antioxidant power was achieved for both native and modified pectin. In this assay, higher reducing power corresponds to higher absorbance [34]. Ferric-reducing antioxidant power for both native and modified pectin was obtained with absorbance at 700 nm and compared with standard ascorbic acid. The results confirmed the findings of previous studies, which indicated a direct relationship between antioxidant activities and the reducing power of pectin [49,50]. An increase in the concentration of native and modified pectin led to an increase in reducing power with the maximum at a concentration of 100 µg/mL [49]. A comparison of ferric-reducing antioxidant power of native and modified pectin and that compared with standard ascorbic acid are shown in Figure 11.

3.4.4. Phosphomolybdenum Assay

The phosphomolybdenum assay is the essential principle to assess the antioxidant capacity of plant extract. In this study, the phosphomolybdenum assay was performed for both the native and modified pectin using ascorbic acid as a standard [35]. The antioxidant capacity of the phosphomolybdenum assay was expressed in millimolar equivalents of ascorbic acid/g of sample dried weight. The calculations were given by comparing values of native and modified pectin with those of standard ascorbic acid (IC₅₀: 72.3 ± 2.2 µg/mL) [51]. During the assay, the reduction of Mo (VI) to Mo (V) happened by the native pectin and modified pectin, confirming the presence of antioxidant compounds in extracts. The analysis of various fractions revealed that the modified pectin exhibited a more efficient reduction of the Mo (VI) to Mo (V) while native pectin showed less efficiency in this process. The phosphomolybdenum antioxidant power assay of native and modified pectin was compared with standard ascorbic acid as shown in Figure 12, indicating the high antioxidant activity of modified pectin.

4. Conclusions

The work was conducted as a part of a green initiative aimed at utilizing agricultural waste for beneficial purposes. Specifically, Punica granatum peel waste was used to meet the increasing demand of pectin worldwide. The fine powder of the desired part of the plant was prepared for extraction purposes, after the initial steps such as washing, drying, and grinding. In order to obtain high yield of native pectin from waste, acid-based extraction was performed and ethanol was used as a precipitating solvent. The native pectin was modified with amidation using the alcoholic ammonia method, which resulted in the increase in antioxidant capacity of modified pectin. In order to compare the structural and functional properties of native and modified pectin, characterizations were carried out using Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) analyses. The antioxidant power of both native and modified pectin was measured by the hydroxyl radical antioxidant assay, DPPH radical scavenging assay, ferric-reducing antioxidant power, and phosphomolybdenum assay, which showed an increase in antioxidant potential of modified pectin in comparison with native pectin. This research provides very valuable information and guidelines for the valorization of pomegranate peels and their value-added utilization, which are otherwise discarded as a waste. Moreover, it also ensures environmental sustainability by helping to reduce waste and clear the environment.