Influence of extraction techniques on the efficiency of pomegranate (Punica granatum L.) peel extracts in oxidative stability of edible oils

Abstract In this study, the effects of pomegranate (Punica granatum L.) peel extract (PPE) on the oxidative stability of soybean oil and ghee were investigated under heat conditions. Three extraction methods (immersion, ultrasound, and combined immersion‐ultrasound) with eight solvents (hot water, cold water, absolute methanol, methanol 50%, absolute ethanol, ethanol 50%, absolute acetone, and acetone 50%) were used for the evaluation of the extracts. Ethanolic extract in maceration method significantly (p ≤ .05) showed the highest DPPH radical scavenging activity (95.018%), reducing power (3.981), and total phenolic content (520 mg GAE/g) compared to the other samples. Then, the effects of PPE in various concentrations (200, 400, 600, and 800 ppm) were compared to the synthetic antioxidant (Butylated hydroxytoluene 200 ppm) in the oxidative stability of soybean oil under 65°C and ghee under 55°C for 24 days with 6‐day intervals, respectively. During storage period, all treatments showed a significant decrease (p ≤ .05) in peroxide value, thiobarbituric acid reactive substances, conjugated dienes value, polar compounds value, and acid value compared to the control. Except for the PPE 200 treatment, the other treatments exhibited superior efficiency to the synthetic antioxidant in a dose‐dependent manner in accelerated stored edible oils. Based on the sensory analyses (flavor, odor, color, and overall acceptability), PPE significantly (p ≤ .05) preserved the sensory features compared to the control group during the entire storage time. PPE 800 ppm was the most efficient treatment in all analyses, followed by PPE 600, 400, and 200 ppm, respectively. Finally, it was concluded that PPE can be introduced as a unique alternative to synthetic antioxidants in edible oils under heating conditions.


| INTRODUC TI ON
Edible oils are among the most consumed food products in the world. Therefore, a large amount of this product is produced and consumed in the world every year. Edible oil producers widely use soybeans for the production and formulation of edible oils. Soybean oil is very susceptible to oxidation reaction during storage time or heat conditions that can be related to its high polyunsaturated fatty acids (PUFA; Rahmati et al., 2022;Tinello & Lante, 2020). Another type of the popular edible oil is ghee. Ghee is typically prepared by simmering butter of sheep or cow milk. Ghee can be used in cooking, frying, and dressing and creates a palatable and pleasant odor and taste in foods (Ahmad & Saleem, 2020). Cow ghee is rich in free fatty acids (FFA), phospholipids, glycerides, sterol esters, sterols, carotenoids, and fat-soluble vitamins (Dhiman et al., 2022).
During storage, the edible oils undergo oxidation reaction that can be correlated to the storage temperature and oxygen availability.
The oxidation phenomenon of foods can lead to the loss of sensory features and customer-friendliness that can be due to the generation of off-flavor and off-odor compounds (such as peroxides, aldehydes, polar compounds, and conjugated dienes). Therefore, the use of antioxidants for preserving their quality is inevitable (Chen et al., 2021). High safety and quality of edible oils can improve consumer health and prevent economic losses. Synthetic antioxidants are considered unpleasant by consumers and they prefer natural alternatives in foods. Herbal products, such as various extracts and essential oils with high health benefits, have been recently considered as food preservatives. Pomegranate with scientific name of Punica granatum L. from the Punicaceae family is a strategic commercial fruit crop that is widely cultivated in the Mediterranean, the Middle East, Asia, and North Africa. This product has many uses that lead to the production of a large amount of waste, including peel, seed, and pulp every year. Pomegranate peel extract is rich in bioactive phenolic and flavonoid compounds, such as anthocyanins, gallotannins, hydroxycinnamic acids, hydroxybenzoic acids, and hydrolyzable tannins, that is, ellagitannins, and gallagyl esters and complex polysaccharides (El-Hadary & Taha, 2020;Rashid et al., 2022). Numerous investigations have reported the antimicrobial, antioxidant, and therapeutic properties of PPE Rashid et al., 2022;Trigo et al., 2020). In addition to the biological activities of pomegranate products, pomegranate derivatives can also be used as food colorants and flavor enhancers (More et al., 2022). Accordingly, this study aimed to measure the antioxidant activities of the produced PPE using immersion (maceration), ultrasound, and combined immersion-ultrasound techniques with various solvents and evaluate their efficiency on chemical and sensory characteristics of soybean oil and ghee stored at 65 and 55°C for 24 days with 6-day intervals in comparison with the chemical antioxidants, respectively.

| Preparation of PPE
Pomegranate peels were provided by a pomegranate processing plant in Hamedan. After drying the samples in environment temperature for 2 weeks, the samples were ground in the grinder (AR110O10, Moulinex) and mixed to the absolute and aqueous (50%) ethanol, methanol, acetone, and water (hot and cold) solvents with the ratio of 1:10 (powder/solvent) and then, were extracted by immersion (maceration), ultrasound, and ultrasound-immersion methods. In the immersion procedure, the samples were shaken by the Earlene shaker (Fan Azma Gostar) at 250 rpm overnight at room temperature. For the immersion method by hot water, ground pomegranate peel (100 g) was refluxed with 1000 mL of the boiling distilled water (DW) for 1 h. In the ultrasound technique, a probe ultrasound (UP400ST, Hielscher) was considered under the power of 50, frequency of 20 kHz, and the temperature of 25°C for 30 min.
In the ultrasound-immersion method, first, the mixtures were subjected to ultrasound waves. Then, the obtained solutions were extracted by the immersion method. After filtering and concentrating the obtained solutions at 40°C by a rotary evaporator apparatus (Lab Tech), the solvent residue was evaporated by the vacuum oven (Fan Azma Gostar) at 50°C in all methods. The prepared extracts were stored in the laboratory freezer (Fan Azma Gostar) at −18°C for the next tests (Albu et al., 2004;Barkhordari & Bazargani-Gilani, 2021;Pan et al., 2008).

| Antioxidant activity of PPE
2.3.1 | DPPH radical scavenging activity DPPH radical scavenging activity (RSA) of the studied extracts was determined based on the method of Blois (1958). A volume of 50 μL of the extracts was mixed with 2 mL of methanol DPPH (24 μg/mL) solution and mixed. The obtained solution was stored in dark at environment temperature for 60 min and the absorbance was read at 517 nm using a spectrophotometer (Thermo Spectronic, Helios Gamma). The DPPH RSA was calculated by the following equation: where A blank and A sample are the absorbance of the blank and extracts, respectively. BHT solution (2 mg/mL) was used as the control.

| Reducing power
For reducing power measurement of the extracts, the method of Oyaizu (1986) was used. A volume of 1 mL of the extracts was mixed with 2.5 mL of the sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%). After 20 min incubation at 50°C, 2.5 mL of trichloroacetic acid (10%) was mixed with the resulting solution. Then, the mixture was centrifuged at 1789 g for 10 min.
In the end, 2.5 mL of the mixture was added to 0.5 mL of ferric chloride (0.1%) and 2.5 mL of the DW and incubated for 10 min. Then, the absorbance was measured at 700 nm using a spectrophotometer (Thermo Spectronic, Helios Gamma), against blanks that contained all used reagents except for the extracts. Higher absorbance showed higher reducing power. The standard solution was prepared by BHT in the concentration of 2 mg/mL.

| Total phenolic content
Folin-Ciocalteu reagent test was used for total phenolic measurement. Briefly, 500 μL of the extracts was mixed with 2.25 mL of DW and then, 250 μL of the Folin-Ciocalteu reagent was added. The mixture was vortexed for 1 min and allowed to react for 5 min. Then, 2 mL of sodium carbonate (7.5%) was added. After incubation at room temperature for 120 min, the absorbance of each mixture was measured at 760 nm using a spectrophotometer (Thermo Spectronic, Helios Gamma). The same procedure was also used for a standard solution of gallic acid. Gallic acid solution was considered for drawing the standard curve. The total phenolic content was determined as milligram of gallic acid/gram of the sample (Machu et al., 2015).

| Conjugated diene value
The method of Saguy et al. (1996) was used for conjugated diene value (CDV) measurement of the samples. The oil sample was diluted (1:600 for the studied oils) with hexane (HPLC grade). An extinction coefficient of 29,000 mol/L (Privett & Plank, 1962) was utilized to quantify the concentration of conjugated dienes formed during oxidation. The absorbance of the diluted oils was read at 234 nm using a spectrophotometer (Thermo Spectronic, Helios Gamma) against hexane as blank.

| Polar compounds value
Nonpolar compounds were determined based on the method of Schulte (2004). A glass column (15 cm in length and 1 cm in diameter) packed with the hydrated silica gel (water/silica gel in the ratio of 5:95) was used for chromatography. The eluent was a mixture of isohexane and diisopropyl ether in the ratio of 85:15 (v/v). The oil sample (0.5 g) was loaded into the packed column and the nonpolar fraction was eluted by the eluent. After collecting the eluent, nonpolar compounds weight was determined. Then, polar compounds value (POV) was calculated by the following equation: where W s is the weight of the sample and W n is the weight of the nonpolar compounds.

| Peroxide value
The peroxide value (PV) test measures the amount of generated peroxides in the samples. According to the method of the International Dairy Federation (Shantha & Decker, 1994), PV was measured. The sample (0.30 g) was mixed with 9.8 mL chloroform-methanol (3:7) in a glass tube. After adding 0.05 mL of the ammonium thiocyanate solution (10 mM), the sample was vortexed. Next, iron solution (II) (0.05 mL) was mixed with the resulting solution and agitated. Then, the samples were incubated at room temperature for 5 min. The absorbance of the mixture was read at 500 nm, using an Ultraviolet-visible spectrophotometer (Thermo Spectronic; Helios Gamma). PV of the treatments was calculated as milliequivalents (meq)

| Statistical analysis
This study was cross-sectional and experimental with controlled trial and replicated twice. All the tests were performed in triplicate for every repetition (n = 2 × 3). The collected data were statistically analyzed by SPSS (IBM SPSS statistics 21) software and considered as mean values ± standard deviations (SD). The analysis of variance (ANOVA) and Tukey test were used at the significance level of p ≤ .05 to compare the means.  released FFAs are very sensitive to the oxidation reaction, following the oil hydrolysis, oxidation is intensified (Rahmati et al., 2022). The oxidation reaction of edible oils is the most common phenomenon, which is accelerated by increasing their storage time. According to (30%) substances in pomegranate peel that exhibit significant antioxidant activity effectively, preventing lipid oxidation (Kaderides et al., 2021;Selahvarzi et al., 2022). In general, PPE has the potential to improve the functional features of various food products (Selahvarzi et al., 2022;Trigo et al., 2020).

| RE SULTS AND D ISCUSS I ON
Due to the high unsaturated fatty acid content in soybean oil, POV and CDV were measured only in the studied soybean oils. POV is dependent on linoleic acid (C18:2) and trans-oleic acid (trans-C18:1) content in the edible oils. The determination of total polar compounds is used as the main indicator in the evaluation of edible oil quality. The highest allowable value of total polar compounds is in the range of 24%-27% in edible oils (Chen et al., 2021). These oxidation products can damage human health and lead to a decrease of body weight and lipid content of tissues, liver, and blood. Therefore, the POV measurement in edible oils F I G U R E 3 Polar compounds value (POV) (a) and conjugated diene (CDV) (b) of the treated oils. Different letters within the same day (a, b, c, etc.) and the same treatment (A, B, C, etc.) show a statistically significant difference (p ≤ .05).
containing unsaturated fatty acids is essential in the evaluation of their quality (Xu et al., 2022). Figure 3a illustrates POV of the studied samples during storage period. There are no significant differences in POV (4.15%-5.75%) among the studied treatments on day 0 of the stor- respectively. In one study, higher significant oxidative stability of sunflower oil containing 800 ppm of PPE was reported in comparison with the synthetic antioxidant (BHT 200 ppm) (Ibrahium, 2010).
In another study, PPE 250, 500, and 1000 ppm could delay corn oil deterioration during storage time, which was positively related to the concentration of PPE. The high amount of phenolic and flavonoid compounds of PPE have been reported to be responsible for this stability (Konsoula, 2016).

| Sensory analysis
Tables 1 and 2 represent the sensory findings (taste, odor, color, and overall acceptability) of the studied samples during

ACK N OWLED G M ENTS
This study was sponsored by the Faculty of Veterinary Science, Bu-Ali Sina University in Hamedan, Iran.

CO N FLI C T O F I NTE R E S T S TATE M E NT
No conflicts of interests were declared by the authors in this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. TA B L E 2 Changes in sensory features of ghee during accelerated storage period.