Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties Vol. 13 (2), December 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 123 Op e n Ac c e s s F u l l L e n g t h A r t i c l e Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties Naila Siraj1*, Saima Tehseen1, Muhammad Asim Shabbir2 1Department of Food Science and Technology, Government College Women University, Faisalabad, 38000, Pakistan. 2National Institute of Food Science and Technology, University of Agriculture, Faisalabad, 38000, Pakistan. A B S T R A C T Background: Pomegranate seeds are the remains of industrial processing of pomegranate fruit into various products such as juice, jam, and jelly that cause environmental pollution and a loss of lipid and water-soluble bioactive compounds. The seeds comprise 15- 25% of the whole fruit weight. Its lipid fraction has a distinct fatty acid profile with high polyunsaturated fatty acid contents such as punicic acid (PA) (50-80%), tocopherols, and phenolic compounds. Objective: The objective of the current study was to characterize the three main Pakistani pomegranate varieties (Desi, Kandhari, and Bedana), supporting waste exploitation strategies. The physicochemical characteristics, fatty acid profile, antioxidant potential of all three varieties were studied. Methodology: The extracted oils samples were investigated for their lipid contents, fatty acid composition, physicochemical properties, and antioxidant potential on the varietal base. Results: Total lipid contents ranged from 8.70-15.9% dry matter with conjugated linolenic acid (68.5-76.7% of total fatty acids) as the predominant fatty acid in all samples, followed by linoleic, oleic acid, stearic acid, and palmitic acid. Further characterization revealed that Desi was superior regarding acidity and oxidation stability over the other two varieties associated with higher total phenolic and tocopherol contents with potent antioxidant activities. Conclusion: The present results demonstrate a preferable fatty acid pattern in pomegranate seed oil (PSO), which may be potential functional ingredient for product development in food and non-food applications in combination with high antioxidant contents. Keywords pomegranate, fatty acids, antioxidant potential, seed oil, waste utilization *Address of Correspondence nailaateeq@gcwuf.edu.pk Article info. Received: January 26, 2022 Accepted: December 27, 2022 Cite this article: Siraj N, Tehseen S, Shabbir MA. Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties. 2022; 13(2):123-133. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. I N T R O D U C T I O N The earliest known fruit plant is Pomegranate (Punica granatum L.), instigating from Ponum granatum, which means seeded or grainy apple, native to Central Asia and Persia1. It is grown in India, Turkmenistan, Pakistan, North Africa, China, Afghanistan, and some Mediterranean countries. USAID (The United States Agency for International Development) estimates global production to be between 3.80 million tons in 2017, with an expected increase in the next years following the risen global awareness of pomegranate fruit’s health benefits2. In Pakistan, the pomegranate’s central cultivation area is in Baluchistan, whereas Punjab and Khyber Pakhtunkhwa O R I G I N A L A R T I C L E Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties Vol. 13 (2), December 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 124 produce pomegranates on a small scale on isolated farms. The best-known varieties in Pakistan are Desi, Kandhari, and Bedana, with the primary harvest season in September but are available around the year when stored under appropriate conditions3. The industrial transformation of fruit and vegetables into food products generates enormous by-products, many of which are considered high value due to their high content of different bioactive compounds4. Pomegranate seeds are the remains of industrial processing of pomegranate fruit into various products such as juice, jam, and jelly that cause environmental pollution and a loss of lipid and water- soluble bioactive compounds. The seeds comprise 15-25% of the whole fruit weight. Its lipid fraction has a distinct fatty acid profile with high polyunsaturated fatty acid contents such as punicic acid (PA) (50-80%), tocopherols, and phenolic compounds. Omega-6 and omega-3 PUFAs are the two primary groups of PUFAs that are relevant to human health. In most diets, linoleic acid (LA, 18:2-6) and α-linolenic acid (ALA, 18:3-3) are the most abundant PUFAs. LA and ALA are considered animals that cannot produce essential fatty acids since these. Because these PUFAs are produced in plants, LA and ALA are mostly found in high concentrations in plant-based foods. Low essential fatty acid intakes have been linked to dermatitis, renal hypertension, mitochondrial activity problems, cardiovascular disease, type 2 diabetes, impaired brain development, arthritis, depression, and decreased body response to infection. Recent research on pomegranate peels has demonstrated good antioxidant potential and antimicrobial activity. Until recently, PSO considered superior nutritional quality for usage in food applications. However, it has been suggested to have great potential to reduce the risk of cardiovascular disease, inflammation, and cancer, as well as relieve menopausal symptoms and support immune functions5. Although the optimization of PSO extraction and characterization of fatty acid profiles have been reported in several studies, there is only limited evidence available on the physicochemical characterization of PSO6. Whereas some data were published on phytochemical and antioxidant characteristics of PSO from varieties grown in the USA, Iran, India, Turkey, and China7, there is a lack of knowledge regarding seed oils from Pakistani pomegranate varieties. Therefore, this study explores the pomegranate seed residues (PSRs) of three Pakistani pomegranate varieties as a material with potential economic value by referring to its lipid yield, fatty acid profile, physicochemical properties, and antioxidant potential. M A T E R I A L S A N D M E T H O D S Materials Approx. 15 kg fresh, fully ripe, and good-quality pomegranate varieties (Desi, Kandhari, and Bedana) were procured from the local market of District Faisalabad, Pakistan. A certified fatty acid methyl ester (FAME) reference standard mixture was obtained from Sigma- Aldrich (Bellefonte, PA, USA). Standards of gallic acid, tocopherols (α (≥96%), β (≥97%), γ (≥96%), δ (≥97%)), Folin-Ciocalteu reagent, 2,2'-azino bis (3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2- carboxylic acid (Trolox), and 2, 2-diphenyl-1 picrylhydrazyl radical (DPPH) purchased from Sigma-Aldrich (St. Louis, MO, USA). All tests used ultra-pure water generated with < 5 ppb TOC and 18.2 m X resistivity (Merck, MA, USA) by integrated Water Purification System Milli-Q. All other chemicals and solvents were obtained from Fisher Scientific Co. (Nepean, Ontario, Canada) of the American Chemical Society (ACS). Pomegranate seed oil extraction Sugars and other clinging components were removed from pomegranate seeds by introducing Ju 2000 Vitae (Moulinex, Barcelona, Spain) in a blender for juice extraction. The pomegranate seed was ground in a regular domestic coffee grinding machine and rinsed and dried in the sun (2-3 days) by passing through 30–40 mesh screens. The pomegranate seed powder was dried at 60ºC for 48 h and kept at -18°C until further use (7±1% moisture content). The oil was extracted from the pomegranate powder using the standard Soxhlet extraction method for 24 h (3 * 8 h) for each variety with hexane as a solvent. A rotary evaporator was used to evaporate the hexane at reduced pressure, followed by drying under nitrogen to remove the remaining hexane. The collected oil samples were preserved at -20oC in amber glass jars for further research. The extraction yield (%) was determined by Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties Vol. 13 (2), December 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 125 dividing the volume of obtained oil (mL) by the original amount of dry matter (g) multiplied by 1008. Fatty acid profile Fatty acid methyl esters (FAME) of oil samples generated utilizing the KOH-methanol method according to Jing et al. (2012)7 procedure. A 20 mg oil sample was combined with 2 mL iso-octane and 0.2 mL KOH-methanol. After 5 minutes at room temperature, add 2 mL iso-octane and 3 mL water to the mixture. The supernatant was then drained and thoroughly rinsed with water. The uppermost iso- octane layer was recovered and used for GC-MS (Model: 14-A, Shimadzu, Japan). A DB-5MS column (30 m x 0.25 mm with a 0.25 µm film thickness) used with carrier gas helium flowed at a 1.0 mL/min rate. The sample was injected as 0.5 µL at a split ratio of 5:1. Initially, the temperature was set to 70°C for 5 minutes, up from 10°C/min to 200°C and then to a final temperature of 275°C at the rate of 5°C/min. MS spectra were obtained with an ion source temperature of 200°C at an ionization energy of 70 eV and an interface temperature of 260°C. An integrator was used to quantify fatty acid contents by estimating the area under individual peaks compared to the total area under all peaks. FAME preparation of PA standard Following Hashempour et al. (2010)9, the methyl esters of the PA standard were produced independently. Trimethylsilyl diazomethane reagent interacted with the standard. 10 mg of PA were combined with 1 mL of methanol/toluene (1:4, v/v), and then 2 mol/L trimethylsilyl diazomethane (100 L) were added. Following 30 minutes at room temperature, the mixture was added to 2 mL iso- octane and 5 mL water, with the supernatant subjected to GC-MS after phase separation. Physicochemical analysis A Sopelem series 3296 refractometer (Sopelem, France) was used to calculate the refractive index (RI) according to the method of Godswill et al. (2018)10 with some modifications. The specific gravity (SG), acid value (AV), iodine value (IV), and saponification value (SV) of recovered oil samples were determined using the AOCS protocols (2006)11. The unsaponifiable matter was estimated following the technique outlined in AOAC12 (2016). The viscosity was measured using a viscometer (model LV DVII-Brookfield, Middleboro, MA, USA). The values recorded as Pa.s. Finally, the melting and flashpoints were determined by following the AOCS (2006) methods11. Tocopherol contents Following Katsanidis and Addis’s method (1999)13, the tocopherol contents were determined using HPLC (Model: Perkin Elmer Series 200, USA). The analysis was carried out using an isocratic mode normal phase HPLC column (250mm x 4.6mm, 5.0m particle size) with a mobile phase of iso-octane/ethyl acetate (96:4 v/v). Simultaneously, the detector was tuned to 290 nm excitation and 400 nm emission wavelengths, with a run time of 30 minutes and a flow rate of 1.0 mL/min. The temperature in the column was 35°C. Individual oil samples or standards were dissolved in hexane, sterile filtered, and injected in a 2.0 µL volume. For every standard (α, β, γ, and δ tocopherol), an external calibration curve was created and utilized to estimate the volume of tocopherol present in the oil samples. After eight (08) injections, the silica column was re-activated using a 10% isopropanol in a hexane solution to increase its efficiency14. Duplicate samples were prepared. Total phenolic content (TPC) After oil extraction, the residual PSRs from all three types were dried at room temperature overnight. Approximately 1 g of dried defatted PSR was extracted in ultrasonic waves with 10 mL of 80% methanol at room temperature and centrifuged for 10 minutes at 2500 g/s. The supernatants were collected and kept at 4°C in the dark until further examination. According to Mohdaly et al. (2011)15, TPC was measured using the Folin-Ciocalteu reagent. The prepared methanolic pomegranate seed extracts (50 mg) were combined with 0.5 mL Folin reagent and 7.5 mL deionized water, then incubated for 10 minutes at room temperature with 20% sodium carbonate (w/v). The mixture was placed in a water bath at 40°C for 20 minutes before cooling in an ice bath. The absorbance was measured using a spectrophotometer at a wavelength of 765 nm (U-2001, Hitachi Instruments Inc. Tokyo, Japan). The TPC was measured using a 10-100 g/mL gallic acid standard curve (R2 = 0.990). Gallic acid equivalents (GAE) were calculated in mg/g of oil. Both samples were tested three times. Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties Vol. 13 (2), December 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 126 Determination of antioxidant activity Radical scavenging activity was measured according to Siddiq et al. (2013)16 using a DPPH solution in methanol. DPPH stock solution (2.4 g/l methanol) was diluted with 800 ml/l methanol to create a working solution (A515 w1.1). Blank, standard, or sample (0.6 ml) was combined with 3.0 ml of DPPH working solution, maintained in the dark for 20 minutes, and measured absorbance. Blank (ethanol acetone, 7:3) was utilized to calculate radical scavenging activity. Using 50-250 mmol/l Trolox solution, a standard curve was created. The Trolox equivalent antioxidant capacity (TEAC) was determined using a modified ABTS radical cation decolorization test16. Briefly, 7 mmol/l ABTS solution and 2.45 mmol/l potassium persulfate were combined in a 1:1 ratio and left in the dark for 12-16 hours to form ABTS.+ radical cation (ABTS.+). This solution was diluted with methanol (800 ml/l) so that its absorbance at 734 nm was between 0.700 and 0.020. The diluted ABTS.+ solution (3 ml) was combined with 30 ml of blank, standard, or sample, and the absorbance was measured at 734 nm using a spectrophotometer after a 6-minute reaction. The blank was processed with ethanole: acetone (7:3). Using Trolox solution (0.3-1.5 m mol/l), the standard curve for calculating antioxidant capacity was generated. The experiment indicated the antioxidant capabilities of the samples as m mol Trolox equivalents (TE)/100 g fresh weight (FW). Oxidative status analysis The peroxide (POV) and para-anisidine (p-AV) values were estimated according to the AOCS (2006) method11. Conjugated dienes (CD) and conjugated trienes (CT) were determined at 232 and 270 nm, respectively. Oil samples were diluted with iso-octane to change the absorbance within limits, as per the standard IUPAC form. Statistical analysis and data interpretation A one-way analysis of variance ANOVA was used in the statistical study, and Duncan’s multiple range test was used to assess the significant difference between means. Differences at p≤0.05 were considered statistically significant. The results are presented as mean±SD of triplicate measurements unless indicated otherwise. R E S U L T S A N D D I S C U S S I O N Lipid contents and fatty acid profile The economically viable PSO exploitation depends on extraction technology, yield, and oil characteristics and quality. That might determine the potential of the oil source for downstream applications, such as food and non-food- based products (Figure 1). The total oil content in the current investigation ranged from 8.7-15.9% in the three varieties (Figure 2), which is comparable to previous studies conducted on PSO from Iranian cultivars (6.6-19.3%)18, Turkish cultivars (7.6- 16.2%)19 and Indian cultivars (7.0-19.0%)20. The kind, number, and placement of fatty acids within the glycerol moiety determine the oil’s nutritional and physicochemical properties. Therefore, they can vary considerably in plant oils, depending on their source. While comparing different extraction methods for PSO extraction, the highest oil yield was observed by the Soxhlet method (15.66%)21. While six primary fatty acids could be identified in PSO samples (Table 1), the predominant fatty acid is conjugated linolenic acid (CLnA, C18:3), also known as PA, considered one of the principal and active PSO components responsible for its antioxidant potential. The average PA values of Bedana (68.5%), Kandhari (75.5%), and Desi (76.7%) are following previous studies in cultivars from Georgia (78-83%)22, China (73-79%)7, Italy (72.20-84.10%)19, India (31.80- 86.60%), Turkey (70-76%)20. Other sources for PA have, on average, a lower PA content, such as seed oils from Catalpa bignonioides (28%), Momordica charantia (59%), Prunus mahaleb (28%), Trichosanthes kirilowii (36%), and Calendula officinalis (30%)23. Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties Vol. 13 (2), December 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 127 Figure 1. External view and cross section of the selected Pakistani pomegranate varieties (a) Desi (b) Kandhari (c) Bedana. Figure 2. Oil recovered from three Pakistani pomegranate varieties (a) Desi (b) Kandhari (c) Bedana Table 1. Lipid contents and fatty acid composition of pomegranate seed oil Values in the same row with different letters are significantly different (p < 0.05). SFA, saturated fatty acids; USFA, unsaturated fatty acids. Oleic (C18:1; 10.4-12.8 %) and linoleic acid (C18:2; 5.4- 12.0 %) are prominent second and third fatty acids and are somewhat higher compared to other studies investigating PMO-composition growing in various countries (Spain, Israel, Turkey, Iran, Tunisia)19. The total saturated fatty acids (SFAs) were between 6.0- 8.5%; the main SFAs are palmitic, stearic, and myristic acid, as seen in table 1, which is significantly lower than other edible oils, such as 15% in soybean oil and pumpkin seed oil24, 25. Stearic and palmitic acid values are comparable with Turkish (1.4-2.8%, 1.4-2.0%), Chinese (2.8-3.6%, 1.6-2.8%), and Georgian pomegranate cultivars (2.8-4.8%, 2.1-3.6%)22, 20. Consequently, saturated to unsaturated FA (SFA/UFA) ratios align with data on cultivars from Spain, Iran, Turkey, Israel, and Tunisia19. Besides cultivar variation, differences in PSO oil content and lipid profiles are most likely due to different climate conditions, local growth, and environmental influence. High Pomegra nate varieties Lipid contents (g/100g) C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 SFA UFA SFA/UF A Desi 15.90±0.0 4 a 0.51±0 .01 a 4.60±0. 04 c 0.75±0. 01 b 10.61±0. 33 a 6.73±0.0 9 b 76.66±1. 12 a 6.00±0. 15 c 94.00±0. 20 a 0.06±0. 02b Kandhari 11.00±0.0 5 b 0.34±0 .02 b 5.30±0. 08 b 2.92±0. 50 a 10.40±0. 19 c 5.42±0.4 0 c 75.50±0. 50 b 8.50±0. 28 a 91.50±0. 78 c 0.09±0. 01ab Bedana 8.70±0.0 c 0.15±0 .01 c 6.18±0. 50 a 0.67±0. 10 c 12.75±0. 22 b 12.00±1. 02 a 68.52±1. 63 c 6.80±0. 13 b 93.20±0 .70 b 0.07±0. 02b Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties Vol. 13 (2), December 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 128 quantities of unsaturated fatty acids, such as linolenic, linoleic acid, and PA, are preferred for human consumption, which may enhance PSO usage in human nutrition. Physicochemical properties Physicochemical characteristics are significant in evaluating edible oil’s present condition and quality. Therefore, several parameters, such as IV, SV, and AV, are used to monitor oil quality and determine the required level of refinement26 (table 2). The iodine value is useful for determining unsaturation but does not identify the individual fatty acid27. A relatively high iodine value was observed in the Desi variety, while other varieties showed a lower iodine value in Kandhari and Bedana, respectively. Due to the high level of punicic acid in oil samples, the higher iodine value indicates a greater number of unsaturated bonds. Besides, the AV, a measure of free fatty acids and inversely proportional to the degree of edibility of an oil25, was significantly lower in Desi than in the other two varieties. The IV and AV of Iranian pomegranate varieties and cold-pressed oil were 179.4- 215.90 and 216.5-220.34g I2/100g and 3.78-8.36% oleic acid, while own data on AV are more comparable with cold- pressed PSO oil with 0.55-3.78% oleic acid28. Short-chain fatty acids have a higher SV than longer-chain fatty acids, with a higher number of carbon atoms in triacylglycerols29. The SV was moderately significant; the highest was observed in Bedana following Kandhari and Desi, similar to Dadashi et al. (2013)27 findings. PSO has a substantial percentage of high molecular weight fatty acids, as seen by the greater SV of oil samples, equivalent to other culinary oils used in everyday life. The differences in unsaponifiable materials reported across three cultivars could be due to the distinctive morphology of pomegranate fruit and the different timing between horticultural maturity for commercial harvesting and physiological ripening. The physical characteristics of oils depend on the chemical and functional bodies present with glycerol moiety that ultimately influence the behavior of lipids in food and the procedures for processing and manipulating these lipids. The RI varies significantly with earlier studies conducted on PSO possessing 1.47-1.51 and 1.46-1.52 RI by Elfalleh et al. (2007)31; Dadashi et al. (2013)27, respectively. SG was observed highest in Bedana, relatively higher than Iranian pomegranate varieties and cold-pressed oil (0.92 to 0.93 and 0.91-0.92 g/cm3)27. Different studies conducted on PSO and evaluated their physicochemical properties agree with our results27. Different extraction methods exert no significant effect on the physicochemical attributes of PSO21. Tocopherol contents Tocopherols (α, β, γ, δ) are naturally occurring antioxidants enriched in vegetable & fruit seed oils with varying contents, depending on their source and with a generally more considerable difference between different oils compared to within cultivar variation. Tocopherol contents in seed oils usually range between 0.20-80 mg/100g, and α-tocopherol is the main tocopherol except for 𝛽 and γ. The total tocopherol content of Pakistani PSO, ranging from 2.5-3.8 µmol/g, is comparable with other natural sources like blueberry, marionberry, onion, cardamom, and milk thistle seed oils23. Table 3 shows that the three cultivars differed in total tocopherol content and composition. Desi has significantly higher overall tocopherol contents than the other cultivars (p≤0.05). The tocopherol contents observed in different pomegranate cultivars worldwide showed different concentrations in seed oil; Elfalleh et al. (2011)31 observed the following pattern α-tocopherol> γ-tocopherol> δ- tocopherol for Tunisian pomegranate seed oils. Jing et al.7 (2012) indicated the order δ-tocopherol> α-tocopherol> γ- tocopherol for seed oils of Chinese pomegranates, and Caligiani et al. (2010)32 found the δ-tocopherol> α- tocopherol> γ-tocopherol pattern in pomegranate oil samples. The varying tocopherol content of Pakistani pomegranate cultivars could be attributable to genetic differences or environmental factors. The α-tocopherol contents are analogous to 718.70-1388.30 µg/g in China- grown pomegranate cultivars seed oil7, 1576-1786 µg/g Tunisian cultivars31, and 1612-1737 µg/g Georgia cultivars21. The γ-tocopherol was much lower than studies conducted earlier; 814-928 µg/g in Georgia-grown cultivars and 957-1154.5 µg/g in Tunisian-grown cultivars31. Also, δ- tocopherol is comparable with Tunisian cultivars (241.90- 299.10 µg/g)31 and Georgia cultivars (214-238 µg/g)7. Results are parallel with Liu et al. (2022), who compare different extraction methods for PSO extraction21. Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties Vol. 13 (2), December 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 129 Table 2. Physicochemical analysis of pomegranate seed oil Pomegranate Varieties Desi Kandhari Bedana Specific gravity 0.98±0.01 0.97±0.03 0.99±0.01 Viscosity (Pa.s; 25ºC) 0.12±0.03 0.13±0.02 0.11±0.01 Refractive index 1.47±0.03b 1.43±0.01c 1.50±0.04a Density (g/cm3) 0.91±0.03b 0.90±0.21b 0.92±0.01a Melting point (ºC) 41.50±0.66b 42.00±0.81b 45.00±0.71a Flash point (ºC) 100.0±1.61c 103.0±1.19b 106.00±2.36a Acid value (% oleic acid) 3.77±0.05b 3.98±0.18a 4.00±0.08a Saponification value (mg KOH/g) 181.10±1.08b 181.50±1.50b 184.30±1.05a Unsaponifiable matter (%) 1.04±0.04a 0.90±0.12b 0.80±0.07bc Iodine value (g I2/100 g oil) 134.90±2.25a 123.50±1.60b 120.40±1.95c Odor Characteristics pomegranate odor Characteristics pomegranate odor Bland Color Dark yellow Dark Yellow Light yellow State at ambient temperature Liquid Liquid Liquid Vapor properties Negligible at ambient temperature Negligible at ambient temperature Negligible at ambient temperature Values in the same row with different letters are significantly different (p < 0.05). Table 3. Tocopherol composition of pomegranate seed oil Pomegranate Varieties Desi Kandhari Bedana α-Tocopherol (µg/g) 1211.10±18.03a 890.80±10.11b 768.63±20.09c γ- Tocopherol (µg/g) 50.21±2.30a 42.50±1.45b 35.10±2.39c δ-Tocopherol (µg/g) 300.51±4.31a 256.90±3.90b 245.71±4.50c Total tocopherols (µg/g) 1561.82±7.90a 1190.20±10.87b 1049.44±7.01c Values in the same row with different letters are significantly different (p < 0.05). Antioxidant activity The natural food sources like fruits and vegetables are enriched with phenolic compounds that function as imperative antioxidants; these exhibit the ability to donate an electron or hydrogen atom to a free radical to form a stable radical and prevent the auto-oxidation chain reactions in biological membranes. Table 4 shows the TPC range between 3.4-4.9 mg GAE per g DW. Desi is offering the highest TPC, following Kandhari and Bedana. Pakistani pomegranate seeds contain higher TPC than other oils, e.g., soybean, sunflowers, maize, grapes, hemp, flax, and pumpkin34. However, Khoddami et al. (2014)35 found higher TPC in cold-pressed PSO of three cultivars of 10.44 mg GAE g-1 oil. The DPPH• radical system is frequently used to evaluate antioxidant activity in vitro and in vivo studies. Rojo-Gutiérrez et al. (2021) found 91.29 and 98.28% scavenging activity against DPPH and ABTS radicals, respectively, in PSO extracted through green extraction methods (microwave-assisted and ultrasound-assisted extractions)36. Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties Vol. 13 (2), December 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 130 Table 4. Antioxidant Potential and oxidative stability of pomegranate seed oils Pomegranate Varieties Desi Kandhari Bedana Total phenolic contents (mg GAE/g oil) 4.94±0.58 a 4.30±0.83 b 3.40±0.38 c DPPH. (µM Trolox/g oil) 67.21±2.82 a 56.36±3.06 b 52.70±2.59 c ABTS.+ (µM Trolox/g oil) 71.90±3.27 a 65.10±3.73 b 61.35±2.03 c Peroxide value (meq of O2/Kg) 1.21±0.05 c 1.54±0.30 b 1.98±0.10 a Para-anisidine value 0.30±0.01 c 0.55±0.15 b 0.77±0.08 a Conjugated dienes (λ232 nm) 0.22±0.24 c 0.75±0.05 b 0.99±0.12 a Conjugated trienes (λ270 nm) 0.04±0.02 b 0.07±0.01 ab 0.09±0.02 a Values in the same row with different letters are significantly different (p < 0.05). Because several approaches might produce significantly divergent results, no single method is suitable for measuring the antioxidant potential of foods. Various methods must be utilized, each based on a different mechanism. Table 4 represents the DPPH. Scavenging activities of seed oil of three Pakistani pomegranate cultivars dependent on the concentration by quenching the DPPH• depicting increased contents increased the DPPH. Scavenging activity of oil samples. Desi had considerably higher DPPH• scavenging activities (p≤0.05) than Kandhari and Bedana. Desi variety showed the highest DPPH. Scavenging activity (67.78±2.82%) follow by Kandhari (56.83±3.06%) and Bedana (52.38±2.59%). Table 4 represents the ABTS•+ scavenging activity of oil samples and shows the antioxidant activity comparable with some artificial antioxidants. Compared to other oil samples, the Desi variety oil samples had the highest ABTS•+ intensity (71.48±3.27%). The variation in results may be due to various factors like radical’s stereo- selectivity or solubility of the sample in different radical scavenging systems reported to upset natural extracts’ ability to scavenge diverse radicals. Pomegranate cultivars from Sri Lanka showed TPC in the range of 1.19-2.39 mg GAE g-1 with a higher 96.70% DPPH, 93.1±2.0% ABTS radical scavenging activity35. He et al. (2012)37 extracted the phenolic compounds from pomegranate seed residues and found the TPC in the range of 3.12-27.52 mg GAE g-1 with 4265.7 mmol/100 g DW DPPH and 2692.0 mmol/100 g DW ABTS radical scavenging activity. The scavenging assay’s consequences recommended that PSO enriched with phenolic contents, gifted to capture the free radicals in a system through hydrogen- or electron-denoting mechanisms. As a result, PSO can scavenge free radicals in vulnerable matrices, such as biological membranes, and prevent fatal free radical arbitrated chain reactions from starting. These studies confirmed the PSO’s ability to capture free radicals in various systems, suggesting that these molecules could be helpful therapeutic mediators for treating free radical-induced pathology. The effect of extraction on PSO using different extraction methods, e.g., Soxhlet, stirring, microwave & ultrasonic irradiation, and supercritical fluid extraction on the phenolic contents, showed significant results with other experimental conditions. The highest phenolic contents were observed in Soxhlet > microwave > stirring > ultrasound with high pressure and lower temperature38. Higher TPC with antioxidant activity of Pakistani pomegranate seeds supported by Jing et al. (2012)7. Oxidative status The POV, p-AV, and CD & CT were used as lipid peroxidation indexes to measure the antioxidant abilities for the present study. Universally, POV is considered a standard method for measuring lipid peroxidation extent in fat/oil-based products. The POV measures the concentration of peroxides and hydro-peroxides generated in the early phases of lipid oxidation. POV is one of the most common methods for determining oxidative rancidity in oils and fats. In our study, the lipid oxidation level of oil samples was determined by placing oil samples at 70ºC for 72 h in a hot air oven. Table 4 shows the POV of oil samples with a significant difference (p≤0.05). The results revealed that the POV of the present study samples were higher than 0.39-0.48 meq O2/kg of four (04) Iranian Assessment of Basic Nutritional Profile and Antioxidant Potential of Pakistani Pomegranate (Punica Granatum L.) Varieties Vol. 13 (2), December 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 131 pomegranate cultivars39 while lower than 1.00-4.40 meq O2/ kg oil in seven (07) Turkish pomegranate cultivars oil. The p-AV for the three cultivars’ oil samples ranged from 0.30±0.01-0.77±0.08% shown in Table 4. Chemical structures can account for the difference in p-AV. The oxidative stability of lipids is improved by the stability of phenoxy radicals, limiting the degree of proliferation40. The results showed that the Desi variety has more antioxidant capability than Kandhari and Bedana varieties. The present findings are supported by Carvalho et al. (2016)41. The CD and CT values of pomegranate oil samples are shown in Table 4, ranging from 0.22±0.24- 0.99±0.12 λ232 and 0.04±0.02-0.09±0.02 λ270, respectively. Because the increase in CD and CT is related to the degree of oxidation, the oil sample’s OS will be lower42. Our study’s oxidative status results showed that Pakistani pomegranate seed has more antioxidant potential than tea leaves. The relatively lower POV, AV, and CD & CT values of oil samples showed that Pakistani PSO could categorize as stable oils. The antioxidant activity of vegetable and fruit-based oils is mainly ascribed to the phenolic contents found in plant seed oils. C O N C L U S I O N Based on the present study results, the seeds of Pakistani pomegranate cultivars as a by-product after juice extraction can be a potential source of essential fatty acid (CLnAs), biologically active compounds. Findings also showed substantial connections between total phenolic contents and antioxidant activity, representing the influence of phenolic compounds in oil antioxidant activity with higher oxidative stability. The lipid content of pomegranate seeds was probably insufficient for commercial use; on the other hand potential constituent for particular consumption or medical usage. Although, it could be an economical source of a natural antioxidant for different food products due to the antioxidant activity of conjugated linolenic acid. The findings support the use of pomegranate seeds in manufacturing pomegranate oil or developing nutraceutical foods for human nutrition. C O N T R I B U T O R S T A T E M E N T All authors contributed equally in preparation and evaluation of the manuscript. C O N F L I C T S O F I N T E R E S T No conflict of interest. F U N D I N G S O U R C E The current research projected was funded by Higher Education Commission, Islamabad, Pakistan. A C K N O W L E D G E M E N T S NS is grateful for the stipend provided by the Higher Education Authorities. L I S T O F A B B R E V I A T I O N S None R E F E R E N C E S 1. Verma N. Mohanty A. and Lal A. 2010. Pomegranate genetic resources and germplasm conservation: A review. Fruit, Vegetable and Cereal Science and Biotechnology. 4(S2): 120-125. 2. Kahramanoglu, I. (2019). Trends in pomegranate sector: production, postharvest handling and marketing. International Journal of Agriculture Forestry and Life Sciences, 3(2), 239-246. 3. Nafees M. Jaskani M.J. Ahmed S. and Awan F.S. 2015. Morpho-molecular characterization and phylogenetic relationship in pomegranate germplasm of pakistan. 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