Pomegranates

ChemicalsAll reagents and solvents were purchased from Merck (Darmstadt, Germany) and Sigma (St. Louis, MO) unless otherwise mentioned. All chemicals used in the experiments were of analytical grade.
Sample preparationNine cultivars of pomegranate were donated from Saveh Agricultural Investigation Center during September 2007. A total of 27 pomegranate fruits (three numbers of each cultivar) were collected and washed three times with distilled water. To prepare pomegranate extract, fresh fruits were peeled and their edible portions (seed coats and juice) were separated. 30 g of pulps and peels were weighted and extracted separately for 4 h by Soxhlet apparatus with acetone, followed by ethyl acetate, methanol and water solvent respectively (four hours for each solvent) to extract different kinds of antioxidant components. The four different extracts of every cultivar were mixed and dried on a water bath. 1 g of pulp and peel extracts were dissolved and diluted with methanol 80% (v/v) to 25 mL. To assay total antioxidant and phenolic content, pulp extracts were diluted 1 : 10 (v/v) where it was 1: 100 (v/v) for peel extracts by 80% methanol.
Total flavonoid assay Total flavonoid content was measured by the aluminum chloride colorimetric assay (21 ). An aliquot (1 mL) of extracts or standard solution of catechin (50, 100, 150, 200, 250 and 300 mg/L) was added to 10 mL volumetric flask containing 4 mL of double distilled water. Then 0.3 mL 5% NaNO₂ was added to the flask and after 5 min, 0.3 mL AlCl₃ (10%) was also added. At 6^th min, 2 mL NaOH (1 M) was added and the total volume was made up to 10 mL with double distilled water. The solution was mixed completely and the absorbance level was measured versus prepared reagent blank at 510 nm. Total flavonoid content was expressed as mg catechin equivalents (CE) per one gram dry extract. The total flavonoid assay was measured three times for each pomegranate extract.
Total phenolic assayTotal phenolics were determined using Folin-Ciocalteu reagent as described by Velioglu et al. (22 ) with slight modifications. The extract (200 μL) was mixed with 1.5 mL of Folin-Ciocalteu reagent (previously diluted 10 times with distilled water) and allowed to stand at room temperature for 5 min. 1.5 mL sodium bicarbonate solution (60 g/L) was added to the mixture and after incubation for 90 min at room temperature, the absorbance level was measured at 750 nm using a UV-Visible spectrophotometer (GBC, Cintra 40). Total phenolics were quantified by calibration curve obtained from measuring the absorbance of the known concentrations of gallic acid standard solutions (25-150 μg/mL in 80% methanol). The results were calculated as gallic acid equivalent (GAE) per one gram dry extract and reported as mean value ± SD.
Total antioxidant activityThe FRAP (ferric reducing antioxidant power) assay was described initially by Benzie and Strain (23 ). It is based on the reduction of a ferric tripyridyl triazine complex to its ferrous blue colored form in the presence of antioxidants. It is a relatively simple method frequently used in the assessment of antioxidant activity of various fruits, vegetables and some biological samples (24 ).
Briefly, the FRAP reagent contained 5 mL of TPTZ (2, 4, 6-tripyridyl-s-triazine) solution (10 mmol/L) in HCl (40 mmol/L) plus 5 mL of FeCl₃ (20 mmol/L) and 50 mL of acetate buffer (0.3 mol/L, pH 3.6). It was freshly prepared and warmed up to 37°C. A volume of 100 μL pulp or peel extract was mixed with 3 mL of FRAP reagent and the absorbance of the reaction mixture was measured at 593 nm after incubation at 37°C for 10 min. To construct calibration curve, five concentrations of FeSO₄ 7H₂O (1000, 750, 500, 250, 125 μmol ∕ L) were used and the absorbance were measured as sample solution. The values were expressed as the concentration of antioxidants having a ferric reducing ability equivalent to that of 1 mmol/L FeSO₄ and also as vitamin E and C equivalent. All the measurements were taken in triplicate and expressed as mean value ± standard deviation (SD).
Statistical analysisThree replicates of each sample were used for statistical analysis and the values were reported as mean ± SD. Pearson’s correlation and regression analysis were carried out using SPSS statistical program to study the relationship between antioxidant activity and total phenolic and flavonoid content. Data were also subjected to the analysis of variance and mean values were compared by Dunnett T3 post-hoc multi-comparison tests. Differences at p < 0.05 were considered to be significant.

EV were isolated with a modified protocol of one described by Shin et al.^{20 (link)}. Isolation procedure was briefly abstracted in Fig. 6.Figure 6

Steps of the ATPS-based EV isolation protocol.

Plants (Punica granatum) were obtained from a local market, were washed and juiced whole in a clean blender. Resulting lysate was passed through a cheesecloth to remove larger contaminants.
20 ml of plant lysate, cell culture media, blood serum, or parasite culture media were then centrifuged at 10,000 g for 10 minutes to remove larger contaminants. Supernatants were then passed through 0.22 μm filters to remove smaller contaminating particles, and to reduce the concentrations of larger extracellular vesicles. Filtered supernatants were then mixed at a 1:1 volume ratio with ATPS-EV isolation solution, and were then centrifuged at 1,000 g for 10 minutes for phase separation. Twice, 80% of the volume was removed from the upper, PEG-rich phase replaced with the upper phase of washing solution, prepared by mixing ATPS-EV isolation solution with distilled water at a 1:1 volume ratio and centrifuging at 1,000 g for 10 minutes, as described in Kim et al.^{21 (link)}.

Pomegranates (Punica granatum L.) were obtained by a local market (Orestiada, Greece). They were washed and processed into juice by blending the seeds for 10 min. Sterilized water was added to adjust the initial sugar concentration to approximately 90 g/L, and the initial pH was adjusted to 3.5 with NaOH 4N. The prepared juice solutions (100 mL) were transferred into 250 mL flasks and pasteurized for 5 min at 80 °C, cooled at room temperature and finally used for the fermentations [26 (link)]. 1 g of harvested (wet weight) Lactobacillus plantarum ATCC 14917 was added to 100 mL of pomegranate juice that was fermented at 30 °C for 24 h. The initial cell viability was determined at 11.42 log cfu/mL of juice. Then, the flaks were kept at 4 °C for 28 days (4 weeks). The fermentations were carried out in triplicate.

‘Manfalouty’ pomegranate (Punica granatum L.) fruits were obtained from a private orchard in Assiut Governorate. This study was conducted in 2021 and 2022. were harvested in mid-October, fully matured as followed in commercial practice, and transported to the postharvest handling lab. at the Horticulture Research Institute (Giza Governorate, Egypt) to study the effect of different postharvest treatments on quality and pomegranate arils. The substances used in this experiment were: chitosan from Acros-organics Company, Morris Plains, NJ, USA. Citric acid, sodium citrate, and sodium hydroxide were obtained from (El Nasr Pharmaceutical Chemicals Company, Cairo, Egypt). Glycerin and calcium hypochlorite were obtained from (El-Gomhouria chemical company, Cairo, Egypt). ZnONPs were obtained from the Nanotechnology & Advanced Material Center Lab, Agriculture Research Center, Giza, Egypt. Ethyl alcohol is 95% produced by El-Gamhoria Company Egypt. Tween was obtained from Win Lab Company (London, UK). Glacial acetic acid was obtained from (Acmatic For Chemicals & Lab. Equipment Company, Cairo, Egypt).

The dipping of fresh pomegranates was peeled and the arils were separated from the tissues. Then, the arils were washed and treated with a disinfecting solution of calcium hypochlorite (0.25 g/L distilled water) for one minute before being air-dried. The arils are created with a disinfecting solution of calcium hypochlorite (0.25 g/L distilled water) for one minute before being air-dried. The arils were treated as follows: Fresh fruits were (A) extracted pomegranate peels (PPE) 5% (0.1%), (B)ZnONPs 1% (0.02%), (C) ZnONPs 2% (0.04%), (D) ZnONPs 3% (0.06%), (E) ZnONPs 1%/PPE1% (0.02%), (F) ZnONPs 2%/PPE2% (0.04%), (G) ZnONPs 3%/PPE3% (0.06%) Chitosan content in packaged coated fruitsPomegranate peeled cubes were drained after dipping and packaged in plastic trays with approximately 250 g. After that, all boxes were cold stored at 2 °C and 90–95% RH for 20 days and kept in carton boxes. All samples were kept after packaging. Samples were taken at weekly intervals to determine the physical and chemical changes during the storage period. Pomegranate samples were withheld for analysis on a regular basis.
Prepared crude phenolic compound extraction from pomegranate peels(PPE):
The air-dried ground (80 mesh) plant material (20 g for each sample) was extracted with each of the solvents ethanol (ethanol: water, 80:20 v/v) and aqueous methanol (methanol: water, 80:20 v/v) (200 mL) for 6 h at room temperature in an orbital shaker in a water bath in separate experiments. The extracts were separated from the residues by filtering through Whitman No.1 filter paper. The sediments were extracted twice with the same fresh solvent, and the combined extracts were used. Using a rotary evaporator, the combined extracts were concentrated and freed of solvent under reduced pressure at 45 °C. According to [16 (link)]. the dried crude concentrated extracts were weighed to calculate the yield and stored in a refrigerator (−4 °C).

Twelve morphological traits of pomegranates were used to study the diversity of genotypes, which included the number of fruit per tree (NFT), mean fruit weight (FMW), mean tree yield (TMY), anthocyanin on the branch of this year (ABTY), petiole color (PC), fruitful flower size (FFS), Fruit albedo color (FAC), fruit bottom shape (FBS), fruit heel shape (FHS), juice content of arils (JA), seed size (SS), and seed color (SC). The number of plants evaluated for each genotype was three trees from which eight samples for each genotype were randomly harvested, and transferred to the laboratory for assessment. Fruit sampling was performed during their ripening stage. Qualitative traits were evaluated visually and then scored (Table 1). As the preliminary analysis of data for two consecutive growing seasons (2017–2019) showed no significant difference, the average of two years of data was used for statistical analysis to increase the accuracy in the analysis.