Effect of Cold Storage of Various Pomegranate Cultivars Fruit Juices on Health Promoting Compounds and Their Activities

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2.1. Chemical Composition Analysis

Water-soluble solids content (Brix), pH and titratable acidity of samples were determined in order to describe the original characteristics of the fruits. Water-soluble solid contents were determined by Bausch & Lomb, USA, refractometer and expressed as oBrix at 20 °C ^[16]. The pH was measured with a digital pH meter (601, Herisau, Switzerland). Following the dilution of 2 ml aliquot of sample with 20 ml distilled water, the total titratable acidity was determined by titrating the sample with 0.1 M NaOH until the pH reached 8.1 and expressed as grams of citric acid per liter ^[17].

Total sugars were determined as described by Ranganna ^[35]. Ascorbic acid content was measured by the method described by Ruck ^[36] with minor modifications and the results were expressed as mg per 100g fw of juice. Total anthocyanins were determined by pH differential method ^[37], and the results were expressed as mg cyaniding-3-glucoside 100 g of juice.

2.1.1. Total Phenolic Assay

Total phenolic content was determined with the Folin-Ciocalteu reagent according to the procedure described by Singleton and Rossi ^[38]. Briefly, 0.50 mL of the diluted sample was reacted with 2.5 mL of 0.2 mol/L Folin-Ciocalteu reagent for 4 min, then 2 mL saturated sodium carbonate solution (about 75 g/L) was added into the reaction mixture. The absorbance was measured at 760 nm after incubation at room temperature for 2 h. Gallic acid was used as a reference standard, and the results were expressed as milligram gallic acid equivalent per grams fresh weight (mg GAE/g fw).

2.1.2. Antioxidant Activity

The TEAC (Trolox Equivalent Antioxidant Capacity) assay was carried out according to the method of Re et al ^[39]. Firstly, to produce the radical cation ABTS^•+, 7 mmol/L ABTS salt and 2.45 mmol/L potassium persulfate were mixed in a volume ratio of 1:1. Next, the reaction mixture was allowed to stay in the dark for 16 h at room temperature and was used within two days of preparation. The ABTS^•+ radical solution was diluted with ethanol to an absorbance of 0.7 ±0.05 at 734 nm. All samples were approximately diluted to provide 20–80% inhibition of the blank absorbance. One hundred microliters of the diluted sample was mixed with 3.8 mL ABTS^•+ working solution, and the reaction mixture was left at room temperature to react for 6 min. Then, the absorbance at 734 nm was measured using the ultraviolet spectrophotometer. Trolox solution was used as a reference standard, and the results were expressed as µmoles of Trolox per gram of sample (µmol TE/g fw). The DPPH (1,1-Diphenyl-2-picryl-hydrazyl) assay was carried out according to the method of Brandwilliams et al. ^[40] with some modiﬁcations. The stock solution was prepared by dissolving 24 mg DPPH with 100 mL methanol and was stored at -20 ^oC. The working solution was obtained by mixing 10 mL stock solution with 45 mL methanol to obtain an absorbance of 1.1±0.02 units at 515 nm. Samples (150 mL) were allowed to react with 2850 mL of the DPPH solution for 24 h in the dark. Then the absorbance was measured at 515 nm. The standard curve was linear between 25 and 800 mM Trolox. Results are expressed in µmoles of Trolox per gram of fresh sample (µmol TE/g fw). Ferric Reducing Antioxidant Power (FRAP) was employed according to the method of Benzie and Strain ^[41]. A portion of an aqueous 10 mmol/l solution of TPTZ reagent in 40 mmol/l HCl was mixed with the same volume of 20 mmol/l FeCl3·6 H₂O and ten times higher volume of acetate buffer of pH 3.6 (3.1 g sodium acetate and 16 ml acetic acid per litre). The mixture was incubated at 37°C for five minutes. A portion (900 μl) of the Fe³⁺-TPTZ mixture and the sample or water (for blank) were adjusted to 1 mL with deionized water, incubated for 30 min, and the absorbance at 593 nm was measured.

2.1.3. Statistical Analysis

Analyses were performed in 3 parallel trials and the descriptive statistics were studied employing Minitab 16.0. One-way ANOVA was performed and Tukey test was used for multiple comparisons of the groups at 0.05 significance level.

3. Results and Discussion

Brix values of pomegranate cultivars were found within the range of 12.1-18.1 while % titratable acidity (TA) of the samples were between 0.27-3.39. pH values varied from 2.87 to 4.65. A wide range of brix and % TA values are expected due to the differences in pomegranate varieties studied. These findings are also consistent with other studies ^{[42, 43, 44]}. These results are important for the understanding of the characteristics of different pomegranate cultivars. Especially, different brix and TA value might result in different solid content that might contribute to the antioxidant capacity. The brix, pH and % TA values were shown in Table 2.

Table 2. Antioxidant Capacity of Pomegranate Varieties

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Total sugars, total anthocyanins, ascorbic acid and total phenolics values of sixteen pomegranate cultivars are shown in Table 3. Total sugars of pomegranate cultivars varied from 9.6 to 22.1 g/100g, while ascorbic acid values were between 2.9 and 13.6 mg/ 100g. Total anthocyanins of pomegranate cultivars were found with a range of 2.6-11.6 mg/100g. The Mersin 23 cultivar showed the highest total anthocyanin value (11.6 mg/100g), followed by Mugla 1267 (10.9 mg/100g) and Antalya 1265 (10.7mg/100g). Although these values vary in a wide rage, they are consistent with other studies ^{[45, 46, 47]}.

Table 3. Total Sugars (TS), Total Anthocyanins (TA), Ascorbic Acid (A) and Total Phenolics (TP) of Juices From Sixteen Pomegranate Cultivars

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The total phenolic content of the pomegranate cultivars was found to fall within a range of 2450.3-956.5 mg/100g. While Mersin 23, Antalya 1265 and Mersin Wonderful had the highest total phenolic content (2450.3, 2367.9 and 2352.9 mg/100g, respectively), Izmir 1265 had the lowest total phenolic content (956.5 mg/100g). Similar to the extensive research conducted on phenolic content of pomegranate cultivars ^{[48, 49, 50, 51, 52]} we found that the phenolic content of our samples varied depending on the region and variety. Specifically, the varieties grown in Izmir have lower phenolic content (956.5-1232 mg/100g) compared to those grown in Antalya, Mersin and Mugla.

The differences in anthocyanins, total phenolics, brix and percent titratable acidity is a likely result of the different cultivars analyzed. Disparities between cultivars analyzed here is mostly influenced by the climate as well as the salinity of the irrigation water. Borochov- Neori et al. ^[53] has indicated that temperature significantly affects the antioxidant capacity of pomegranate through modification of anthocyanin content. Despite the pomegranate cultivars analyzed here were subjected to similar climates, temperature fluctuations during handling might change their composition. Likewise, salinity of the irrigation water has been reported to affect the accumulation of phenolic compounds in different pomegranate cultivars ^[53].

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Figure 1. Correlation of ABTS, DPPH and FRAP assays for total antioxidant capacity measurement

ABTS, DPPH and FRAP assays were used to determine the total antioxidant capacity of pomegranate cultivars and the results were between 2.54-12.4, 5.5-36.2 and 3.9-26.7 µmol TE/g fw, respectively (Table 2). According to the results, Antalya Tirbey and Mersin 23 showed the highest antioxidant capacity, followed by Mersin Wonderful and Antalya 1265, whereas Izmir 1264, Izmir 1479 and Izmir 01N07 varieties had the lowest antioxidant capacity. The correlation coefficient between the assays, ABTS-DPPH, ABTS-FRAP and DPPH- FRAP, were found to be 0.96, 0.96 and 0.94, respectively (Figure 1). High correlation between the assays suggest they were consistent in total antioxidant capacity measurement. However, our results are slightly higher than ^{[54, 55]} the previous studies.

The correlation of total antioxidant capacity with pH, ⁰BRIX, titratable acidity, total sugars, ascorbic acid, total anthocyanins and total phenolics are shown in Table 4. According to the results, high linear correlation between total antioxidant capacity and total anthocyanins (r²= 0.94), ascorbic acid (r²=0.75), and phenolic content (r²=0.94) were observed. These results demonstrated that total anthocyanins, ascorbic acid and phenolic compounds are the main contributors for total antioxidant capacity. Many studies have been reported on the correlation of antioxidant capacity with these values ^{[16, 33, 48, 56, 57, 58]}. Correlation of antioxidant capacity with anthocyanins and phenolic content determined here are similar to these studies while our regression analysis yielded slightly lower correlation with ascorbic acid.

Table 4. Correlation Coefficients (r) of pH, ⁰BRIX, titratable acidity (TA), total sugars (TS), ascorbic acid (A), total anthocyanins (TAs), total phenolics (TPs) and antioxidant activity (AA) of juice from sixteen pomegranate cultivars

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