Punica Granatum L.

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Comparison of the Antioxidant and Antiradical Activity of Pomegranate (Punica Granatum L.) by UltrasoundAssisted and Classical Extraction a

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Dimitra Z. Lantzouraki , Vassilia J. Sinanoglou , Panagiotis Zoumpoulakis & Charalampos Proestos

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Food Chemistry Laboratory, Department of Chemistry, National and Kapodistrian University of Athens, Athens, Greece b

Instrumental Food Analysis Laboratory, Department of Food Technology, Technological Educational Institution of Athens, Egaleo, Greece c

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Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, Athens, Greece Accepted author version posted online: 08 May 2015.

To cite this article: Dimitra Z. Lantzouraki, Vassilia J. Sinanoglou, Panagiotis Zoumpoulakis & Charalampos Proestos (2015): Comparison of the Antioxidant and Antiradical Activity of Pomegranate (Punica Granatum L.) by Ultrasound-Assisted and Classical Extraction, Analytical Letters, DOI: 10.1080/00032719.2015.1038550 To link to this article: http://dx.doi.org/10.1080/00032719.2015.1038550

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Comparison of the Antioxidant and Antiradical Activity of Pomegranate (Punica granatum L.) by Ultrasound-assisted and Classical Extraction

Downloaded by [Nat and Kapodistran University of Athens] at 02:03 13 May 2015

Dimitra Z. Lantzouraki1, Vassilia J. Sinanoglou2, Panagiotis Zoumpoulakis3, and Charalampos Proestos1 1

Food Chemistry Laboratory, Department of Chemistry, National and Kapodistrian University of

Athens, Athens, Greece 2

Instrumental Food Analysis Laboratory, Department of Food Technology, Technological

Educational Institution of Athens, Egaleo, Greece 3

Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research

Foundation, Athens, Greece Address correspondence to Dr. Vassilia J. Sinanoglou, Instrumental Food Analysis Laboratory, Department of Food Technology, Technological Educational Institution of Athens, Ag. Spyridonos, 12210 Aigaleo, Greece. Tel +30210 5385523. E-mail: [email protected]; [email protected] Received 12 January 2015; revised 01 April 2015; accepted 01 April 2015.

Abstract The antiradical and antioxidant properties of pomegranate (Punica granatum L.) extracts from the arils, juice, and seeds were examined and compared for classical and ultrasound-assisted 1

extraction. The total phenolic concentration, 2, 2-diphenyl-1-picryl-hydrazyl, 2, 2-azino-bis-(3ethylbenzothiazoline-6-sulfonic acid) radical scavenging, and ferric reducing antioxidant power assays were compared. High correlations were found between total phenolic concentration and antiradical activities. Juice extracts exhibited the highest total phenolic concentrations and antiradical activity compared to seed and aril extracts. However, juice extracts provided the


lowest antioxidant capacities because phenolics present in juice may scavenge free radicals rather than reduce Fe(III). Arils were similar due to their high juice content. Only seed extracts exhibited statistically significant higher scavenging and antioxidant activities with the use of ultrasound-assisted extraction that may be attributed to the antioxidant character of the phenolics to scavenge free radicals. The total phenolic concentrations in aril and juice extracts by ultrasound-assisted extraction were similar to results obtained by classical extraction. The results show that pomegranate juice and seeds have high nutritional value. Keywords antioxidant activity, antiradical capacity, extraction, phenolic compounds, Punica granatum L.

Introduction Pomegranate (Punica granatum L.) was one of the first fruit crops to be domesticated and is among one of the oldest known edible fruits (Fawole and Opara 2013). Pomegranate has been dubbed as “nature's power fruit”, as the recently increasing interest for this fruit is not only because of its pleasant taste, but also due to its scientifically demonstrated therapeutic properties such as anti-atherogenic, antiparasitic, antimicrobial, antioxidant, anticarcinogenic, and antiinflammatory activities (Adhami, Khan, and Mukhtar 2009; Akpinar-Bayizit, Ozcan, and Yilmaz-Ersan 2012). Pomegranate fruit is a source of biologically active compounds, such as 2

vitamin C, and phenolics, which are known to act as natural antioxidants (Zaouay et al. 2012). High correlations between phenolic composition and antioxidant activities of pomegranate extracts have been previously reported (Kaneria, Bapodara, and Chanda 2011). Currently, pomegranate is ranked eighteenth in terms of annual global fruit consumption, with


increasing demand especially in developed countries. Pomegranate fruit is valued for its juicecontaining arils, the percentage of which ranges from 50 to 70 percent of the total fruit weight (Goula and Adamopoulos 2012; Fawole and Opara 2013). Pomegranate is consumed and marketed as whole fresh fruit, extracted arils, and fresh and fermented juice. The arils have long been valued for their flavor, and consumers’ preference for the sweet acidic and refreshing juice (Fawole and Opara 2013). Fresh pomegranate juice contains many bioactive compounds, including phenolic acids, ascorbic acid, and flavonoids (Banihani, Swedan, and Alguraan 2013). Commercial pomegranate juices show an antioxidant activity three times higher than those of red wine and green tea (Gil et al. 2000). Pomegranates’ seeds range from 40 to 100 grams per kilogram of fruit weight depending on P. granatum's cultivar, and have antidiarrhoeal, hypoglycaemic, and antioxidant activities (He et al. 2012). Pomegranate seed residue may therefore be utilized in food applications as a nutraceutical resource (Jing et al. 2012). In recent years, novel techniques have been developed for the extraction of nutraceuticals from plants, including ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, and high hydrostatic pressure extraction (Ignat, Volf, and Popa 2011). Ultrasound-assisted extraction is a fast and efficient method for extracting phenolic compounds from fruits. The increase in phenolics’ extraction by this technique is due to cell wall disruption,

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particle size reduction, and mass transfer enhancement of the cell contents into the solvent, caused by the collapse of the bubbles produced by cavitation (Haminiuk et al. 2012). In this study, total phenolic content as well as antiradical and antioxidant properties of pomegranate extracts from arils, juice, and seeds using classical and ultrasound-assisted


extraction were examined. The results by the different assays were compared in respect to the extraction techniques and the pomegranate matrices. Because of the increased economical, commercial, and nutritional interest in pomegranates, an extensive comparison among arils, juice, and seeds on the bases of extraction methodology and antiradical/antioxidant capacity is important.

Materials and Methods Chemicals, Standards, and Solvents All reagents, standards, and solvents were used as previously described by Lantzouraki et al. (2015). Sodium persulfate, 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ), ferrous sulfate heptahydrate, and iron(III) chloride hexahydrate were obtained from Sigma Chemical (St. Louis, MO, USA).

Sampling and Sample Preparation Ripe fresh fruits of pomegranate (Punica granatum L.) from Volos (Magnesia Regional Unit, Greece) were purchased on five separate occasions from October to December 2013 from a local market. Approximately 3 kilograms (about ten fruits) of fruit without defects were randomly selected in each sampling and immediately transported to the laboratory. Fruits (N = 53) were weighted (308.40  29.12 grams) and their length (76.7  3.5 millimeters) and diameter (84.2  3.7 millimeters) were measured. Fruits were washed and drained, while the top and 4

bottom of the husks were removed with a stainless steel knife. The fruits were hand peeled and each was used to provide either arils or seeds and juice. The juice was obtained using a kitchen mortar and pestle to keep the seeds intact. The mixture was filtered to remove large particles and collect the seeds. Arils, juice, and seeds were freeze-dried in a Modulyo D unit equipped with a Thermo Savant ValuPump VLP200, (Thermo Electron Corporation, Thermo Fischer, USA). The


resulting separated samples (N = 5 for arils, juice, and seeds) were stored at 20 degree celsius until extraction. All analytical procedures and determinations were carried out three times per sample.

Extraction Classical extraction procedure was performed as follows. Approximately 3 grams of freeze-dried sample (arils, juice, and seeds) were weighted, ground, and methanol was added to a final volume of 50.00 milliliters. The extraction took place for twenty-four hours at 10 degree celsius. The extracts were passed through a Buchner filter and were diluted to a final volume of 50.00 milliliters. For ultrasound–assisted extraction (UAE), 3 grams of lyophilized sampleswere extracted according to the method described by Lantzouraki et al. (2015). All extracts were stored at 4 degree celsius until analysis.

Determination of Total Phenolic Content (TPC) The total phenolic content (TPC) of each sample was determined applying the micromethod of Folin–Ciocalteu's colorimetric assay as described by Lantzouraki et al. (2015).

Antiradical and Antioxidant Activity 5

Scavenging Activity on 2, 2-Diphenyl-1-picrylhydrazyl Radical (DPPH) The antiradical activity of methanolic extract solutions was evaluated by using the 2, 2-diphenyl1-picryl-hydrazyl radical (DPPH) by a modification of the method of Brand-Williams, Cuvelier, and Berset (1995) as described by Lantzouraki et al. (2015). The decolorization reaction of the


radical was studied for a wide range of the dry matter/2,2-diphenyl-1-picryl-hydrazyl radical ratio (2:1–200:1, w/w). Scavenging Activity on 2,2-Azino-bis-(3-Ethylbenzothiazoline-6-Sulfonic Acid) Radical (ABTS+) The antiradical activity of methanolic extract solutions was determined according to a minor modification of the method of Re et al. (1999), as described by Lantzouraki et al. (2015). Ferric Reducing/Antioxidant Power Assay (FRAP) In the ferric reducing antioxidant power (FRAP) assay, antioxidants were evaluated as reductants of Fe(III) to Fe(II). The ferric reducing antioxidant power assay, based on the reduction of a ferric-2,4,6-tripyridyl-s-triazine complex to the ferrous form, was carried out according to Benzie and Strain (1996) with minor modifications. The ferric reducing antioxidant power (FRAP) solution was prepared by mixing 500 microliters of a 10 millimolar TPTZ [2, 4, 6-tris(2-pyridyl)S-triazine] in 40 millimolar HCl, plus 500 microliters of 20 millimolar FeCl3 × 6H2O and 5.0 milliliters of 0.3 molar acetate buffer at pH 3.6. In 96 well plates, 60 microliters of extract solutions (50–70 milligrams dry matter/milliliters) or standard aqueous solutions of FeSO4 × 7H2O were mixed with 52 microliters of acetate buffer (300 millimolar, pH 3.6) and 88 microliters of the FRAP reagent. The mixture was incubated for twenty minutes at 37 degree

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celsius and the absorbance was measured at 593 nanometers, every sixty seconds, for thirty minutes, on a Thermo Scientific Varioskan Flash Multimode Reader. A standard curve (y = 0.0035x + 0.0074, R2 = 0.9903) was prepared using various concentrations (N = 10) of FeSO4 × 7H2O stock solutions (10–300 micromolar). The results were expressed as mg


FeSO4 × 7H2O per gram of dry matter.

Statistical Analysis All measurements were carried out in triplicate. Values were averaged and reported along with the standard deviations. As variables were treated as dependent and analyzed using a two-way analysis of variance including the type of matrix and the type of extraction as fixed factors in the model. The matrix type had three levels (juice, arils, and seeds) and of extraction type had two levels (classical and ultrasound–assisted extraction). The Bonferroni’s test was used for pairwise multiple comparisons. Probabilities less than 5 percent were considered to be significant (P < 0.05). Statistical calculations including partial correlations were performed with the Spss statistical software (IBM Spss Statistics, version 19.0, Chicago, IL, USA). Curve fitting was conducted with the GNU General Public Licensed QtiPlot software.

Results and Discussion Total Phenolics of Pomegranate Arils, Juice, and Seds The total phenolic concentrations of pomegranate arils, juice, and seeds are presented in Table 1. The total phenolics of juices were significantly (P < 0.05) higher compared to seeds and arils, irrespective of the extraction method. The classical extraction (CE) exhibited similar (P > 0.05) phenolic concentrations for arils, juice, and seeds compared to ultrasound-assisted extraction 7

(UAE) irrespective of matrix (Table 1). Nevertheless, since ultrasound-assisted extraction (UAE) is faster (fifteen minutes duration) and similarly efficient, it may be preferred to the classical approach. Fu et al. (2014) compared different extraction methodologies and concluded that ultrasound-assisted extraction (UAE) accelerates molecular movements, causing rapid and immediate contact with the solvent, in comparison to classical extraction where simple wetting


of the matrix with the solvent is performed. The total phenolic concentrations of juice extracts are in agreement with those reported in a previous study on pomegranate cultivars from Greece. More specifically, juice extracts using ultrasound-assisted extraction (UAE) were between 10.95 and 13.60 milligrams gallic acid equivalents/gram dry matter (Lantzouraki et al. 2015).

Antiradical Activity of Pomegranate Arils, Juice, and Seeds The percentage of the radical scavenging activity (RSA, percent) for each concentration of the samples studied was calculated as RSA = (A516nmDPPH − A516nmTplateau × 100 percent)/A516nmDPPH, where A516nmTplateau is the absorbance of the remaining 2, 2-diphenyl-1-picryl-hydrazyl radical (DPPH) after the reaction has been stabilized (Tplateau), and A516nmDPPH is the absorbance of the blank DPPH solution at Tplateau. The RSA at steady state was plotted against the ratio dry matter/DPPH and sigmoidal (Boltzmann) functions {y = A2 + (A1-A2)/[1 + exp[(x-x0)/dx)]} were fitted to the data. Different points on the sigmoidal plots (Figure 1) represent the DPPH radical percentages which were scavenged from the samples. The point of each curve which corresponds to RSA = 50 percent determines the necessary amount of sample to inhibit half of the initial quantity of DPPH radical (EC50 value). The lower the value of EC50, the higher the antiradical activity of the sample towards the DPPH radical. The correlation factor value (R) for 8

each curve (N = 12), which was used for the extraction of results, was excellent (R > 0.99). According to Figure 1, the extracts of pomegranate juices, demonstrated higher scavenging capacity for the 2, 2-diphenyl-1-picryl-hydrazyl radical (DPPH), followed by the aril extracts, while seed extracts showed the lowest scavenging capacity. These findings are in accordance with results from the Folin–Ciocalteu method (Table 1), since the juice extracts which had the


highest phenolic concentrations are those with the highest antiradical capacity against 2, 2diphenyl-1-picryl-hydrazyl radical (DPPH), while aril and seed extracts with lower phenolic concentrations have lower antiradical activity. The antiradical power (ARP), in per milligram, was calculated from ARP = 1/EC50. The reaction time needed to reach the steady state for the efficient concentration (EC50) (TEC50, minute) was estimated, and antiradical efficiency (AE = 1/EC50 × TEC50) expressed in per gram per minute was also calculated for the samples. The lower the efficient concentration, the higher the antiradical activity of the sample, while high values for the antiradical power and antiradical efficiency indicated strong activity of a sample with respect to 2, 2-diphenyl-1-picryl-hydrazyl radical (DPPH). The order of antiradical activity for the studied extracts in terms of the EC50; the dry matter/DPPH percent (w/w) to reach EC50; and the ARP was juice classical > juice ultrasound-assisted > aril classical > aril ultrasound-assisted > seed ultrasound-assisted > seed classical (Table 2). The order concerning the matrix type is in agreement with Folin–Ciocalteu results. More specifically, the higher the total phenolic concentration of the extracts, the higher the antiradical activity against 2,2-diphenyl-1-picryl-hydrazyl radical that was observed. However, the order of samples according to their antiradical efficiency was not identical. Particularly, the time parameter measured in the antiradical efficiency (AE) expression results in

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the higher antiradical activity of the ultrasound-assisted extracts by pomegranate arils, juice, and seeds compared to those by the classical extraction. Thus, the order from higher to lower AE values

is

juice

ultrasound-assisted > juice

classical > aril

ultrasound-assisted > aril

classical > seed ultrasound-assisted > seed classical.


The extracts from pomegranate juice and arils by classical extraction exhibited higher antiradical power (ARP) values but required longer times to complete their reaction with the 2, 2-diphenyl1-picryl-hydrazyl radical compared to ultrasound-assisted extraction. Since the above results cannot be correlated with the total phenolic concentrations, it is probable that the extracts from juice and aril ultrasound-assisted extraction have different phenolic compositions compared to juice and aril classical extracts. Similar results have been reported in various cases using different extraction matrices (Spranger et al. 2008). It was also reported that the antiradical efficiency, which considers antiradical power (ARP = 1/EC50) and TEC50 parameters, may be more appropriate to compare different phenolic extracts than ARP (Sendra, Sentandreu, and Navarro 2006; Spranger et al. 2008). From the above results, it may be concluded that ultrasound waves may cause chemical bond cleavage between phenolic and sugar molecules, and lead to the formation of aglycon structures with different antiradical and antioxidative activities compared to their glycosilated analogs as reported by Le and Le (2012). Aglycones are more potent antioxidants than their corresponding glycosides and react faster with 2, 2-diphenyl-1-picrylhydrazyl radical without affecting the reaction yield (Londoño-Londoño et al. 2010). Another possible explanation is that ultrasound causes hydroxylation of flavonoids at the ortho-, meta-, or para-positions by hydroxyl radicals produced during the sonolysis and increasing their antiradical activity (Alighourchi et al. 2013).

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Pomegranate juice extracts, from both extraction methods, presented significantly higher (P < 0.05) antiradical activity, as determined by the 2, 2-diphenyl-1-picryl-hydrazyl radical measurement, in comparison to aril and seed extracts (Table 3). These results are in accordance to Folin–Ciocalteu’s results, where pomegranate juice extracts had almost two (P < 0.05) and


four (P < 0.05) times higher total phenolic content than aril and seed extracts, respectively. From Figure 1 and Table 3, classical extraction of arils and juice provided compounds with significantly higher antiradical activity compared to ultrasound-assisted extraction in respect to the 2, 2-diphenyl-1-picryl-hydrazyl radical measurement. Contrary results were observed for seed extracts where ultrasound-assisted extraction provided 12.6 percent higher DPPH values compared to the classical method. Possibly, seeds after freeze drying remained as relatively large solids not fully milled to optimize the phenolics’ extraction, in contrast to the freeze dried arils and juice which were powdered. Ultrasound may reduce seed particle size and cause enhancement of the mass transfer of cells to the solvent, caused by bubbles produced by cavitation (Haminiuk et al. 2012). For aril and juice, wetting and vortexing with the extraction solvent for twenty-four hours were sufficient to extract the phenolics compared to ultrasound. For seeds, where the disruption of cells was necessary for the optimum extraction, ultrasound provided better results. In conclusion, aril classical, juice classical, and seed ultrasound-assisted extracts exhibited higher values (P < 0.05) with respect to ascorbic acid equivalents. Regarding 2, 2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS+) assay (Table 4), pomegranate juice extracts provided two to four times (P < 0.05) higher antiradical activity compared to aril and seed extracts. These results are in agreement with Folin–Ciocalteu (Table 1) and 2, 2-diphenyl-1-picryl-hydrazyl radical results (Table 3). Furthermore, in agreement with Tables 1 and 3, extracts from pomegranate arils using the classical extraction 11

demonstrated higher values for trolox equivalent (P < 0.05) in comparison to those from ultrasound-assisted extraction. In contrast, no significant difference was observed for the pomegranate juice and seed extracts in respect to the extraction methods.

Antioxidant Activity of Pomegranate Arils, Juice, and Seeds


Pomegranate aril and seed extracts presented significantly higher (P < 0.05) antioxidant activity (expressed as milligram of FeSO4 × 7H2O/gram dry matter) compared to juice (Table 5). These results are contradictory to those for the Folin–Ciocalteu assay, where the pomegranate juices had higher total phenolics than arils and seeds (Table 1). This may be explained because this assay, which measures the ferric reducing power of a sample, is different from the 2, 2-diphenyl1-picryl-hydrazyl radical (Table 3) and 2, 2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical (Table 4) assay mechanisms. Lahouar et al. (2014) reported that DPPH radical inhibitory activity of barley varieties changed in an opposite manner to ferric-reducing power. Cao and Prior (1998) reported that the antioxidant capacity does not necessarily match the reduction of Fe(III) to Fe(II). Furthermore, the use of Fe(II) as an indicator in the ferric-reducing power assay may cause problems when an antioxidant not only reduces Fe(III) to Fe(II), but also reacts with Fe(II) to generate additional free radicals.

Correlation and Conclusions High Pearson correlation values (R > 0.95) among Folin–Ciocalteu, 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) and 2, 2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS+) results were observed (Table 6). It is apparent that as the total phenolic concentration increases, the antiradical activity against DPPH and ABTS+ radicals increases irrespectively of

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the matrix (arils, juice, and seeds) and the extraction methodology. This implies that the phenolic compounds in the examined extracts are those presenting the antiradical properties in the DPPH and ABTS assays. Negative correlation of the ferric reducing antioxidant power (FRAP) to the other three assays was expected since its values are correlated to the reduction power of a sample and not the scavenging activity over radicals. Moreover, the above-mentioned results imply that


other constituents in the extracts (apart from phenolics) act as Fe(III) reducing agents. In conclusion, since natural phenolics do not exert their antiradical and antioxidant activity under the same mechanisms, it is crucial to perform in vitro comparative characterization of pomegranate extracts, using different assays, in order to give a comprehensive prediction of their antioxidant efficacy. In the current study, juice extracts exhibited the highest total phenolic concentrations and antiradical activities, but the lowest antioxidant capacity compared to aril and seed extracts. Notably, pomegranate seed extracts demonstrated significantly higher antioxidant activity than the other extracts, indicating their potential use in the food industry as a source of bioactive compounds. Concerning the extraction methodologies employed, only seed extracts exhibited statistically significant higher scavenging and antioxidant activities. Nevertheless, the major advantage of ultrasound remains the rapid and efficient extraction of phenolics from different matrices.

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Lahouar, L., A. El Arem, F. Ghrairi, H. Chahdoura, H. B. Salem, M. El Felah, and L. Achour. 2014. Phytochemical content and antioxidant properties of diverse varieties of whole barley (Hordeum vulgare L.) grown in Tunisia. Food Chemistry 145: 578–83. doi:10.1016/j.foodchem.2013.08.102 Lantzouraki, D. Z., V. J. Sinanoglou, P. G. Zoumpoulakis, J. Glamoclija, A. Ciric, M. Sokovic, G. Heropoulos, and C. Proestos. 2015. Antiradical–antimicrobial activity and phenolic profile of pomegranate (Punica granatum L.) juices from different cultivars: A comparative study. RSC Advances 5: 2602–14. doi:10.1039/c4ra11795f Le, H. V., and V. V. M. Le. 2012. Comparison of enzyme-assisted and ultrasound-assisted extraction of vitamin C and phenolic compounds from acerola (Malpighia emarginata DC.) fruit. International Journal of Food Science & Technology 47: 1206–14. doi:10.1111/j.13652621.2012.02960.x Londoño-Londoño, J., V. R. de Lima, O. Lara, A. Gil, T. B. C. Pasa, G. J. Arango, and J. R. R. Pineda. 2010. Clean recovery of antioxidant flavonoids from citrus peel: Optimizing an aqueous ultrasound-assisted extraction method. Food Chemistry 119: 81–87. doi:10.1016/j.foodchem.2009.05.075 Re, R., N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, and C. Rice-Evans. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine 26(9–10): 1231–37. doi:10.1016/s0891-5849(98)00315-3 Sendra, J. M., E. Sentandreu, and J. L. Navarro. 2006. Reduction kinetics of the free stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•) for determination of the antiradical activity of citrus juices. European Food Research and Technology 223: 615–24. doi:10.1007/s00217-0050243-3 Spranger, I., B. Sun, A. M. Mateus, V. de Freitas, and J. M. Ricardo-da-Silva. 2008. Chemical characterization and antioxidant activities of oligomeric and polymeric procyanidin fractions from grape seeds. Food Chemistry 108: 519–32. doi:10.1016/j.foodchem.2007.11.004 Vasilef, I. QtiPlot – Data analysis and scientific visualisation. http://www.qtiplot.com/index.html (accessed February 2015). Zaouay, F., P. Mena, C. Garcia-Viguera, and M. Mars. 2012. Antioxidant activity and physicochemical properties of Tunisian grown pomegranate (Punica granatum L.) cultivars. Industrial Crops and Products 40: 81–89. doi:10.1016/j.indcrop.2012.02.045

15

Table 1. Total phenolic content expressed as gallic acid equivalents in pomegranate arils, juice, and seeds (n = 15)


Sample – extraction

Milligram

gallic

acid Effect

within Effect

equivalent/gram dry matter

extraction

matrices

Aril - classical

5.66  1.14

Not significant

*

Aril - ultrasound-assisted

4.92  0.72

Juice - classical

12.01  1.64

Juice - ultrasound-assisted

11.50  1.00

Seed - classical

2.85  0.56

Seed - ultrasound-assisted

3.34  0.90

** Not significant

* **

Not significant

* **

*,**Values in the same column at corresponding extraction type differ significantly with P < 0.05.

16

within

Table 2. Efficient Concentration (EC50) in milligram for certain quantity of DPPH●, dry matter/DPPH● (w/w ratio) where EC50 is observed, antiradical power (ARP) in per milligram, and antiradical efficiency in per gram per minute for pomegranate (n = 15). Sample


extraction type

– Efficient

Dry

Concentration

matter/2,2- Antiradical

diphenyl-1-

(milligram) for picryl-hydrazyl 1500

power

Antiradical

(per efficiency

milligram)

gram·per minute)

radical (w/w) for

microliters 2,2- efficient diphenyl-1-

concentration

picryl-hydrazyl radical

100

micromolar 1.32  0.04a

22.36  0.60a

0.76  0.02a

4.20  0.11a

Aril - ultrasound- 1.61  0.04b

27.29  0.62b

0.62  0.01b

4.43  0.10b

0.51  0.01c

8.62  0.11c

1.96  0.03c

13.08  0.17c

Juice - ultrasound- 0.55  0.01d

9.28  0.15d

1.82  0.03d

15.18  0.25d

2.96  0.06e

50.04  1.02e

0.33  0.01e

1.89  0.04e

Seed - ultrasound- 2.79  0.06f

47.17  1.03f

0.36  0.01f

1.99  0.04f

Aril - classical

assisted Juice - classical

assisted Seed - classical

17

(per

assisted


Means in the same column bearing different superscripts differ significantly (P < 0.05).

18

Table 3. Antiradical activity expressed as ascorbic acid equivalents of pomegranate arils, juice, and seeds (n = 15)


Sample –extraction type

mg

ascorbic

acid Effect

within Effect within

equivalents/gram dry matter

extraction

matrices

Aril - classical

4.62  0.30

***

*


3.80  0.43

Juice - classical

13.80  0.79


12.07  0.61

Seed - classical

2.34  0.15


2.79  0.23

** ***

* **

***

* **

*,**Values in the same column at the corresponding extraction type differ significantly with P < 0.05.

***Values in the same column differ significantly between the extraction type with P < 0.05.

19

Table 4. Antiradical activity expressed as Trolox equivalents of pomegranate arils, juice, and seeds (n = 15)


Sample –extraction type

Milligram

Trolox Effect


equivalents/gram dry matter extraction

matrices

Aril - classical

7.50  0.93

*


5.80  0.31

Juice - classical

13.17  0.67


13.81  0.25

Seed - classical

3.34  0.16


3.85  0.41

***

** ns

* **

ns

* **

*,**Values in the same column at the corresponding extraction type differ significantly among matrices with P < 0.05.

***Values in the same column differ significantly between extraction type with P < 0.05.

20

Table 5. Antioxidant activity by ferric reducing antioxidant power assay expressed as milligram of FeSO4 × 7H2O/gram dry matter of pomegranate arils, juice, and seeds (n = 15) Sample –extraction type

Milligram

Effect

FeSO4 × 7H2O/gram


dry extraction

matrices


matter Aril - classical

2.42  0.04


2.27  0.08

Juice - classical

1.80  0.07


1.89  0.01

Seed - classical

2.14  0.01


3.11  0.11

***

* **

***

* **

***

* **

*,**Values in the same column at the corresponding extraction type differ significantly with P < 0.05.

***Values in the same column differ significantly between extraction type with P < 0.05.

21

Table 6. Statistical correlation among the assays by matrix (arils, juice, and seeds) and extraction type (classical and ultrasound-assisted extraction) Folin–

Statistical correlation

2,

2- 2,

Ciocalteu diphenyl-


1-picryl-

ethylbenzothiazoline- reducing 6-sulfonic acid)

hydrazyl Folin–Ciocalteu

Type

2-azino-bis-(3- Ferric

antioxidant power

of 1

0.955

0.960

0.669

of 1

0.953

0.961

0.724

of

1

0.970

0.690

of

1

0.961

0.718

of

1

0.669

of

1

0.734

matrix Type

extraction 2,

2-diphenyl-1- Type

picryl-hydrazyl

matrix Type

extraction 2,

2-azino-bis-(3- Type

ethylbenzothiazoline- matrix 6-sulfonic acid)

Type

extraction

22

Figure 1. Comparison of the radical scavenging activity of 2, 2-diphenyl-1-picryl-hydrazyl radical for pomegranate aril, juice, and seed extracts using different extraction methods as a


function of the ratio of dry matter/2, 2-diphenyl-1-picryl-hydrazyl radical at 18 degree celsius.

23

Punica Granatum L.

Punica Granatum L.

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