Food Chemistry 118 (2010) 1116

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Anthocyanins and polyphenol oxidase from dried arils of pomegranate (Punica granatum L.)
Vidhan Jaiswal a, Ara DerMarderosian a,b,*, John R. Porter a,b
a b

Program in Pharmacognosy, Department of Chemistry and Biochemistry, University of the Sciences in Philadelphia, Philadelphia, PA 19104, United States Department of Biological Sciences, University of the Sciences in Philadelphia, Philadelphia, PA 19104, United States

a r t i c l e

i n f o

a b s t r a c t
Anthocyanins are natural pigments responsible for red, purple, and blue colouration in plants. Human consumption of anthocyanins is increasing because of the rising awareness and interest in their potential health benets. Pomegranate is one of the major sources of polyphenolic phytochemicals, such as anthocyanins. Dried pomegranate raisins (anardana) are consumed in large quantities in Asian countries, and contain substantial amounts of anthocyanins. Five anthocyanins were found to be present in pomegranate raisins. Drying adversely affected the amount of anthocyanins, with polyphenol oxidase (PPO) playing a possible role in oxidative degradation of anthocyanins. Anthocyanins are heat-stable compounds, and inactivation of PPO by processing at high temperature for short periods may prevent PPO-catalysed anthocyanin oxidation in pomegranate arils. Pomegranate PPO kinetics were partially characterised by investigating the effect of substrate (catechol) concentration; optimum pH for PPO activity was found to be 6.0. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 30 July 2008 Received in revised form 27 January 2009 Accepted 29 January 2009

Keywords: Pomegranate Punica granatum L. Polyphenol oxidase Anthocyanin

1. Introduction Anthocyanins are the largest and most important group of water-soluble pigments in nature. In plant tissues, they are responsible for producing red, purple, blue, and intermediate hues, depending upon the vacuolar pH and the presence of copigments (Brouillard, Figueiredo, Elhabiri, & Dangles, 1997; Clifford, 2000; Rapeanu, Loey, Smout, & Hendrickx, 2006). Anthocyanins are of considerable importance in the co-evolution of plantanimal interactions, as they contribute to the colourful appearance of owers, fruits and vegetables, helping them to attract animals, leading to seed dispersal and pollination (Kong, Chia, Goh, Chia, & Brouillard, 2003). Anthocyanin stability is inuenced by various factors such as temperature, pH, light, and oxygen. Anthocyanins also may be susceptible to degradation by oxidising enzymes. Polyphenol oxidases (PPOs), or tyrosinases, are nuclear-coded enzymes with a dinuclear copper centre, which are able to insert oxygen in a position ortho to an existing hydroxyl group in an aromatic ring, followed by the oxidation of the diphenol to the corresponding quinone. These o-quinone intermediates are highly reactive and quickly polymerise to dark-coloured melanins (Rapeanu et al., 2006). PPO is widely distributed in animals, plants, fungi,

* Corresponding author. Address: Program in Pharmacognosy, Department of Chemistry and Biochemistry, University of the Sciences in Philadelphia, Philadelphia, PA 19104, United States. Tel.: +1 215 596 8915; fax: +1 215 596 8710. E-mail address: a.dermar@usp.edu (A. DerMarderosian). 0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.01.095

and bacteria and plays an important role in various physiological and defence reactions (Mayer, 2006). PPO is inducible by both biotic and abiotic stresses, and is implicated in several physiological processes, including photoreduction of molecular oxygen by photosystem I (PSI), regulation of plastidic oxygen levels, aurone biosynthesis, and the phenylpropanoid pathway (Thipyapong, Stout, & Attajarusit, 2007). The pomegranate (Punica granatum L.), from the Latin words pomus and granatus, meaning a seeded or granular apple, is native from Iran to the Himalayas in northern India, where it has been cultivated for thousands of years. There are over 1000 cultivars of Punica granatum (Levin, 1994) that are grown from Iran, eastward to China and India and westward through the Mediterranean region, on to the American Southwest, California and Mexico (Lansky & Newman, 2007). There has been a virtual explosion of interest in pomegranate as a medicinal and nutritional product because of its multifunctionality, and, as a result, the eld of pomegranate research has experienced tremendous growth in the past decade. Polyphenols are the major class of pomegranate fruit phytochemicals, including avonoids (anthocyanins), condensed tannins (proanthocyanidins) and hydrolysable tannins (ellagitannins and gallotannins) (Gil, Tomas-Barberan, Hess-Pierce, Holcroft, & Kader, 2000; Hernandez, Melgarejo, Tomas-Barberan, & Artes, 1999; Santagati, Duro, & Duro, 1984). Pomegranate juice may provide protection against cardiovascular diseases and stroke, by acting as a potent antioxidant against LDL oxidation and inhibition of atherosclerosis development (Aviram et al., 2002a, 2002b). Pomegranate

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phytochemicals also show potential in chemoprevention of various types of cancers, by exerting antiproliferative effects on tumour cells (Kim et al., 2002; Shishodia, Adams, Bhatt, & Aggarwal, 2006). Pomegranates are popularly consumed as fresh fruit, beverages (juice and wine) and other food products (jams and jellies). The presence of anthocyanins is responsible for the appealing bright red colour of juice and other products of pomegranate fruit. Pomegranate raisins (anardana) are dried arils of wild pomegranates that are manually separated from the rind and septa of the fruit and sun- or air-dried. Pomegranate raisins have a distinct sour or tart avour, and are commercially available in many West and East Asian countries, where they are consumed in large quantities. The raisins are also used in the ayurvedic system of medicine, and are claimed to be digestive and stomachic (Pruthi & Saxena, 1984; Singh, Kingly, & Jain, 2007). Pomegranate juice, which is prepared by hydrostatic pressing of whole fruits, has been found to contain more than 26 different chemical compounds, whereas the seeds contain various fatty acids and steroidal compounds (Seeram, Zhang, Reed, Krueger, & Vaya, 2006). The aril (the seed and surrounding esh) contains anthocyanins, which might be affected by the severity of the drying process employed for the raisin preparation. Scarce information exists on the chemical constituents of anardana. This study was designed to evaluate the effect of drying on pomegranate aril anthocyanins and to investigate the role of PPO in aril anthocyanin oxidation. 2. Materials and methods 2.1. Plant material Fresh pomegranates (P. granatum L. cv. Wonderful) were supplied by POM Wonderful company (Del Rey, CA). Arils were separated manually and kept in dark and cold (80 C) storage until analysed. Pomegranate raisins (anardana) were purchased from Deep Foods, Inc. (Union, NJ). 2.2. Chemicals Cyanidin 3-glucoside (kuromanin chloride) standard for HPLC analysis, catechol, triuoroacetic acid (TFA), acetone, acetonitrile, ethyl acetate, hexane, and methanol were purchased from SigmaAldrich (St. Louis, MO). Unless stated otherwise, all solvents were HPLC-grade. Diaion HP-20SS resin was purchased from Supelco (Bellefonte, PA). Triton X-100 was purchased from Bio-Rad (Richmond, CA). 2.3. Preparation of pomegranate raisins (anardana) Commercially available anardana contains 2.7% moisture, whereas fresh pomegranate arils contain about 78.7% moisture. One batch of fresh pomegranate arils was dried in a cabinet dryer at 90 C for 90 min, followed by 70 C for 2 h, and nally 50 C for an additional 9 h to remove the required 76% moisture. This drying regime was used following a variety of drying methods at various temperatures to achieve material that was similar to the commercial product. A second batch of fresh pomegranate arils was sundried in shallow trays in the summer months in the USP research greenhouse; the average temperature was 3243 C. The drying was continued until the arils lost the required 76% moisture. 2.4. Anthocyanin isolation and purication Fresh and dried pomegranate arils were extracted exhaustively by maceration with acidic methanol (1:10, w/v) containing 1%

0.1 M HCl over 24 h at room temperature. The extract was ltered through a sintered glass funnel and concentrated in vacuo at 37 C. The extract was partitioned sequentially with hexane and ethyl acetate, and the aqueous fraction containing the anthocyanins was lyophilised. Removal of other water-soluble constituents, such as sugars and ascorbic acid, was accomplished by chromatography over Diaion HP-20SS resin (Einbond, Reynertson, Luo, Basile, & Kennelly, 2004). The lyophilised, semi-puried anthocyanin extract was resuspended in 25 ml water, applied to the Diaion HP20SS resin column (60 cm 5 cm ID, 120 g) and allowed to adsorb for 20 min. The column was sequentially eluted with 500 ml water to remove sugars and then with 300 ml acidic methanol containing 0.1% 0.1 M HCl to elute all of the anthocyanins. Finally, the column was washed clean with 300 ml methanol:acetone (1:1) mixture. The methanol fraction containing anthocyanins was concentrated in vacuo at 37 C and lyophilised. 2.5. Anthocyanin analysis by quantitative HPLC and LCMS Quantitative HPLC analysis was conducted on an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA) consisting of a low pressure quaternary pump, an autosampler and a photodiode array detector controlled by Agilent ChemStation [Rev. B. 01.03(204)] software. LCMS analysis was conducted on a Shimadzu liquid chromatographmass spectrometer (Shimadzu, Columbia, MD), consisting of a dual-plunger parallel-ow solvent delivery module (LC-20AD), an autosampler (SIL-20AC), and a single-stage quadrupole mass analyser (LCMS-2010EV) monitored by LCMSsolution (ver.3.30) software (Shimadzu). The anthocyanin separation was done on a Zorbax SB-C8 column (4.6 mm ID 75 mm, 3.5 lm) (Agilent). Anthocyanin samples were dissolved in HPLC-grade water containing 0.1% TFA and ltered through a 0.45 lm nylon lter (Fisher Scientic, Pittsburgh, PA) prior to the analysis. The binary mobile phase consisted of solvents A, 0.1% TFA in water, and B, acetonitrile acidied with 0.1% TFA. The gradient method was 010 min, 1320% B; 1018 min, 20 40% B; 1821 min, 40% B; and post-run time of 5 min with 13% B. Solvent ow rate was 0.2 ml/min, and the sample injection volume was 10 ll. Detection was at 520 nm. The amounts of individual and total anthocyanins in samples extracted from fresh, ovendried, and sun-dried pomegranate arils were quantied using a cyanidin 3-glucoside standard calibration curve (r2 P 0.99). 2.6. Heat stability of anthocyanin Cyanidin 3-glucoside was dissolved in distilled water at a concentration of 0.01 mg/ml. Two samples of 1 ml each were transferred into 5 ml glass vials. Air was replaced with nitrogen in one vial, and both vials were capped and sealed immediately. These samples were subjected to heat treatment as mentioned in the drying protocol for pomegranate raisins, i.e. heated in a cabinet dryer at 90 C for 90 min, followed by 70 C for 2 h, and nally at 50 C for 9 h. The samples were cooled and analysed by HPLC to determine the amount of cyanidin 3-glucoside degraded by heat treatment. The results were compared to a freshly prepared 0.01 mg/ ml standard sample. 2.7. Polyphenol oxidase enzyme extraction Polyphenol oxidase enzyme extract was prepared following a modication of the procedure described by Coseteng and Lee (1987). Fresh and dried pomegranate arils (30 g each) were homogenised for 2 min in a prechilled blender with 50 ml ice-cold 0.1 M potassium phosphate buffer, pH 7.2, containing 1% Triton X-100. The homogenate was ltered through a sintered glass funnel and centrifuged (2 C, 5000g) for 15 min in a Sorvall RC-5B+

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centrifuge (Thermo Fisher Scientic, Inc. Waltham, MA). The supernatant was decanted and used as a crude enzyme preparation in the PPO activity assay. 2.8. PPO activity assay The PPO activity was determined by measuring the initial rate of increase in absorbance at 420 nm as described by Gonzalez, de Ancos, and Cano (1999). The activity was assayed in 3 ml of reaction mixture consisting of 2.5 ml potassium phosphate buffer (pH 6.0), 0.3 ml substrate (0.5 M catechol) and 0.2 ml crude enzyme. The blank consisted of 3.0 ml potassium phosphate buffer (pH 6.0). The rst control cuvette contained 2.7 ml buffer solution and 0.3 ml substrate, whereas the second control cuvette contained 2.8 ml buffer and 0.2 ml enzyme preparation. Absorbance values of these controls were subtracted from that of the sample. The enzyme activity was dened as the change in absorbance of 0.001 per min per ml enzyme. 2.9. Optimum pH for PPO activity The effect of pH on pomegranate PPO activity was determined by catechol oxidation in 0.1 M sodium acetate buffer (pH 5.0 and 5.5) and 0.1 M potassium phosphate buffer (pH 6.0, 6.5, 7.0, 7.5, and 8.0). The sample cuvette contained 2.5 ml buffer solution (pH 58), 0.3 ml substrate and 0.2 ml enzyme. 2.10. Effect of boiling on PPO activity PPO was extracted from fresh pomegranate arils and divided into two parts. The rst sample was boiled for 2 min at 100 C, and the PPO activity was determined using catechol as a substrate. The activity of boiled aril PPO was compared to that of untreated PPO from the second sample. 2.11. PPO kinetics: the MichaelisMenten constant Seven catechol solutions varying in concentration from 0.1 to 0.9 M were prepared, and the effect of substrate concentration on pomegranate PPO activity was investigated to partially characterise the enzyme kinetics. The sample cuvette contained 0.3 ml substrate (0.10.9 M catechol), 2.5 ml 0.1 M potassium phosphate buffer, pH 6.0, and 0.2 ml undiluted enzyme preparation. The MichaelisMenten constant (Km) and maximum velocity (Vmax) were calculated from a double reciprocal plot of 1/velocity vs. 1/ substrate concentration by the method of Lineweaver and Burk (1934). The reaction velocity was dened as change in optical density (DOD) of reaction mixture per min per ml of enzyme. 2.12. Effect of drying on anthocyanins and PPO activity Anthocyanins were extracted from fresh, oven-dried and sundried pomegranate arils and quantied by HPLC using the cyanidin 3-glucoside standard calibration curve. The PPO was extracted from fresh, oven-dried and sun-dried pomegranate arils and activity determined by catechol oxidation assay. 2.13. Effect of boiling on anthocyanins and PPO activity Two 25 g samples of fresh, frozen pomegranate arils were placed in glass bottles and immersed in an oil-bath at 100 C. It took 14 min to bring the frozen arils (80 C) to 100 C. The samples were maintained at 100 C for 2 min (total 16 min). Anthocyanins were extracted from the rst sample and quantied by HPLC using the cyanidin 3-glucoside standard calibration curve. PPO

activity was determined in the second sample by the catechol oxidation assay. The results were compared to total anthocyanins and PPO activity of 25 g samples of fresh, untreated arils, extracted simultaneously. 2.14. Data analysis Values are averages of three determinations. The results were analysed for variation (ANOVA) and statistical signicance by t-test. Error bars shown in gures are standard deviations of the values.

3. Results and discussion 3.1. Anthocyanin analysis by quantitative HPLC and LCMS We determined the presence of anthocyanins delphinidin 3,5diglucoside (m/z 627), cyanidin 3,5-diglucoside (m/z 611), delphinidin 3-glucoside (m/z 465), cyanidin 3-glucoside (m/z 449) and pelargonidin 3-glucoside (m/z 433) by LCMS analysis (Fig. 1: peaks 15). These anthocyanins have been reported previously to be present in pomegranates (Hernandez et al., 1999; Santagati et al., 1984). The mass spectrum showed the presence of the molecular ion for the respective anthocyanin along with that of its aglycone. Depending on the variety of the pomegranates, the amount of total anthocyanins varies from 35 to 350 mg/kg fresh weight of arils (Hernandez et al., 1999). Quantitative HPLC analysis indicated that arils of the fresh pomegranates of Wonderful variety contained 250 mg/kg anthocyanins. 3.2. Heat stability of anthocyanin The cyanidin 3-glucoside sample maintained under nitrogen showed only a small degradation that was not statistically different from the control of freshly prepared cyanidin 3-glucoside standard sample (Fig. 2). There was about 65% loss of cyanidin 3glucoside in the sample maintained under air, signifying the role of oxygen in anthocyanin degradation. Pure anthocyanins were stable at high temperatures in the absence of oxygen, but they quickly degraded in the presence of oxygen. 3.3. Optimum pH for PPO activity Depending upon the enzyme source and substrate, the optimum pH for browning reactions catalysed by PPO is between pH 4.0 and pH 7.0 (Severini, Baiano, De Pilli, Romaniello, & Derossi, 2003). The effect of pH on PPO activity was investigated using catechol as a substrate in the pH 5.08.0 range (Fig. 3). The optimum pH for pomegranate PPO activity was found to be about pH 6.0. The PPO activity decreased rapidly at pH below or above this optimum. 3.4. Effect of boiling on PPO activity There was about a 73% decrease in activity when the PPO extract was boiled. PPO is relatively heat-labile, and temperatures above 50 C for sufcient time result in a decrease of activity (Vmos-Vigyz, 1981). In fact, the heat inactivation of PPO by blanching treatment is the most common method of preventing the browning reaction in fruits and vegetables (Chen, Collins, McCarty, & Johnston, 1971). However, PPO heat resistance depends on the species and cultivar of the plant from which it is extracted. The ability of the PPO assay to measure this decrease in activity indicates, to some degree, that the assay is capable of distinguishing the enzyme reaction from any chemical reactions that may be present.

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V. Jaiswal et al. / Food Chemistry 118 (2010) 1116

Fig. 1. Typical LCMS chromatograms of anthocyanins from fresh, oven-dried and sun-dried pomegranate arils. Peak identities: 1 delphinidin 3,5-diglucoside; 2 cyanidin 3,5-diglucoside; 3 delphinidin 3-glucoside; 4 cyanidin 3-glucoside; 5 pelargonidin 3-glucoside. Identity of the sixth peak is unknown.

3.5. PPO kinetics: the MichaelisMenten constant Pomegranate PPO kinetics were partially characterised by investigation of the effect of substrate concentration on PPO activity. The MichaelisMenten constant (Km) and Vmax values were determined from a LineweaverBurk plot (Fig. 4). The Km value was 635 mM, and the Vmax value was 1.045 DOD/min/ml. Spinach PPO was considerably inhibited above its Km value when dopamine was used

as a substrate, whereas catechol is also shown to inhibit PPO activity at higher concentrations (Golbeck & Cammarata, 1981). Our results indicate a similar phenomenon; pomegranate PPO was inhibited above the catechol Km value in a progressive manner proportional to the catechol concentration. Using catechol as a substrate in the PPO assay may be responsible for these non-classic enzyme kinetics. The high Km value of 635 mM can be explained by the fact that catechol is a non-physiological substrate for pomegranate PPO.

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Table 1 Anthocyanin amount and PPO activity in pomegranate arils. Values are averages of three determinations and the number in parentheses is the standard deviation of the corresponding value. Characteristic Total anthocyanins (lg/g SD) PPO activity (units/ml SD) Fresh arils 250.5 (10.9) 647.7 (9.9) Oven-dried arils 97.4 (1.8) 205.7 (5.1) Sun-dried arils 42.2 (0.3) 356 (23.6)

Fig. 2. Effect of temperature on anthocyanin stability. Values are averages of three determinations. Error bars are standard deviations of the values.

but only 45% loss was recorded in the PPO activity. Anthocyanins are stable at high temperatures, whereas PPO is heat-labile and is considerably inhibited above 80 C (Severini et al., 2003). Inhibition of PPO by oven-drying at high temperature may be responsible for protecting the anthocyanins from oxidation by PPO. Sundrying was apparently unable to inhibit PPO activity as much, resulting in enhanced anthocyanin oxidation. 3.7. Effect of boiling on anthocyanins and PPO activity To further investigate the role of PPO in anthocyanin degradation, the effect of boiling on anthocyanins and PPO activity in pomegranate arils was determined. PPO activity in boiled arils was reduced by 75% from that of fresh arils, whereas total anthocyanins were reduced by only 2.5% (Fig. 5). Anthocyanins were protected from oxidative degradation because of inactivation of PPO by boiling. The results of this experiment again indicate the possible involvement of PPO in anthocyanin degradation. The role of PPO has always been speculated in anthocyanin degradation with mixed results. Although, our investigations were carried out on crude enzyme preparations, the results indicate a possible involvement of PPO in anthocyanin oxidation. In the presence of very low levels of hydrogen peroxide, peroxidase (POD; EC 1.11.1.7) also catalyses the formation of quinones from phenolic compounds (Vaughn & Duke, 1984). To adequately understand the physiology of an enzyme, it is important that the assay used can distinguish that enzyme from all others. Unfortunately, however, the catechol oxidation assay is not sufcient to measure the true PPO activity and needs to be rened, in order to distinguish PPO activity from POD or other similar enzyme activities. Our results strongly indicate a likely involvement of PPO in anthocyanin oxidation in pomegranate arils, although further investigations are required. Instead of a non-physiological substrate, such as catechol, anthocyanin may be the most suitable substrate to evaluate the role of PPO in anthocyanin oxidative degradation.

0.3

0.25

Absorbance at 420 nm

0.2

0.15

0.1

0.05

0 5 5.5 6 6.5 7 7.5 8

pH
Fig. 3. Effect of pH on pomegranate PPO activity. Values are averages of three determinations. Error bars are standard deviations of the values.

8
Km = 0.635 M Vmax = 1.045 OD/min/ml

7 6

1/OD/min/ml

5 4 3 2 1 0

-1/Km

1/Vmax

-2 -1

10

12

1/[Catechol] M

Fig. 4. Effect of substrate concentration on pomegranate PPO activity (Lineweaver Burk plot of the reaction data). Average of three determinations.

3.6. Effect of drying on anthocyanins and PPO activity Drying reduced the amount of anthocyanins and the PPO activity in fresh pomegranate arils (Table 1). Oven-drying resulted in 61% loss of anthocyanins and 68% loss of PPO activity. Sun-drying was more destructive to anthocyanins, resulting in about 83% loss,

Fig. 5. Effect of boiling on total anthocyanins and PPO activity in pomegranate arils. Average of three determinations and error bars are standard deviations of the values.

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V. Jaiswal et al. / Food Chemistry 118 (2010) 1116 Kong, J., Chia, L., Goh, N., Chia, T., & Brouillard, R. (2003). Analysis and biological activities of anthocyanins. Phytochemistry, 64, 923933. Lansky, E. P., & Newman, R. A. (2007). Punica granatum (pomegranate) and its potential for prevention and treatment of inammation and cancer. Journal of Ethnopharmacology, 109, 177206. Levin, G. M. (1994). Pomegranate (Punica granatum) plant genetic resources in Turkmenistan. Plant Genetic Resource. Newsletter, 97, 3137. Lineweaver, H., & Burk, D. (1934). The determination of enzyme dissociation constant. Journal of the American Chemical Society, 56, 658661. Mayer, A. M. (2006). Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry, 67, 23182331. Pruthi, J. S., & Saxena, A. K. (1984). Studies on anardana (dried pomegranate seeds). Journal of Food Science and Technology, 21, 296299. Rapeanu, G., Loey, A. V., Smout, C., & Hendrickx, M. (2006). Thermal and high pressure inactivation kinetics of victoria grape polyphenol oxidase: From model systems to grape must. Journal of Food Process Engineering, 29, 269286. Santagati, N. A., Duro, R., & Duro, F. (1984). Study on pigments present in pomegranate seeds. Journal of Commodity Science, 23, 247254. Seeram, N. P., Zhang, Y., Reed, J. D., Krueger, C. G., & Vaya, J. (2006). Pomegranate phytochemicals. In N. P. Seeram, R. N. Schulman, & D. Heber (Eds.), Pomegranates: Ancient roots to modern medicine (pp. 329). Boca Raton, FL: Taylor and Francis. Severini, C., Baiano, A., De Pilli, T., Romaniello, R., & Derossi, A. (2003). Prevention of enzymatic browning in sliced potatoes by blanching in boiling saline solutions. Lebensmittel-Wissenschaft und - Technologie, 36, 657665. Shishodia, S., Adams, L., Bhatt, I. D., & Aggarwal, B. B. (2006). Anticancer potential of pomegranate. In N. P. Seeram, R. N. Schulman, & D. Heber (Eds.), Pomegranates: Ancient roots to modern medicine (pp. 107116). Boca Raton, FL: Taylor and Francis. Singh, D. B., Kingly, A. R. P., & Jain, R. K. (2007). Studies on separation techniques of pomegranate arils and their effect on quality of anardana. Journal of Food Engineering, 79, 671674. Thipyapong, P., Stout, M. J., & Attajarusit, J. (2007). Functional analysis of polyphenol oxidases by antisense/sense technology. Molecules, 12, 1569 1595. Vmos-Vigyz, L. (1981). Polyphenol oxidase and peroxidase in fruits and vegetables. Critical Reviews in Food Science and Nutrition, 15, 49127. Vaughn, K. C., & Duke, S. O. (1984). Function of polyphenol oxidase in higher plants. Physiologia Plantarum, 60, 106112.

References
Aviram, M., Dornfeld, L., Kaplan, M., Coleman, R., Gaitini, D., Nitecki, S., et al. (2002a). Pomegranate juice avonoids inhibit low-density lipoprotein oxidation and cardiovascular diseases: Studies in atherosclerotic mice and in humans. Drugs under Experimental and Clinical Research, 28, 4962. Aviram, M., Fuhrman, B., Rosenblat, M., Volkova, N., Kaplan, M., Hayek, T., et al. (2002b). Pomegranate juice polyphenols decreases oxidative stress, low-density lipoprotein atherogenic modications and atherosclerosis. Free Radical Research, 36(Suppl. 1), 7273. Brouillard, R., Figueiredo, P., Elhabiri, M., & Dangles, O. (1997). Molecular interactions of phenolic compounds in relation to the color of fruit and vegetables. Proceedings of Phytochemical Society of Europe, 41, 2949. Chen, S. C., Collins, J. L., McCarty, I. E., & Johnston, M. R. (1971). Blanching of white potatoes by microwave energy followed by boiling water. Journal of Food Science, 36, 742743. Clifford, M. N. (2000). Anthocyanins Nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture, 80, 10631072. Coseteng, M. Y., & Lee, C. Y. (1987). Changes in apple polyphenoloxidase and polyphenol concentrations in relation to degree of browning. Journal of Food Science, 52, 985989. Einbond, L. S., Reynertson, K. A., Luo, X., Basile, M. J., & Kennelly, E. J. (2004). Anthocyanin antioxidants from edible fruits. Food Chemistry, 84, 2328. Gil, M. I., Tomas-Barberan, F. A., Hess-Pierce, B., Holcroft, D. M., & Kader, A. A. (2000). Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. Journal of Agricultural and Food Chemistry, 48, 45814589. Golbeck, J. H., & Cammarata, K. V. (1981). Spinach thylakoid polyphenol oxidase isolation, activation and properties of the native chloroplast enzyme. Plant Physiology, 67, 977984. Gonzalez, E. M., de Ancos, B., & Cano, M. P. (1999). Partial characterization of polyphenol oxidase activity in raspberry fruits. Journal of Agricultural and Food Chemistry, 47, 40684072. Hernandez, F., Melgarejo, P., Tomas-Barberan, F. A., & Artes, F. (1999). Evolution of juice anthocyanins during ripening of new selected pomegranate (Punica granatum) clones. European Food Research and Technology, 210, 3942. Kim, N. D., Mehta, R., Yu, W., Neeman, I., Livney, T., Amichay, A., et al. (2002). Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer. Breast Cancer Research and Treatment, 71, 203217.