Food Chemistry 118 (2010) 11–16

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Food Chemistry

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

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

Keywords:PomegranatePunica granatum L.Polyphenol oxidaseAnthocyanin

0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.01.095

* Corresponding author. Address: Program in PhaChemistry and Biochemistry, University of the Scienphia, PA 19104, United States. Tel.: +1 215 596 8915;

E-mail address: a.dermar@usp.edu (A. DerMardero

a b s t r a c t

Anthocyanins are natural pigments responsible for red, purple, and blue colouration in plants. Humanconsumption of anthocyanins is increasing because of the rising awareness and interest in their potentialhealth benefits. Pomegranate is one of the major sources of polyphenolic phytochemicals, such as antho-cyanins. Dried pomegranate raisins (anardana) are consumed in large quantities in Asian countries, andcontain substantial amounts of anthocyanins. Five anthocyanins were found to be present in pomegran-ate raisins. Drying adversely affected the amount of anthocyanins, with polyphenol oxidase (PPO) playinga possible role in oxidative degradation of anthocyanins. Anthocyanins are heat-stable compounds, andinactivation of PPO by processing at high temperature for short periods may prevent PPO-catalysedanthocyanin oxidation in pomegranate arils. Pomegranate PPO kinetics were partially characterised byinvestigating the effect of substrate (catechol) concentration; optimum pH for PPO activity was foundto be 6.0.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Anthocyanins are the largest and most important group ofwater-soluble pigments in nature. In plant tissues, they are respon-sible 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 ofconsiderable importance in the co-evolution of plant–animal inter-actions, as they contribute to the colourful appearance of flowers,fruits and vegetables, helping them to attract animals, leading toseed dispersal and pollination (Kong, Chia, Goh, Chia, & Brouillard,2003). Anthocyanin stability is influenced by various factors suchas temperature, pH, light, and oxygen. Anthocyanins also may besusceptible to degradation by oxidising enzymes.

Polyphenol oxidases (PPOs), or tyrosinases, are nuclear-codedenzymes with a dinuclear copper centre, which are able to insertoxygen in a position ortho to an existing hydroxyl group in an aro-matic ring, followed by the oxidation of the diphenol to the corre-sponding quinone. These o-quinone intermediates are highlyreactive and quickly polymerise to dark-coloured melanins (Rape-anu et al., 2006). PPO is widely distributed in animals, plants, fungi,

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rmacognosy, Department ofces in Philadelphia, Philadel-

fax: +1 215 596 8710.sian).

and bacteria and plays an important role in various physiologicaland defence reactions (Mayer, 2006). PPO is inducible by both bio-tic and abiotic stresses, and is implicated in several physiologicalprocesses, including photoreduction of molecular oxygen by pho-tosystem I (PSI), regulation of plastidic oxygen levels, aurone bio-synthesis, and the phenylpropanoid pathway (Thipyapong, Stout,& Attajarusit, 2007).

The pomegranate (Punica granatum L.), from the Latin wordspomus and granatus, meaning a seeded or granular apple, is nativefrom Iran to the Himalayas in northern India, where it has beencultivated for thousands of years. There are over 1000 cultivarsof Punica granatum (Levin, 1994) that are grown from Iran, east-ward to China and India and westward through the Mediterraneanregion, on to the American Southwest, California and Mexico (Lan-sky & Newman, 2007). There has been a virtual explosion of inter-est in pomegranate as a medicinal and nutritional product becauseof its multifunctionality, and, as a result, the field of pomegranateresearch has experienced tremendous growth in the past decade.Polyphenols are the major class of pomegranate fruit phytochemi-cals, including flavonoids (anthocyanins), condensed tannins (pro-anthocyanidins) and hydrolysable tannins (ellagitannins andgallotannins) (Gil, Tomas-Barberan, Hess-Pierce, Holcroft, & Kader,2000; Hernandez, Melgarejo, Tomas-Barberan, & Artes, 1999; San-tagati, Duro, & Duro, 1984). Pomegranate juice may provide protec-tion against cardiovascular diseases and stroke, by acting as apotent antioxidant against LDL oxidation and inhibition of athero-sclerosis development (Aviram et al., 2002a, 2002b). Pomegranate

12 V. Jaiswal et al. / Food Chemistry 118 (2010) 11–16

phytochemicals also show potential in chemoprevention of varioustypes of cancers, by exerting antiproliferative effects on tumourcells (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). Thepresence of anthocyanins is responsible for the appealing brightred colour of juice and other products of pomegranate fruit. Pome-granate raisins (anardana) are dried arils of wild pomegranates thatare manually separated from the rind and septa of the fruit andsun- or air-dried. Pomegranate raisins have a distinct sour or tartflavour, and are commercially available in many West and EastAsian 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 pre-pared by hydrostatic pressing of whole fruits, has been found tocontain more than 26 different chemical compounds, whereasthe seeds contain various fatty acids and steroidal compounds(Seeram, Zhang, Reed, Krueger, & Vaya, 2006). The aril (the seedand surrounding flesh) contains anthocyanins, which might be af-fected by the severity of the drying process employed for the raisinpreparation.

Scarce information exists on the chemical constituents of anar-dana. This study was designed to evaluate the effect of drying onpomegranate aril anthocyanins and to investigate the role of PPOin aril anthocyanin oxidation.

2. Materials and methods

2.1. Plant material

Fresh pomegranates (P. granatum L. cv. Wonderful) were sup-plied by POM Wonderful company (Del Rey, CA). Arils were sepa-rated manually and kept in dark and cold (�80 �C) storage untilanalysed. Pomegranate raisins (anardana) were purchased fromDeep Foods, Inc. (Union, NJ).

2.2. Chemicals

Cyanidin 3-glucoside (kuromanin chloride) standard for HPLCanalysis, catechol, trifluoroacetic acid (TFA), acetone, acetonitrile,ethyl acetate, hexane, and methanol were purchased from Sig-ma–Aldrich (St. Louis, MO). Unless stated otherwise, all solventswere HPLC-grade. Diaion HP-20SS resin was purchased from Supe-lco (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 dryerat 90 �C for 90 min, followed by 70 �C for 2 h, and finally 50 �C foran additional 9 h to remove the required 76% moisture. This dryingregime was used following a variety of drying methods at varioustemperatures to achieve material that was similar to the commer-cial product. A second batch of fresh pomegranate arils was sun-dried in shallow trays in the summer months in the USP researchgreenhouse; the average temperature was 32–43 �C. The dryingwas continued until the arils lost the required 76% moisture.

2.4. Anthocyanin isolation and purification

Fresh and dried pomegranate arils were extracted exhaustivelyby maceration with acidic methanol (1:10, w/v) containing 1%

0.1 M HCl over 24 h at room temperature. The extract was filteredthrough a sintered glass funnel and concentrated in vacuo at 37 �C.The extract was partitioned sequentially with hexane and ethylacetate, and the aqueous fraction containing the anthocyaninswas lyophilised. Removal of other water-soluble constituents, suchas sugars and ascorbic acid, was accomplished by chromatographyover Diaion HP-20SS resin (Einbond, Reynertson, Luo, Basile, &Kennelly, 2004). The lyophilised, semi-purified anthocyanin ex-tract was resuspended in 25 ml water, applied to the Diaion HP-20SS resin column (60 cm � 5 cm ID, 120 g) and allowed to adsorbfor 20 min. The column was sequentially eluted with 500 ml waterto remove sugars and then with 300 ml acidic methanol containing0.1% 0.1 M HCl to elute all of the anthocyanins. Finally, the columnwas washed clean with 300 ml methanol:acetone (1:1) mixture.The methanol fraction containing anthocyanins was concentratedin vacuo at 37 �C and lyophilised.

2.5. Anthocyanin analysis by quantitative HPLC and LC–MS

Quantitative HPLC analysis was conducted on an Agilent 1100HPLC system (Agilent Technologies, Santa Clara, CA) consisting ofa low pressure quaternary pump, an autosampler and a photodiodearray detector controlled by Agilent ChemStation [Rev. B.01.03(204)] software. LC–MS analysis was conducted on a Shima-dzu liquid chromatograph–mass spectrometer (Shimadzu, Colum-bia, MD), consisting of a dual-plunger parallel-flow solventdelivery module (LC-20AD), an autosampler (SIL-20AC), and a sin-gle-stage quadrupole mass analyser (LCMS-2010EV) monitored byLCMSsolution (ver.3.30) software (Shimadzu). The anthocyaninseparation was done on a Zorbax SB-C8 column (4.6 mmID � 75 mm, 3.5 lm) (Agilent). Anthocyanin samples were dis-solved in HPLC-grade water containing 0.1% TFA and filteredthrough a 0.45 lm nylon filter (Fisher Scientific, Pittsburgh, PA)prior to the analysis. The binary mobile phase consisted of solventsA, 0.1% TFA in water, and B, acetonitrile acidified with 0.1% TFA.The gradient method was 0–10 min, 13–20% B; 10–18 min, 20–40% B; 18–21 min, 40% B; and post-run time of 5 min with 13%B. Solvent flow rate was 0.2 ml/min, and the sample injection vol-ume was 10 ll. Detection was at 520 nm. The amounts of individ-ual and total anthocyanins in samples extracted from fresh, oven-dried, and sun-dried pomegranate arils were quantified using acyanidin 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 con-centration of 0.01 mg/ml. Two samples of 1 ml each were trans-ferred into 5 ml glass vials. Air was replaced with nitrogen in onevial, and both vials were capped and sealed immediately. Thesesamples were subjected to heat treatment as mentioned in the dry-ing protocol for pomegranate raisins, i.e. heated in a cabinet dryerat 90 �C for 90 min, followed by 70 �C for 2 h, and finally at 50 �Cfor 9 h. The samples were cooled and analysed by HPLC to deter-mine the amount of cyanidin 3-glucoside degraded by heat treat-ment. 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 amodification of the procedure described by Coseteng and Lee(1987). Fresh and dried pomegranate arils (30 g each) werehomogenised for 2 min in a prechilled blender with 50 ml ice-cold0.1 M potassium phosphate buffer, pH 7.2, containing 1% TritonX-100. The homogenate was filtered through a sintered glass fun-nel and centrifuged (2 �C, 5000g) for 15 min in a Sorvall RC-5B+

V. Jaiswal et al. / Food Chemistry 118 (2010) 11–16 13

centrifuge (Thermo Fisher Scientific, Inc. Waltham, MA). The super-natant was decanted and used as a crude enzyme preparation inthe PPO activity assay.

2.8. PPO activity assay

The PPO activity was determined by measuring the initial rateof increase in absorbance at 420 nm as described by Gonzalez, deAncos, and Cano (1999). The activity was assayed in 3 ml of reac-tion mixture consisting of 2.5 ml potassium phosphate buffer (pH6.0), 0.3 ml substrate (0.5 M catechol) and 0.2 ml crude enzyme.The blank consisted of 3.0 ml potassium phosphate buffer (pH6.0). The first control cuvette contained 2.7 ml buffer solutionand 0.3 ml substrate, whereas the second control cuvette con-tained 2.8 ml buffer and 0.2 ml enzyme preparation. Absorbancevalues of these controls were subtracted from that of the sample.The enzyme activity was defined as the change in absorbance of0.001 per min per ml enzyme.

2.9. Optimum pH for PPO activity

The effect of pH on pomegranate PPO activity was determinedby catechol oxidation in 0.1 M sodium acetate buffer (pH 5.0 and5.5) 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 5–8), 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 dividedinto two parts. The first 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 untreatedPPO from the second sample.

2.11. PPO kinetics: the Michaelis–Menten constant

Seven catechol solutions varying in concentration from 0.1 to0.9 M were prepared, and the effect of substrate concentration onpomegranate PPO activity was investigated to partially character-ise the enzyme kinetics. The sample cuvette contained 0.3 ml sub-strate (0.1–0.9 M catechol), 2.5 ml 0.1 M potassium phosphatebuffer, pH 6.0, and 0.2 ml undiluted enzyme preparation. TheMichaelis–Menten 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 defined as change in optical den-sity (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 sun-dried pomegranate arils and quantified by HPLC using the cyanidin3-glucoside standard calibration curve. The PPO was extractedfrom fresh, oven-dried and sun-dried pomegranate arils and activ-ity determined by catechol oxidation assay.

2.13. Effect of boiling on anthocyanins and PPO activity

Two 25 g samples of fresh, frozen pomegranate arils wereplaced in glass bottles and immersed in an oil-bath at 100 �C. Ittook 14 min to bring the frozen arils (�80 �C) to 100 �C. The sam-ples were maintained at 100 �C for 2 min (total 16 min). Anthocy-anins were extracted from the first sample and quantified by HPLCusing the cyanidin 3-glucoside standard calibration curve. PPO

activity was determined in the second sample by the catecholoxidation assay. The results were compared to total anthocyaninsand PPO activity of 25 g samples of fresh, untreated arils, extractedsimultaneously.

2.14. Data analysis

Values are averages of three determinations. The results wereanalysed for variation (ANOVA) and statistical significance by t-test.Error bars shown in figures are standard deviations of the values.

3. Results and discussion

3.1. Anthocyanin analysis by quantitative HPLC and LC–MS

We determined the presence of anthocyanins delphinidin 3,5-diglucoside (m/z 627), cyanidin 3,5-diglucoside (m/z 611), delphin-idin 3-glucoside (m/z 465), cyanidin 3-glucoside (m/z 449) andpelargonidin 3-glucoside (m/z 433) by LC–MS analysis (Fig. 1:peaks 1–5). These anthocyanins have been reported previously tobe present in pomegranates (Hernandez et al., 1999; Santagatiet al., 1984). The mass spectrum showed the presence of themolecular ion for the respective anthocyanin along with that ofits aglycone. Depending on the variety of the pomegranates, theamount of total anthocyanins varies from 35 to 350 mg/kg freshweight of arils (Hernandez et al., 1999). Quantitative HPLC analysisindicated that arils of the fresh pomegranates of Wonderful varietycontained 250 mg/kg anthocyanins.

3.2. Heat stability of anthocyanin

The cyanidin 3-glucoside sample maintained under nitrogenshowed only a small degradation that was not statistically differ-ent from the control of freshly prepared cyanidin 3-glucoside stan-dard sample (Fig. 2). There was about 65% loss of cyanidin 3-glucoside in the sample maintained under air, signifying the roleof oxygen in anthocyanin degradation. Pure anthocyanins werestable at high temperatures in the absence of oxygen, but theyquickly degraded in the presence of oxygen.

3.3. Optimum pH for PPO activity

Depending upon the enzyme source and substrate, the opti-mum pH for browning reactions catalysed by PPO is between pH4.0 and pH 7.0 (Severini, Baiano, De Pilli, Romaniello, & Derossi,2003). The effect of pH on PPO activity was investigated usingcatechol as a substrate in the pH 5.0–8.0 range (Fig. 3). The opti-mum pH for pomegranate PPO activity was found to be about pH6.0. The PPO activity decreased rapidly at pH below or above thisoptimum.

3.4. Effect of boiling on PPO activity

There was about a 73% decrease in activity when the PPO ex-tract was boiled. PPO is relatively heat-labile, and temperaturesabove 50 �C for sufficient time result in a decrease of activity(Vámos-Vigyázó, 1981). In fact, the heat inactivation of PPO byblanching treatment is the most common method of preventingthe browning reaction in fruits and vegetables (Chen, Collins,McCarty, & Johnston, 1971). However, PPO heat resistance dependson the species and cultivar of the plant from which it is extracted.The ability of the PPO assay to measure this decrease in activityindicates, to some degree, that the assay is capable of distinguish-ing the enzyme reaction from any chemical reactions that may bepresent.

Fig. 1. Typical LC–MS chromatograms of anthocyanins from fresh, oven-dried and sun-dried pomegranate arils. Peak identities: 1 – delphinidin 3,5-diglucoside; 2 – cyanidin3,5-diglucoside; 3 – delphinidin 3-glucoside; 4 – cyanidin 3-glucoside; 5 – pelargonidin 3-glucoside. Identity of the sixth peak is unknown.

14 V. Jaiswal et al. / Food Chemistry 118 (2010) 11–16

3.5. PPO kinetics: the Michaelis–Menten constant

Pomegranate PPO kinetics were partially characterised by inves-tigation of the effect of substrate concentration on PPO activity. TheMichaelis–Menten constant (Km) and Vmax values were determinedfrom a Lineweaver–Burk plot (Fig. 4). The Km value was 635 mM,and the Vmax value was 1.045 DOD/min/ml. Spinach PPO was con-siderably inhibited above its Km value when dopamine was used

as a substrate, whereas catechol is also shown to inhibit PPO activityat higher concentrations (Golbeck & Cammarata, 1981). Our resultsindicate a similar phenomenon; pomegranate PPO was inhibitedabove the catechol Km value in a progressive manner proportionalto the catechol concentration. Using catechol as a substrate in thePPO assay may be responsible for these non-classic enzyme kinet-ics. The high Km value of 635 mM can be explained by the fact thatcatechol is a non-physiological substrate for pomegranate PPO.

Km = 0.635 M Vmax = 1.045 ΔOD/min/ml

1/Vmax -1/Km

-1

0

1

2

3

4

5

6

7

8

-2 0 2 4 6 8 10 12

1/[Catechol] M

1/ΔO

D/m

in/m

l

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

Table 1Anthocyanin amount and PPO activity in pomegranate arils. Values are averages ofthree determinations and the number in parentheses is the standard deviation of thecorresponding value.

Characteristic Fresh arils Oven-dried arils Sun-dried arils

Total anthocyanins (lg/g ± SD) 250.5 (±10.9) 97.4 (±1.8) 42.2 (±0.3)PPO activity (units/ml ± SD) 647.7 (±9.9) 205.7 (±5.1) 356 (±23.6)

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 thevalues.

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

0

0.05

0.1

0.15

0.2

0.25

0.3

5 5.5 6 6.5 7 7.5 8

pH

Abs

orba

nce

at 4

20 n

m

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

V. Jaiswal et al. / Food Chemistry 118 (2010) 11–16 15

3.6. Effect of drying on anthocyanins and PPO activity

Drying reduced the amount of anthocyanins and the PPO activ-ity in fresh pomegranate arils (Table 1). Oven-drying resulted in61% loss of anthocyanins and 68% loss of PPO activity. Sun-dryingwas more destructive to anthocyanins, resulting in about 83% loss,

but only 45% loss was recorded in the PPO activity. Anthocyaninsare stable at high temperatures, whereas PPO is heat-labile andis considerably inhibited above 80 �C (Severini et al., 2003). Inhibi-tion of PPO by oven-drying at high temperature may be responsi-ble for protecting the anthocyanins from oxidation by PPO. Sun-drying 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 degrada-tion, the effect of boiling on anthocyanins and PPO activity inpomegranate arils was determined. PPO activity in boiled arilswas reduced by 75% from that of fresh arils, whereas total antho-cyanins were reduced by only 2.5% (Fig. 5). Anthocyanins were pro-tected from oxidative degradation because of inactivation of PPOby boiling. The results of this experiment again indicate the possi-ble involvement of PPO in anthocyanin degradation.

The role of PPO has always been speculated in anthocyanin deg-radation with mixed results. Although, our investigations were car-ried out on crude enzyme preparations, the results indicate apossible involvement of PPO in anthocyanin oxidation.

In the presence of very low levels of hydrogen peroxide, peroxi-dase (POD; EC 1.11.1.7) also catalyses the formation of quinonesfrom phenolic compounds (Vaughn & Duke, 1984). To adequatelyunderstand the physiology of an enzyme, it is important that the as-say used can distinguish that enzyme from all others. Unfortunately,however, the catechol oxidation assay is not sufficient to measurethe true PPO activity and needs to be refined, in order to distinguishPPO activity from POD or other similar enzyme activities. Our re-sults strongly indicate a likely involvement of PPO in anthocyaninoxidation in pomegranate arils, although further investigationsare required. Instead of a non-physiological substrate, such as cate-chol, anthocyanin may be the most suitable substrate to evaluatethe role of PPO in anthocyanin oxidative degradation.

16 V. Jaiswal et al. / Food Chemistry 118 (2010) 11–16

References

Aviram, M., Dornfeld, L., Kaplan, M., Coleman, R., Gaitini, D., Nitecki, S., et al.(2002a). Pomegranate juice flavonoids inhibit low-density lipoprotein oxidationand cardiovascular diseases: Studies in atherosclerotic mice and in humans.Drugs under Experimental and Clinical Research, 28, 49–62.

Aviram, M., Fuhrman, B., Rosenblat, M., Volkova, N., Kaplan, M., Hayek, T., et al.(2002b). Pomegranate juice polyphenols decreases oxidative stress, low-densitylipoprotein atherogenic modifications and atherosclerosis. Free Radical Research,36(Suppl. 1), 72–73.

Brouillard, R., Figueiredo, P., Elhabiri, M., & Dangles, O. (1997). Molecularinteractions of phenolic compounds in relation to the color of fruit andvegetables. Proceedings of Phytochemical Society of Europe, 41, 29–49.

Chen, S. C., Collins, J. L., McCarty, I. E., & Johnston, M. R. (1971). Blanching of whitepotatoes by microwave energy followed by boiling water. Journal of FoodScience, 36, 742–743.

Clifford, M. N. (2000). Anthocyanins – Nature, occurrence and dietary burden.Journal of the Science of Food and Agriculture, 80, 1063–1072.

Coseteng, M. Y., & Lee, C. Y. (1987). Changes in apple polyphenoloxidase andpolyphenol concentrations in relation to degree of browning. Journal of FoodScience, 52, 985–989.

Einbond, L. S., Reynertson, K. A., Luo, X., Basile, M. J., & Kennelly, E. J. (2004).Anthocyanin antioxidants from edible fruits. Food Chemistry, 84, 23–28.

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 phenoliccomposition and processing. Journal of Agricultural and Food Chemistry, 48,4581–4589.

Golbeck, J. H., & Cammarata, K. V. (1981). Spinach thylakoid polyphenol oxidaseisolation, activation and properties of the native chloroplast enzyme. PlantPhysiology, 67, 977–984.

Gonzalez, E. M., de Ancos, B., & Cano, M. P. (1999). Partial characterization ofpolyphenol oxidase activity in raspberry fruits. Journal of Agricultural and FoodChemistry, 47, 4068–4072.

Hernandez, F., Melgarejo, P., Tomas-Barberan, F. A., & Artes, F. (1999). Evolution ofjuice anthocyanins during ripening of new selected pomegranate (Punicagranatum) clones. European Food Research and Technology, 210, 39–42.

Kim, N. D., Mehta, R., Yu, W., Neeman, I., Livney, T., Amichay, A., et al. (2002).Chemopreventive and adjuvant therapeutic potential of pomegranate (Punicagranatum) for human breast cancer. Breast Cancer Research and Treatment, 71,203–217.

Kong, J., Chia, L., Goh, N., Chia, T., & Brouillard, R. (2003). Analysis and biologicalactivities of anthocyanins. Phytochemistry, 64, 923–933.

Lansky, E. P., & Newman, R. A. (2007). Punica granatum (pomegranate) and itspotential for prevention and treatment of inflammation and cancer. Journal ofEthnopharmacology, 109, 177–206.

Levin, G. M. (1994). Pomegranate (Punica granatum) plant genetic resources inTurkmenistan. Plant Genetic Resource. Newsletter, 97, 31–37.

Lineweaver, H., & Burk, D. (1934). The determination of enzyme dissociationconstant. Journal of the American Chemical Society, 56, 658–661.

Mayer, A. M. (2006). Polyphenol oxidases in plants and fungi: Going places? Areview. Phytochemistry, 67, 2318–2331.

Pruthi, J. S., & Saxena, A. K. (1984). Studies on anardana (dried pomegranate seeds).Journal of Food Science and Technology, 21, 296–299.

Rapeanu, G., Loey, A. V., Smout, C., & Hendrickx, M. (2006). Thermal and highpressure inactivation kinetics of victoria grape polyphenol oxidase: Frommodel systems to grape must. Journal of Food Process Engineering, 29,269–286.

Santagati, N. A., Duro, R., & Duro, F. (1984). Study on pigments present inpomegranate seeds. Journal of Commodity Science, 23, 247–254.

Seeram, N. P., Zhang, Y., Reed, J. D., Krueger, C. G., & Vaya, J. (2006). Pomegranatephytochemicals. In N. P. Seeram, R. N. Schulman, & D. Heber (Eds.),Pomegranates: Ancient roots to modern medicine (pp. 3–29). Boca Raton, FL:Taylor and Francis.

Severini, C., Baiano, A., De Pilli, T., Romaniello, R., & Derossi, A. (2003). Prevention ofenzymatic browning in sliced potatoes by blanching in boiling saline solutions.Lebensmittel-Wissenschaft und - Technologie, 36, 657–665.

Shishodia, S., Adams, L., Bhatt, I. D., & Aggarwal, B. B. (2006). Anticancer potential ofpomegranate. In N. P. Seeram, R. N. Schulman, & D. Heber (Eds.), Pomegranates:Ancient roots to modern medicine (pp. 107–116). Boca Raton, FL: Taylor andFrancis.

Singh, D. B., Kingly, A. R. P., & Jain, R. K. (2007). Studies on separation techniques ofpomegranate arils and their effect on quality of anardana. Journal of FoodEngineering, 79, 671–674.

Thipyapong, P., Stout, M. J., & Attajarusit, J. (2007). Functional analysis ofpolyphenol oxidases by antisense/sense technology. Molecules, 12, 1569–1595.

Vámos-Vigyázó, L. (1981). Polyphenol oxidase and peroxidase in fruits andvegetables. Critical Reviews in Food Science and Nutrition, 15, 49–127.

Vaughn, K. C., & Duke, S. O. (1984). Function of polyphenol oxidase in higher plants.Physiologia Plantarum, 60, 106–112.