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Original Article

Effect of storage on the content of polyphenols, vitamin C and theantioxidant activity of orange juices

Inga Klimczak�, Maria Ma"ecka, Miros"awa Szlachta, Anna Gliszczynska-Swig"o

Faculty of Commodity Science, The Poznan University of Economics, Al. Niepodleg!osci 10, 60-967 Poznan, Poland

Received 23 November 2005; received in revised form 9 February 2006; accepted 16 February 2006

Abstract

The effect of time and temperature on the content of vitamin C, total polyphenols and individual phenolic compounds as well as on the

antioxidant activity of two commercial orange juices was studied. The polyphenol content was determined using Folin–Ciocalteu and

HPLC methods. The two methods, SPE versus direct injection following a simple treatment of samples, were compared to assess two

techniques of sample preparation. For antioxidant capacity determination, DPPH and FRAP assays were used. All analyses were carried

out for fresh juices and after storage at 18, 28 and 38 1C for 2, 4 and 6 months. It was found that vitamin C and free and conjugated

hydroxycinnamic acids were the most affected by both duration and temperature of storage. The decrease in the content of polyphenols

and vitamin C upon storage was reflected by the decrease in the antioxidant capacity of orange juices. Small changes in flavanone content

were observed, indicating high stability of these compounds upon storage.

Keywords: Orange juice; Polyphenols; Phenolic acids; Flavanones; Vitamin C; Antioxidant activity; FRAP assay; DPPH assay; Storage

1. Introduction

Phenolic compounds—especially phenolic acids andflavonoids—are ubiquitously present in vegetables, fruits,seeds, tea, wines and juices; thus they are an integral part ofthe human diet. Recently, they have received muchattention since many epidemiological studies suggest thatconsumption of polyphenol-rich foods and beverages isassociated with a reduced risk of cardiovascular diseases,stroke and certain forms of cancer. These protective effectshave partly been ascribed to the antioxidant propertiesespecially of flavonoids (Middleton and Kandaswami,1993; Hollman and Katan, 1997; Prior and Cao,2000a, b; Kaur and Kapoor, 2001). Among fruits and fruitproducts, oranges and orange juices—because of theirrelatively high consumption—may be highlighted as animportant source of vitamin C and polyphenolic com-pounds. Vitamin C is considered as a most importantwater-soluble antioxidant. It protects compounds in

ing author. Tel.: +4861 8569035; fax: +48 61 8543993.

ess: inga.klimczak@ae.poznan.pl (I. Klimczak).

extracellular and intracellular spaces in most biologicalsystems and reduces tocopherol radicals back to theiractive form at the cellular membranes (Kaur and Kapoor,2001). It can directly scavenge superoxide radical, singletoxygen, hydrogen peroxide and hydroxyl radical. Cur-rently ascorbic acid is the most widely used vitaminsupplement worldwide. Based on available biochemical,clinical, and epidemiological studies, the current recom-mended daily acceptance (RDA) for ascorbic acid issuggested to be 100–120mg/day to achieve cellular satura-tion and optimum risk reduction of heart diseases, strokeand cancer in healthy individuals (Naidu, 2003). Thevitamin C content in orange juices range from 150 to450mg/l; one glass of orange juice (200mL) can deliverabout 30–80% of recommended daily intake of vitamin C(Gliszczynska-Swig"o et al., 2004).A major part of phenolic compounds of oranges and

orange juices are hydroxycinnamic acids (HCA) andflavonoids, among which flavanones are predominant.Hydroxycinnamic acids occur mainly as esters of ferulic,p-coumaric, sinapic and caffeic acids. Flavanones inoranges occur mainly as glycosides; glycosilation takes

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place at position 7 either by rutinose (6-O-a-L-rhamnosyl-D-glucosides) or neohesperidose (2-O-a-L-rhamnosyl-D-glucosides) (Tomas-Barberan and Clifford, 2000). Amongflavanone aglycons, naringenin, hesperetin, eriodictyol andisosakuranetin are the most common, but they are presentin much smaller quantities than are glycosides. Citrusflavonoids, especially hesperidin, have shown a wide rangeof therapeutical properties such as anti-inflammatory,antihypertensive, diuretic, analgesic and hypolipidemicactivities (Da Silva et al., 1994; Galati et al., 1994, 1996;Monforte et al., 1995).

Some literature data on hydroxycinnamic acids andflavanone content in commercial orange juices are available(Peleg et al., 1991; Robards et al., 1997; Tomas-Barberanand Clifford, 2000; Kanitsar et al., 2001; Gil-Izquierdoet al., 2002; Belajova and Suhaj, 2004), but they aremarginal and differentiated because of differences invarieties of oranges, their ripeness and the technologicalprocesses used to obtained commercial juice. Moreover, thedifferences in published data may be due to the applicationof different methods of sample preparation and polyphe-nolic compounds determination. The most useful andcommon in polyphenol analysis is reversed-phase high-performance liquid chromatography (RP-HPLC) because ofits high specificity, sensitivity and simplicity. However, thetreatment of samples before injection onto HPLC columnmay affect polyphenol content significantly (Tura andRobards, 2002; Robards, 2003). Moreover, due to thecomplexity of polyphenols, isolation and quantitativeanalysis are often difficult and interferences among pheno-lics and other compounds may affect the HPLC analysis.Thus, separation of polyphenols using solid-phase extrac-tion (SPE) with C18 cartridges is recommended as a fast andeasy procedure for the isolation, purification and pre-concentration of organic compounds present in biologicalmaterial (Buldini et al., 2002; Tura and Robards, 2002).

Moreover, although some data on the content ofhydroxycinnamic acids derivatives in fresh and storedorange juices are available (Naim et al., 1988; Fallico et al.,1996), there is little information on the influence of storageduration and temperature on flavanone content (Del Caroet al., 2004).

The objectives of the present study were: (1) evaluationof the effect of time and temperature on the content ofvitamin C, total polyphenols and individual flavanones andfree and conjugated HCA as well as on the antioxidantactivity of commercial orange juices and (2) comparison oftwo techniques of sample preparation—SPE versus directinjection following a simple treatment of samples of freshand stored orange juices.

2. Materials and methods

2.1. Materials

Thirty one-litre cartons (1 L) of each of two differentbrands of pure orange juice were obtained from the local

juice manufacturers. Three cartons of each brand of juicewere analysed immediately for polyphenols, vitamin C andantioxidant activity (fresh juices). Three cartons each of thesame brand were stored for 2, 4, and 6 months at 18, 28and 38 1C (in all, 27 cartons of each brand of juice werestored). The temperature of 18 1C was used in our study asthe boundary, in accordance with the requirements of thePolish norm. In order to accelerate the ongoing changes inthe cartons of juices, temperatures were increased by 10and 20 1C in relation to the value permitted by the norm.Phenolic acids (caffeic, ferulic, p-coumaric and sinapic

acids), hesperidin, neohesperidin, Folin–Ciocalteu reagentand DPPH (1,1-diphenyl-2-picrylhydrazyl) were purchasedfrom Sigma-Aldrich (Steinheim, Germany). Narirutin waspurchased from Roth (Karlsruhe, Germany); naringin,didymin and TPTZ (2,4,6-tripyridyl-s-triazine) were pur-chased from Fluka (Buchs, Switzerland); and ascorbic acidwas purchased from Merck (Darmstadt, Germany).

2.2. HPLC analysis of free phenolic acids and flavanones

after direct injection

Direct determination of free phenolic acids and flava-nones in orange juice was carried out after centrifugationof samples at room temperature at 10,000 rpm for 5min.Twenty microlitres of the supernatant obtained wereinjected onto the HPLC column. Each sample was injectedat least three times. The HPLC analyses of phenolic acidsand flavanones were performed at room temperature on aWaters 600 high-performance liquid chromatograph(Waters, Millford, MA, USA) equipped with SymmetryC18 column (150� 3.9mm, 5 mm, Waters, Millford, MA,USA) fitted with mBondapak C18 cartridge guard column(Waters, Millford, MA, USA). For simultaneous determi-nation of phenolic acids and flavanones in orange juices agradient of mobile phase: acetonitrile (solvent A) andtrifluoroacetic acid pH 2.5 (solvent B) was developed andused according to the following program: isocratically 10%A for 15min, linear increment starting with 10–33%A in10min, 35% A during next 5min and the return to theinitial conditions within 10min with a flow rate of 1mL/min. The eluate was detected using a Waters 996photodiode-array detector. The phenolic acids and flava-nones were identified by comparing their UV spectra andretention times with those of corresponding standards andby the spiking of samples with the appropriate standard.Quantification of phenolic acids was carried out at 320 nmand flavanones at 280 nm using the external standardmethod.

2.3. HPLC determination of free phenolic acids after SPE

Free phenolic acids were extracted from the juiceaccording to the procedure of Suarez et al. (1996) withsome modifications. Briefly, 5mL of juice adjusted to pH2.0 was passed through the C18 columns (J. T. Baker,Philipsburg, NJ, USA) which had been pre-conditioned

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with 12mL of methanol and 5mL of 0.01M HCl. Thecolumn was then washed with 3mL of 0.01M HCl.Adsorbed fractions were eluted with 6mL of methanoland evaporated to dryness under reduced pressure. Theresidue was dissolved in 1mL of methanol and 10 mL wasinjected onto the HPLC column. The recoveries of caffeic,ferulic, sinapic and p-coumaric acids were 96%, 99%, 91%and 90%, respectively.

The HPLC analyses of free phenolic acids after separa-tion of them by SPE were performed on Waters 600E high-performance liquid chromatograph equipped with Nova-Pak C18 column (150� 3.9mm, 5mm, Waters, Millford,MA, USA) and mBondapack C18 cartridge guard column.A gradient of mobile phase: acetonitrile (solvent A) anddemineralized water containing 20mL of acetic acid perlitre (solvent B) was developed and used according to thefollowing program: linear increment starting with 5–15% Ain 35min, from 15% to 100% A in 2min, 100% A duringnext 7min, and the return to the initial conditions withinnext 10min. Flow rate was 0.6mL/min. The eluate wasdetected using a Waters 916 photodiode-array detector. Thephenolic acids were identified by comparing their UVspectra and retention times with that of correspondingstandards and by the spiking of samples with appropriatestandard. Quantification of phenolic acids was carried outat 320nm using the external standard method.

2.4. HPLC determination of total phenolic acids

In order to determine the total phenolic acids (free andbound), juice samples were hydrolyzed in 2N NaOH for4 h at room temperature in the darkness in an argonatmosphere, as described by Rouseff et al. (1992). Thereaction mixture was acidified to pH 2.6 with concentratedphosphoric acid and centrifuged at 4500 rpm for 15min.Then supernatant was extracted twice for 15min with10mL of ethyl acetate. Afterwards, extracts were combinedand evaporated to the dryness and dissolved in 1mL ofmethanol. The HPLC injection volume was 10 mL. TheHPLC analysis was carried out as described for freephenolic acids after SPE technique. The recoveries ofcaffeic, ferulic, sinapic and p-coumaric acids were 85%,93%, 94% and 89%, respectively.

2.5. Determination of total polyphenol content using

Folin–Ciocalteu assay

Total polyphenols were determined by the Folin–Cio-calteu method (Singleton and Rossi, 1965) using caffeicacid as the standard. The results are expressed in mg ofcaffeic acid equivalents per 1 L of juice.

2.6. HPLC determination of vitamin C

The extraction method was adapted from Ross (1994).A juice sample (0.5mL) and 10% metaphosphoric acid(MPA) (0.5mL) were mixed using vortex (5min), centri-

fuged at 10,000 rpm for 5min and the supernatant wasinjected onto the HPLC column. Vitamin C was deter-mined as described previously (Gliszczynska-Swig"o andTyrakowska, 2003) using Waters 600 high-performanceliquid chromatograph (Waters, Millford, MA, USA)equipped with LiChrospher C18 (250� 3.9mm, 5mm,Merck, Darmstadt, Germany) fitted with the same guardcolumn. A gradient of mobile phase methanol (solvent A)and 5mM KH2PO4 pH 2.65 (solvent B) was used accordingto the following program: linear increment starting with5–22% A in 6min and the return to the initial conditionswithin next 9min with the flow rate 0.8mL/min. The eluatewas detected using a Waters 996 photodiode-array detectorset at 245 nm. Vitamin C was identified by comparing itsUV spectrum and retention time with that of standard.Quantification of vitamin C was carried out using theexternal standard method.

2.7. Determination of antioxidant activity

The antioxidant activity of the orange juices wasevaluated by DPPH free radical-scavenging method andferric reducing antioxidant power (FRAP) assay.The DPPHd free radical-scavenging activity measure-

ments were carried out according to the procedure ofSanchez-Moreno et al. (1998) with some modifications.Briefly, 0.1mL of juice sample (diluted with distilled waterand centrifuged) was added to 2.46mL of 1,1-diphenyl-2-picrylhydrazyl radical (DPPHd; 0.025 gL�1 in 50% etha-nol) and mixed by vortex for 5min. The absorbance of thesamples was measured at 515 nm every 1min for 5minusing the spectrophotometer Genesys 2 (Milton Roy,USA). For each sample, three separate determinationswere carried out. The antioxidant activity was expressed asthe percentage of decline of the absorbance after 1min,relative to the control, corresponding to the percentage ofDPPHd that was scavenged. The percentage of DPPHd,which was scavenged (%DPPHd

sc) was calculated using

%DPPHdsc ¼ ðAcont � AsampÞ � 100=Acont,

where Acont is the absorbance of the control, and Asamp theabsorbance of the sample.The FRAP assay is based on the reduction of Fe3+-

TPTZ complex to the Fe2+-TPTZ by the presence ofantioxidants (Benzie and Strain, 1999). The juices werediluted 15-fold, and 50 mL of samples were added to 950 mLof FRAP reagent. The absorbance at 593 nm was measuredafter 4min. A standard curve was prepared using differentconcentrations of FeSO4. The results were expressed as mM

of FeSO4.

2.8. Statistical analysis

Data are presented as mean7SD of at least threeseparate determinations for each juice sample. Statisticalanalyses were performed using Statistica 5.5 (StatSoft, Inc.,2000) program). All data were submitted to two-way

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analysis of variance (ANOVA). Differences between freshand stored juice were evaluated by the Duncan test at 5%levels of significance.

3. Results and discussion

3.1. Polyphenol and vitamin C content of fresh juices

The content of phenolic acids (free and total) infresh orange juice of two different brands are presentedin Table 1. Free hydroxycinnamic acids (HCA) wereanalysed quantitatively by HPLC method without andwith fractionation and cleaning juice samples using a solid-phase extraction (SPE) technique. To the best of ourknowledge, this is the first study in which the comparisonof these two methods used in determination of phenolicacids in orange juice has been presented.

As expected from previous studies (Peleg et al., 1991;Fallico et al., 1996), four HCA were identified: caffeic,p-coumaric, ferulic and sinapic acids. The amount of thesecompounds was differentiated depending on the method of

Table 1

Changes in the content of hydroxycinnamic acids (free and total) in orange ju

Treatment Months of

storageCaffeic p-couma

18 1C 28 1C 38 1C 18 1C 28 1C

Orange juice 1

Direct injection (free) 0 8.2a 0.5a

2 7.8a 7.1b 4.4b 0.5a 0.5a

4 7.9a 7.2b 2.8c 0.6b 0.9b

6 7.5b 6.0c 2.1d 0.8c 1.5c

SPE (free) 0 1.5a 0.4a

2 1.3b 1.2b 0.5b 0.4a 0.5a

4 1.2c 1.2b 0.2c 0.6b 0.7b

6 1.2d 0.9c 0.2d 0.8c 1.5c

Hydrolysis (free and bound) 0 1.6a 8.3a

2 2.0b 2.1b 2.1b 8.1a 8.1b

4 2.2c 2.3c 2.1b 7.2b 6.1c

6 2.3c 2.5d 2.0b 6.4c 6.0c

Orange juice 2

Direct injection (free) 0 8.4a 0.4a

2 7.9b 7.2b 5.2b 0.4a 0.4a

4 7.2c 7.1b 3.3c 0.4a 0.8b

6 7.2c 5.9c 1.7d 0.6b 1.5c

SPE (free) 0 1.3a 0.4a

2 1.2a 1.1b 0.7b 0.5a 0.5b

4 1.1b 1.1b 0.4c 0.5a 1.2c

6 1.0b 0.8c 0.2d 0.8b 2.0d

Hydrolysis (free and bound) 0 2.2a 10.3a

2 2.6b 2.8b 2.9b 10.0b 9.3b

4 2.8c 3.0c 3.1b 9.9b 8.2c

6 3.0d 3.3d 2.3c 8.9c 8.0c

a–d: Means in each column within each treatment and product with different le

Po0.05; 3 determinations were done for HCA, flavanones, total polyphenol co

vitamin C.

sample preparation used. Content of total free phenolicacids in fresh juices after SPE technique and withoutcleaning of sample ranged from 2.6 to 3.7 and from 8.9 to10.0mg/L (Table 1), respectively. A few samples of juiceswithout cleaning and after SPE were analysed using bothHPLC methods (Sections 2.2 and 2.3) to exclude that thedifferences in the content of phenolic acids in juices injectedonto HPLC column directly and after SPE are due todifferent HPLC conditions. Similar results obtained usingboth HPLC methods (results not shown) indicate that onlysample preparation significantly affect the results ofphenolic acid analysis. Caffeic acid predominated in alljuices analysed. Its concentration was significantly higherin juice samples analysed by direct injection, coveringabout 82–94% of the total amount of phenolic acids,whereas samples after SPE technique showed only45–50%. This difference could be caused by the lack ofseparation of caffeic acid from the other compounds withsimilar UV spectrum that may have been present insamples analysed by direct injection. This may possiblyhave resulted in an overestimation of this acid.

ices during 6 months of storage at 18, 28 and 38 1C

Hydroxycinnamic acids (mg/L)

ric Ferulic Sinapic Total acids

38 1C 18 1C 28 1C 38 1C 18 1C 28 1C 38 1C 18 1C 28 1C 38 1C

0.6a 0.7a 10.0a

0.5a 0.6a 0.7a 0.8b 0.7a 0.9b 3.6b 9.6b 9.2b 9.3b

1.6b 0.7b 0.9b 1.0c 0.8b 1.5c 4.9c 10.0a 10.4c 10.4c

3.6c 1.4c 2.5c 5.6d 0.4c 0.4d 0.4d 10.1a 10.5d 11.7d

0.8a 0.9a 3.7a

0.5b 1.0b 1.1b 1.2b 1.2b 1.2b 2.5b 3.9b 4.0b 4.7b

1.2c 1.1c 1.8c 2.3c 1.7c 2.8c 4.0c 4.6c 6.5c 7.8c

3.7d 3.1d 3.4d 6.3d 0.6d 0.5d 0.5d 5.7d 6.3d 10.7d

52.4a 7.4a 69.6a

7.5b 51.2a 50.0a 48.0b 7.4a 6.6b 5.7b 68.7a 66.9a 63.3b

4.3c 50.5a 43.1b 40.1c 7.4a 5.6c 4.5c 67.4a 57.1b 51.0c

4.3c 47.3b 40.3c 38.3c 6.5b 4.6d 3.3d 62.5b 53.4c 47.86d

0.1a 0.1a 8.9a

0.4b 0.1a 0.1a 0.1b 0.1a 0.2b 1.2b 8.4b 7.9b 7.01b

1.8c 0.1a 0.2b 0.7c 0.1a 1.1c 3.0c 7.8c 9.2c 8.8c

3.7d 1.4b 2.2c 4.5d 0.4b 0.3d 0.3d 9.6d 10.0d 10.3d

0.4a 0.5a 2.6a

0.5b 0.4b 0.5b 0.6b 0.6b 0.7b 1.1b 2.7b 2.8b 3.0b

2.4c 2.3c 2.6c 4.1c 1.1c 1.5c 2.3c 5.0c 6.3c 9.2c

4.1d 2.7d 3.8d 4.1c 0.8d 0.6d 0.5d 5.4d 7.3d 9.0d

47.5a 9.3a 69.2a

9.3b 46.1a 44.0b 41.1b 8.8a 8.5b 6.7b 67.4a 64.6b 59.9b

6.9c 41.1b 37.5c 33.2c 8.3b 7.2c 6.1c 62.1b 55.9c 49.3c

6.5d 37.9c 36.1c 32.1c 7.6c 6.8d 4.9d 57.5c 54.5c 45.8d

tters are significantly different according to Duncan’s multiple range test at

ntent and antioxidant activity per carton; 4 determinations were done for

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It is well known that HCA in citrus juice occur mainly inester forms, and that they can be released by alkalinehydrolysis. The concentration of total phenolic acids (freeand bound) in the first brand of orange juice was69.672.0mg/L, and in the second brand 69.271.7mg/L(Table 1). Ferulic acid was the major compound of thetotal phenolic acids fraction followed by p-coumaric,sinapic and caffeic acid, which is in agreement with theliterature data (Peleg et al., 1991; Fallico et al., 1996;Rapisarda et al., 1998). It was found that the level of totalcaffeic acid in the first juice is about 4-fold lower than thatof free caffeic acid analysed by direct injection (Table 1).There are two possible reasons for these different results.The first one is that the oxidation of o-diphenols such ascaffeic acid in alkaline solution may lead to the loss of thesecompounds. Thus, the reported amounts of caffeic acidafter alkaline hydrolysis may be lower than the realconcentration (Swatsitang et al., 2000). The second reasoncould be the overestimation of free caffeic acid usingHPLC with direct injection due to the lack of separation ofcaffeic acid from the other compound with similar UVspectrum. The second explanation seems to be moreprobable, taking into account that the content of totalcaffeic acid in the second juice was, as expected, higherthan the content of free caffeic acid.

The major flavonoid compounds in citrus juices areflavanone glycosides. According to Kanitsar et al. (2001)and our studies (results not shown), determination offlavanones in citrus juices does not require pre-concentra-tion and cleaning of samples; thus the SPE technique wasnot used. The results of the qualitative and quantitativeanalyses of flavanones in fresh orange juice of two differentbrands are presented in Table 2. Flavanones were presentin the orange juice at higher concentrations than hydro-xycinnamic acids. Of the five flavanones analysed (narir-utin, hesperidin, didymin, naringin and neohesperidin),naringin and neohesperidin were not detected. The total

Table 2

Changes in the content of flavanones in orange juices during 6 months of stor

Months of storage

Narirutin Hesperidin

18 1C 28 1C 38 1C 18 1C 28 1C 3

Orange juice 1

0 70.2a 76.9a

2 69.3a 69.6a 70.7a 68.6b 68.2b 7

4 70.2a 69.7a 69.3a 70.5b 69.6b 7

6 67.6b 67.4b 67.5b 68.0b 69.8b 7

Orange juice 2

0 50.6a 75.9a

2 49.6a 50.4a 50.0a 68.1b 68.3b 6

4 49.6a 50.9a 50.7a 68.2b 68.9b 6

6 48.4b 49.2b 48.7b 69.1b 66.2b 6

a–c: Means in each column within each product with different letters are sign

determinations were done for HCA, flavanones, total polyphenol content and

content of flavanones in the first juice was 157.074.4mg/L;in the second juice it was 133.475.1mg/L. The concentra-tion of narirutin (70.271.5 and 50.671.8mg/L) is in goodagreement with previous findings (Tomas-Barberan andClifford, 2000; Gil-Izquierdo et al., 2002), while didymincontent (9.9070.5 and 6.970.3) was found to be lowerthan that previously reported by Gil-Izquierdo et al.(2002), namely 37–70mg/L. The level of hesperidinreported here (76.972.8 in the first juice, and 75.973.2mg/L in the second juice) is lower than the rangereported by Kanitsar et al. (2001), 127–219mg/L, and byTomas-Barberan and Clifford (2000), 104–637mg/L. How-ever, these authors reported concentration of hesperidin infresh, hand-squeezed and unpasteurized orange juices.Belajova and Suhaj (2004) published a hesperidin level of44–56mg/L in commercial orange juices. Robards et al.(1997) and Tomas-Barberan and Clifford (2000) reportedthat a hesperidin level in the orange juice depends on anextraction method, technological treatment and storage.It was suggested that the content of this compound in juiceobtained from concentrated products may be lower than infresh unpasteurized juice.The content of total polyphenols in fresh orange juices

(Table 3), calculated simply as the sum of total HCA andflavanones determined by HPLC, was found to be226.776.4mg/L (juice 1) and 202.776.8mg/L (juice 2).The concentration of total polyphenols determined byFolin–Ciocalteu method was higher than the concentrationobtained by HPLC method—684.271.0 for juice 1 and634.670.9mg of caffeic acid equivalents/L for juice 2. Ourresults are in good agreement with those reported in theliterature (Rapisarda et al., 1999; Gardner et al., 2000);however there is evidence that the spectrophotometricmethod overestimates the polyphenolic content as com-pared to the chromatographic method. This can beexplained by the lack of selectivity of Folin–Ciocalteureagent (Escarpa and Gonzalez, 2001), which reacts not

age at 18, 28 and 38 1C

Flavanones (mg/L)

Didymin Total flavanones

8 1C 18 1C 28 1C 38 1C 18 1C 28 1C 38 1C

9.9a 157.0a

0.2b 9.9a 9.6a 9.7a 147.9bc 147.4b 150.6b

1.2b 10.2a 9.9a 9.8a 150.9b 149.2b 150.3b

1.1b 10.1a 9.8a 10.1a 145.7c 147.1b 148.6b

6.9a 133.4a

8.7b 6.7a 6.8a 6.7a 124.4b 125.5b 125.5b

8.8b 6.7a 6.9a 6.6a 124.5b 126.6b 126.1b

6.9b 6.9a 6.9a 6.9a 123.4b 122.3b 122.5b

ificantly different according to Duncan’s multiple range test at Po0.05; 3

antioxidant activity per carton; 4 determinations were done for vitamin C.

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Table 3

Changes in the content of vitamin C, total polyphenols measured by Folin-Ciocalteu method, total polyphenols measured by HPLC and sum of total

polyphenols measured by HPLC and vitamin C in orange juices during 6 months of storage at 18, 28 and 38 1C

Months of storage Content (mg/L)

Vitamin C Total polyphenols

(Folin–Ciocalteu)aTotal polyphenols

(HPLC)bTotal polyphenols

(HPLC) and vitamin Cc

18 1C 28 1C 38 1C 18 1C 28 1C 38 1C 18 1C 28 1C 38 1C 18 1C 28 1C 38 1C

Orange juice 1

0 408.5a 684.2a 226.7a 635.2a

2 385.4b 336.6b 265.6b 639.5b 619.5b 582.8b 216.6b 214.5b 213.9b 602.0b 551.1b 479.6b

4 354.0c 302.0c 145.1c 633.4c 603.4c 551.2c 218.2b 206.3c 204.3c 572.3c 508.3c 349.4c

6 333.0d 283.6d 83.5d 656.6d 622.8 573.2d 208.3c 200.5d 196.5d 541.3d 484.0d 279.9d

Orange juice 2

0 361.5a 634.6a 202.7a 564.2a

2 340.2b 311.3b 202.4b 615.0b 594.2b 568.6b 191.8b 190.1b 185.4b 532.0b 501.4b 387.7b

4 303.5c 270.4c 127.4c 595.6b 570.0c 506.8c 186.5c 182.4c 175.5c 490.0c 452.8c 302.9c

6 280.2d 244.2d 63.1d 622.5c 573.9c 519.8d 180.9d 176.8d 168.2d 461.1d 420.9d 231.3d

a–d: Means in each column within each product with different letters are significantly different according to Duncan’s multiple range test at Po0.05; 3

determinations were done for HCA, flavanones, total polyphenol content and antioxidant activity per carton; 4 determinations were done for vitamin C.aTotal polyphenol content measured by Folin–Ciocalteu method, expressed as caffeic acid equivalents.bTotal polyphenol content measured by HPLC, calculated as sum of total HCA and flavanone content.cSum of total polyphenols content measured by HPLC and vitamin C.

Table 4

Changes in the antioxidant activity (measured by DPPH and FRAP

assays) in orange juices during 6 months of storage at 18, 28 and 38 1C

Months of storage Antioxidant activity

DPPH (%) FRAP (mM)

18 1C 28 1C 38 1C 18 1C 28 1C 38 1C

Orange juice 1

0 49.2a 7.5a

2 50.9b 53.2b 42.5b 7.2b 6.5b 5.5b

4 46.7c 41.1c 28.9c 6.4c 6.0c 4.6c

6 42.2d 27.4d 9.5d 6.1d 5.1d 3.3d

Orange juice 2

0 47.5a 7.1a

2 48.6b 45.6b 41.6 6.9a 6.1b 5.5b

4 43.1c 37.3c 26.1c 6.0b 5.4c 4.4c

6 36.9d 25.5d 8.7d 5.4c 4.5d 3.0d

a–d: Means in each column within each product with different letters are

significantly different according to Duncan’s multiple range test at

Po0.05; 3 determinations were done for HCA, flavanones, total

polyphenol content and antioxidant activity per carton; 4 determinations

were done for vitamin C.

only with phenols but also with other reducing compoundssuch as carotenoids, amino acids, sugars and vitamin C(Vinson et al., 2001). However, this method has beenshown to be a useful analytical tool for the routine analysisof polyphenols and it is widely used in many laboratoriesfor the determination of differences among fruits andvegetables and their products (Bartolome et al., 1998).

Orange juices are a rich source of vitamin C, which is animportant antioxidant in these juices (Miller and Rice-Evans, 1997; Rapisarda et al., 1999; Gardner et al., 2000;Arena et al., 2001). Concentration of vitamin C is asignificant indicator of orange juice quality, and it mayserve as an indicator that all processes, which ensure a highquality of the product, have been applied in the productionprocesses (Post, 1998). In both juices analysed in this study,the vitamin C content was found to be similar: 408.570.9and 361.571.8mg/L in juice 1 and 2, respectively (Table 3).Our previous study, where 14 commercial orange juicesdelivered by different producers were assessed, showed thatthey may contain from 150 to 440mg of vitamin C in 1L ofjuice (Gliszczynska-Swig"o et al., 2004).

3.2. Antioxidant activity of fresh juices

In our study, the antioxidant activity of orange juice wasevaluated using DPPH free radical-scavenging and ferricreducing antioxidant power (FRAP) assays. Both methodsare recommended by many authors as easy and accurateassays for measuring the antioxidant activity of fruit juices.Table 4 presents the results of the antioxidant activityobtained for orange juices tested in the present study. Inthe DPPH assay, the scavenging activity of juice was49.270.4% juice (1) and 47.570.3% juice (2) scavenged

DPPHd radical. These results are in agreement with thosereported in our previous study (Ma"ecka et al., 2003), inwhich orange juices scavenged 33–49% of DPPHd radical.The antioxidant activity of the orange juices tested was alsodetermined using FRAP assay. Juices analysed in thisstudy had ability to reduce Fe3+ to Fe2+. The FRAPvalues of fresh juices was 7.570.2mM of FeSO4 for the firstjuice and 7.170.1mM for the second one. Juice whichshowed a higher antioxidant activity contained higherconcentration of vitamin C and phenolic compounds.

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Gardner et al. (2000) observed that both vitamin C andtotal polyphenols in fruit juices strongly correlate with theantioxidant capacity determined in FRAP assay.

3.3. Effect of time and temperature of storage on the content

of hydroxycinnamic acids and flavanones

In the literature, little information is available on thechanges in individual phenolic constituents of orange juiceduring storage. For orange juices, mostly experimental, aninfluence of storage time and temperature on the content offerulic and p-coumaric acids was investigated (Naim et al.,1988; Fallico et al., 1996). To the best of our knowledge,this is the first study showing the changes in flavanonecontent in commercial orange juice during storage over along time period.

The content of phenolic acids, flavanones and totalphenolics in stored orange juice is presented in Tables 1–3.It was found that free and total phenolic acids wereaffected most by duration and temperature of storage(Table 1). As with fresh juice, changes in free HCA presentin stored juices were analysed after direct injection and SPEtechnique. The changes in HCA content in both storedjuices using both methods were found to be similar. During6 months of storage at 18, 28 and 38 1C, the free caffeic acidcontent in orange juices significantly decreased, while anincrease in the concentration of p-coumaric and ferulicacids was observed during the same period. This increaseresulted from the release of free acids from their boundforms during storage. The concentration of sinapic acid injuices stored for 4 months increased, whereas after this timeits concentration significantly decreased. The results pre-sented for ferulic and p-coumaric acids are in line with thedata obtained by Fallico et al. (1996) and Naim et al.(1988). Fallico et al. (1996) showed that the content offerulic and p-coumaric acids in freshly squeezed bloodorange juices stored for 4 months at 25 1C increased from1.0 to 3.2 and from 0 to 5.2mg/L, respectively. In the studyof Naim et al. (1988) who stored single-strength orangejuice for 28 days at 35 1C, the concentration of ferulic acidincreased up to 3.7mg/L. In our previous study (Klimczakand Ma"ecka, 2001) fresh orange juice contained 0.8mg/Lof free ferulic acid. After 6 months of storage at 18, 28 and38 1C the content of this compound increased to 1.5, 3.2and to 4.5mg/L, respectively.

As regards total HCA (free and bound) (Table 1), onlythe amount of total caffeic acid in stored juices increased ascompared to fresh juices. The total content of the otherindividual HCA (free and bound) in stored juices as well astotal HCA (calculated as a sum of individual total acids)decreased upon storage. The decrease in total phenolicacids was observed, whereas the content of free phenolicacids increased as a result of their release from the esterforms. After 6 months of storage at 18, 28 and 38 1C thecontent of total HCA decreased by about 13%, 22% and32%, respectively. These results are in agreement with thedata obtained by Fallico et al. (1996); however they

presented only changes in total ferulic and p-coumaricacids. In their study, a 35% reduction in concentration ofthese compounds in blood-orange juices stored for 4months at 25 1C was observed.In the literature, little information is available on the

changes of flavanone content in orange juice duringstorage. The effect of chilled storage (4 1C over 10–15days) of freshly squeezed orange juice on flavonoid contenthas been already reported (Del Caro et al., 2004; Sanchez-Moreno et al., 2003). However, there is lack of dataconcerning the influence of long-term storage on thesecompounds in commercial orange juices. Table 2 shows thechanges in flavanone concentrations upon storage. A slightdecrease in narirutin and hesperidin content was observed.Storage conditions appeared to have no significant effecton the didymin content in analysed samples. None of theflavanones changed with temperature. Only the influence oftime was significant, although the small loss (about 6.5%)of total flavanone content after 6-months of storage at alltemperatures was observed. The small changes in indivi-dual and total flavanone content could be explained by thehigh stability of these compounds.

3.4. Effect of time and temperature of storage on the total

phenolic content

As it was shown in Table 3, time and temperature ofstorage significantly affected the total polyphenol content asdetermined by Folin–Ciocalteu assay. There was a signifi-cant decrease in total polyphenols during 4 months underthe experimental conditions applied. After 4 months ofstorage at 18, 28 and 38 1C the content of these compoundsdecreased by 7%, 11% and 20%, respectively. At the end ofstorage, both brands of juices showed a significant increasein total phenolic content (Fig. 1A). It is possible that duringjuice storage, some compounds are formed that react withFolin–Ciocalteu reagent and significantly enhance totalphenolic content (Vinson et al., 2001).In the light of the above limitations, it seems to be more

appropriate to determinate total phenolic content, as wellas their changes upon juice storage, by HPLC method. Inour study, in contrast to the total phenolic contentmeasured by Folin–Ciocalteu method, total phenolic(calculated simply as the sum of the total HCA andflavanones) and vitamin C content decreased significantlywith time and temperature of storage (Fig. 1B and Table 3).A 6-month storage of orange juices at various temperaturesresulted in an approximately 10–22% decline in totalphenolic and vitamin C content.

3.5. Effect of time and temperature of storage on vitamin C

content

According to the literature data, the content of vitaminC in different juices decreases during storage, dependingon storage conditions, such as temperature, oxygen andlight access (Kabasakalis et al., 2000; Zerdin et al., 2003).

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0 2 4 6

0 2 4 6

300

400

500

600

700

800

d

c

c

cb

b

b

b

b

a

Tota

l pol

yphe

nols

[m

g/l]

Time [months]

300

400

500

600

700

800

d

dc

c

d

b

c

b

ba

Tota

l pol

yphe

nols

+ v

itam

in C

[m

g/l]

Time [months](B)

(A)

Fig. 1. Changes in (A) total polyphenols (measured by Folin–Ciocalteu)

and (B) sum of total polyphenols (measured by HPLC) and vitamin C in

orange juices upon storage at: 18 1C (K), 28 1C (’) and 38 1C (m).

Among all compounds analysed, vitamin C was the onemost affected by temperature and time of storage (Table 3).Increase of temperature by each 10 1C caused a distinctdecrease in the concentration of this compound. After 6months of storage at 18, 28 and 38 1C the content ofvitamin C decreased by 21%, 31% and 81%, respectively.However, the content of ascorbic acid in juices after 6months of storage at 18 and 28 1C and even after 12months at 18 1C (results not shown) was as the producerdeclared. The vitamin C content dropped below this valuein juices stored at 38 1C for 4 months. The results presentedare in line with the data obtained by Kabasakalis et al.(2000) and Arena et al. (2001) who reported a decrease inascorbic acid content in commercial orange juices after 6months of storage at room temperature by 29% and inreconstituted from concentrate blood-orange juice storedat 25 1C during 60 days by about 25%, respectively.

3.6. Antioxidant activity of stored juices

Table 4 presents the changes in the antioxidant activityof orange juice samples measured by DPPH and FRAP

assays. In the test with DPPHd radical, there was a slightincrease in antioxidant capacity during the first 2 months ofstorage at 18 and 28 1C. In the same period at 38 1C, theantioxidant activity decreased by 13%. The resultspresented are in line with the data obtained by Arena etal. (1999) and Piga et al. (2002). Arena et al. (1999) showedthe increase in the antioxidant activity after 2 months ofstorage in orange juices reconstituted from concentrate.According to Piga et al. (2002), storage of mandarin juicesduring 15 days at 4 1C also resulted in the increase in theDPPHd antioxidant activity. In contrast to Piga et al.(2002), Del Caro et al. (2004) described a slight decrease inthe TEAC (trolox equivalent antioxidant capacity) valueobtained by DPPH method for orange juice stored in thesame conditions. If the decrease in the antioxidant activitymay be linked to a lower content of phenolic compoundsand vitamin C in stored juice as compared to fresh, theincrease in the antioxidant activity is usually ascribed toMaillard’s reaction products (Anese et al., 1999). Theantioxidant activity of juices analysed in the present studydecreased by 18%, 45% and 84% after 6 months of storageat 18, 28 and 38 1C, respectively.The FRAP values also declined upon storage; but for

juices stored at 28 1C and 38 1C the changes were lower ascompared to DPPH assay. At the end of storage at 18, 28and 38 1C the FRAP values were reduced by 23%, 34%and 57%, respectively.The reduction of the antioxidant activity, measured with

the use of ABTS �+ radical cation, in orange juice stored upto 60 days at 20 1C, was reported by Arena et al. (2001).From the data presented by Gliszczynska-Swig"o andTyrakowska (2003), it follows that storage of apple juicefor 11 months at room temperature results in the decreasein the TEAC value by about 6–14%.Fig. 2 illustrates the rate of decline of the FRAP value,

vitamin C and total phenolic content determined by HPLCmethod in orange juices upon storage as compared to freshjuices. As can be seen from this figure, the decrease invitamin C level (Fig. 2B) upon storage followed the sametrend as that obtained for the FRAP value (Fig. 2A). Thelosses for both parameters were 20–80%. At the same time,polyphenol retention was significantly better: their contentdecreased only 8–15% (Fig. 2C). These results may confirmthat the antioxidant activity of orange juice evaluated bythe FRAP method is significantly related to the vitamin Cconcentration.

4. Conclusion

The differences in quantitative determinations of freephenolic acids in tested juices were observed, indicatingthat sample preparation may affect the results obtained.For quantitative determinations of free phenolic acids, theapplied SPE technique of extraction and cleaning of thesecompounds seems to be more accurate than directinjection. The results obtained indicate that, from amongall compounds analysed, vitamin C and next free and total

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0 2 4 60

20

40

60

80

100

Cha

nges

in F

RA

P va

lue

[%]

Time [months]

0 2 4 6

Time [months]

0 2 4 6Time [months]

0

20

40

60

80

100

Cha

nges

in v

itam

in C

[%

]

0

20

40

60

80

100

Cha

nges

in to

tal p

olyp

heno

ls [

%]

(A) (B)

(C)

Fig. 2. Changes in (A) FRAP value, (B) vitamin C and (C) total polyphenols (measured by HPLC) in orange juice upon storage at: 18 1C (K), 28 1C (’)

and 38 1C (m). Changes were calculated as the percentage of the values obtained for fresh juice.

phenolic acids were most affected by time and temperatureof storage. Small changes in flavanone content wereobserved indicating a high stability of these compoundsupon storage. The decrease in polyphenol and vitamin Ccontent upon storage is reflected by the decrease in DPPHand FRAP antioxidant capacities of orange juices.Although, significant decrease in vitamin C in orangejuices stored at 18 1C was observed, these juices still retainappropriate quality with respect to vitamin C content,which is a normalized parameter.

Acknowledgements

The grant from the State Committee for ScientificResearch (Poland), 2 P06T04826, is gratefully acknowl-edged.

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